Proteins of the Fibrinolytic System in Human Thrombi

Summary

The proteins of fibrinolysis have been quantified in human thrombi, to assess the balance between plasminogen activators and their major inhibitor PAI-1. The relative roles of PAI-1 and α₂-AP were also examined since we have previously shown that both platelet PAI-1 and plasma α₂-AP are important determinants of clot lysis in vitro. Extracts and sections were prepared from human thrombi for quantitative immunoassay and immunohistochemical staining respectively. PAI-1 and α₂-AP were present at high concentrations. Levels of t-PA and t-PA-PAI-1 complex were relatively low. Staining confirmed the presence of abundant PAI-1, associated primarily with platelet material within the thrombus and also with fibrin. Staining for α₂-AP was also intense and demonstrated strong association with fibrin. The α₂-AP concentration was similar to its high plasma concentration, whereas PAI-1 levels were up to 30 times greater than that in circulating blood, suggesting that active recruitment of platelets contributes to the high PAI-1 concentration in thrombi.

• References

• 1 Kruithof EKO. Plasminogen activator inhibitor type 1: Biochemical, biological and clinical aspects. Fibrinolysis 1988; 2 (02) 59-70
• 2 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Br J Haematol 1988; 70: 327-333
• 3 Declerck PJ, Alessi M-C, Verstreken MV, Kruithof EKO, Juhan-Vague I, Collen D. Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 1988; 71: 220-225
• 4 Murayama H, Bang NU. Incorporation of plasminogen activator inhibitor into fibrin, an alternative regulatory pathway of fibrinolysis. Thromb Haemost 1987; 58: 446 (abstract 1643)
• 5 Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). J Clin Invest 1989; 84: 647-655
• 6 Reilly CF, Hutzelmann JE. Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution. J Biol Chem 1992; 267: 7128-7135
• 7 Collen D, Juhan-Vague I. Fibrinolysis and atherosclerosis. Semin Thromb Haemost 1988; 14: 180-183
• 8 Prisco D, Chiarantini E, Boddi M, Rostagno C, Colella A, Gensini GF. Predictive value for thrombotic disease of plasminogen activator inhibitor-1 plasma levels. Int J Clin Lab Res 1993; 23: 78-82
• 9 Plow EF, Collen D. The presence and release of α₂-antiplasmin from human platelets. Blood 1981; 58: 1069-1074
• 10 Sakata Y, Aoki N. Cross-linking of α₂-plasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest 1980; 65: 290-297
• 11 Sakata Y, Aoki N. Significance of cross-linking of α₂-plasmin inhibitor to fibrin in inhibition of fibrinolysis and in haemostasis. J Clin Invest 1982; 69: 536-542
• 12 Robbie LA, Booth NA, Croll AM, Bennett B. The roles of α₂-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) in the inhibition of clot lysis. Thromb Haemost 1993; 70: 301-306
• 13 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Br J Haematol 1988; 70: 327-333
• 14 MacGregor IR, MacDonald S, Dawes J, Mickelm LR, James K. A monoclonal antibody enzyme-linked immunosorbent assay (ELISA) directed towards a fibrin binding region of tissue-type plasminogen activator. Fibrinolysis 1987; 1: 247-253
• 15 Booth NA, Croll AM, Bennett B. The activity of plasminogen activator inhibitor-1 (PAI-1) of human platelets. Fibrinolysis 1990; 4 (suppl) (Suppl. 02) 138-140
• 16 Nakane PK, Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091
• 17 Laurell C-B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochem 1966; 15: 45-52
• 18 Booth NA, Bennett B. Plasmin α₂-antiplasmin complex as an indicator of in vivo fibrinolysis. Br J Haematol 1982; 50: 595-600
• 19 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford A, Stein H, Mason DY. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984; 32: 219-229
• 20 Simpson AJ, Booth NA, Moore NR, Bennett B. Distribution of plasminogen activator (PAI-1) in tissues. J Clin Pathol 1991; 44: 139-143
• 21 MacGregor IR, Tonner AM, Micklem LR, James K, Booth NA. Murine monoclonal antibodies against active site epitopes on human endothelial plasminogen activator inhibitor (PAI-1). Fibrinolysis 1990; 4: 27-34
• 22 Booth NA, Anderson JA, Bennett B. Plasminogen activators in alcoholic cirrhosis: demonstration of increased tissue-type and urokinase-type activator. J Clin Pathol 1984; 37: 772-777
• 23 Booth NA, Anderson JA, Bennett B. The plasma inhibitors of plasminogen activator, studied by a zymographic technique. Thromb Res 1985; 38: 261-267
• 24 Potter van Loon BJ, Rijken DC, Brommer EJP, van der Maas APC. The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex vivo lysability. Thromb Haemost 1992; 67: 101-105
• 25 Levi M, Biemond BJ, van Zonneveld A-J, ten Cate JW, Pannekoek H. Inhibiton of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85: 305-312
• 26 Ogston D, Ogston CM, Fullerton HW. The plasminogen content of thrombi. Thromb Diath Haemorrh 1966; 15: 220-230
• 27 Hedner U, Nilsson IM, Robertson B. Determination of plasminogen in clots and thrombi. Thromb Diath Haemorrh 1966; 16: 38-50
• 28 Reed GL, Matsueda GR, Haber E. Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the cross-linking of α₂-antiplasmin to fibrin. Thromb Haemost 1992; 68: 315-320
• 29 Declerck PJ, De Mol M, Alessi M-C, Baudner S, Pâques E-P, Preissner KT, Müller-Berghaus G, Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. J Biol Chem 1988; 263: 15454-15461
• 30 Mimuro J, Loskutoff DJ. Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix: evidence that the protein is vitronectin. J Biol Chem 1989; 264: 936-939
• 31 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem 1982; 257: 2912-2919
• 32 Kruithof EKO, Nicoloso G, Bachmann F. Plasminogen activator inhibitor 1: development of radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation. Blood 1987; 70: 1645-1653
• 33 Stringer HAR, van Swieten P, Heijnen HFG, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor 1 (PAI-1) released from activated platelets has a key role in thrombolysis resistance: studies with thrombi generated in the Chandler loop. Arterioscl Thrombos 1994; 14: 1452-1458
• 34 Fenech A, Hussey JK, Smith FW, Dendy PP, Bennett B, Douglas AS. Diagnosis of aortic aneurysm using autologous platelets labelled with indium-III. Br Med J 1981; 282: 1122
• 35 Peters RF, Lees CM, Mitchell KA, Tweed MF, Talbot MD, Wallis RB. The characterization of thrombus development in an improved model of arteriovenous shunt thrombosis in the rat and the effects of recombinant desul-phatohirudin (CGP 39393), heparin and illoprost. Thromb Haemost 1991; 65: 268-274
• 36 Endenburg SC, Hantgan RR, Sixma JJ, de Groot PhG, Zwaginga JJ. Platelet adhesion to fibrin(ogen). Blood Coag Fib 1993; 4: 139-142
• 37 Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC, Hantgan RR. Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: Light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus. Blood 1993; 81: 1290-1299
• 38 Kooistra T, Sprengers ED, van Hinsbergh VWH. Rapid inactivation of the plasminogen activator-inhibitor upon secretion from cultured endothelial cells. Biochem J 1986; 239: 497-503
• 39 Loskutoff DJ, van Mourik JA, Erickson LA, Lawrence D. Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc Natl Acad Sci USA 1983; 80: 2956-2960
• 40 Fujii S, Sobel BE. Induction of plasminogen activator inhibitor by products released from platelets. Circulation 1990; 82: 1485-1493
• 41 Handt S, Jerome WG, Braaten JV, Lewis JC, Kirkpatrick CJ, Hantgan RR. PAI-1 released from cultured human endothelial cells delays fibrinolysis and is incorporated into the developing fibrin clot. Fibrinolysis 1994; 8: 104-112
• 42 Kakkar VV, Flanc C, Howe CT, Clarke MB. Natural history of postoperative deep vein thrombosis. Lancet 1969; 2: 230-232