Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma

Judith Leurs; Britt-Marie Wissing; Viveca Nerme; Katinka Schatteman; Petter Björquist; Dirk Hendriks

doi:10.1055/s-0037-1613441

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 89(02): 264-271
DOI: 10.1055/s-0037-1613441

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma

Judith Leurs

¹Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium

,

Britt-Marie Wissing

²Department of Cell Biology and Biochemistry, AstraZeneca R&D, Mölndal, Sweden

,

Viveca Nerme

³Department of Integrative Pharmacology, AstraZeneca R&D, Mölndal, Sweden

,

Katinka Schatteman

¹Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium

,

Petter Björquist

²Department of Cell Biology and Biochemistry, AstraZeneca R&D, Mölndal, Sweden

,

Dirk Hendriks

¹Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium

› Author Affiliations

Abstract

Summary

Carboxypeptidase U (CPU, TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this study was to investigate the time course of the generation of CPU activity during coagulation and fibrinolysis using an in vitro clot lysis model in human plasma. A first peak of CPU activity appeared after initiation of the coagulation phase and a second rise in CPU activity was observed during the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation during coagulation but not during fibrinolysis. Addition of the plasmin inhibitor aprotinin during fibrinolysis resulted in a decrease in CPU activation during the lysis phase. These results demonstrate that proCPU was activated during coagulation by thrombin and during fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.

Keywords

Carboxypeptidase U - TAFI - clot lysis - inogatran - fibrinolysis

Full Text

References

References
1 Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273: 27176-81.
2 Fleury V, Angles-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry 1991; 30: 7630-8.
3 Hendriks D, Scharpe S, van Sande M, Lommaert MP. Characterisation of a carboxy-peptidase in human serum distinct from car-boxypeptidase N. J Clin Chem Clin Biochem 1989; 27: 277-85.
4 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.
5 Campbell W, Okada H. An arginine specific carboxypeptidase generated in blood during coagulation or inflammation which is unrelated to carboxypeptidase N or its subunits. Biochem Biophys Res Commun 1989; 162: 933-9.
6 Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 1995; 96: 2534-8.
7 Hemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb Haemost 1995; 74: 134-8.
8 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.
9 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.
10 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-84.
11 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271: 16603-8.
12 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.
13 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombincatalyzed activation of protein C. J Biol Chem 1982; 257: 859-64.
14 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.
15 Mosnier LO, Meijers JC, Bouma BN. Regulation of fibrinolysis in plasma by TAFI and protein C is dependent on the concentration of thrombomodulin. Thromb Haemost 2001; 85: 5-11.
16 Hosaka Y, Takahashi Y, Ishii H. Thrombomodulin in human plasma contributes to inhibit fibrinolysis through acceleration of thrombin-dependent activation of plasma pro-carboxypeptidase B. Thromb Haemost 1998; 79: 371-7.
17 Schatteman K, Goossens F, Scharpe S, Hendriks D. Proteolytic activation of purified human procarboxypeptidase U. Clinica Chimica Acta 2000; 292: 25-40.
18 Marx PF, Dawson PE, Bouma BN, Meijers JC. Plasmin-mediated activation and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry 2002; 41: 6688-96.
19 Mao SS, Cooper CM, Wood T, Shafer JA, Gardell SJ. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem 1999; 274: 35046-52.
20 Boffa MB, Bell R, Stevens WK, Nesheim ME. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombinactivable fibrinolysis inhibitor. J Biol Chem 2000; 275: 12868-78.
21 Teger-Nilsson AC, Bylund R, Gustafsson D, Gyzander E, Eriksson U. In vitro effects of inogatran, a selective low molecular weight thrombin inhibitor. Thromb Res 1997; 85: 133-45.
22 Urano T, Ihara H, Suzuki Y, Nagai N, Takada Y, Takada A. Soluble thrombomodulin quen-ches thrombin-mediated neutralization of PAI-1 activity and inhibits fibrinolysis through a TAFI independent mechanism. Fibrinolysis & Proteolysis 1999; 13: 264-71.
23 Nagashima M, Werner M, Wang M, Zhao L, Light DR, Pagila R, Morser J, Verhallen P. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis Model. Thromb Res 2000; 98: 333-42.
24 Wang W, Hendriks DF, Scharpe SS. Carboxy-peptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994; 269: 15937-44.
25 Hendriks D, Scharpe S, van Sande M. Assay of carboxypeptidase N activity in serum by liquid-chromatographic determination of hip-puric acid. Clin Chem 1985; 31: 1936-9.
26 Matsui T, Fujimura Y, Titani K. Snake venom proteases affecting hemostasis and thrombosis. Biochim Biophys Acta 2000; 1477: 146-56.
27 Fritz H, Wunderer G. Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneimittelforschung 1983; 33: 479-94.
28 Kettner C, Shaw E. D-Phe-Pro-ArgCH2C1-A selective affinity label for thrombin. Thromb Res 1979; 14: 969-73.
29 Schatteman KA, Goossens FJ, Scharpe SS, Hendriks DF. Activation of plasma procarboxy-peptidase U in different mammalian species points to a conserved pathway of inhibition of fibrinolysis. Thromb Haemost 1999; 82: 1718-21.
30 Stromqvist M, Schatteman K, Leurs J, Verkerk R, Andersson JO, Johansson T, Scharpe S, Hendriks D. Immunological assay for the determination of procarboxypeptidase U antigen levels in human plasma. Thromb Haemost 2001; 85: 12-7.
31 Schatteman KA, Goossens FJ, Scharpe SS, Neels HM, Hendriks DF. Assay of procarboxypeptidase U, a novel determinant of the fibrinolytic cascade, in human plasma. Clin Chem 1999; 45: 807-13.
32 Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998; 97: 544-52.
33 Francis CW, Markham Jr. RE, Barlow GH, Florack TM, Dobrzynski DM, Marder VJ. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med 1983; 102: 220-30.
34 Mutch NJ, Robbie LA, Booth NA. Human thrombi contain an abundance of active thrombin. Thromb Haemost 2001; 86: 1028-34.
35 Stewart RJ, Fredenburgh JC, Rischke JA, Bajzar L, Weitz JI. Thrombin activatable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator: A potential mechanism for enhancing the fibrin-specificity of tissue plasminogen activator. J Biol Chem 2000; 275: 36612-20.