Plasma TAFI Levels Influence the Clot Lysis Time in Healthy Individuals in the Presence of an Intact Intrinsic Pathway of Coagulation^[*]

Summary

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.

Abbreviations: The abbreviations used are: TAFI, Thrombin Activatable Fibrinolysis Inhibitor; TAFIa, activated TAFI; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; tPA, tissue-type plasminogen activator.

^* This study was supported in part by grant 96.088 of The Netherlands Heart Foundation. JCMM is an Established Investigator of The Nederlands Heart Foundation.

• References

• 1 Mosesson MW. Thrombin interactions with fibrinogen and fibrin. Semin Thromb Hemost 1993; 19: 361-7.
• 2 Mosesson MW. The roles of fibrinogen and fibrin in hemostasis and thrombosis. Semin Hematol 1992; 29: 177-88.
• 3 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991; 78: 3114-24.
• 4 Vassalli JD, Sappino AP, Berlin D. The plasminogen activator/plasmin system. J Clin Invest 1991; 88: 1067-72.
• 5 Collen D. On the regulation and control of fibrinolysis. Thromb Haemost 1980; 43: 77-89.
• 6 Carmeliet P, Collen D. Gene targeting and gene transfer studies of the plasminogen/plasmin system: implications in thrombosis, hemostasis, neointima formation and atherosclerosis. FASEB J 1995; 9: 934-8.
• 7 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-84.
• 8 Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833-8.
• 9 Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994; 269: 15937-44.
• 10 Nesheim M, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 1997; 78: 386-91.
• 11 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombinthrombomodulin complex. J Biol Chem 1996; 271: 16603-8.
• 12 Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem 1998; 273: 2127-35.
• 13 Nesheim ME, Boffa MB, Wang W. The effects of temperature, GEMSA and EACA on the stability of activated TAFI. Fibrinolysis 1996; 10: 126 (Abstract).
• 14 Wang W, Boffa MB, Walker J. et al. Reciprocal inactivation of TAFIa and plasmin. Fibrinolysis 1996; 10: 124 (Abstract).
• 15 von dem Borne PA, Meijers JC, Bouma BN. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis. Blood 1995; 86: 3035-42.
• 16 von dem Borne PA, Bajzar L, Meijers JC, Nesheim ME, Bouma BN. Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997; 99: 2323-7.
• 17 Minnema MC, Friederich PW, Levi M, Von dem Borne PAK, Mosnier LO, Meijers JCM, Biemond BJ, Hack CE, Bouma BN, ten Cate H. Enhancement of rabbit jugular vein thrombolysis by neutralization of factor XI – In vivo evidence for a role of factor XI as an anti-fibrinolytic factor. J Clin Invest 1998; 101: 10-4.
• 18 Gresele P, Momi S, Nenci GG, Schwarz HP, Semeraro N, Colucci M. Activated human protein C prevents thrombin-induced thromboembolism in mice. J Clin Invest 1998; 101: 667-76.
• 19 Naito K, Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem 1991; 266: 7353-8.
• 20 Gailani D, Broze Jr. GJ. Factor XI activation in a revised model of blood coagulation. Science 1991; 253: 909-12.
• 21 von dem Borne PA, Koppelman SJ, Bouma BN, Meijers JC. Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen. Thromb Haemost 1994; 72: 397-402.
• 22 von dem Borne PA, Mosnier LO, Tans G, Meijers JC, Bouma BN. Factor XI activation by meizothrombin: stimulation by phospholipid vesicles containing both phosphatidylserine and phosphatidylethanolamine. Thromb Haemost 1997; 78: 834-9.
• 23 Hendriks D, Wang W, Scharpe S, Lommaert MP, van Sande M. Purification and characterization of a new arginine carboxypeptidase in human serum. Biochim Biophys Acta 1990; 1034: 86-92.
• 24 Hendriks D, Scharpe S, van Sande M, Lommaert MP. Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N. J Clin Chem Clin Biochem 1989; 27: 277-85.
• 25 Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 1995; 96: 2534-8.
• 26 Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem 1984; 140: 513-22.
• 27 Norrman B, Wallen P, Ranby M. Fibrinolysis mediated by tissue plasminogen activator. Disclosure of a kinetic transition. Eur J Biochem 1985; 149: 193-200.
• 28 Bajzar L, Nesheim ME, Tracy PB. The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood 1996; 88: 2093-100.
• 29 Bajzar L, Nesheim M, Morser J, Tracy PB. Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273: 2792-8.
• 30 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-6.
• 31 Meijers JCM. Regulation of the blood coagulation mechanism by plasma proteinase inhibitors. Ph D Thesis State University of Utrecht; 1988: 93-108.
• 32 Laurell CB, McKay EJ. Electroimmunoassay. Methods Enzymol 1981; 73: 339-69.
• 33 Brunner J, Skrabal P, Hauser H. Single bilayer vesicles prepared without sonication. Physico-chemical properties. Biochim Biophys Acta 1976; 455: 322-31.
• 34 Rouser G, Fkeischer S, Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970; 5: 494-6.
• 35 Hurst PL, Lovell Smith CJ. Optimized assay for serum angiotensin-converting enzyme activity. Clin Chem 1981; 27: 2048-52.
• 36 Hayakari M, Kondo Y, Izumi H. A rapid and simple spectrophotometric assay of angiotensin-converting enzyme. Anal Biochem 1978; 84: 361-9.
• 37 Suzuki S, Hachimori Y, Yaoeda U. Spectrophotometric determination of glycine with 2,4,6-trichloro-s-triazine. Anal Chem 1970; 42: 101-3.
• 38 Taylor Jr. FB, Lockhart MS. A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes-platelet suspensions stimulated by phorbol diester. Thromb Res 1985; 37: 155-64.
• 39 Burdick MD, Schaub RG. Human protein C induces anticoagulation and increased fibrinolytic activity in the cat. Thromb Res 1987; 45: 413-9.
• 40 de Fouw NJ, Haverkate F, Bertina RM. Protein C and fibrinolysis: a link between coagulation and fibrinolysis. Adv Exp Med Biol 1990; 281: 235-43.
• 41 Bajzar L, Fredenburgh JC, Nesheim ME. The activated protein C-mediated enhancement of tissue-type plasminogen activator induced fibrinolysis in a cell-free system. J Biol Chem 1990; 265: 16948-54.
• 42 Von dem Borne PAK. The role of factor XI in blood coagulation and fibrinolysis. Ph D Thesis State University of Utrecht; 1996. chapter 7 127-45.
• 43 Redlitz A, Nicolini FA, Malycky JL, Topol EJ, Plow EF. Inducible carboxypeptidase activity. A role in clot lysis in vivo. Circulation 1996; 93: 1328-30.
• 44 Broze Jr. GJ, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. Blood 1996; 88: 3815-23.