Effects of leukocytes, plasmin and thrombin on clotting factors a comparative in vitro study

THFtOmosIs RESEARCH 16: 301-312 @Pergamon Press Ltd.1979. Printed in Great Britain

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EFFECTS OF LEUKOCYTES, PLASMIN AND THROMBIN ON CLOTTING FACTORS A COMPARATIVE JN VITRO STUDY Per Henriksson and Inga Marie Nilsson Department of Paediatrics and Coagulation Laboratory, Allmtina Sjukhuset, University of Lund, Malmti, Sweden (Received 2.4.1979; in revised form 10.5.79. Accepted by Editor H.C. Godal) _ __

ABSTRACT

In an in vitro investigation the influence of leukocyte extract, plasmin and thrombin on various clotting factors was compared. The clotting factor preparations used as substrates were fraction I-O (fibrinogen, factor VIII and factor XIII) and Preconativ (factors II, VII, IX and X) (Kabi, Sweden). The various factor VIII activities (VIII:C, VIIIR:Ag - electroimmuno aSSay, VIIIR:Ag immunoradiometric assay and VIIIR:RCF) and factor XIII subunit 2 showed a degradation by leukocyte extract resembling that of plasmln. During incubation thrombin first activated and then destroyed VIII:C. The prothrombin complex (factors II, VII and X) and the separate activities of factor VII and factor IX were degraded by the leukocyte extract as well as by plasmin. A fibrin/ fibrinogenolytic activity by the leukocyte extract was demonstrated. Multiple defects in the coagulation and fibrinolytic systems complicating many disorders might well be produced by proteases frcm leukocytes and injured tissue cells.

INTRODUCTION The roles played by the leukocytes and their lysosomal constituents in ha-static disturbances have been receiving increasing attention during the last few years. Procoagulant activity (l-6), fibrinogenolytic/fibrinolytic activity (7-111 as well-as kinin activating activity (12) have been described. Also a direct action of leukocyte proteases on isolated coagulation factors, such as fibrinogen (13, 14) and factors V, VIII, XII and XIII (15) has recently been reported. In certain clinical conditions, haemostatic defects have been 301

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explained by a direct effect of proteases liberated from leukocytes rather than of a thrombin and/or plasmin activity, namely in acute leukaemia and septicaemia (16), in erosive gastritis (17), in Weber-Christian disease (18) and in Henoch-Sch&lein's purpura (19). This paper reports a series of in vitro experiments designed to show the effect,of leukocyte extract on isolated clotting factors and to compare that effect with plasmin and trace mts of thrombin.

MATERIALS Human fraction I-O @iH1_K_&&+_gSggeL prepared according to Blo~~iS~'~8'6i~~~~~-(20), Nilsson et al. (21). usincr fresh frozen platelet-poor plasma as starting material. As shown by Nilsson and Hedner (22) fraction I-O carries about 3 u/ml of VIII:C, about 6 u/ml of VIIIR:Ag (e~ectroimIuno assay and immunoradiometric assay), about 5 u/ml of VIIIR:RCF, about 17 g/ 1 of fibrinogen and about 150 FSF u/ml of factor XIII (amine incorporating u/ml of titrated plasma /23/) corresponding to about 8 u/ml. Preconativ Sweden1 a human plasma fraction containing ___________lKabi ,,,,~,,,,,,, prothrombln 25 u/ml, factor VII 10 u/ml, factor IX 25 u/ml and factor X 25 u/ml. HumanLElasmin ~Plaamin,_Ea& Swefiepl,dissolved in 50 % glyEGz;f gI?3[fiz'a ~~~~~&atIon GP'T.5 CTA u/ml in the stock solution. Bovigf thrombin (Topostasin Roche, Switzerland) stock solutioii'&t~l;iIii~'~~ONIH u/ml. The separation technique of !ia&afe_n%fr~ct kwwratQn. Bayum (24) was &~~'M'I&a'i~;ikocyte preparations were obtained by layering leukocyte-rich blood from healthy donors on top of a medium containing equal volumes of dextran and sodium-metrizoate (3 % dextran T-500 Pharmacia, Sweden, 10.9 % Na metrizoate solution, Nyegaard a Co, Norway). After sedimentation of the erythrocytes the cell-suspension was centrifuged at 700 p for 15 min. The remaining erythrocytes were haemolysed by resuspension of the cell button in 0.03 MNaCl for 30 sec. me suapnsian was brought to isotonicity by adding an equal volume of 0.28 M saline. This procedure was repeated four times and was followed by washing three times in isotonic saline. The cells were then resuspended in 0.1 M Tris (pH 7.4) giving a leukocyte count of approximately 150 x 109/l. The cell suspension contained also platelets, approximately 3000 x 109/l. The leukocyte extract was obtained by disrupting the cells by freezing and thawing 8 times followed by centrifugation at 3000 3 for 30 min in order to remove cellular debris (8). The supernatant was stored at -700C until used. A pure preparation of platelets disrupted by freezing and thawing 8 times was used for testing whether the platelets con-minatingthe leukocyte preparations and known to carry various proteases (25, 26) could significantly contribute to the proteolytic activity. No leukocytes were observed in the platelet

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suspension.

METHODS Fibrlnolytic activity on human fibrin plates was determined according to Nilsson and Olow (27). Plasminogen-rich as well as plasmlnogen-free fibrin was used. Fibrinogen, factor VII, VIII:C, factor IX and P&P (factors II, VII, X1 was measured according to Nilsson et al. (28). VIIIR:Ag (electroimnuno aSSay1 according to Holmberg and Nilsson (291, VIIIR:Ag (immunoradiometric assay) according to Ruggeri et al. (30) and Holmberg and Ljung (31), and VIIIR:RCF according to Zuzel et al. (32). Factor XIII subunits a and b were determined immunologically with the Laurel1 tech:ique 733) using antisera from Behringwerke, West Germany. Crossed immunoelectrophoresis was done on factor VIII and on factor XIII a according to the technique described by Ganrot (34).

EXPERIMENTS

AND

RESULTS

All the experiments with fraction I-O and Preconatlv were performed at 37OC and the incubations were terminated after 1, 4, and 24 h by rapid freezing in a cooling bath. The samples were then stored at -7OOC until examined.

Incubation mixtures containing fraction I-O 1. Fraction I-O and leukocyte extracts. One part of fraction __________________-_------------I-O, one part of 0.1 M Tris buffer pH 7.4 and 2 parts of leukocyte extract giving the following approximate levels of VIII:C of 0.7 u/ml, VIIIR:Ag of 2 u/ml, VIIIR:RCF of 5 u/ml, fibrinogen of 4 g/l and factor XIII of 2 u/ml. 2. Fraction I-O and plasmin. ---------------------- One part of I-O, three parts of 0.1 M Tris buffer pH 7.4 and 3 different amounts of plasmin, resulting in the final plasmin concentrations of 0.03, 0.96 and 1.92 CTA u/ml. 3. Eranrioa_I=O_nnQ_tbr~~~~. One part of fraction I-O, three parts of 0.1 M Tris buffer DH 7.4 and thrombin sivins a final thrombin concentration of 1-x 10'4 NIH u/ml. This was the highest thrombin concentration that did not produce a clot within 4 h when incubated with fraction I-O. A mixture of one part of I-O and 3 parts of 0.1 M Tris buffer DH 7.4 was used as a control. The results of the incubation studies with fraction I-O are shown in Fig. 1 and 2. VIII:C was rapidly extinguished by leukocyte extract and by plasmin. Thrombin, however, caused an initial activation of VIII:C followed by a rapid decrease.

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= I-o*LE 0 Tris 1-O 0 1-O ??

---I-OTrts

buffer A I-O*Plasmin 0.96 CTA u/ml ??

LE Plasmin 003 CTA u/ml Thmmbin 1x10“ u/ml

?? ?? ??

”

--A A 0

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I-O*LE I-O+Tris buffer I-O Ptasmin 0.96 CTA u/ml Rasmin 1.92 --I-O ?? I-O Thrombin 1 *l(r4 u/ml ??

??

WR:RCF u/M

VlllR:RCF u/N

VISIR:RCF u/@

I .

1

b*?iLt

b”‘,

b’-xh

FIG. 1 Influence of leukocyte extract (x), plasmin 0.03 CTA u/ml (o), 0.96 CTA u/ml (a), 1.92 CTA u/ml (A) and thrombln 1 x 10'4 NIH u/ml (I) on VIII:C, VIIIR:Ag (EI), VIIIR:Ag (IRMA) and VIIIR:RCF determined at 1, 4 and 24 hours' incubation at 370C. Dashed line denotes the control. (o denotes VIIIR:Ag (EI) levels in leukocyte extract and Tris buffer and represents the platelet contamination. In the determinations of the plasmin effect on VIIIR:RCF (0 A) a higher concentration of fraCtion I-O was used.)

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= I-O*LE ---I-O *Tris buftr . I- 0. Ptasnin 403 CTA u/ml

305

. I-O*Plrsmin 0.96 CfA ulmt b I-O*PLaslnin 1.92 --0 I-O*Thrcmbin lmlO*u/ml

FIG. 2 Influence of leukocyte extract (x), plasmin 0.03 CTA u/ml (o), 0.96 CTA u/ml (4, 1.92 CTA u/ml (rJ and thrombin 1 x 10'4 NIH u/ml ((=I)on factor XIII a determined at 1, 4 and 24 hours' incubation at 370C. Dazhed line denotes the control. (The plasmin incubations were done with a higher concentration of fraction I-O.)

.

The leukocyte extract Itself contained VIIIR:Ag (EI) owing to the contribution of platelet VIIIR:Ag , and during incubation of the extract alone the levels of VIIIR:Ag (EI) were raised. But the VIIIR:Ag _- (EI) concentration in the mixture of leukocyte extract and fraction I-O did not rise significantly during incubation. The concentration of VIIIR:Ag (IRMA), however, decreased during incubation of I-O with leukocyte extract. Plasmln markedly raised the VIIIR:Ag (EI) concentration, but lowered the levels of VIIIR:Ag (IRMA). The immunoradiometrlc assay was performed at various dilutions. The dose-response curves did not run parallel with those of the controls. During leukocyte extract incubation the VIII protein displayed a slight change in rate of migration on crossed immunoelectrophoresis resulting in a quotient of 1.3 and 1.4 In relatlon to the control (normal value <1.2). The plasmin already in the lower of the three concentrations used produced quotients around 1.6. VIIIR:RCF was lowered by leukocyte extract, and by the higher concentration of plasmin. Thrombin, however, caused no changes in the VIIIR:Ag (EI) or VIIIR:RCF. The fibrinogen values at 1 and 4 hours during incubation with leukocyte extract were 4.5 and 0 g/l while the controls were 4.3 and 4.4 g/l respectively. The lowest plasmin concentration reduced the fibrinogen level from 6.2 to 6.3 g/l at 1 hour and to 2.2 g/l at 4 hours (control

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FIG. 3 Influence of leukocyte extract (x1 and plasmin 0.03 CTA u[ml on P&P (factors II, (01, 0.96 CTA u/ml (a), 1.92 CTA u/ml 0 VII, X1, factor VII and factor IX determined at 1, 4 and 24 hours' incubation at 37OC. Dashed line denotes the control. (The effects of plasmin on PIP and factor IX were studied with a higher concentration of Preconativ.]

values 8.2, 8.3 and 8.3 g/l respectively). The higher concentrations of plasmin reduced the fibrinogen level to zero at one hour. (In the plasmin experiments only a twofold dilution of fraction I-O was used.) Factor XIII g concentration was reduced by the leukocyte extract as well as by the two higher concentrations of plasmin, while thrombin had no demonstrable effect (Fig. 2). Crossed immunoelectrophoresls of XIII 2 revealed no difference from the control. No change in the XIII b concentration was observed. an some of the incubatxons with plasmin a lower dilution of I-O was used. This is mentioned in the legends.1

Incubation mixtures containing Preconativ 1. Preconativ and &eukocy~~_~~$~act. One part of Preconativ, 4 part~'~Z'~Ti'fi'??~~ %?% pH 7.4'GEd 5 parts of leukocyte extract. The final l/10 dilution of Preconativ gave the following approximate activities of prothrombin of 2.5 u/ml, factor VII of 1 u/ml, factor IX of 2.5 u/ml and factor X of 2.5 u/ml in the incubation mixture. 2. -------_-_----Preconativ and p&asmin. One part of Preconativ, 9 parts of 0.1 M Tris buffer pEI 7.2";;; plasmin resulting in 0.03, 0.96 and 1.92 CTA u/ml in the final incubation mixtures. One part of Preconativ and 9 parts of 0.1 M Tris buffer pH 7.4 was used as a control. (In 2 experiments

with Preconativ

and plasmin

only a 5-fold

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dilution of Pteconativ was used.) Results from the Preconativ incubation studies are presented in Fia. 3. The leukocvtes produced a marked and rapid decrease in th; activities of -tie prothrombin complex (II, VII, X1 and in the separate activities of factors VII and IX. Plasmln, however, did not produce such a marked reduction of the prothrombin complex or of the factor VII activity. The platelets did not cause any significant changes when incubated with fraction I-O or with Preconatlv. Fibrinolytic studies. The leukocyte extract produced lysis on unheated human plasminogen-rich fibrin plates. The lytic area was 105 arm*. Only a weak lysis was seen when tested on plasminogen-free fibrin plates.

DISCUSSION Thorough investigations have been made on the ability of leukocyte proteases to produce both a direct flbrinogenolytic and fibrinolytic activity, unaffected by RACA or Trasylol, and a plasm2nogen activator activity inhibitable by EACA (11, 35). A differentiation between the two alternative pathways of fibrlnogeno-fibrinolysis has also been made possible by differences in pattern of cleavage (8, 14). We found that fibrinogen was lncoagulable after 4 h's incubation with leukocyte extract, an effect resembling that of the low plasmin concentration. The extract produced lysis on unheated plasminogen-rich human fibrin plates, .butwas practically inactive on plasminogen-free plates. These effects might represent both the direct fibrinogenolytic activity and the plasminogen activator activity which is resistent to the extraction process, as documented earlier (35, 36). VIII:C was rapidly degraded by leukocyte extract, which is in accord with the in vitro observation by Schmidt et al (15). Also the effects of plasmin and thrombin on VIII:C in our study agreed with those on record (37, 38, 39). An apparent increase of VIIIR:Ag measured by electroimmuno assay was noted during leukocyte extract Incubation. This does not represent a real increase of the protein concentration, but is a result of a conformal change of the molecule which was further illustrated on crossed lxununoelectrophoresis.The change resembled that brought about by plasmln although this was more pronounced. That VIIIR:Ag (IRMA) and VIIIR:RCF decreases during the same conditions are probably further expressions of the partial degradation of the VIII molecule. This means a diminishing functional capacity, measured as the ristocetin induced aggregation of platelets (VIIIR:RCF) and destruction of the major antlgenic sites, which have the greatest affinity to the antibodies during the lnnnunoradiometricassay (VIIIR:Ag /IRMA/). No noteworthy differences were seen between the effect of leukocyte extracts and that of plasmln. Owing to the long incubation time used in the assay, it must, however, be pointed out that remaining proteases in the mixtures, although very diluted, might influence the antibodies coated on the tube walls

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and result in false low VIIIR:Ag (IRMA) levels. As shown in earlier in vitro studies, the factor XIII activity was reduced by leukocyte proteases (15). We found that the concentration of factor XIII subunit 5, which carries the plasma transamidase activity, was reduced both by the leukocyte extract and by plasmin, while factor XIII subunit b was not affected. Other fibrlnolytic studies have shown the -factor XIII activity to be preserved, both In vitro (40, 41) and in vivo (41, 42). In one in vitro study a low temperature and a short incubation time were used; in the other , the use of plasma as incubation medium, might explain the discrepancy between the findings. Both the leukocyte extract and plasmin produced a reduction of the prothrombin complex (factors II, VII, X) and of factors VII and IX when determined separately. In a previous study elastase-like and chymotrypsin-like proteases were shown to have no effect on bovine prothrombin but a moderate effect on factor VII (15). According to a recent report by the same authors, human prothrombin is degraded by elastase-like granulocytlc proteases (16). In our plasmin experiments we used plasmin concentrations within a "physiological potential". Since the ffbrinolytic potential in the circulation Is reflected by the plasminogen concentration it will not exceed approximately 5 CTA u/ml, In practice about l/3 of that activity due to the Instant a2-antiplasmin-plasmin reaction and their molar relationship (43). During the last 15 years a variety of clinical disorders have been shown to be complicated by the secondary phenomenon of disseminated intravascular coagulation (DICl(441, which implies thrombin activation in the circulation and secondary fibrinolysis. However, proteases different from thrombin and plasmin and liberated from leukocytes and damaged tissue cells in various diseases mentioned in the introduction can degrade many coagulation factors, as shown in our experiments, and may cause laboratory manifestations similar to those In the DIC syndrome. The conditions in vivo are, however, more complex owing to the potent inhibitor system, mainly a2-macroglobulln and alantitrypsin (45). These inhibitors limit the effect of the abnormal proteolytlc activity to local tisoue damage or inflammation where the inhibitory capacity will soon be over-saturated. It is difficult to produce laboratory evidence for such mechanisms since the complexes formed are eliminated so fast (46). In patients with leukaemia and septicaemia with coagulation defects complexes between al-antitrypsin and elastase-like granulocytic protease have, however, been demonstrated (16). The pattern in vivo will also be modified by the various coagulation factors' property of being reactive proteins. The underlying disease enhances their production, thus counterbalancing their destruction. Factor XIII, however, does not seem to behave in the same way which may explain why factor XIII is the only factor that is low in certain conditions. Flbrinolytlc inhibitors (EACA, ANA, Trasylol) have no effect in the absence of plasmin activity. Therapy must be directed against the underlying disorder. In certain cases with factor XIII deficiency and bleeding symptoms substitutional therapy is Indicated (17, 19, 47).

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ACKNOWLEDGEMENT This investigation was supported by grants from the Swedish Medical Research Council (B79-19X-00087-15B) and the Medical Faculty, University of Lund, Sweden.

REFERENCES 1. SABA,H.I., HERION,J.C., WALKER,R.I., and ROBERTS,H.R. The prOCOagUli¶nt activity of granulocytes. Proc. Sot. Exp. Biol. Med. N.Y. 142, 614. 1973. 2. HILLER,E., SAAL,J.G., and RIETHMULLER,G. Procoagulant activity activity of activated monocytes. Haemostasis. 6, 347, 1977. 3. LERNER,R.G., GOLDSTEIN,R., and CUMMINGS,G.Endotoxin induced disseminated intravascular clotting: evidence that it is mediated by neutrophil production of tissue factor. Thromb. Res. 11, 253, 1977. 4. MULLER-BERGHAUS,G. and ECKHARDT,T. The role of granulocytes in the activation of intravascular coagulation and the precipitation of soluble fibrin by endotoxin. Blood. 4fr 631, 1975. 5. RIVERS,R.P.A. and HATHAWAY,W.E. Studies on tissue factor activity and production by leukocytes of human umbilical cord and adult origin. Pediat. Res. 9, 167, 1975. 6. RIVERS,R.P.A., HATHAWAY,W.E., and WESTON,W.L. The endotoxininduced coagulant activity of human monocytes. Br. J. Haematol. 30, 311, 1975. 7. HAVERKATE,F., NOGRDHOEK-HEGT,V., van de PUTTE,L.B.A., and van GINKEL,C.J.W. Fibrinolytic activity of human leukocytes. Third Internat. Conf. on Synthetic Fibrinolytic Thrombolytic Agents. Progr. in Fibrinolysis, Glasgow, Sept 1976 (Abstr no 35). 8. BILEZIKIAN,S.B. and NOSSEL,H.L. Unique pattern of fibrinogen cleavage by human leukocyte proteases. Blood. 50, 21, 1977. 9. MERSKEY,C. Defibrination syndrome or ...? Blood. 41, 599, 1973. lO.PLOW,E.F. and EDGINGTON,T.S. An alternative pathway for fibrinolysis. 1. The cleavage of fibrinogen by leukocyte proteases at physiologic pH. J. Clin. Invest. 56, 30, 1975.

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11. S?iBA,H.I., EiERION,J.C.,WALXER,R.I., and ROBERTS,H.R. Effect of lysosomal cationic proteins from polymorphonuclear leukocytes upon the fibrinogen and fibrinolysis system. Thromb. Res. 7, 543, 1975. 12. MOVAT,H.Z., STEINBERG,S.G., HABAL,F.M., and RANADIVE,N.S. Demonstration of a kinin-generating enzyme in the lysosomes of human polymorphonuclear leukocytes. Lab. Invest. 29, 669, 1973. 13. OHLSSON,X. Properties of leukocytic protease. Clin. Chim. Acta. 32, 399, 1971. 14. PLOW,E.F. and EDGINGTON,T.S. Comparative immunochemical characterization of products of plasmln and leukocyte protease cleavage of human fibrinogen. Thromb. Res. 12, 653, 1978. . 15. SCHMIDT,W., EGBRING,R., and HAVEMANN ,X. Effect of elastaselike and chymotrypaln-like neutral proteases from human granulocytes on isolated clotting factors. Thrcxab. Rer. 6, 315, 1975. 16. EGBRING,R., SCHMIDT,W., FUCHS,G., and HAVEMANN ,X. Demonstration of granulocytic proteases in plasma of patients with acute leukemia and septicemia with coagulation defects. Blood. 49, 219, 1977. 17. NILSSON,I.M., BERGENTZ,S.-E., WIXLANDER,O., and HEDNER,U. Erosive hemorrhagfc gastroduodenitis with fibrinolysis and low factor XIII. Ann. Surq. 182, 677, 1975. 18. HENRIXSSON,P., HEDNER,U., NILSSON,I.M., and NILSSON,P.-G. Generalized proteolysfs in a young woman with WeberChristian disease (nodular nonsuppuratlve panniculitis). Stand. J. Haematol. 14, 355, 1975. 19. HENRIKSSON,P., HEDNER,U., and NILSSGN,I.M. Factor XIII (fibrin stabilising factor) in Henoch-Scwnlein's purpura. Acta Paediat. Scsnd. 66, 273, 1977. 20. BLOMBACK,B. and BLOMB#CK,M. Purification of human and bovine fibrinogen. Arkiv Xemi. 10, 415, 1956. 21. NILSSON,I.M., BLOMB&CX,M., and RAMGRKN,O. Haemophilia in Sweden. VI. Treatment of haemophilia A with the human antihaemophilic factor preparation- (fraction I-O). Acta Med. Stand. Suppl. 379, 61, 1962. 22. NILSSON,I.M. and HKNDER,U. Characteristics of various factor VIII concentrates used in treatment of haemophilia A. Br. J. Haematol. 37, 543, 1977. 23. LORAND,L., URAYAMA,T., de XIEWIET,J.W.C., and NOSSEL,H.L. Diagnostic and genetic studies on fibrin-stabilizing factor ;;t.i,: nz a:;; ,bm;; on amine incorporation. J. Clin. ..I

??

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24. BdYUM,A. Isolation of leukocytes fram human blood. Stand. J. Haematol. Suppl. 97, 9, 1968. 25. LEGRAND,Y., PIGNAUD,G., and CAEN,J.P. Human blood platelet elastase and proelastase. Haemostasis. 6, 180, 1977. 26. NACHMAN,R.L. and FERRIS,B. Studies on human platelet protease activity. J. Clin. Invest. 47, 2530, 1968. 27. NILSSON,I.M. and OLOW,B. Determination of fibrinogen and fibrinogenolytic activity. Thromb. Diathes. Haarnorrh. 8, 297, 1962. 28. NILSSON,I.M., BLOMB#CK,M., and RAMGREN,O. Haemophilia in Sweden. I. Coagulation studies. Acta Med. Stand. 170, 665, 1961. 29. HOLMBERG,L. and NILSSON,I.M. Immunologic studies in haemophilia A. Stand. J. Haematol. 10, 12, 1973. 30. RUGGERI,Z.M., MANNUCCI,P.M., JEFFCOATE,S.L., and INGRAM, G.I.C. Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand's disease. Br. J. Haematol. 33, 221, 1976. 31. HOLMBERG,L. and LJUNG,R. Purification of F. VIII:C by antigen-antibody chromatography. Thromb. Res. 12,,667, 1978. 32. ZUZEL,M., NILSSON,I.M., and hESG,M. A method for measuring plasma ristocetln cofactor activity. Normal distribution and stability during storage. Thromb. Res. 12, 745, 1978. 33. LAURELL,C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Bio*. 15, 45, 1966. 34. GANROT,P.O. Crossed immuno electrophoresis. Stand. J. Clin. Lab. Invest. 29, 39, 1972. 35. PROKOPOWICZ,J. Distribution of fibrinolytic and proteolytic enzymeS in subcellular fractions of human granulocytes. Thromb. Dlathes. Haemorrh. 19, 84, 1968. 36. WUNSCHMANN-HENDERSON,B., HORWITZ,D.L., and ASTRUP,T. Release of plasminogen activator from viable leukocytes of man, baboon, dog and rabbit. Proc. Sot. Exp. Biol. Med. 141, 634, 1972. 37. HENRIKSSON,P. and HOLMBERG,L. Factor VIII activity and antigen in sick newborns with pathological proteolysis in blood. Acta Paediat. Stand. 67, 83, 1978. 38. RAPAPORT,S.I., SCHIFFMAN,S_, PATCH,M.J., and AMES,S.B. The Importance of activation of antlhaemophilic globulin and proaccelerin by traces of thrombin in the generation of

312

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intrinsic prothrombinase activity. Blood 21, 221, 1963. 39. WEISS,H.J. and HOYER,L.W. von Willebrand factor: dissociation from antihaesmphilic factor procoagulant activity. Science. 182, 1149, 1973. 40. KOPEC,M., LATALLO,Z.S., STAHL,M., and WEGRZYNOWICZ,Z. The effect of proteolytic enzymes on fibrin stabilizing factor. Biochim. Biophys. Acta. 181, 437, 1969. 41. MILOSZEWSKI,K., SHELTAWY,M.J., and LOSOWSKY,M.S. Fibrinolysis and factor XIII. Acta Haemat. 52, 40, 1974. 42. HEDNER,U., JOHANSSON,L., and NILSSON,I.M. Effects of porcine plasmin on the coagulation and fibrinolytic system in humans. Blood.51, 157, 1978 43. COLLEN,D. and WIMAN,B. Fast-acting plasmin inhibitor in human plasma. Blood. 51, 563, 1978. 44. McKAY,D.G. Disseminated intravascular coagulation. An intermediary mechanism of disease. Harper and Row, New York 1965. 45. OHLSSON,K. al-antitrypsin and a2-macroglobulin. Interactions with human neutrophil collagenase and elastase. Ann. N.Y. Acad. Sci. 256, 409, 1975. 46. OHLSSON,K. and LAURELL.C.-B. The disappearance of enzymeinhibitor complexes from the circulation of man. Clin. Sci. Mol. Med. 51, 87, 1976. 47. HAVEMANN,K., EGBRING,R., GROPP,C., SCHMIDTM., and SODOMANN, C.P. Faktor XIII Mange1 bei akuter LeukCLeaie des Erwachsenen: Ergebnisse einer Substitution mit Faktor XIII. Klin. Wschr. 55. 801. 2977.