Products of fibrinolysis

Many haemorrhagic complications have been attributed to increased intravascular fibrinolytic activity. The degradation products resulting from fibrinolysis have been shown to interfere in fibrin polymerization and have been observed in defective clot formation., Although considerable knowledge has accrued concerning the mechanism of filarinolysis and the resulting products, areas of controversy and intermittent obscurity remain» The study presented herein was conducted to resolve, if possible, existing discrepancies and characterize with certainty intermediate and end proteolytic products, An effort was made to correlate experimental findings with clinical disease states. The products of serial digestion of fibrinogen by plasmin were studied by immunodiffusion, immunoelectrophoresis and gel exclusion. Two products were observed. These products were identified as the D and E fragments, originally reported by Nussenweig et al. (1960). The D fragment was represented by a band located in the beta area of the electrophoretic field. This product was formed early in the digestion process and underwent no further degradation. The second product, a migratory band, probably representing a plasmin susceptible precursor of the E fragment, was initially observed within the locus of the D fragment. However, as digestion progressed the band migrated across the electrophoretic field to the prealbumin area, remaining in this position with exhaustive proteolysis. This band represented the plasmin resistant E fragment end product, Under certain experimental conditions the hydrolysis of fibrinogen into the E fragment may apparently be reversed. Serum samples from 196 hospitalized patients were studied for the presence of fibrinolytic intermediates and end products. Pathologic products were found in 10 of the 196 samples. Of these la samples, 8 contained D fragments, whereas Z samples contained both the D and E fragments. Clinical-pathologic correlation indicates that demonstrable fibrinolytic products were associated with injury to the hepatic, renal and vascular systems. The small molecular weight E fragment was encountered only in instances of renal failure.

PRODUCTS OF FIBRINOLYSIS by Molly Tucker Maxwell A thesis submitted to the faculty of the UniveI'Sity of Utah in partial fulflllment of the requirements for the degree of Master of Science Medical Technology Program Universi ty of Utah August t 1969 Tbis Thesis for the Master of Science Degree by Molly Tucker Maxwell has been approved July, 196,S Supervisory Committee Reader. Canmittee Committee Reader, ��� c ' Head, Maj or Dep.ar-tment Jg�.���' Dean, Gr The author wishes to express her appreciation to Miss; LOI'a Allred and Dr. DeWit't T" liunter for guidance and encouI'agement in the preparation of this thes.is o Grateful acknowle.dgement is made to Dro J J" Eichwald and Dr 0 G> LaMar Robbins, Dro Ernst Edward VI" Hanly for their suggestions and criticisms" She would also like to acknowledge the assistance of MrsO' Ann Thomas- and Mrs. Teda Rollins for typing this. thes.is" Appreciation is expressed for the use of the laboratozy facilities at the Unive·rsi.ty of Utah Medical Center. Work described has been made possible by the Public Health Service, Allied Health Professions Advanced Traineeship Grant, AHT-6.8=043 o Acknowledgement is; made especially to her husband, Dr W.o Maxwell. 0 Kameron Without his s.upport and encouragement this work could not have been undertaken. iii TABLE OF CONTENTS ABSTRACT • • • • • • • G ~ .. .. 0 .. .00 INTRODUCTION o • • • 0 0 0 • 0 0 0 0 • • • 0 000 • 0 0 .. REVIEW or I.. II. III. IV. V. VI. LITERAT.UR& CLarTING MECHANISM FIBRINOLYSIS • .. o 000 000 .. 0 0 0 0 0 .. 0 0 0 000 0 0 ~ • 0 000 1 0 .. 0 .. 0 • 0 • 0 • 00. 3 • .. 0 0 0 00. • 0 0 00. 3 • 0 0 0 0 0 0 0 0 0 00.0. 0 0 000 DEGRADATION PRODUCTS OF FIBRINOGEN • 0 0 vi 0 0 0 11 • 18 EFFECTS OF FIBRINOGEN BREAKDOWN PRODUCTS ON CLOTTING CRY oFIBRIN OGEN .. • • 0 0 0 0 0 0 INCREASED FIBRINOLYSIS IN DISEASE MATERIALS AND M&THODS .. • 0 0 o .. 0 • . .. • 0 0 0 • ~ • 0 0 o 0 eo 21 • 25 0 28 31 • • .0. • • • • .. • 0 000 SERA AND PLASMA o .. 0 eo .. • 0 0 0 0 0 0 .. 0 eo 0 00 .. 31 II. FIBRINOGEN 0 0 .. 0 eo • • 0 .. .. 0 000 0 • 0 0 eo 0 32 Ill. ANTISERA .. I. ~ .. • • 0 0 ~ .. 0 • .. • .. .. • .. PLASMINOGEN ACTIVATORS AND INHIBITORS V. VI. VII. VIII. IX. x. XI. FIBRINOGEN ASSAY 0 • .. .. • .. • CRYOFIBRINOGEN DETERMINATION .. .. 0 .00 EUGLOBULIN LYSIS TIME • • o • • .. 0 IMMUNODIFFUSION • • .. 0 0 .. .. 0 • • 0 e 0 o 0 • 0 0 .. • .. 0 000 0 0 0 .. 0 0 0 0 • 0 34 o 0 000 34 0 0 o 0 0 0 .. .. 37 0 .. 39 o 0 .. 0 • GEL FILTRATION eo 0 0 0 .. .. 0 0 0 iv 0 .. .. 0 0 36 0 0 33 • o • .. 0 • • • 0 o 0 32 .00 IMMUNOELECTROPHORESI S IN AGAR GEL .. 32 00. .. • 00. IMMUNO&L&CTROPHORESIS ON CELLULOSE ACETATE 0 42 EXPERIMENTAL RESULTS I. IMMUNODIFFUSION IY. INHIB.ITION VII. 0 o 0 • 0 0 0 00. 0 000 45 0 0 0 00. 45 0 0 0 • 0 0 47 • 0 • 0 00. 47 o 000 0 0 0 • • • 000 0 0 0 0 • 0 0 0 0 0 .00 • 0 0 000 PROTEOLYTIC ACTI VIn 0 0 0 o • 000 • 0 000 0 0 .0. .00 o 0 0 ASSAYC FOR FIBRINOGEN AND CRYOFIRRINOGEN EUGLOBULIN LYSIS TIME VIII. IX. PATIENT DATA DISCUSSION 0 • 0 0 0 0 51 • 53 000 • • 58 o • • 0 0 • 0 0 0 0 • • • • 0 S,8 0 0 • 0 0 0 o 0 • • • • 000 0 • • 0 • • 0 60 0 • 0 0 0 0 0 • • 0 • • • 0 0 0 65 0 • 0 0 0 0 76 0 • 78 • • • 000 • • • • • 0 0 • 0 0 • 0 000 0 0 0 0 • 0 000 • • • 0 0 00. • • 0 • 0 0 0 • • 0 .00 0 0 • • 0 0 • 0 0 0 0 0 0 • 0 0 0 • 0 000 0 • 00. • 55 00000 • InRLIOGRAPHY • or GEL FILTRATION VI. • 0 THERMAL PROPERTIES OF FIBRINOLYTIC PRODUCTS V. VITA .00 I.MMUNOELECTROPHORESIS III. 0 000 CROSS-REACTIVltl OF PLASMIN II. SUMMARY o • 0 v • 88 ABSTRACT Many haemorrhagic complications: have been attributed to increased intravasculal' fibrinolytic activity. The- degradation products: resulting from fibrinolysis have been shown to interfere in fibrin polymerization and nave been observed in defective clot formationo Although considerable knowledge of fibrinolysis and tne h~ accrued concerning the mecnanism resulting products. areas of controver,sy and intermittent ol)scuri:ty remain. The study presented herein was: conducted to resolve. if poss;ible. existing di"screpancies and character$ze with. certainty intermediate and end proteolytic products. An effort was made to correlate experimental findings; with clinical disease states;. The products; of serial digestion of fibrinogen by plasmin were studied by immunodiffusion. immunoelectrophoresis: and gel exclusion. Two products were observed. These products were identified as- the D and E fragmenta, originally reported by Nussenweig .!!..!!~ {l960)o The D fragment was repr-esented by a band located in the beta area of the electrophoretic fieldo This product was: formed early in tbe digestion process; and underwent no further degradationo The second product. a migrtatoxy band, probably representing a plasmin susceptible. precursor of the. E fragment, was initially observed within the locus of the D fragment. However, as digestion proptessed the band migrated across. the electropboretic field t.o the pre albumin area t remaiuing in this position with exhaustive proteolysis" This band represent.ed the plasmin resistant & fragment end product. Undel' certain experimental conditions the hydl'olysis of fibrinogen into the E. fragmen.t may apparently :De reversed. Sel'um samples: from 19&, nospitallzed patients were studied fO'l.' the presence. of, Patho~ogic f~inolytic intermediates and end products. products. were found in 10 of. the 196 samples. Of tbese 10. aample&, 8 contained D fragments, whereas 2, samples contained both the D and :& fr,apenta. Clinical-patbologic correlation indicates that demonstl'able fibrinolytic products were associated wi th inj ury to the hepatic, renal and vascular systems. The small molecular weigbt E fragment was encountered only· in instances of renal failure. vii TABLES AND FIGURES TABLES .. . .. . . . 1. PROPERTIES OF D AND E FRACTIONS ... 2. IMMUNOELECTROPHORESIS OF PLASMIN . . .. . .. 3. • • • • •• 22 • • • •• 46 INHIBITION- OF THE PROTEOLYTIC ACTIVITY OF PLASMIN BY c-AMINO CAPROIC ACID • • • • • • • • • • • • • • • • • • • ... 56 4. ASSAY FOR F1BRllNOGEN AND CRYOFIBRINOGEH • • • • • • • • • •• 59 5. EUGLOBULIN LYSIS • • • • • • • • • • • • • • • • • • • • • • • 61 .. .. .. .. • FIGURES 1. SCHEMATIC REPRESENTATION OF BLOOD COAGULATION • • • • • • • • 2. A STRUCTURAL MODEL OF THE FIBRINOGEN MOLECULE •• • • • • • • 7 3. MODEL OF FULLY POLYMERIZED FIBRIN MOLECULE .. • • • • • • • • • 8 ... TERMINAL AMINO ACID RESIDUES OF FIBRINOGEN AND FIBRIN • • • • 9 ~. MULTIPLE ACTIONS OF PLASMIN • •• 14 i. DEGRADATION P-RODUCTS OF FIBRINOGEN • • • • •• 20 7. IMMUNODIFFUSION PLATE ••••••• • • • • • • • • • • • •• 38 s. IMMUNOELt.CTROPHORESlS PATTERN OF FIBRINOGEN RREAKDOWN PRODUCTS • • • • • • • • • • • .. • 9. •••••• . ..... • • • .. . . . • • .. . ... ... .. S.TR&PTOJ.<.INASE ACTIVATION OF NORMAL HUMAN PLASMA 10.. SERIAL DIG&STION OF FIB,RINOGEN BY PLASMIN • • •• 49 • •• 56 .. ........... 0 S2 11. PLASMIN DIGESTED NORMAL RUMAN PLASMA SUBJECTED TO 56 C .. • • 0 54 .. • ... 57 • ... 63 MODEL OF FIBRINOGEN DIGESTION BY PLASMIN • • • • • • • • • •• &8 16. SERIAL FIRRINOGEN ASSAYS ON P-LASMIN TREATED PLASMA • • • • •• 71 17. SERIAL FlaRINOGEN ASSAYS ON STREPTOKINASE TREATED PLASMA • • • 72 12. PATTERN OB.TAINEll ArI'ER GEL FILTRATION • 0 13. A CLASSIFICATION OF DISEASE STATES .. • • • 0 0 • • • ... . .. 14. PHYSIOLOGIC SITES INVOLVED IN DISEASE STATE,S. 1~. 0 0 • 0 .0. INTRODUCTION Bleeding diathesis secondary to intravascular thranbosis have recently been attributed to increased fibrinolytic activity~ Fibrinolysis has been defined as the dissolution of the human blood clot or fibrin by a proteolytic enzyme; whereas t fibrinogenolysis refers to the enzymatic digestion of human f'ibrinogen. Accumulati~ knowl~dge concerning a group of naturally occuring blood components primarily functioning in, fibrinolysis or fibrinogenolysis has greatly facilitated the understanding of the fibrinolytic mechanismo The, basic component of this mechanism is plasminogen. a proenzyme normally circulating as part of the globulin moiety of the plasma proteins. Plasminogen activated to plasmin t a proteolytic enzyme, is capable of degrading both fibrinogen and fibrino The products. resulting from the degradation of fibrinogen and ~' fibrin and the relationship of these products to disease states have stimulated considerable interest. Fibrinolytic products and fibrin ogen oly tic products appear to be immunologically indist 1nguishable and are, therefore, often referred to synonymously as fibrinolytic products. The end products of fibrinolysis have been defined as: (1) small polypeptide fragments accounting for approximately 3Q% of the parent fibrinogen molecule and, (2) two large molecular wei~t frasment& accounting fortne remaining 70\0 Recent studies have focused upon the characterization of the two 2 larger molecular weight fragments labelled liD" and "E"., These products have been implicat.ed in the inhibition of the enzymatic conversion of fibri~ogen to fibrin and appear to account for the abnormal clot formation often demonstrated in pet"Sons exh.i.bi ting increased fibrinolytic activity., A well defined correlation between the products of fibrinolysis and the bleeding diathesis often encountered as a result of increased fibrinolytic activity haa not been cleaX"ly delineatedo Therefore. further studies are needed to elucidate fihX"inolysis. the products of fibrinolys.is and the relationship of fibrinolytic products to disease., Immunological procedures and other methods have been employed to study (.1) the products. of fibrinolysis resulting from tbe d.egradation of fibrinogen by commercial plasmin and. '-2) the fibrinolytic products appearing spontaneously in the serum of hospitalized patientso The results of these studies t together with additional findings are presented 0 REVIEW OF LITERATURE I CLOTTING MECHANISM In recent years, it has become apparent that the fibrinolytic Pl'ocess in man is in a m01"e dynamic state than pI'eviously Ncognized and that al}el"rations in its mechanisms may contribute significantly to the pa~genes.i.s invea~igative: of disease. Such considuation bas stimulated interest that has resulted in an increased number of publications. In as much as fibrin formation' is the end product of coagulation process, consideration should he' given to the clotting mechanism. According to Hiale (1967) the clotting process, leading to the fibrin clot, is represented by four interdependent phases (see Figure 1): Phase I t in which-platelet factor 3 is. released following the breakdown of platelets during tbe iuitiatOP or'contact reaction; Phase II, in wbich plasma. thl"omhoplastin is formed; Phase III, in which thrombin is, formed from, prothrombin; and' Phase IV, in which fibrin is fomed from. fibrinogen. Many' workers. pNfel" to combine Phase I and Phase Il. since hoth phases thromboplastin. l'es.u~t in the genel'ation of Potential %'esources. for thromboplastin are the blood {intrinsicl and .tbe.~tis.sues.,'e.x:trin&ic) (Seegers, 1962). Phase I and II involve the generation of intrinsic thromboplastin from factors X'eadily- available in the blood. Following contact with a fONign surface inactive Hageman factor (.factor XII) 4 PHASE I: THE INITIATOR (CONTACT) REACTION PLATELETS PLATELET FACTOR 3 (PHOSPHOLIPID) ConlOCI (ThrombinJ "INACTlVE II CONTACT II FACTORS (Jl • XlI) ACTIVATED" CONTACT FACTORS (Xl .. Xll) INHIBITOR: NORMAL ] [ ENOOTHEL'AL SURFACE PHASE JI: THROMBOPLASTOGENESIS PLATELET PHOSPHOLIPIO .) Foctor It FACTOR Actlvoted n Co·· JZDt - - - - - - PLASMA THROMBOPLASTIN CT",olftbi"~ FACTOR IX .m: J INHIII TOR: [ ANTITHROMIOPLASTIN (S) \ FACTOR X PHASE • .D. THROMBOGENESIS T"rombopIO.,jft Co ... • PROTHROMBIN - - - - Factor lC. Focror tal Foctor % 'TiltOmb;lI) THROMBIN J INHIIITOR : [ ANTITHROM81N (S) PHASEDl: FIBRIN FORMATION --"i'""-ThrOMbin FIBRINOGEN FIBRIN [STABIl.IZER: FACTOR nIl INHIBITOR: FIBRINOGENOLYSINS} [ ANO FIBRINOl.YSINS Fig. 1. Schematic representation of the process of blood coagulation. The division into 4 phases is arbitrary and is used only as a convenient scheme for classifying the hemorrhagic disorders and the tests used to study them. (Miale, 1967) 5 is CODvertedto itaactiva fOl'ln. The. activation of factor XII is followed by tne activation afplasma thromooplastin antecedent (PTA or factor XI). In the presence of this product, plasma tbrOll'Jbo- plastin component (,pTe. or factor IX) is changed to its active form. Activated factor IX reacts with- antihemopnilfac . factor (AHF or factor VIII)., Stuart-h-ower factor 'factor X) and ionized calcium (.factor IV) to form a suilstance called intermediate. product I. Intermediate product,! reacts with-platelet pbospholipid to form intermediate pl"oduct II. Finally, intermediate product II combines wi tb proaccelerin ,Uabile:factor.OI'. f·actor V). to . form intrinsic lllood tbromboplastin '-Rodman,. 19.68}.. The generati.on of intrinsic thromboplastin is complex, time con$uming and usually involves about 60\' of the total time required. for coagulation. An altemate, more rapid route to clot formation occurs following injury to tisaues. whereby blood is. exposed to body tissues rich in intrinsic thromboplastin. Prothrombin is an' alpha globulin produced .in the liver , requiring vitamin clotting process.. l.{ for its,_ synthesis, jID.~ is consumed during the One. molecule of prothromlJinprobabl.y yields two molecules. of .thrombin, '-Rodman, liS 8 ).. Activated, purif!ad protbroml.j)in yields.thromin-at a concentration of about 2,70.0 unitslmg of dry material. Estimates indicate the concentration of prothrombin in nonnal plasma .is ahout 12 mg/100 ml (Seegers, 1967). The activation. of prothrombin t then, results in the formation 6 of thrombin, a highly specific esterase fibr~nogen acts upon the monomer. (McKay'~ 1965} 0 This: enzyme molecule to produce the resultant fibrin Fibrinogen {.factor I ~ is a plasma protein normally present at concentrations of '300. to SOO mg/100 ml plasma (Quick. 1966)0 The molecular weigh-t of this protein in man has been estimated at 350,000 (Schultze, 1966)" Electron micrographs have been obtained of the shadow cast bovine fibrinogen molecule establishing the general mOl'phology and dimensions in the dry state (see Figure 2).. The molecule consists of a linear arra:y of three nodules held together by a very tbin thread~ The two end nodules are alike. but the center one is slightly small8%'," When polymerization occurs, the fibl'inogen molecule is altered 'see Figure 3). Electron microgl'sphs of fibrin indicate the fibrinogen molecule shrinks in length during the fibrinogen-fibrin conversion. The cross bands binding the fibrinogen molecules together are also apparent (Jiall and Slayter, 1955l). Using fluorescent antibody techniques, Forman' and Barbard (1964) have shown that fibrinogen is synthesized in the Iivel' and have identified the liver parenchymal cells, as the exact site of production. Fibrinogen is formed in the liver at a rate governed by its concentrati.OD in plasma" ,When circulating fibrinogen levels are depressed .... synthesis can be enhanced by as much as eigb.t times the normal rate~ Fibrinogen and fibrin are' eanposed of six peptide chains 'see Figure 4). During the conversion' of fibrinogen to tbe o 475±25 A o 65 A o 150 A o 150 A o 65 A Fig -- 2. A structural model of" tin:! fibrinaqenll\Cre:C1"a-Ie: base-don data derived from eie'Ctron in±cro~opi-c' antl-other- p'hys-icochemical observations. (H'al1. and8:iayter, 1959) "'-J o Ii Fig. 3. 240 A .. ~ Model of fully polymerized fibrin molecule. (Hall and Slayter, 1959) CD 0 0 ALA ALA TYR TYR FIBRINOGEN I THROMBIN > t=== GLY GLY GLY GLY TYR TYR FIBRIN Fig. 4. A schematic drawing o£~the, :t:axnd.~ :.ain±uoacid-:r.eS±dttes." on't:he'p-eptide chains of fibrinogen fractions isolated from human plasma. The action of thrombin produces fibrin with identical terminal amino acid residues. The end groups found on the polypeptide chains include alanine (Ala), tyrosine (Tyr), glycine (Gly), and unidentified amino acid groups (0). (Adapted from Searcy, 1969) \0 10 fibriD mcnomer, foUX' of the teminal amino acid :residues in fibrinogen are replaced wi'tll glycine {Searcy, 1969}. action of thrombin. fihr~ogen ~gi.nyl-glycine During the bonds are disrupted in the molecu.le: aDd paptides are separated from the molecule -- {Gladnv at a1., 196.41. This action causes a loss of negative cnarges frOID tIle remaining fibrin monaners. Polymerization of the molecules occursbf electrostatic attraction. Fibrinogen-fibrin conversion takes place in stages and depends on environmental conditions such as pH and ionic strength. The fibrin monomers fil'St form an intermediate polymer i:Jy lining up' end t.o end. These polymers then line' up side to side to f'orm the subsequent course -- fibrin strands Uaatallo et. a1.. 1962al .. In 196Q, Laki !l.!!:, suggested that the tbrombin...induced conversion of fihrinogen to fibI'in clot., is a' two phase process: first, an enzyme. action modifying fibrinogen ·to. fibrin. and second, the cross linkage of fibrin molecules. Lorand and Jacobsen '-19S8) have described a factor known as the fibrin stabilizing factor' (rsr). These investigators contend that the naturally occurri:og insoluble fibrin clot is a co-polymer of fibrin and the fibrin stabilizing factor. These workers demonstrated that when fibrinogen, is clotted. ~.vitro.by thrombin in the pI'esence of calcium ions and fibrin stabilizing factor a clot is formed resembling the fibrin clot formed!2.,!!.!.2,. In the absence of fibrin atabiU.zing factor, a . ,mechanically weaker' clot is formed that can he 11. dissolved in 5M urea or monochlol'acetic acid. Upon further s.tudy t Lorand (19i5) suggested that tbe enzyme. thrombin.·baa The first a dual role in catalyzing two overlapping reactions. ~action inducest.be polymerization of fil)rinogen t and the second activates the fibrin stabilizing factor causing cross- linkage of the fibrin molecule, thus supplying the" stabilizing effect. Nussbaum and Morse (.1904) determined the fibrin stabilizing factor (FSF) aotivity in the plasma of patients with various disease states. These investigators found diminished fibrin stabilizing factor actlvity in ZS of 86 patients wi tb 11 vel' disease, 16 of which had severe cirrhosis in the tennin.al pbase of the disease. Diminished FSF activity was also found in 21 of 28 patients with metastasestotbe liver. -- Other workers (Alimi et '. a1., 1968) have., shown that f !brin clots formed in the presence of fibrin stabilizing factO%', Factor XIII, are more resistant to plasmin and other lytic enzymes than clots formed in tbe absence of Factor XIII. These workers also noted reduced fibrin stabilizing factor activity in patients with liver 4isease. suggesting 'that Factor XIII may be synthesized in the liver .. II FIBRINOLYSIS The process of fibl'in clot digestion is called fibrinolysis. Fibrinolysis undoubtedly Npresents an important mechanism in the 12 maintenance of the in't:ep"ity of the vascular lumen (McKay, 1965) • T11e dissolution of human blood clots bas been reported in the literature as • .-ly as 179-4, but. It. was not. until 1905 that Nolf t ODe of the great pioneers iD blood coagulation,. introduced tbe view that fibrinolysis was due to a proteolytic "enzyme in plasma, and that indeed the coagulation process itself· was of a proteolytic ~igin. with fibrinolysis repNSenting the end stages of the pX'Ocess. Later, Ratnolf (lQS5) modified Nolf's concept by suggesting that the fibrin proteolytic activity of plasma is activated simultaneously with the clott~ process, thus pennitting the destruction of fibrin. It is now,. however., geDerally agreed that tile processes involved in clotting are. distinct from those involved in fibrinolysis (Sherry and Alkjaersig,19S7).. A review of current concepts concerning fibrinolytic mechanisms" -follaws. -- Fibrinolysis is. contl'ol1ed in vivo by anenz.ymati.c process .. This process" involves. the conversion of the proenzyme, plasminogen, (.143.00Q molecular weight). into a proteolyti.c enz.yme, plasmin (molecular wei.gb.t l08,flOG1. inacti.ve form as part of '-Radman. 196.8l. tb~ Plasmin:ogen normally circulates in an globulin moiety of the plasma proteins When plasminogen is converted to the proteolytic enzyme, plasmin, soluble pept.ide fragments are released (Alkjaersig -- et al. t 1958a1. Actlvated plasminogen or plasmin was origiDally called f ibl'inolysin, but it bas heen demonstrated to be a potent proteolytic enzyme l'esemDifDg pepsin. This enzyme can digest a bost 13 of protein sullatancea including fihrinog8ll,fUrin, FactOl' V. FactOI' VIII. some components of complement, corticotropin, growth, hormone and glucagon.. Addi"tioaal protein subsuates hydX'olyzed by plasm.in and used for asaayi.ngplastnin are:, casein •. -gelatin, betalactoglobulin. azocal, Md. power and protamina complexes. (.Fletcher et al •• -...... ---- 1962a. ShelTy et .al •. , 19&6). Although generated plaSmin can participate .in at least three distinct reactions, pb.ysiologically the lytic action upon fibrin is favored (see Figure 5).. capable of competing with antiplasmins for plasmin. Fibrin is The latter attacks proteina other than fibrin as previ.ously described, but only when t,he lytic factor is produced in great excess (Searcy. 1969). The conversion of plaaminogen to plasmin is' an enzymatic "action rnedia'ted Ji)y activatOl' substances called kinases. Many tissues of the body .• particularly vascular intima. lung. pancreas, and prostate contain activator sullstances capable of splitting tne inactive plasminogen from the globulin molecule and converting it to its active form, plasmin 'Rodman, 1968l. Activators,' as des:cribed today. were originally considered to be. fibrinolysins. These fibrinolysins were first demonstrated in certain strains of hemolytic streptococci and later. correlated with the activation of tbe proen~ plasminogen. A substance from, filtrates of broth, cultures of certain strains of hemolytic streptococci. capable of inducing rapid fibrinolysis of human blood clots- was f 1r8t oiserved by Tillett and Garner in 1933. PLASM'INOGEN 1l88U€ l ¥SOKINASES ACTIVATORS ,BACfiEftIAL KINASES + ANT IPLASM INS, INACTIVATION' + .otf-(--~~- PLASMA PROTEINS PLASMIN""" + ' ,. PROlEOLYS.I S FIBRIN FIBRINOLYSIS Fig. 5. A simplified scheme of the multiple actions of pla'smin. (Searcy, 1969') ~ 15 Thi$ substance tIlatl named streptococcal fiDXtinolysin. Later, Milstone (19.41) demon&trated that the streptococcal fibXtinolysin would not lyse thl'ombin induced fibrin clots when the subs,trate was' highly purified human fibrinogen. Milstone noted, however, that if a small amount of euslohulin waa added from human seXtum, rapid lysis of the clot resulted. Milatone named this substance the plasma lysinS factor and reported that it interacted with s'treptococcal fibrfnelysin to form an active lysing system. Later, ot.her worKers (,Kaplan, 19.44; Christenaen,19.4S1 showed that the plasma euslobulin which Milstone had described was an inactive precursors (plasminogen) of a proteolytic enzyme (plasmin) which was rapidly activated by &tXteptecoccal fibrinolysin. CbXtistensen (1945) suggested that since tbe streptococcal substance was not a fibrinolysin, but instead an activator, that streptococcal fibrinolysin be.Xtenamed streptokinase. To date, plasminogen activator bas been.found in the plasma (plasma activator) of v8l'ious tissues, as well as in the urine (urokinase). Alkjaersig !1.!!.., (1958b) demonstrated that plasminogen is activated by uXtokinase, streptokinase, trypsin an.d autocatalytically by plasmin. Although the possible identity 01' relationship of these act! vators ispr.esent,ly unknown, the r letcher -- et al., (1962a) suggested that urokinase may represent excreted plasma activator .and that a similarity exists between tissue activatorand urokinase. This observation led to the theory tbat tissue activator is the primary source, that plasma activator is its pXtoduct 16 and that urokinase is its excretion form. Mullertz (1955) maintains there is evidence that streptokinase does not react directly with plasminogen, instead a chain reaction is initiated. Plasminogen interacts with a "proactivator", which is converted to an activator, which in turn catalyzes the plasmi~ogen-plasmin reaction. Numerous studies have been performed to characterize the plasmin activator. Troll and Sherry (1955) have shown that the activation of human plasminogen by streptokinase involves two steps: first, the stoichiometric interaction of streptokinase with a plasma activator; and second, the enzymatic activation of plasminogen by this activator. A,lkjaersig ~ al.(l959b) investigated the respective roles of plasminogen activators and plasmin by means of test systems using 1311: labeled clots containing various concentrations of plasminogen. Other studies on plasminogen activators in human plasma clots indicate that the quantity of plasminogen activators varies in response to stress, drug administration, and disease (Alkjaers.ig et Sawyer et al., 1960a; Sherry, 1959b). !.!.., 1959a; Further studies, Sawyer ~~., 1960b; Beck and Jackson, 1966, have compared the effects of plasminogen activators and proteolytic enzymes on fibrinolysis. These investigators discovered that plasminogen activators added to a fibrin clot would initiate the lysis of more fibrin per unit fibri~ogen than the addition of either plasmin or trypsin. A counter effect to proteolysis is evident in the competition for plasmin by antiplasmin (see Figure 5). Inhibitors of plasmin are found 17 in the circulating blood as well as in other tissues. McFarlane and Bi.ggs (1948) found plasmin inhibitors in saline extracts of human liver, kidney, spleen, adrenal, thyroid, muscle, lung, heart, and brain. Fletcher ~~. (1959a) showed that the concentration of plasma antiplasmin ave~ages approximately five casin inhibitory units per milliliter and exceedstheplasmi~ogen is 3.8 casein units per milliliter. demonstrated plasmi~ content of plasma, which Gallimore and Shaw (1967) inhibitors in serum by studying the lysis of fibrin clots prepared from plasminogen-deficient fibrinogen. In a system containing no plasminogen, the clot lysis time was inversely proportional to the concentration of added plasmin. When serum was included, a reduction of reciprocal lysis time resulted, the extent of which varied linearly with the amount of serum added. Recently, Jacobson (1968a) demonstrated other controls of plasmin inactivators in his description of a modified caseinolytic method to measure the proteolytic activity in plasma. His studies indicated that in normal populations there was a variation between l~rge individual plasma samples, probably due to plasminogen-plasmin levels. Of significance was the variation produced as a result of hormone influence. Increased proteolytic activity was found in pregnant women and women receiving oral contraceptives. Further studies (Jacobsen, 1968b) were made concerni.ng "proteolytic capacity" (i.e. the maximal proteolytic activity that can be generated in plasma with the inhibitors present) in several persons 18 of the same family • These studies s.ll:ggested that the property of "low proteolytic capacity" was inherited as an autosomal dominant trai t. However, no clear cut inheritance could be found concerni.ng the property of Ith:igh proteolytic capacity". III DEGRADATION PRODUCTS OF FIBRINOGEN Godal and Helle (1963) compared the influence of fibrinogenolytic and fibrinolytic products in the last st.ages of coagulation and reported that fibrinogen breakdown products prolo.ng the thrombin time of plasma more effectively than the breakdown products of fibrin. However, this may be an erroneous assumption. compared the proteolysis products of Hirsh fibri~ogen ~2.!. (1965) and of fibrin and found no qualitative or quantitative differences in their actions. From these studies it would appear that dist~nguish~ng fibrinogen breakdown products from the breakdown products of fibrin is difficult. Later work by Beller and Mak, (1967) indicated that proteolytic products derived from fibri~ogen are immuno~ogically indistinguishable from the proteolytic products of fibrin. During the past decade, ~ great deal of research has been done in an effort to characterize·the'proteolytic products of fibrinogen. Nussenwieg and co-workers (1960) described at least four antigenic . groups resulti.ng from plasmin proteolysis of fibrinogen. .!! Fletcher al. (1962) s.u.ggested that- only two of the four fractions ( D and E located in the alpha-beta electrophoretic regions) are antigenically reliable, i.e., demonstrable immunologically. During plasmin 19 digestion, intermediate breakdoWn products are formed, but the end result is plasmin-resistant fragments. fragments contained the'an~igenic smaller fragments exhibited 80me of the plasmin-resistant Group D and some Group E. nO'an~igenic qualities, Other Jamieson (1963) suggested that the two la,rger fragments (D and E) are aggr,egates of these lower molecular weight pieces. Other workers have shown by density gradient centrifugation that during proteolysis of fibrinogen molecular components were formed with respective values of 5.68,6.278, 3.08, and 1.48 (Fischer 1963; and Alkjaersig and Fischer, 1964). ~~., The two larger fragments (D and E) contain individual antigenic determinants that are resistant to further action by plasmin. These fragments have been isolated by a number of workers using':various techniques: continuous flow paper electrophoresis, (Triantaphyllopoulas and Triantaphyllopoulas, 1962); gel titration chromat.ography.on 8ephadex(R) (Laursen and Gormsen, 1967; Nilehn, 1967a); simple radial diffusion, turbidimetric assays (Beller' and Maki,1967); immunoelectrophoresis (Lewis and Wilson, 1964; Nilehn and Nilsson, 1964;'and Nilehn, 1967b) and plasma electrophoresis in narrow pore acrylamide, gel (Fletcher, 1965). Recently, Kowalski (1968) summarized these studies characterizing the physical and immunologic properties of the ~egradation products of fibrinogen and proposed a simplified scheme of the reactions representing the sequence of events occurri,ng during plasmin fibrinolysis. (see Figure 6) Two main groups of products were PL F ---.;. x + PPI PL Y+ F = PL = PP = -PLPP2 D + E PP3 'PL --- D+ E + PP4 fibrinogen plasmin small polypeptides or peptides X AND Y ='< Fig. 6. Adapted from Kowalski (1968). N o 21 recognized: small polypeptide fragments, soluble in trichloroacetic acid (TCA), and high molecular weight fragments. fragments, formed in the course of fibri~ogen The TCA-soluble proteolysis by plasmin, were released at a steady rate during the reaction. After exhaustive proteolysis, these fragments contained about 30 per cent of the total nitrogen derived from fibrinogen. The fragments of high molecular weight underwent a number of changes leading to the formation of several intermediates. These intermediates underwent further proteo- lysis to form the two fragments referred to by Nussenwieg as "D" and "E". (see Table I) Fragment D is thermolabile and has a reported molecular weight of 80,000. Fragment E is thermostabile and has a molecular weight of 30,000. One mole of fibrinogen (molecular weight 350,000) during proteolysis yields two moles each of D and E fragments. IV EFFECTS OF FIBRINOGEN BREAKDOWN PRODUCTS ON CLOTTING In 1959b Fletcher !!~. demonstrated that purified fibrinogen, partially digested by plasmin, clotted slowly on treatment with thrombin, and when added to normal fibrinogen inhibited clotting by thrombin. These investigators suggested that the accumulation of fibrinogen breakdown products and the development of defective fibrin polymers is a significant abnormality, and that the biochemical lesion of defective fibrin polymerization may be comparatively common and relevant 'to the hemorrhagic complication encountered in many diseases. Table 1. properties of D and EFractions, MOLECULAR WEIGHT SED IMENTAT IOff , COEFFICIENT THERMAL PROPERTIES D FRAcrrON 80,000 5.6 S THERMOLABILE E FRACTION 30,000 5.27 S THERMOSTABILE DEGRADATION PRODUCTS' ~ ~ 23 Kopec et ale (1960)· associated fibriD:ogen breakdown products with antithrombin activity and su.ggested that an increase of degradation products is the·· intermediate cause of delayed clotti.ng. These investigators proposed· that the nature of the inhibitory action of degradation products may· be two-fold: in the strict sense, i.e~ (1) antithrombin activity inhibition of the activity of thrombin; and (2) inhibition of the pOlymerization of fibrin monomers. Latallo et ale (1962c) further confirmed that fibrinogen proteolysis products interfere with fibrinogen-fibrin conversion at the stage of fibrin polymerization and gel formation. Alkjaersig et ale (1962) suggested that a single large molecular weight fibrinogen fragment, resistant to enzymatic digestion by plasmin, is predominantly responsible for the inhibition of fibrin polymerization. Fibrinogen degradation products appear to influence platelet activity as well as fibrin formation. In 1964 Kowalski et ale reported that digestion of" platelet rich plasma with streptokinase and plasmin diminished the aggregation of platelets. This treatment also diminished the adhesive properties of platelets to glass and connective tissue fibers (collagen). Platelet aggregation and adhesiveness is elicited by thrombin (Kowalski, 1968). (Kopec ~ !!.!.., Later studies 1966) indicated that platelet .aggr.egation could also be induced by solutions. of fibrinogen preparations; however, the addition of fibrinogen degradation products to platelet suspensions inhibited fibrinogen-induced aggregation. Jerushalmy and Zucker 24 (1966) dist~nguished.inhibitory effects of early and late products of fibrinogen degradation on platelets. These workers have shown that str~nger early degradation products produced inhibitory effects on platelet aggregation than-late degradation products. Release of adenine nucleotides from platelets upon addition of thrombin was also found to'be--decreased in the presence of fibrinogen degradation products. In this instance the early products also appeared to be more effective 'inhibitors than the late products (Kopec !! ~., 1966; Wilson!!!.!.., 1968). Kowalski ~ al. (1946b) worked with dogs infused with -strepto- kinase-plasminogen to induce the formation of fibrinogen breakdown products. The appearance of early found circulating in the blood. profuse bleeding and pronounced fibri~ogen breakdown products was These products were correlated with prol~ngation of the bleeding time. The effect of early fibrinogen breakdown products was more pronounced than the effects of late-'breakdown products. Later work has shown the fibrinogen degradation products exhibit a maximum of anticoagulant activity at nine minutes (Nanninga, 1966). Latallo ~!!!.. (1964) clarified the role of early and late fibrinogen degradation products. These invest,igators demonstrated that the early breakdown products of fibri~ogen are plasmin susceptible and interfere with the action-of-thrombin, i.e. the enzymatic conversion of fibrinogen to fibrin monomer. The late fibri~ogen breakdown products interfered with the polymerization of the fibrin monomer and 25 are plasmin resistant. Recent investigation has indicated that the presence of fibrinogen degradation products may be responsible for defective fibrin clot formation. In 1962, B~g .!!..!!: using the electron' microscope, studied the effects of fibrinogen proteolysis products on the fibrin clot and demonstrated" defecti.ve fibrin polymerization. Low concentrations of fibrinogend.egr-adationpr-oducts produced" striking alterations in clot atZ'Ucture. Not only do pr-oteolytic products produce defective clots, but evidence also indlcates these products are actually incoZ'porated into the clot. In 1962, Latallo !!!!.. demonstrated that fibrin,ogen proteolysis products were, in fact, incorpor-ated into ., structurally abnomal clots. These workers suggested that proteolysis products inhibit polymerization' of the fibrin monomer by bonding with monoMr- units and in tbis way mask the sites essential for- nonnal polymerization. Hirsch !!!!... This t,heory was l.ater supported by {19(5) ,who indicated that large molecular weight products of fibrinogen proteolysis link with f !brin monomers and inhibit fibrin polymerization. This inhibition produced clots with abnwmal structural characteristics, including a" defect in the tensile strength of the fibrin clot. V CRYOFIBRINOGEN Shainoff and Page (1960) isolated a'product from the plasma of endotoxin tr-eated r-abbits that' corztesponded,on'the basis of its 26 peptide composition, to an intermediate product of the fibrinogenfibrin conversion. 'Since the product described precipitated in tbe cold at 4C)C, these workers suggested the term Ifcry'oprofibrin" ~ Later work (Shainoff and P,age, 1962; Sasaki ..!!.~., 1966) suggested thattbe liberation, of peptides'occurred during 'the fibrinogen exposure to ,thrombin. to form fibrin. The .,resultant fibrinogen monaner polymerizes In certain disease states the fibrinogen altered by the rele_e' of peptides, may combine with native fibrinogen to form a cold precipitable complex. Weerdt and Vreeker (1965) concluded that traces of thrombin formed ~ !!!.2..resulted in the formation of intermediate products of cryoprofibrin. Thrombin in trace quanti ti.es induces the formatIon of intermediate products, but not the formation of f !brin Itself. Therefore, . the presence of intermediate products results in the formation" of cold precipitable cry oprof!brin0 From the invest igat. ors , description, it seems possible that the cold precipitable 'protein described as cryoprofibrin is analagous to cryofibrinogen. Cryofihrinogen bas been demonstrated in the plasma of patients with carcinoma, leukemia, secondary polycythemia,,' and abdominal aneurysm in association with surgical procedures 0' This cold precipitable protein has" also been found, in small amounts, in samples from pregnant women, especially in the eighth month of pregnancy... When pregnancy was complicated by phlebitis, the amount of cryofibrinogen was increased. This protein was not evident in 27 control s-amples from normal pe'l'Sons: (Glueck and Her'l'l'ftan, 1964) .. Lipinski !l.!!. (1957) indicated that the: cryoprecipitate described by previous workers may be soluble complexes formed in the presence of excess. fibrinogen degradation products. These soluble complexes: may he precipitated by protamine sulfate or by cooling to 4°C. Lipinski and coworkers described these complexes as a result of .investigative studies using 1311 labeled fibrinogen. Samples of normal human blood were mixed with 131r fibrinogen and breakdown products of fibrinogen ~ The mixture was allowed to clot at 37°C for QQ minutes, and the resultant serum tested for radioactivity. The amount of radioactivity in the serum was directly proportional to the amount of fIbrinogen breakdown products pNsent. Aliquots of the serum were cooled to 4°C and a cryoprecipitate occurred. From . these experiments, Lipinski !!, £0 suggested tbat the cryoprecipi,tate termed by other workers astlcryofibrinogentt is a soluble complex formed by fibrinogen, fibrinogen degradation products, and intermedi.ates of . the en~ymatic- conversion of fibrinogen to fibrin. The electrophoretic and immunoelectrophoretic behavior of cryofibrinogen has been found to' resemble fibrinogen (Korst and Kratcbvil, 1955). Recently, immunoelectrophoretic studies were done on a cryoprecipitate found in tbe plasma, but not the serum, of eight patients with cryofibrinogenemia (Zlo!neck and Landau, 196&) .. These studies demonstrated that the cryoprecipitate primarily 28 contained fibrin.ogen; h.anver, albumin and alpha, beta, and gamma . globulins were also -preaent. VI . INCREASED. FIBRINOLYSIS IN DISEASE As. early as: 19.1... , Gcodpasture repOl"'ted that blood specimens from patients witb atrophic hepatic ciwhosis exhibited clot diaeation w:ithin a few hours- at body temperature, while a clot of normal blood did notdigeat for days or weeks.. Goodpasture suggested that dissolution of the clot in the blood of ci:rrhotic patients is due to an enz.yme inhibited by nOl"'mal serum. Later, Ratnoff (19~9) confirmed thi .. finding and substantially-extended the study of this phenomenon. He noted that rapid plas.,ma clot lysis was a frequent accompaniment of Laennec's- cirrhos-is and waa sometimes observed in patients who experienced. hepatic -damage during the course of some other illness. However, rapid plasma clot lysis was not observed in either acute hepatitis or obs:tructivejaundice. unless. hepatic was~ injury associated. In recent years, several authors tFinkhinner GroBsi !l.!!" !l !,!:' 1901, 1962 j lewaan !l.!!., .!!..!.!... t 1959; 1956, 1957; and Bergstrom 19.6Q) have reported that a large proportion' of patients suffering from advanced hepatic ciIThosis exhibited rapid spontaneous lysis of whole hlood, siaortened euglobulin lysis times and other evidence of enhanced plaaminoaen-p lasmin activity. Other autbors have shown bepatic cirrhosis' is often accompanied by an ill-defined 29 coagulation disorder. Patients s.uffering from this anomaly may. when subjected to opel'at.ive stress., develop a severe hemorrhagic diathesis. This diathesis is often associated with pathological plasma proteolysis (Zucker !l.!!... 1957 i Ratnoff, 1954; Purcell aDd Phi llips, 196 3). Nocola and Soardi in 1958 associated increased fibrinolytic activity with all liver disease, particularly cirrhosis. Fletcher -- et 81. USe4) showed that patients suffering fl'om liver cirrhosis had an enhanced plasma fibrinolytic activity after the administration of ni.cotinic acid or after electroshock treatment. The increase in activity of cirrhotic patients was five times tbat of normal controls. Menon (1969) conducted a study onsubj ects randomly admitted to a medical ward with a variety of illnesses. Based on euglobulin lysis time, increased fibrinolytic activity was ascertained.. This investigator found tbat fibrinolytic activity was increased in 15 per cent (two patientsl of the instances of coronaX"Y thrombosis, 10 per cent of the instances of acquired valvular heart disease (10 patients). and 18 per cent (.five patients) of congenital heart disease. In the instances of congenital and valvular heart disease. the li ver function tests were normal. Thus t i t appears that increased fibrinolytic activity extends beyond liver involvement. Further suDstantiation of this involvement may be postulated from the wOJ'k of Todd. lysis in tissues. '~tS.9l who studied anatomic sites for fibrina- Zones of fibrinolysis on a fibrin plate were 30 obsu-ved in all tlsaues except the live!'. Tha liver did. bowever. manifest fiDrinolytic activl.ty when ina diseased state. 'The zones of fibrinolysis were related . exclusively. to veins aD.d venules, except in the. lU:D1 .where these were related to pulmonary, arteries and arterioles. It would De expected thatiffiDrinolytic activity can De demonstrated. in tlle tissues a relationship between increased fibrinolyt.ic products and disease states ,.may he demon&trated. with the extenaionof tbe study of fibrinolytic prod.ucts. MATERIALS AND METHODS I SERA AND PLASMA Normal human serum and plasma were used to s'tandaroize the various test and experimental systems'. Pathologic serum and plasma wem studied for abnonnal f!bl'inogen degl'adation products. The patient sera used in this s,tOOy were collected fl'om hospitalized patients at the University Hospital and the Veteran's Administration Hospital. Specimen collection followed the routine procedure of each cUnical chemistry laboratory. The se:t"um was removed from the cells illlllediately after clotting. and stored at -20 C until immunological studies were performed. Samples of serum used as controls we:t"e collected from healthy persons previously screened as blood dono:t"S by the Blood Bank laboratory at the University Hospital. Specimens of blood were drawn according to tbe blood bankp:t"ocedure of this laboratory_ Tbe serum was i1llllediately removed f:t"Otn the cells and stored at -20 C. Samples of plasma were randomly collected from the outpatient laboratory of the University Hospital and from employees of tbe hospital. citrate. alood specimens were drawn in tubes containing sodium The plumawas X'emoved. pooled (8 to 10 samples per pool) and stored at -20 C until, needed. Pooled normal plasma was used as a fibrinogen control in immunological studies designed to identify fibrinogen anei/or fibrinogen products. 32 _II FIBlUHOG&H The fibrinogen used for this study· was dried buman fibrinogen procured from the AmerieanRed Cross (processed by Cutler Laboratories, Bel'keley, California}. FUty-five millip_ of the dried preparation _as Nconstituted in one milliliter of distilled water. Tbe resultant solution contained 10 mg/ml of clottahle protein. III ANTISERA The antisera used throughout these studies was obtained from HylaDCi Laboratories, Los Angeles. CallfOl'Dia. The polyvalent antisera were commercialpNparations obtained from animals (goat and rabbit) byperiuunized with poolednOl'Ul human sera. The antifibrinogen (.rabbit) or ant if ibrin (rabbit) antisera were prepared by hyperiDlDunization with washed -fibrin, processed from dilute solutions of purified human fibrinogen. IV- PLASMDlOGEN ACTIVATORS AND -INHIBITORS Proteolyses of fi»rinogen was studied by varying the concentration of plasminogen activators. used were a& follows: The proteolytic agents {l). Streptokinase-Streptoclomase varidase(R), a streptokinase pNparation obtained conmeJ:lciallyfrcm Lederle Laboratories, Pearl River, New York, and (2) Tbrombolysin(R), an activate4 human fibrinolysin obtained· cOI'Dmercially from Merck, Sharp, and Dobae. West PointtPennsy~vania. 33 The plasmin inhibitor used was Amicar (R}, a pl'eparation of e-aminocaproic acid Obtained commercially from Lederle Laboratories, Pearl River, Hew Yo:rk. "V, FllUUNOGEN ASSAY A modified,turDidimetric fibrinogen' assay was performed according to the me.tl.od of Hunter and Allensworth (1965). A. -Sera Fresh plasma samples were. obtained frail two patients. Serum samplea from these patients previously demonstrated a positive test for fibrinogen and/or fibrinogen derivatives by immunediffusion. Plasma aamples from normal persons were used as. control samples. B. Reagents The precipitating reagent contained 14\ (w/v) ammonium sulfate 3% (v/v) sodium. citrate and 1\ (w/v) sodium Chloride in distilled water. Five-hundredths milliliter of 17 per cent Zephiran was added to lQG ml of solution. The blank reagent contained 3% sodium citrate, 1% tv/v} sodium chloride and a.os, ml tw Iv) of Zepbiran in 100 ml. distilled water. C. Procedure Using Beckman cuvettes, 0.2 ml of patient plasma vas added to 1.'" ml of blank reagent and inserted into a well of a Coleman Jr. apectrophotometer ,",Coleman Instruments Inc., Maywood, Illinois). At exactly three minutes. the apectrophotometer was adjusted to 100% 34 tl'anS1Dission at a wave length. of 400, mil. Using a matching cuvette to. 2 ml of plasma was added to 1.4 ml of precipitating Nagent. transmission was recorded.. At exactly three minutes. the per cent Each test was performed in duplicate. The fibrinogen level in milligrams per cent was determined from a previously prepared curve. VI The CRJOFIBRINOGEN DETERMINATION c~ofibrinogen method used was proposed by Kalbfleisch and Bird (1960). A. - Sera Fresh plasma samples were obtained from two. patients. Serum samples from these patients. previously demonstrated a positive teat for fibrinogen and lor fibrinogen derivatives: byimmunodiffus;i.on. Plasma samples: from normal persons were used as control samples. ll. Procedure A fresh sample of plasma was refrigerated" for 48 hours' at 4 C. The cryoprecipitate was washed in cold s,aline and resuspended in saline to the original plasma volume. A fibrinogen assay was performed on tbe original plasma sample. and, the Nsuspended cryoprecipitate. VII EUGLOaULIN ,LYSIS TIME Euglobulin lya!$ time was 'performed according to the method proposed 1ly B.iggs and Mcfarlane (1963). 35 A. Plasma Samples Tbe following plasma samples were Obtained from the Clinical LaboratOX'iea: thNe samples with a prolonged prothrombin time and two samples- showing a positive serum test fOX' fibrin.ogen and/O'I.' fibrinogen derivatives by immunodiffusion. Plasma samples from normal pemona were obtained fl'om the outpatient department of the hospital and used fO%' controls:. Do. Regents The Nagents used for the test were 0.025 M calcium chloride. 1% acetic acid t and 0.1\ bOl'ate a-olution. The horate solution was prepared hy dissolving 9; 8 sodium chloride and 1-8 sodium borate in one liter of distilled water. The solution was. adjus-ted to pH 9:.Q. C. Procedure The blood w_ collected in s:odium citrate t kept on melting ice, and the test carried out within 2Q minutes after obtaining the specimen. Five-tenths. milliliter of the plasma was added to centrifuge tubes containing adjusted to 5.3 by addi~g 9,. ml of distilled water. 0..1 ml of 1% acetic acid. The pH was The tubes were allowed to stand at 4 C for 30 minutes. to permit the plasma euglo1)ulln fraction to pl'ecipitate. The tubes· were then centrifuged for five minutes. and the supernatant decanted. The euglobulin fraction was re&uspended by adding to eacb. tube Q.5 ml of the bOl'ate solut.ion. After the tubes were placed in a 37 C water bath, 36 0.5 ml of O.Q25 M calcium chloride was added and the clotting time recorded. After clotting, the tubes were inspected at intenals and the lysis time recorded. Lysis time longer than two hours was considered normal. VIlI A. IMMUNODIFFUSION Controls Samples of serum Obtained from normal blood donors screened by the University Hospltal Blood Bank Laboratories were used as controls. Samples of nonnal human plasma were obtained from the outpatient department of the University Hospital and were pooled (eight to ten samples per pool) and used as a plasma control. B. Patient Sera ..... -_.A total of 198 sel'UJD samples from hospitalized patients were collected fr~ the Clinical Chemistry Laboratories of the University Hospital and the Veteran's Administration. Hospital. The specimens were frozen at -20 C until immunological studies were performed. C. Procedure Patient sera were screened immunologically on immunodiffusion plates commercially prepared by My land Laboratories (Los Angeles, Californial. ren microliters of the test samples. were loaded in the wells on the immunodiffusion plates. Each sample was run in triplicate. A control sample of normal. human plasma was used on each plate. Ten microliters of fibrinogen vas placed in the aentel' 37 well. After loading It the plates were allowed to. incubate for 48 hoUl'S at room tempe.l'at.uI'e in a moist chamber. read and recorded at 24. and 72 hours GO The results were Since there was. no significant difference in these readings, only the 24-bour reading was recorded. The presence of fibrinogen and/or fibrinogen degrad.ation. products was determined . by the presence of a precipitin band (0 (see Figure 7) IX A. IMMUNO£LECTROPHORESIS ON CELLULOSE ACETATE Fibrinolytic Agent Tbl:'ee di.lutions of commercial plasmin were prepared in tbe (1) 100 mg of plasmin (10,000 units) was following manner: reconstituted in 0.1 ml distilled water; '-2) 10 mg of plasmin (1,000 unit.s). W.as reconstituted in 0.1 ml of distilled water; and (.3) 1.0 ag of plasmin (.10·0 units) was reconstituted in 0",1 ml of distilled water. B. Ruffer The buffer solution. 'pH 8.6, ionic strength.. 00075) was prepared »y dissolving 2 76 & of 5,5-diethylbarbituric acid and 0 15.40 g sodium S,S-diethylbarbituric acid in distilled water to make one liter. e. Protein Stain Ponceau-S fixative dye solution was used as the protein stain. The dye solution contained 0.2\ (w/v) Ponceau-S stain, 300% (w/v) trichloroaceti.c acid, and 3.0% (w/v) sulfosalicylic acid in water. 38 Fig. 7. Immunodiffusion plate. The double diffusion test was carried" out in buffered agar. Antifibrinogen was applied" in" the center. Applied in the five different spots were: normal human plasma (starting from top, counterclockwise) and four serum samples from hospitalized patients. The precipitin bands represents th~ presence of fibrinogen breakdown products. 39 D. Procedure Cross-reactivity of plasmin with respect to commercial antifihrin,ogen was studied using cellulose acetate strips and Microzone equipment (.Beckman Instruments. Fullerton t California) in accordance with.. preswiDed pl"Otocol (RyeX'S, 1966). The power supply used was a .Spinco Duostat: (Beckman Instl"UDlents., Fullerton, California). Normal human plasma was used as a control. Cellulose acetate membranes were presaturated with buffer and placed in at) embosser that embossed tbe membrane with·two wells for the 88q)le and three troughs for the antisera. The membrane was then mounted on the Microzone (Rl electI'ophol"8tic cell bridge and 0.3 ",1 of plasmin dilutlona were applied. Electrophoresis was performed at 150 volts for SO minutes at room temperature. The membrane.was then removed from the bridge, antifibrinogen (2Q lJll was applied, to each of the three troughs. followed by immersion in light mineral 01.1 and incubation for 66 to 72 boul'S at room temperature. After incubation. the membrane was rinsed two times in petroleum ether and four times in normal saline • stained in the fixative dye solutionfatt seven minutes. rinsed three more times in st aqueous acetic acid anddJ'ied in a drying frame before a hot-air dryer for 20 minutes. X IMMUNOELECTROPHORESIS. IN AGAR GEL Immunoelectrophoresis was performed on camnercial illDuno- 40 electrophoretic equipment (Gelman Inst.ruments~ Ann Arbor, Michl,anl. The pOlffer supply used was a Spinco Duostat (.Beckman Instruments, Fullerton, Califomial. The method used was the microtecbnique of Scheide"er {lQSS} as. modified by Hirschfield (1960). A. !e!:. auffer The a,ar auffer .as prepared by disaolvio, 1.&6 , :barbituric acid, lCL.$l I sodium bar»-iturate, and 1.5.4 , calcium lactate in one liter of distilled water. The al>ove .,olution was adjusted to a pH of 8.6. B. ---TanJc B.uffer The tank buffer was prepared by dissolving 1.38 , barbituric acid, 8.7 g sodium harbiturate, and 0.399 g of calcium lactate in ene liter of distilled water and adjusted to pH 8.i. c. Agar The two per cent. agar medium was. prepared Dy dissolvin, 20 g of conmercially prepared Ion Alar (Difco Laboratories, Detroit, Michi,an) in one liter of distilled water. The agar s.olution was slowly brought to a boil and. allowed to boil until the agar was canpletely dissolved. SO D. The medium was then allowed to s,olidify in ml aliquots. Protein Stain The dye used for staining the slides, was. prepared by dissolving one gram .acb of thiazine red R, amidoschwarz 10. B, light green SF, mercuric chloride, and 2Q g of acetic acid in one liter of distilled 41 water (.CrONle •. lUll. E. Procedure Three horizontal fl"arDes were: used, each holding six microscope s.lides. previously cleaned wi tIl. alcohol and coated wi tl- agar by inmersion in a llotling a.2\ 'w/vl i.on agar. Fifty mi.llillteN· of tne· previously prepared.ag. . was melted by hoiling and then allowed to cool to approximately 56 C. While still warm, the agar auffer mixture was applied to each series: of six slides in the hO%'izontal frames:. ••. needed for each; frame.) {Approximately 20 ml of agar After the agar was s.olidified. eaen slide was, punched with. a die des:igned to cut two wells for the &ample and a center trench between the wells for the antiserum. The horizontal frame. were placed in the electrophoresis: cell previously filled with buffer. Cellulose acetate wicks. presaturated in. buffer'. were used to connect the sUdes· with the buffer. minutes.. A constantcuXTent of 35 ma was:· applied for about 20 to 3tl The frames were. removed from the cell and the agar was aspirated fran the wella. In each case. 5 pl. of the control 8-ample marked with Bromphenol &lue {Hartman. Leddon Company, Philadelphia. Pennsylvania), was: loaded in the top well and the bottom well was filled with 5 111 of the sample to be tes."ted; the frames were returned to the cell and the current '35 mal applied for approximately one and one ...half to two hours;. The cell was again. opened and the agar was aspirated from the ECCLES HEALTN 42 center tl'encl\., tie trench was then filled wi:t1l 100. 111 of ant ifibrin osen • The slides. were allowed to incubate at room temperature in a moist diffuaion chamber for 24 hours:. After incubation, the al1dea were washed intbNe 24-hour rinses of normal saline and one 24-AoUl" rinse of distilled water. Tne slides were dried in a hot-air dxyer and stained for approximately 30 minutes. The excess, s:tain was removed by rinsing hrief 1,. several times in cold tap water. A final rinse in 5% aqueous acetic acid was· used. for differentiation of the stain. After drying, the stained slidea were laheled and the results. recorded. Xl A. GEL FILTRATION Preparation. 2!. DeEmation Products Dried hwaan flbrinOien was reconstituted to 55 mg/ml in distilled. water &ivinS a final concentration of 10 mg/m1 of clottable protein. To 5 ml of tne reconstituted fibrinogen was added 0.15 ml of plasmiD, giving a final dilution of 300 units of plasmin per milliliter of fibrinogen. Proteolysis' was ·allc.ed to proceed far 48 hoUN. B. Preparat ion. 2!!!!!. Column G-75 and G-2QO. Sepbadex (R} (Phal"lDacia Fine Chemicals, Incorporated, Piscat_ay t New Jersey). were -allowed to swell in demineralized wa'ter for three days with periodic. water chanaes dl.Jlling this ti_ and:. the- fine materials raaoved by- decantation. 43 The water w_ then Nplaced wi'th,O.85\ sodi_ chJ.oride. The Sepbalex vas allONeci to equilibrate with.the saline solution befON })ainl packed into a column 2 .. S em in cliUleter by 45 an in height, or a column 2 .. 5 em in diameter by 100 em in helpt. Coltal packing was accomplisbed by pouring a sllU'TY of Sephadex and saline into the column. The outlet of the column was adjusted to avoid excessive packiDl' of tbe lel beads. The slurry was cClOtinually NplaceG .. pacJdng proceeded until the packed gel bed was the desired height. The column was fitted with upward. flow adaptOl'S and. cODneC'ted to a saline l'eservoir. to c. l'Un Saline was allowed thl'ouah· the column overnight. PJ:tocedU1'e Five milliliters of the sample were .appliedt.o the column usin, a tb........,ay valve andsyTlnge and-the pressure was adjusted to allow a flow rate of 9· ml/br through .the col\RD. A total of three fractiCDa. containiDa tbJ;tee·1Rillilit.ers of effluent were collected each hoUl' at 2.QmiDute intervills with an '''ISCO(Rl automatic fraction collector (.Ill8tJ:tu11entation Specialties Company, IncOl'porated, Lincoln, Hebl'aska). The tubes were analyzed for protein content uaiD& a BeciJDan DU .ultravi,olet llghtspecU'ophotometep (.Beclaaan Inatrw.nts, Fullerton t California), with a wavelength setting of 280- mll. The aptlcal density was graphed as the OI'di.nate aDd the volume of the effluent was plotted on the abaci••a. The effluent fran -the tubes defined within tbe absorbance 44 peaks iD the above plot were pooled and concentrated ten-fold using LypbOgell,'Rl a polyacrylamide gel acting as a dehydrating aaent (.Gelman Inatl'UJDen'ts. Ann Arbor, Michigan). EXPERIMENTAL RESULTS 1. CROSS-REACTIVITY OF PLASMIN Throughout these studies plasmin was consistently used to effect the proteolysis of fibrinogen. In order to correctly identify the breakdown products resulting from fibroinogen proteolysis, the necessity of ruling out cross-reactivity between plasmin. and antiflbI'inogen<became evident. Agar gel immunoelectrophoresis was performed on the following dilutions of plasmin and expressed in MSD unitsl lO,OOOunits/ml, 1,000 unitslml, and 100 units/ml. Table 2 indicates that no cross- reaction occurred using 100 unitslml and 1,000 unitslml of plasmin. However, when 10,000 units/ml of plasmin were tested with antifibrinogen, a light pl'8cipitin band occurred in the alpha area of the electrophoretic pattern extending into theprealbumln region. To eliminate the further possibllity of cross-reactivity of plasmin wi.th the agar gel t cellulose acetate immunoelectrophoresis was performed on the above dilutions of plasmin, using antifibrinogen as the antisera and normal buman plasma as a fibrinosen control. A light band, iOOlcatiDg cross-reactivity, was again· seen with this concentrati.OD of plasmin (10 ,000 units), but was notevldent at lower concentrations 'see Table 2). Therefore, dilutions of plasmin were maintained below 300 unitalml in subsequent studies to avoid observable Dcmapecificprecipitin bands. Table 2. Immunoelectrophoresis of Plasmin UNITS OF PLASr·1IN 100 1,000 10,000 ._. AGAR GEL CELLULOSE ACETATE NEGATIVE NEGATIVE NEGATIVE LIGHT PRECIPITIN NEGATIVE LIGHT PRECIPITIN BAND BAND I ~ 47 ,1.1 IMMUlODI.FFUSION IaunodiffusiOll was used to. screen patient sera for the presen.ce of fibrinogen and/or'derivatives of fibrinogen'.' A total of 196 samples of serum frOlllhospi,talized patients was- collected from the Clinical-eheads.try· LabOl'atOl'ies of the· University' Haspital and the Veterau~s Admin.i:atration,Hospital. 'Altb.ough most of this population had a history of abnormal liver function.. a widespread spectrum of diseasewa repNSented •. ,I.mmunodiffusion was. performed in triplicate with ,all serum samp les. Fl'om' the 196 serum s . .ples sCNened, ten aample& (approximately 5\1 gave visiDle immunological Nactiona with antlfibrinogeD. lndicating" the presence of fibrinogen and/or braakdorm, products of fi»rinogen. Immunocli.ffusion was also performed on control sera collected from So. persons' previously screened for' blood. donation- by •the Universi.ty Rospit.al B.lood Bank Laboratory. Neither fibrinogen J1OI' fibrinogen breakdOlll'l products were demonstrated. in the sera of the controls.. Using 1) iagnostic Flasma (Hyland Laboratories, Los AngelestCalifornia) as a fibrinogen ref8l'e1lce. studies indicated that fibrinogen in amounts exceeding J.Q JOg was demonstrable by immunodiffusion. III IMMUHOELECTROPHORESIS From the various teat systems employed, it appeared that immunoelectrophoretic technique" was most satisfactory for identifying and Cbaracterizfng the procluctsresulting from the proteolysis of human fibrinogen. Preliminary studies were made 48 using two different ,agents- to induce fihrinolysis. These studi.es were conducted. to detemine which agent would be best suited for study of fibrinogen. breakdosm··products. These .agents were Thrombolyain '-cOIDIIle.rcial plasmin)' and Streptokinase '.a plasminogen activator1. When" immunoelectroph.oNs;is was performed on normal liwnan plasma after beiDa suDjecteci iothe 'proteolytic activity of plasmin, two distinct bands were observed in the alpha-beta zone (see Figure 8). Both hands. differed from the nOZ'lllal immunoelectrophoretic position of fibrinolen. For purpoaa. of this discussion.. the hand migrating neareat to the origin was labeled Band A, while the band with the faster mipation rate 'Was labeled Band B.. The following experiments 'WeX'CJ based on the assumption,that tile_ products represent the D and E fractions respectively, as. originally reported by Mussenweig and co-workers, {19601. Streptokinase •.as used in preliminary experiments. as a plaaminogen activator. poo~d This activator was added to samples of normal human plasma, and proteolysis was allowed to proceed for perioda of ti_ from Z8Z:-0 to three hours. The timing was synchronized to allOill all samples to be innoculated on the agar sel aimultaneoualy. After loacllng, a constant current was i-.diately applied to the cell. Tbe results; of this, expez:-iment are &i ven in FilUl'e 9,. Ualn& ant.if~ino,en as the antiserum, two products were ORIGIN 0 ~ FIBRINOGEN AtfTIFIBRINOGEfJ· _ _ _ _ _ _ _ ____ BAND B Q Fig. 0.. A diagramatic representation of the immtlnoelectroph-oretic pattern of human fibrinogen before digestion (upper well) and after (lower well) exhaustive plasmin. .r: U) 50 NHP ANTIFIBRINOGEN TIME 0 NHP ANTIFIBRINOGEN 15 MIN NHP ANTIFIBRINOGEN 30 MIN NHP ANTIFIBRINOGEN 1 HR. NHP ANTIFIBRINOGEN 2 HR Fig. 9. Streptokinase activation of normal human plasma (NHP) at varying periods of time. 51 aug.eated in .a~ case.})!, tile presence of two precipitin D,anc.\S. Band A. nearest tbe _11. Nmained in a s.table position regardless: of timing. HoIrrevel'. Band B., migrating fUX'thest from the: well, varied in mObility depending upon the time. allowed for proteolysis; to . proceed. Streptokinase.as added to normal human plas.ma iJ'lll'D8d.lately ])efore loading. Figure 9 illustrates tnat proteolysis . ..,. baveoccw:-red intttantaneous;,ly '-note time zerol, or that the pluminogen may have :Men activated sometime during the electrophoretic procedure. Tae above treatment of normal human plasma was repeated substituting lO~ ~/ml of plasmin in lieu of streptOkinase. In Figure 10 it can be seen that the action of plasmin differs from atreptokinaae. mobility of 'the Although the same two products; are evident, the E components differ. Comparing Figm'e 9 with Figure lo,it appeax's that proteolyais of f.£brin,ogen occW!'& at a alower rate when plasmin is used. this finding and als~ Repeated experiments supported indicated that plasmin gave more reliable and reproducilale reaults·. Fozt these reasons, plasmin was used in subsequent experiments. I V THERMAL PROPERTIES OF FIBRINOL1TIC PRODUCTS Usina a.imple radial diffusion technique. B.eller and Malei '1961) c:1emonatratec:1 two products resulting from the degradation of fibrinogen lay plasmin and ident.ifiec:1 these products as the D and E fractions originally reported It.ly Nussenweig (1960). The above 52 NORMAL HUMAN SERUM ANTIFIBRINOGEN TIME 0 1 HR ANTIFIBRINOGEN 15 MIN 30 MIN ANTIFIBRINOGEN 30 MIN NHP ANTIFIBRINOGEN 1 HR 6 HR ANTIFIBRINOGEN 48 HR Fig. 10. Serial digestion of fibrinogen in normal human plasma (NHP) by plasmin. 53 !Dvestigators alao reported tbat after beati~g the, filiz'inolytic products, the pl'oduct deS:ignated by these workers: as the D fZ'acticn, could no longer be demonstZ'ated. The thermolabile propeZ'ties of fraction D andtfte thermoatabile properties' of ~action E were later substantiated by Kowalski '1968). Plasmin '-100. lA/mll was added to -two samples of pooled normal lU.uoan plasma. B.otL samples: were allowed to incubate at x-oom temperature for one D.Ot.lX'. Following incubation, one of the samples (sample 2) was heated in a 56 C water b.ath for 30 minutes. was used as. a control. samples 'see Fi.gure ll}. Sample 1 Immunoelectrophoresis: was:perfonned on both. The control sample was placed in tbe upper well, whereas'·"'tb.e beated sample was; placed in the lower well. Two products were demonatrated in the upper well and only one procluct was demonstrated in the lower well, &uggesting that the heavier hand t Band At misrat~g nearer the origin, represents tbe proteolysis procluct previously described as the D fraction. V INHIBITION OF PROtEOLYTIC ACTlVIn -- Allc.j aersig et ale (1959a) "ported that the proteolytic action of plasmin is inbii.ited. by e-amino caproic acid. bowever, attempts in this study to inhibit the proteolytic acti,on of plasmin in normal human plasma were UllSuccesaful. Thus, an experiment was designed to determtnetbe threshold of c-amino caproic acid plasmin inhibitory activity. purified human fibrinogen was reconstituted to SS mg/ml in distilled water living a fil1al concentration. of 10 mg/ml of 54 PNHP ANTIFIBRINOGEN PNHP (56. C) 30 MIN PLASMIN DIGESTION PNHP ANT IF IBRINOGEN PNHP (56 C) 60 MIN PLASMIN DIGESTION Fig. 11. Plasmin digested normal human plasmin (PNHP) without subjection to 56 C and with 56 C treatment. 5S clottable protein. £-amino caproic acid was added to each of seven tubes containing one milliliter of fibri~ogen,. giving a final concentration of 50 mg/ml of the fibrinogen solution. Vary~ng dilutions of plasmin, as indicated in Table 3, were then respectively added to each tube. Control tubes contain~ng fibri~ogen, but not £-amino caproic acid, were prepared to indicate the proteolytic action of plasmin at each respective dilution. After the tubes were incubated at room temperature for one hour, immunoelectrophoresis was performed on all samples us~ng antifibri~ogen as the antisera. When two bands representing fibrinogen breakdown products were observed, the sample was described as not inhibited. It may be noted from Table 3 that proteolysis did occur in each of the control samples. However, when less than 40 units of plasmin was used, the proteolytic action was inhibited by the €-amino caproic acid. VI GEL FILTRATION Normal human plasma treated with plasmin for 48 hours was applied to a column of G-75 Sephadex gel. content. gel and a column of G-200 Fractions were collected and analyzed for protein The peaks ascertained by Sephade~ represent~ng the distribution of protein were plott~ngoptical .. density on ~ graph (see ~igure 12). The tubes containing the absorbance peaks were pooled and concentrated and immunoelectrophoresis was performed on the resultant concentrates. It .was observed by inspection of the immunoelectrophoresis slides that partial separation had been accomplished. Samples from the 56 Table 3. Inhibition of the proteolyte Activity of Plasmin on Fibrinogen by ~Amino Caproic Acid (EACA) EACA PLASMIli (mg/ml) Control 1 Sample 1 (mg/ml) 10 10 (in units) 100 100 Control 2 Sample 2 10 10 -- Control 3 Sample 3 10 10 -- Control 4 Sample 4 10 10 -- Control 5 Sample 5 10 10 -- Control 6 Sample 6 10 10 -- Control 7 Sample 7 10 10 TUBE FIBRINOGEN * NI = Not ~nhibited -SO SO SO 50 SO 50 -50 PROTE-' OLYSIS* HI NI 80 80 NI NI 60 60 NI NI SO SO NI NI 40 40 NI NI 30 30 NI Inhibited 20 20 NI Inhibited 0.25 0.20 - 0.15 ;::1. ~ 0 CX) N 0.10 QJ 0 s::: '" ..Q J..I 0 1'8 0.05 ~ o" 500 100'0" 15'00 Elution Volume (rol) Fig. '12. Pattern obtained after filtration of fibrinogen degradation produ'ets through Sephadex G 200. ~ 58 absorbance peak first eluted from the column contained both the D and the E fractions while samples from the second peak contained only the E fraction. VII ASSAY FOR FIBRINOGEN AND CRYOFIBRINOGEN A modified ,turbidimetric fibri~ogen assay (Hunter and Allensworth, 1965) was utilized'to determine the on plasma samples. fibri~ogen levels Inasmuch'as fresh plasma was required for the analysis, two persons with. nopmal plasma fibrinolytic activity and two patients with increased plasma fibrinolysis were selected because of availability at the time of testi.ng. Fibrinogenand/ or fibrinogen breakdown products had been demonstrated immuno~ogically from serum samples previously obtained from these patients. The normal range of fibrinogen for this method is 180 to 415 mg/IOO mI. Although the patient values were considerably the control, only one of the considered as having a fo~ s~ightly ~igher than those of patients (see Table 4) may be increased i fibri~ogen level. Samples of plasma from the same. group:.of patients weve refrIgerated at 4 C and observed for cryofibrinogen (Kalbfleisch and Bird, 1960). After 48 hours, no cvyoprecipitate was observed, in any of the samples. VIII EUGLOBULIN-LYSIS TIME Increased fibrinolytic activity has been determined by the euglobulin lysis time in patients with a history of abnormal liver function tests and coagulation disorders (B.iggs and McFarlane, 1963). Table 4. Assay for Fibrinogen and Cryofibrinogen, !I SAMPLE FIBRINOGEN (MG%) CRYOPRECIPITATE !: I CONTROL A CONTROL B t!ONE NONE 270 180 , I , 1 ! , f PATIENT A PATIENT B J~ONE 450 365 I NONE .. :: 6a The' followi,ng fresh plasma samples were studied: five samples with coagulation test values in' the ",normal r~n~e, three samples with prolonged prothrombin times', '.and.two samples showi,ngthe presence of fibrinogen and/or fibri~osen'derivativesby immunodiffusion. The clotting time was recorded ',as: well as the time required for the lysis of the clot. Clot' lysis' times lO,nger than two hours were considered to be in the normal r~nge. Only one plasma sample (see Table' 5) showed increased'fibrinolytic activity as demonstrated by the Euglobulin lysis test'~ .,. A.' positive test for fibrin:ogen and/or fibrinogen derivatives by immunodiffusion was previously demonstrated in the serum obtained from this patient. IX' 'PATIENT DATA As previously stated", 196 samples of sera were collected ,from patients hospitalized at'the"University Hospital and the Veteran's Administration Hospital." Thirty samples of sera were collected from persons previously screened as blood donors by the University Hospital Blood Bank Laboratory and used as normal controls. All' samples were screened by immunodiffusion'for the-presence of fibrinogen and/or fibrinogen breakdown products. These products were not demonstrated immunologically in anyof'the control sera; however, in ten of the sera obtained from hospit,alized patients fibrin:ogen and/or fibrinogen products were demonstrated~ 'Immunoelectrophoresis was performed on each of these ten samples,' usi,ng normal human plasma degradated with plasmin (100 units/ml) as a'control and antifibriIl:ogen as 'the Table 5. Euglobulin Lysis -- SERA SAf,'PLE ... . ....... CONTROL " A CONTROL B CONTROLS CONTROL C CONTROL LYSIS CLOTTING D CONTROL E ... .... , .TIME " . ....... TIME 2 MIN • .. 3 MIN. 2 MIN. 5 MIN. 3 MIN. 2 HR., 30 MIN. 2 HR., 50 MIN. - 2 HR. ,30 MIN •. 2 HR., 40 MIN. 2 HR., 10 MIN. 2 HR., 10 MIN. \ PATIENT 1 PROTHROMBIN PATIENT 2 9 MIN • 7 MIN. TIME PATIENT 3 23 MIN. PROLONGED .. POSITIVE " IMMUNODIFFUSION PATIENT A 3 MIN. ... PATlENT .. B··.. . . . ... 2.. MIN·.. ··· 2 HR., 40 MIN. 2 HR., 5 MIN. 2HRt, 5 MIN. .' . l .. HR·t.,· 50 ·Ml~ • G\ ..... 52 antisera. One band, corresponding to the hand desC1'iDed as the D fraction. was demonstrated in e,iglit of the· ten samples:, and in the other two sel'Umsamplea bands representing the D and E fractions were demonstrated .. Medical bis.tories of these patients were: oJ)tained and a diagnostic claaaifi.cation was made. Tile di:agnosis of the patients' illness at the ti_ of this study was, performed could be olassif ied in to three categories: (.1); hepatic disease, (.2} kidney disease, and (.3) intravaacular thrombosis,. A s.uhclasaif lcation of categories is: presented in Figure 13. eac~ of these three Eight of the ten patients were being treated for liver aiImenta. These included viral hepatitis, cirrhosis. of the liver,cbronic passive conges.tion. and biliary disease. The kidney ailments afflicting five patients included kidney -infection, myeloma nephrosis:, kidney infarction, and nephroscleroai,s. Six patients had intravascular thrombosis, which included one patient with an abruptio placentae .. In s,ome ins,taJI·ces,. evidence of more than one dis:e·ase process wi thin a single patient was found. i.e., three patients presented with a bistory of all three disease entities.• while one patient had kidney disease accompanied by intravascular thromboais and one patient had. both. hepatic and kidney disease. A diagranmatic pres.entation of the overlap of diseasea. is given in Figure 14.. indicate the number of patients, in each The arabic numerals categ~. Itmay De noted that there were no case a of kidney problems without the added eanplication of either liver disease or intravascular thrombosis:. 63 VIRAL HEPATITIS (1) CIRRHOSIS (2) LIVER (8) CHRONIC PASSIVE CONJESTION (3) BILIARY DISEASE (2) MYELOMA NEPHROSIS (1) INFECTION (2) KIDHEY (5) INFARCTION (1) NEPHROSCLEROSIS (1) I IlTHAVASCU LAR THROr.1BOS I S (6) ABRUPTIO PLACENTAE (1) Fig. 13. A classification of disease states seen in patients with fibrinogen breakdown products in the serum. RENAL 3 HEPATIC - INTRAVXS"CULAR THROMBOSIS Fig. 14. A diagrammatic repr.esen-tatci.an of ·the oVer'l.ap of' phys'iei.-egic sites involved in' the diseasep'iatl:S'of' pati:ents· wi'th t'ibrln'O"g'e11 brea:1atown products in<the- serUm. • .J: DISCUSSION Immunological methods have been employed to demonstrate and identify the proteolysis products resul ti.ng from the degradation of human fibrinogen by plasmin~ The proteolytic breakdown products of fibrinogen studied include' products occurri.ng spontaneously and products induced by treatment with streptokinase ,and plasmin. The fibrinogen degradation products studied were derive~ from: (1) normal human plasma treated with the fibrinolytic .agent plasmin, (2) normal human plasma treated with streptokinase, -an activator that converts the proenzyme plasminogen to plasmin, and (3) spontaneous products occurring in the serum of ten hospitalized patients. Direct observation of the'digestion process was facilitated when immunoelectrophoresis was performed on normal human plasma subjected to varying periods of exposure to the proteolytic .agent, plasmin. At time zero, the fibrinogen molecule had already begun to split, indicating the occurrence of rapid proteolysis that may have occurred during the electrophoretic procedure. At fifteen minutes Band A representing the D fraction'wasobserved. This band remained stable and did not differ in immunoelectrophoretic position regardless of the time allowed for proteolysis to proceed. The second band, Band B, varied in immunoelectrophoretic position depending on the length of exposure to the proteolytic agent. From these results it appeared that this band may represent an intermediate product containing the E fragment rather than a plasmin resistant product. Furthermore, it 66 appeared" that this intermediate ,product is still susceptible to the action'of plasmin. However,"exhaustive:proteolysis (48 hours) produces an E fragment resistant to'further action by plasmin. This postulate is further substantiated by the increased electrophoretic mobility after prolonged"exposure~of,this proteolytic enzyme. intermediate to the The appearance'of a spur on the anodal end of the precipitin band of the E fragment" with extended digestion suggests that polypeptides are being continually split from the intermediate product containing the E fragment. With exhaustive proteolysis of 48 nours, the spur disappears and the E fr,agment, resistant to plasmin, now appears in the prealbumin electrophoretic region rather than the original alpha-beta position. The ~oncordant opinion as to the exact rate and mechanism involved in the formation of" the D and E fractions from the parent fibrinogen molecule has not" been-established. Kowalski (1968) has indicated' that dur~ng The recent work of plasmin proteolysis of fibrinogen',' fractions D and' E' are formed at the same rate (see Figure 6). However, the" study' presented herein indicates that fragment D is formed more" rapidly and" remains stable, while E is formed ata slower rate. Kowalski formulated his composite hypothesis of this'mechanism (see Figure' 6)'by' compiling several discreet experimental studies. In the present study, a sequential degradation of the parent molecule was serially observed. This digestion yielded a rapid formation of the D fragment and a sequential alteration of 67 theE'moiety. This produced'a'~igratory band o~igiriat~ng in the locus of the D fragment and' p~ogressi,ng across the electrophoretic field to the prealbumin regionat'the completion of the ~igestion. A diagrammatic representation'of'this process is, given in Figure 15. This mechanism is supported'bythe earlier work'of Fisher (1967)~ !!!!., Using zone electrophoresis in polyacrylamide, gel these investigators indicated that'the electrophoretic mobility of the E moiety continues to change'until there is a clear separation of both precipitin lines. Complete'separation appears to require a digestion period of approximately 24 hours, This work substantiates the postulate that the bands seen in Figure 10 (note 48 hour) represent the final products resulting from the 'proteolysis of fibriIl:ogen'byplasmin, and indicates that the rapid-formation of the D fragment may be a more reliable observation of plasmin'digestion. Recent studies performed by Beller and Maki (1967) using radial diffusion confirm an earlier'proposal, i.e., when fragment D is sUbjected to temperc;ltures' of' 56' C or altering its molecular structure~' ~ighe:r;', the fr,agment' precipitates, To substantiate the identification of Band A, previously described in this work as the D fragment, the thermolabile properties were studied by heat~ng treated plasma to 56 C for 15 minutes. Immunoelectrophoresis of this plasma and plasmin sample demonstrated that' the product migrati.ng near the well was no longer visible, thus establishing the identification of Band A as fragment D. (Refer to Figure 11) FIBRINOGEN (MW ,350',000) 000 PLASMIN RESISTANT PLASMIN RESISTANT D FRAG~lEr~TS £ FRAfiMfrlTS (MW~ '80',000) / + PLAsmN SUSCEPTIBLE pp IN1ERf·1ED IATES ---'" /' <f.1W 30,000) + pp + pp Fig. 15. Model of fibrinogen digestion by plasmin. Plasmin digestion appears to give immediate rise to two D. fragments and a. sequential degradation to two E fragments. (pp. indica't'es"'l3tna'1i' p61yPepti.tl:e'S~··~ peptittes"and~ amino acid residues. ) en m 69 Gel filtration chromat.ography usi,ng Sephadex was· employed to separate and further identify the l~rger fr.agments formed during the fibrinogen,-i~e.~-f~agment D (molecular weight 80,000) and fr,agment- E (molecular- we.ight- 30;000). Followi,ng- fractionation on proteolysis of the column, immunoelectrophoresis was performed on the-two absorbance peaks. Both Band A and Band B'wereobserved in elution samples contained in the first absorbance peak. The D fragment appeared to be present in a greater concentration than the E fragment. Only Band B was demonstrated in the elution samples contained in the second absorbance peak, indicating that partial separation had been accomplished. The larger molecular weIght fragments would be expected to be eluted from the column before the smaller molecular weight fragments. Since partial separation was obtained and Band B appeared as the exclusive'component in the second peak, Band B most probably represents the Ef~agment. When electrophoresis'was performed on normal human plasma following the addition of-streptokinase (refer to Figure 9) a different pattern was observed~ Two precipitin bands were'visible, one appearing in the beta-r,egion.,just above theor,igin, and the secondanerging initially'from-the anodal end of the first'into the alpha region. The presence of· two precipitin bands at time zero, instead of one, would indicate'that the action of streptokinase is faster than the action of'plasmin~ However, at 30 minutes the band representing the E moiety'demonstratesa slower electrophoretic 70 mobility and greater identity with f~agment D. This spur could possibly result from recontugation of the E moiety into an intermediate product, a "reversal" effect. Further d.egradation of the D fragment would not be a plausible· explanation for the spur, in that the plasmin resistant end product of theE fraction is not seen. The reversal effect exhibited by streptokinase has not been explained from the data presented herein. However,· the occurrence of this effect has been substantiated by Hunter and' Allensworth (1966). These workers demonstrated by serial fibrinogen assays a biphasic effect on fibrinogenolysis result~ng from the· addition· of streptokinase, i.e., after one hour fibrinogen levels' were approximately 25 to 33% of the starting material, whereas withprol~nged streptokinase treatment fibrinogen levels returned· to approximately 75% of the (see Figures 16 and 17) o~iginal assay. This'effect was not seen when fibrinogenolysis was initiated using commercial. plasmin in excess. A possible explanation for the differences observed in the type of reactivity of plasmin and streptokinase may relate to the qualitative and quantitative kinetics of the enzyme' systems. experimental studies by Li~iger Mathematical and and. Rll:egs.e.gger (1967) showed that when streptokinase (an activator) enzymatically activates plasminogen generating the active enzyme plasmin, an antiactivator inhibits streptokinase and plasmin' is' inhibited by reacting stoichiometrically with antiplasmin. It appears, however, the excessive extrinsic plasmin will completely hydrolyze fibri~ogen and/or fibrin to stable 600 NORMAL 500 # PLASMA ,~-------------. -- ,..~ me __ ----- 1r z = oz 400 •a:~ ..J 300, :II 8 '" e 200 :II EACA PLASMA ----.-------ADDED-;LAs~;--,. r------- DECREASE IN FIBRINOGEN PLAtN PLASMA 100 o ~ HOURS 2 3 4 23 24 Fig. 16. Fibrinogenolysis is demonstrated by serial fibrinogen assays in a' specimen of plasma containing added commercial plasmin. .....:I t-r 600· NORMAL PLASMA -1 I,.--- - - - - --""---------------. 500 ---_~-----~~:.:~:...---.." z 8'"z: c • u: .J a 1------- .___----r.,',--------- T 300 DECREASE IN FIBRINOGEN 8 ~ • a 200· ADDED STREPTOKINASE 100 o ...... 2 3 4 23 24 Fig. 17. Seria1f'ibrinogen"assClysdemonstrate a'biphasic eff-ecton fibrinogenolysis" resulting from the addition of--excess -streptokin'ase. ...... p.J 73 D and E end'products. Serum samples from 196" patients were collected at random without prior knowle,dge of the admitti,ng- diagnosis. Immunodiffusion was employed to screen these sera for the presence of breakdown products of fibrin,ogen. fibri~ogen and/or Since fibrin,ogen is completely converted during the clotting'process, (Rodman, 1968) the presence of fibrinogen and/or products of,fibri~ogen in the serum in traceable amounts were considered abnormaL, indicat i,ng a disease state. Fibrinogen and/or breakdown-products of fibri~ogen were found in the serum of ten patients,or approximately 5% of the 196'serumsamples. Immunoelectrophoresis was'employed to further characterize the products demonstrated by immunodiffusion. A band appeari.ng in the same immunoelectrophoretic'position as the D fraction was demonstrated in eight of the ten sera,-whilebandsoccupy~ng the immunoelectrophoretic positions previously observed for both the D and E fractions were demonstrated in the remaini.ng two sera. Since the filteri,ng threshold of the glomerulus has been reported at' a molecule w~ight of approximately 40,000 (Fulton, 1956), the demonstration of only the D fraction in 80% of the sera tested is not surprisi,ng. The molecular weight'of this fraction is around 80,000, whereas the molecular weight of the E fraction is 30,000. If kidney excretion of the E fragment is consistent with the behavior-patterns of other proteins of similar molecular weight, a clearance from the blood stream of the E fraction by the kidney would be predicted and expected. It is interesting to 74 note that the seI1um samples containi,ng, both the D and E fI1,agments weI1e collected fI10m patients havi,ng' a' histoI1Y of I1enal disease. A modified fibI1iD:ogen'assay was used to cOI1I1elate incI1eased levels of plasma fibrinogen with the pI1esence of fibI1inogen pI1oteolytic pI10ducts in the seI1um, wheI1eas the euglobulin lysis time was employed to cOI1I1elate incI1eased fiPI1inolytic activity with the pI1esence of degI1adation products in the serum. mateI1ial pI1ecl~ded The lack of patient an adequate evaluation of fibrinolytic activity and fibI1inogen levels; however, the presence of fibrinogen bI1eakdown pI10ducts in the serum does not'necessarily accompany incI1eased fibrinogen levels 011 prolonged' e,uglobulin lysis time. Only the plasma from one of the two-available.patients exhibited an'increased fiPI1inogen level and prolo,nged" e,uglobulin lysis time, Fresh plasma samples fI1om" the two patients just described were cooled to 4 C. and observed' for the appeaI1ance of a cryoprecipitate. CI1yoprecipitation was not evident in the plasma of either patient, suggesting'that in addition to incI1eased fibI1in,ogen levels and fibrinolytiC activity cryofibriD:0gen is not necessarily cOI1related with fibI1inogen breakdown'products demonstrated in the serum. McKee et al. (1962) studied samples of plasma from 670 hospitalized patients. Twenty-nine patients in this study demonstrated a cI1yopI1ecipitate termed by' these workeI1s as cryofibI1inogen. Later work by' Lipinski et ale (1967). s,u.ggested that the cI1yoprecipitate is a soluble complex formed in-the"pI1esence of incI1eased fibI1inogen 75 bl'eakdown pl'oducts and intermediate-products formed in-the-enzymatic convel'sion of fibl'in,ogen- to- fibl'in.' 'The studies that> have been l'epol'ted on the d,egl'adation" produt!ts:-of:fibl'inolysis do not confine incl'eased fibl'inolytic activity,to-any single disease entity (Menon, 1969) Ol' physiologic site. Indeed', :fibl'inolytic activity has been demonstl'ated to be distl'ibuted-throughout the tissue components of the body, with the exception of,the' liver (Todd, 1959). the greatest preponderance-of'~igestionproducts However, appeal's in instances where the liver, renal and" intravascular systems are involved. Doubtlessly, as further studies'areundertaken and procedures for studying fib~inogen bl'eakdownproducts'become more refined, the relationship of these products,to'specific disease entities will be elucidated- and the di,agnostic-,and. therapeutic implications will be understood. Altho,ugh current'method01:0gy for detect~ng fibrinogen breakdown products in the'serum-cannot.readily be employed as a diagnostic tool, thoseinstances-,of- bleedi,ng diathesis secondary to intravascular fibrinolysis' could-be'rationally and specifically treated if-fibrinolytic products were identified. SUMMARY 1. Human fik'mogen was degraded by the addition of caamercial fii-rlinolytlc agents to pooled normal human plasma. Agar gel immunoelactropAoNs.i:a was employed to identify and cIiaracterize the products. resulting from the proteolytic action. Two products are. oliserve.d and identified. as the D and E fl'apaents. Tbe D fragment, represented by a band ill tIle beta are·a of tbe electroph.oNti:c ftald. i& formed rapidly and remains a:table. The & moiety is ·fOl'1ll8d at a slCMer rate and is Npztesented as a mgratory ))and origJinating from the locus of the D fragment at the onset of digestion and progressing across the electroph.ONtic field to the prealbumin region. at the' caopletion of digestion. 2. Gel filtration was used for the separation of the D and E fractions. These fractions were identified By immunoelectro- phoretic tecb:nicque. 3. Serum sup.lea from 199 hos.pitalized patients: were studied Dy immunodiffus.ion. for the presence of spontaneous fibrinolytic products. Tb.e. spontaneous products were detected in the serum of 1G of the 19i patients tested. '+. Immunoelectrophoresis was, employed to further characterize the filf,rino1ytic products occurring spontaneously in the serum of 1Q., patients. Evidence of the D fraction was:' demonstrated in aQ% of the sera, whereas in 20\ of tne s'era both, the D andE 77 fracti.cm.a 5. 1tJ8.N obaeI'ved. Fihrinogen levels, euglobulin. lysis time andczyofilrinogen weJ:'e determined in p-J.asma samples frail 2 of the 10 patien'ts. Only ene patieDt ex'fUl)lted an increased fibrinogen level and euglobuUn .lysis time. i. The clinical evaluation of tbe patient:ta .disease state was correla:ted in the. iDstances where fihl-inogen··breakdown products were present in the serum. Physiologic site. predominently associated with fibrinogen. breakdownpl'oducts included tbe li.vel". the lddney.<. and the' intravascular system. BIBLI.OGRAPHt Alillli, S.Y., J~Wo Hampton, GoJ. Race, and R.J. Speer. stabillzing factor {Factor XIII}. Amer. J co Med. 1968. Fibrin 44~ 1-7. Alkjaersig, No, A. P. Fletcher, and S" Sberry. 1958a. The activation of human plasminogen. I. Spontaneous activation in glycerol. J Biol. Chem. 233: 8l-8S. p Alkjaersig. No. AoPo Fletcher, and S. Sherry co 19S5b. The act"t.vation of human plasminogen. II •. Akinetic study of activation wid txypsin, urokinase, anc:l-st:reptoJdnase. J. Biol. Chem. 2.332 86-900 Alkjaersig, H•• A.Po-Fletcher, and S. Sherry. 1959a. £-Amino caproi.c acid: an inbibitor of plasminogen acti-vation. J. Biol. Chem. 234: 832-837. Alkjaersig. N., A.P o Pletcher, and So Sherry. 1959b. The mecbanism of clot dissolution ))y plasmin. J. Clin. Invest. 38: 1086-1095. Alkjaersig. N., A.P. Fletcher. and S. Sherry. 1962. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic '''fibrinolytic'' 1 states. II. The significance. Dl8cbanism and consequences of defective fibrin polymerization. J" ClinG Invest. 41: 917-934. Alkjaersig. N. and S. Fischer. 1964. Proteolysis of fibrinogen by plasmin. Thromboso Di.athes" Haemorrho 11: 284-285. Bang, NoU •• A.P. Fletcher. N. Alkjaersig. and So Sherry. 1962. Pathogenesis of the coagulation defect developing during pathological proteolytic {"fibrino1yti.c"1 states. 11140 Demonstration of abnormal clot structure by electron microscopy. J .. Clin. Invest. .41: 935-948. . Beck, EoA. and D.. P. Jackson. 1966. Studies on the degl'adation of human fibrinogen by plasmin and trypsin. Thrombos4O Dlathea. Haemorrh. 16: 526-540. Beller. F .K. and M. Maki. 1967", Properties of fibrinogenolysis and fibrinolysis products in immune assaySo Thrombos. Diathes. Haemown. lS: 114-132. Bergstrom, K., B. Blomback. and G. Kleen.. 1960. Studies. on the plasma fibrinolytic activity in a case of liver cirrhosis. Acta. Mad. Scand.168t 291-305. 19 Bigp, R. and R. G. HeF_lane. 1963. Human blood coagulation. 3rd Ed. Blackwell Scientific PuDllcations., Oxford. Byers. Ro 19664t Model R-103 i1llllunoelectrophoresis. accessory ins.truction manual. Beckman Instruments, Inc., Fullerton, California. Christensen, L.R. 19.45.. Streptococcal fibrinolysisn a proteolytic reaction due to a serum en~ activated ~ streptococcal fibrinolysin. J. Gen. Physiol. 28: 363-383. Crowle, A.J. 1961. Immunodiffusion. Academic Press, Nev YorK. Dellelbach., H.R. and S.E. Ritzmann. 1~7. LaD synopses, Vol. 2. Hoechst Pbannaceutical Co.,. Kansas City. DeNicola, P. and F. Soard!. 1958. Fibrinolysis in liver diseases. Study of· 10"9 cases. by means of the fibrin plate methocl. Throml>os. Diathes. HaemorrD. 2: 290-299. Finkbiner, R. B.., J. J. McGovern, R. Goldstein.. and J.P. Bunker. 1959. Coagulation defects in liver disease and response to transfusion dUf'ingsurgery. Amer. J. Med. 26: 199-213. Fischer, S., A.P. Fletcher, N. Alkjaersig, and S. Sbel":ry. 1963. Fibrinogenolysis in vivo: Identification,. occulTence~, and. characterization 'Ii ~ J. Clin. Invest, '+2: 931. (Abstl".) Fisher, S., A.P. Fletcher, N. Alkjaersig, and S. Sherry. 1967. Immunoelectrophoretic cliaracterization of plasma fihrinogen derivatives in patients with pathological plasma proteolysis. J. Lab. Clin. Med. 7G: 903-922. Fletcher, A.P., N. Alkjaersig, and S. Sherry. 19590. The maintenance of a sustained thrombolytic state in man. I. Induction and ·effects.- J. Clin. Invest. 38: lOge-lllO. Fletcher, A.P., N. Alkjaersig, and S. Sherl"Y. 195.9h. Pathogenesis of hemonbapc diathesis developing during· .tfibrinolytic" states: Th8 s1pificance of defective fibrin polymerization. J. Clin. Invest. 3S: 1005. 4Abstr.) Fletcher, A.P., N. Alkjaersig, and S. Sbel'l"J'. 19.6.2a. Fibrinolytic mechanisms and the development of thrombolytic therapy. ArneI' If J. Med. 33: 738-752. 80 Fletcher. A.P •• N. Alkjaersig. and S. Sherry. 1962b. Patbogeneaia of the coagulation defect developing duriul pathological plasma proteolytic (.·'fiDrinolytictr ): states. I. The significance. of fibrinogen proteolysis and circulating fibrinogen breakdown products. J. Clin. Invest. 41: 89.-916. o. Fletcher. A.F o. Biederman. D. Moore, N. Alkj aeraig. and S. Sherry. 1964. A])normal plasminogen-plasmin s.ystem activity (fibri.nalyaisl. in patients: witli nepatic cirrhosis: Its cause and consequences41 J Clin. Invest. 43: 681-69.5. Q Fletcher. A.P. 19.iS. 24: 822-82f.. FiD't'inolytic dysfihrinogenemias. Fed. Prac. Fletcher. A.P41, K41 Alkjaarsig. S. Fisher-. and S.Sherry. 1966. The proteolysis of. fnrinogen hy pla.sm.tn: The identification of tnromDin clottable.fi.brinogen.derivatives which polymerize abnormally. J. LaD. Clin. Ked &8: 7 8G-8G2. 0 Forman. Well •• and M.J aarnhart. 1964. Cellular site for synt1lesis. J. Amar. Med. Assoc. 187: 128-132. 0 Fulton. J.r. 18.56. Textb.ook of Physiology. Philadelphia p. 923. ffl)r~nogen W.B. Saunders, Co. Gallimol'8. M.J. and J. T .B'41 Sh_. 1967 Some aspects of f1arin clot lysis. and ita inb.ib:itl;,on hyhuman serum. Thrombos. Diathes. Q HaemOl'l"A. 13: 101-113. Gladner, A. t P.A., Murtaugh, J.E. Folle. and K. La1ci. 1963. NatUN of peptides released hy"thrombin. Ann. N.Y. Acad. S.cl. 1041 47-52. Glueck. H.Jo and L.G ... Herrmann. 196-It. Gold precipitah.le~ fiIJrinogen ftcryofibrinogent ... Arch. Int. Med.· 113: 748-757. Godal, H.C. and I. Helle. 196,3. The influence of f!Jarinogenolytic and fibrinolytic split products. on the last stage of coagulation. Scand. J. Clin. Lab. Invest. 15: 327-·330. Goodpasture. E e W. 1911t. F.ibrinolysis; i.n chronic hepatic insufficiency. Jonns Hopk. Hasp. Bull. 25: 330-336. Gormsen, J. and :a. Laursen. 1967. Fibrinogen breakdown products and clotting parameters. Thrombos. Diathes,. Haemorrh. 17 : 467-481. 81 Grossi, C.E. t A.H. Mareno, and L.M. Rausse10t. 1961. Studies on spontaneous fibrinolytic activity in patients with cirrhosis of the liver and its innibition by epsilon amino caproi.c acid. Ann. Surge 153: 383-3930 Grossi t C.E. t L. Mg. Rausselot, and W. F. Panke. 1962. Coagulaticn defecta in patients witn- cirrhosis-of· tile liver undergoing portasystemic shunts. Amer. J .. SUI'g. 104: '512-526. Hall, C.E.. and H..S. Slayter. 1959. The fihl'inogen molecule: Its size,shape,and mode of polymerization. J. Siaphys. Biochem. eytol. 5: Il-lS. Hl:rscbfi-eld, J 1960.. IDlmunoe1ectl'opboresis.J Procedure and application to the study of group-specif ic·variati.ODS in sera. Sci. Tools 1: 18-2.5. 0 SiNh, J., A.P. Fletcber. and S. Sherry. 1965. Effect 'of fih·rin_ and fibrinogen proteolysis products in clot physical pt'opel't·1es. Amel'. J. Physiol. 209: 4l5~424. Hunter, D. T. and J .Lo Allensworth. 1965. A modified fibrino,en assay. Amer. J. Clin. Path. 44: 359-363. Hunter, D. T. and J .. L., Allensworth. 1966. Plasmin. A new method for the estimation of physiologically active plasmin. Hemostase 1: 201-206. Jacobsen, C.Do 1968a. Proteolytic capacity in human plasma. Scand.J ClinG Labci> Invest. 21: 21&-226. 0 Jacobsen, C.D. 1968b. Proteolytic capacity in human plasma. II. Genetics and clinical study. Scand-.J. Clin. Lab .. Invest. 21: 221-231. Jamieson, G.A. and J oH. Pert. 1963. Studies on the digestion of human fibrinogen with plasmin. Vox Sang. 8: 460 (Abstr.) Jerushalmy, Z. and M.S. Zucker. 1966. Some effects of fibrinogen degradation proc:lucts CroP) on blood platelets. Thrombos. Diathes. Haemorrb. lSI 413-419. Kalbfleisch, J.M. and R.M. Bird. 1960. England J .. Med, 2a3: 881-·886. Cryofibrinogenemia. New Kaplan, M.H. 19,44. Nature and role of the lytic factor in hemolytic stl'eptoooccal fibrinolysis. P1:'OCh Soc. Exp. Biol. (N.Y.) 57: 40~3. 82 Kaulla, K.N. von and R.L. Schultz. 1958. Methods for the evaluation of human fibrinolysis. Amer. J. Path. 29: l05-~11. Kopec, M., E. Kowalski, and J. Stachurska. 19&0,. Studies. on peacoagulation. Role of antithrombin III. Thrombos. Diathes. Haemorrn. 5: 285-295. Kopec, M., A.Z. Budzynski, J. ItachUNka, Z" Wegrynowicz, and E. Kowalski. 1966. Studies on the mechanism of interference by fibrinogen degradation products (rep 1 with the platelet· funct ion. Role of fillrinogen in the platelet atmo&phere. ThrOmDos. Diathes. Haemowh. 15: 476-490. Kowalski, E. 19.0,8. Fibrinogen derivatives. and their biologic activities. Hematology 51 45-SS. Kowalski, E., M. Kopec, and Z. Wegrzynowicz~ 1964&. Influence of fibrinogen degradation products (FDP) on platelet aaregation, &dbesi veness and viscous metamorphosis. Thrombos. Diathes. Haemorrb. 10: 406-423. Kowalski, E., A.Z. Budzynski, M. Kopec, Z.S. Latallo, B. Lipinski, and Z. Wegrzynowicz. 1964b. Studies on the molecular pathology and pathogenesis ·of bleeding in severe. fibrinolytic states in dogs. Tb:rombos. Dlathes .. Haemorrb. 12: 69-86 • .Kwaan, H.C., A.J .S. McFadzean, and J. Cook. 1956" Plasma fibrinolytic activity in cirrbosis of the liver. Lancet 1: 132-131. Kwaan, H.C., A.J.S. MeFadzean, and J. Cook. 1957. Onplasma fibrinolytic activity in cryptogenic splenomegaly. Scot.. Ked. J. 2: 137-150. . wi, K., J.A. Gladner, and J.E. Folk. 1960. Some aspects of the fibrinogen-fibrin t.ranaition. Nature 187: 758-761. Latallo, Z.S., A.P. Fletcher, N. Alkjaersig, and S. Sherry. 1962a. Influence of pH, ionic strength, neutral ions, and thl'ombin on fibrin polymerization. Amer. J. Physiol. 202: 615-680. Latallo, Z.S., A.P. Fletcher, N. Alkjaersig, and S. Sherry. 1962b. Inhibition of fibrin polymerization by fibrinogen proteolysis products. Amer. J. Physiol. 2021 681-686. Latallo, Z.S •• A.Z. Budzynski. B. Lipinski, and E. Kowalski. 1964. Iabi»i tion of thrombin and of fibrin polymerization, two activities derived from plasmin-digested fibrinogen. Nature 203: 118'+-1185. 83 Laursen, B. and J. Gonnsen. 1967. Spontaneous fibrinolysis demonstrated by immunological technique. Tbrombos, Diatbea. Haemorrh.. 17:' 42-50. Lewis, J.R. and J.H. Wilaon. 1964. Fibrinogen breakdown products. Amer. J. Physiol. 207: 1053-1057. Liniger, W. and P. Ruegsegger. 1967. Diathes. Haemorrh •. 17: 412-417. Fibrino~ysis.. Tbrombos. Lipinski, B., Z. Wegrzynowicz, A. Z. Budzynski, M. Kopec, Z.S •. Latallo, E. Kowalski. 1967. Soluble. unclottable complexes formed in the presence of fibrinogen degradation products. (FDP) during the fibrinogen-fibrin conversion and their potential signiflcance in pathology. Thrombos. Diathes. Haemorrh. 17: 65-77. Lorand, L. and A. Jacobsen. 1958. StudiesOD the polymerizatioD of fibrin. The role of the globulin.: Fibrin-stabilizing factor. J. Biol. Chem. 230: 421-434. Lorand, L. 1965. Physiological roles of fibrinogen and fibrin. Fed. Proc. 24: 784-793. McFarlane, R.G. and R. Biggs, 1948. FibrinOlysis: Its mechanism and significance. Blood 3: 1167-1187. Menon, J.S. 1969. A study of fibrinolytic activity in" subjects with different diseases. Lab. Pract. 18: 427-428. Herskey, C., G.J. Kluner, and A.J. Johnson. 1966. Quantitative estimation of split products of fibrinogen in human serum related to diagnosis and treatment~ Blood 28: 1-18. Miale, J.B. 1967. Laboratory medicine hematology. Mosby Co., Saint Louis. 3rd Ed. C,V. Milstone, Haskell. 1957. A factor in normal human blood wbich participates in streptococci fibrinolysis. Thrombos. Diatbes. Haemorrh. 1: 264-286. Mullertz, S. 1955. Formation and properties of the activator of plasminogen and of human and bovine plasmin. Riochem. J. 61: 424-434. 84 McKay, D.G., A. Ki~, and B. Alexander. 1959. Experimental producticnof afDl-illopaemia and Aemor'l'hagtcpDenomena comDined filil-molyaia aDd disseminated intravascular coagulation. Newo England Jo Medo- 261: 1150-1154 I),. 0 McKay, D.G. 19&5. Dfaaemmated intravascular coagulation. Harper and RCK, New pp. 8-9. Yo~i.. McKee, P.A., J.M. lC.a1Bfleisch. ad R.M. Bird. 1962.. TOe incidence aDd &ip.iftcance of cryof~tnogemia. C1in. Res. 10: 56. GAaair.l Hanninga, L.ll. 19&&. Pl'eparation of anticoqu1ant split-product of farinogen and i'ta determ:tnation ill p1_. Fed. Proc. 25: Uktl'.). "'' 5. Hileb, J.&. and J.M. Nelsson. 1964. Demonstration of fil)riDolytic split products :in human S81'UII liyan immuno101fca1 methoi-h spontaneous and .induced fi1)rino1ytic states. Scand. J. Haemat. 1: 313-330.' Hi~hQ, J .E. 1967a. Split products. of fil)rinOien after" pro1onlec:l interaction with plasmin. . Thrombos. Oiathea. Haemor'l'h. 18: S9-1ee. Kllehn, J.E. 1967:0.. Separation and estimation of split "products" of fibrinogen and. f11lrin in human serum. Tb..rombos. Oiathes. Haemol'l'n. 18: 487-49.8. Nolf, P. 190.5. Des. mcdlfications.· de 18 coagulation du sang cliez le clUen apres exterpation du foie. Arch. Int. Physio1. 3: 1 (.as reported h.1 Sherry, S. and N. Alkjaersig. 1957. Studies on the fiirino1ytic enzyme of human p1as.ma. Haemorrh. 1: 261f-296.) Thrombos. Oiathes. Nussbaum, M. and a.s. Morse. 1964. Plasma f!brinstahi1izing factor activity in var-ioua diseases. B,l00ci 23: 669-678. 1960. Analyse par des, methodes imUDocbemiquea. de 18 degradation par la pasmine du fibrinogene humain et de 18 f~ine, a differentea stages. Rev. Hemat. 15: 451 t- reported Dy Gormsen. J. and - B.. Laursen. 1967 • Fibrinogen brea1c4own products and clotting parameters-. Thromboa. Diathea. Haemorrh. 11: 461-481.) Nussenweig, V. and M. Selegman. 85 Osbabr, A.J •• J .A. Gladner. and. 1(.. LaId" 19fi4. Studies on the pblsi-ological activity of tbe peptide released during the fibrinogen-fiDrin cOIlversion. Siocnem. :BLaphys,. Acta. 86: &35-5420 PhanDacia Fine Chemicals Incorporated" 1966. Sephadex-gel filtration ir1 tneoX"Y' and practice" Pescataway, Hew Jersey. Purcell, G. and L.L. Phillips. 1963. Fibrinolytic activity in cirz'1iosis oftbe liver. Surg. Gynec. Obstet. 117: 139-144. Quick, A.J. 19&6. HemoZ'rhagic disease and thrombosis. Fellelex-, Philadelphia. Lea and Ratnoff, O.D. 194~. Studies on a pZ'oteolytic enzyme in plasma. IV. The Z'ate of lysis of plasma clots in normal and diseased individuals with particular reference to hepatic disease. Johns Hopk. Soap. Bull. 84: 29-42. Ratnoff, O.D. 1954. An accelerating property"" of, plasma for the, coagulatioo of fibrinogen by thrombin.. J. "Clin. Invest. 33 : 1115-1182. Ratnoff, O. D. 1955. The effect of clotting on the spontaneous activation of plasmin.' J .Clin. Invest. 34: 9:58-959. Rodman, T. 1968. Blood c08aulation, and' clot, lysis. pp. 113-153. In J1" L. Rabinowitz and R.N. Myerson Topics in Medicinal Chemistry. Interscience Pub., New York. Sasaki, T., I.H. Page and J .R. Shain,off. 1966. Stahle complex of fibrinogen and. fibl"in. Science 152: 1069-1011. Sawyer, W.O., N. Alkjaersig, A.P. Fletcher, and S. Sherry. Studies on the thrombolytic activity of human plasma. Clin. Invest. .' 39: 426-434. 1960a. J• Sawyer, W.D., N. AlkjaeZ'Sig, A.P. Fletcher, and S. SheJ:'l'lY. 1960b. A comparison of fibZ'inolytic and fibrinogenolytic,. effects. of plasminogen activatoNand proteQlyti,c enzymes in plasma. Thrombos. Dtathe•• Haemorrh. 5: 149-161. Sebe1deaer, J.J. lt55. Une micro.methode de l'immunoelectZ'ophorese. Intern. Arch.• Allergy Appl. Immunol. 7:. 103-110. Schultze, H.E. and J. F. Heztemans. 1966. MoJ.ecul.ar biology of human proteins. Elsevier, Amsterdam, London, New YOZ'k1I' 86 Searcy t R.L. 1969-. Dlagnostlc biochemistry '" Company. New Yo~k. Seegers, W.H" 19.62. Seegers, W.R. 1967. New York. Prothrombin" McGraw-Hil1 Book Harvard, Cambridge .. Blood clotting enzymology. Academic Press, Shainoff, J .R. and T .H. Page. 19GB. .Cofibrins.and fibrin!'" in.termed-lates as iDdicators of thrombin activity- in vivo. Circulation Research 8: 1013....1022. -Shainoff, J .R., and T .H. P-age. 1962. Significance of cryofihrin in fibrinogen....fihriD conversion. J. Exp. Med. - 116: &87-707. Sberry, S. and N. Alkjaersig. enzyme of human plasma. 19S7. Studies on -the- fibrinolvticThrombos. Diathes. Haemorro. 1: 264-288. Sherry, S., A.P. Fletcher, and N. Alkjaersig. 1959a. and fibrinolytic activity in man. Physiol. Rev. Fibrinolysis 391 31+3-382. Sherry, S., R.T. Lindemeyer, A.P. Fletcher, and N. Alkjaersig. 19S9h. S'tudies on enhanced fibrinolytic activity in man. Clin. lnveat. 38a 810-822. J. Solum, N.O. 1966. Platelet aurelation during fibrin polymerization. Scand. J. Clin. Lab. Invest. 18: 577-587. Tillett, W.S. and R.L. Gamer. 1933. The fibrinolytic activity of hemolytic s.treptococci. J. Exp. Med S8: 485-502. (i Todd, A.S. 1959. Tbehistological localization of fibrinogen activator. J. Patb. and Bact. 78: 281-283. Triant opby llopoulos-. &. and D. C. Triant ophy llopoulos. 1962. Coagulation _s.tudied _on the electrophoretic fraction AFIF. J. Physiol. 203: S.95-5~9. Arner. Troll, W. and S. Sbel'ry. 1955. The activation of human plasminogen by streptokinase. J. Bfol. Chem. 213: 881-,891. WeeX'dt, C.M. van del" and J. Vreeken. 1965. Influence of cryofiMin on the antiglobulin consumption test with platelets. Vox. Sang. 10: 53&.-5-42. 87 Wilson, P.A., G.P. McNicol, and A.S. Douglas., 19.6.8. Effect of fib%'inogen degradation pZ'oducts on platelet aggl'egation. J. Clin. Path. 21& 147--1S-3. Zlotnick t A. and S. Landau" 1966. IlIIIlunoelectrophoret!c s1:uciies in patients with cryofibrinogenemia. J .. Lab. Clin.· Med. 68: 70-80.. Zucker, M.D.., M. Siegel, E.&. Cliff tOll, J.W., Bellville, LS. Rowand, ad C.E. Grosalo 1957 \) Tile effect of liepatic lohctomy on some blood clotting factors and on fibrinolysis. Ann. Surg" 14.: 112-181.