Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms

Summary

Thrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFITI- wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eightfold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MATCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MATCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.

Keywords

Epitope mapping - fibrinolysis - human TAFI - monoclonal antibody - TAFIinhibitors

• References

• 1 Bouma BN, Meijers JC. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). J Thromb Haemost 2003; 1: 1566-1574.
• 2 Bouma BN, Marx PF, Mosnier LO. et al. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). Thromb Res 2001; 101: 329-354.
• 3 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: 16603-16608.
• 4 Hendriks D, Wang W, Scharpé S. et al. Purification and characterization of a new arginine carboxypeptidase in human serum. Biochim Biophys Acta 1990; 1034: 86-92.
• 5 Eaton DL, Malloy BE, Tsai SP. et al. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833-21838.
• 6 Tan AK, Eaton DL. Activation and characterization of procarboxypeptidase B from human plasma. Biochemistry 1995; 34: 5811-5816.
• 7 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-14484.
• 8 Boffa MB, Bell R, Stevens WK. et al. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2000; 275: 12868-12878.
• 9 Marx PF, Hackeng TM, Dawson PE. et al. Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage. J Biol Chem 2000; 275: 12410-12415.
• 10 Marx PF, Dawson PE, Bouma BN. et al. Plasmin-mediated activation and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry 2002; 41: 6688-6696.
• 11 Hillmayer K, Macovei A, Pauwels D. et al. Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI) - a comparative study assessing the biological equivalence of rat, murine and human TAFI. J ThrombHaemost 2006; 4: 2470-2477.
• 12 Schneider M, Boffa MB, Stewart R. et al. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem 2002; 277: 1021-1030.
• 13 Leurs J, Hendriks D. Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease. Thromb Haemost 2005; 94: 471-487.
• 14 Redlitz A, Tan AK, Eaton DL. et al. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 1995; 96: 2534-2538.
• 15 Campbell WD, Lazoura E, Okada N. et al. Inactivation of C3a and C5a octapep- tides by carboxypeptidase Rand carboxypeptidase N. Microbiol Immunol 2002; 46: 131-134.
• 16 Myles T, Nishimura T, Yun TH. et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem 2003; 278: 51059-51067.
• 17 te Velde EA, Wagenaar GTM, Reijerkerk A. et al. Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2003; 1: 2087-2096.
• 18 Asai S, Sato T, Tada T. et al. Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in lipopolysaccharide primed mice. J Immunol 2004; 173: 4669-4674.
• 19 Boffa MB, Koschinsky ML. Curiouser and curiouser: recent advances in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene regulation, and biological roles. Clin Biochem 2007; 40: 431-442.
• 20 Willemse JL, Heylen E, Nesheim ME. et al. Carboxypeptidase U (TAFIa): a new drug target for fibrinolytic therapy?. J Thromb Haemost 2009; 7: 1962-1971.
• 21 Boffa MB, Wang W, Bajzar L. et al. 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: 2127-2135.
• 22 Rawlings ND, Morton FR, Barrett AJ. MEROPS: the peptidase database. Nucleic Acids Res 2006; 34: 270-272.
• 23 Vendrell J, Querol E, Aviles FX. Metallocarboxypeptidases and their protein inhibitors: Structure, function and biomedical properties. Biochim Biophys Acta 2000; 1477: 284-298.
• 24 Schneider M, Nesheim M. Reversible inhibitors of TAFIa can both promote and inhibit fibrinolysis. J ThrombHaemost 2003; 1: 147-154.
• 25 Walker JB, Hughes B, James I. et al. Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro. J Biol Chem 2003; 278: 8913-8921.
• 26 Hillmayer K, Vancraenenbroeck R, De Maeyer M. et al. Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor. J Thromb Haemost 2008; 6: 1892-1899.
• 27 Bajzar L, Nesheim ME, Tracy PB. The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood 1996; 88: 2093-2100.
• 28 Bouma BN, Mosnier LO, Meijers JC. et al. FactorXI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation. Thromb Haemost 1999; 82: 1703-1708.
• 29 Gils A, Ceresa E, Macovei AM. et al. Modulation of TAFI function through different pathways: implications for the development of TAFI inhibitors. J Thromb Haemost 2005; 3: 2745-2753.
• 30 Ceresa E, Peeters M, Declerck PJ. et al. Announcing a TAFIamutant with a 180-fold increased half-life and concomitantly a strongly increased antifibrinolytic potential. J Thromb Haemost 2007; 5: 418-420.
• 31 Gils A, Alessi MC, Brouwers E. et al. Development of a genotype 325-specific proCPU/TAFI ELISA. Arterioscler Thromb Vasc Biol 2003; 23: 1122-1127.
• 32 Hillmayer K, Brouwers E, León-Tamariz F. et al. Development of sandwich-type ELISAs for the quantification of rat and murine thrombin activatable fibrinolysis inhibitor in plasma. J Thromb Haemost 2008; 6: 132-138.
• 33 Ceresa E, Brouwers E, Peeters M. et al. Development of ELISAs measuring the extent of TAFI activation. Arterioscler Thromb Vasc Biol 2006; 26: 423-428.
• 34 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 1978; 15: 429-436.
• 35 Nakane PK, Kawaoi A. Peroxidase-labeled antibody: a new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091.
• 36 Buelens K, Hassanzadeh-Ghassabeh G, Muyldermans S. et al. Generation and characterization of inhibitory nanobodies towards Thrombin Activatable Fibrinolysis Inhibitor. J Thromb Haemost 2010; 8: 1302-1312.
• 37 Morange PE, Juhan-Vague I, Scarabin PY. et al. Association between TAFI antigen and Ala147Thr polymorphism of the TAFI gene and the angina pectoris incidence. Thromb Haemost 2003; 89: 554-560.
• 38 Silveira A, Schatteman K, Goossens F. et al. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease. Thromb Haemost 2000; 84: 364-368.
• 39 Montaner J, Ribo M, Monasterio J. et al. Thrombin-activable fibrinolysis inhibitor levels in the acute phase of ischemic stroke. Stroke 2003; 34: 1038-1040.
• 40 Juhan-Vague I, Morange PE, Aubert H. et al. Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe. Arterioscler Thromb Vasc Biol 2002; 22: 867-873.
• 41 van-Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 2000; 95: 2855-2859.
• 42 Polla MO, Tottie L, Norden C. et al. Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa). Bioorg Med Chem 2004; 12: 1151-1175.
• 43 Do YH, Gifford-Moore DS, Beight DW. et al. Inhibition of thrombin activatable fibrinolysis inhibitor by cysteine derivatives. Thromb Res 2005; 116: 265-271.
• 44 Mosnier LO, von-dem-Borne PA, Meijers JC. et al. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb Haemost 1998; 80: 829-835.
• 45 Mutch NJ, Moore NR, Wang E. et al. Thrombus lysis by uPA, scuPA and tPA is regulated by plasma TAFI. J ThrombHaemost 2003; 1: 2000-2007.
• 46 Guimaraes AH, Rijken DC. Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner. Study of seven plasminogen activators in an internal clot lysis model. Thromb Haemost 2004; 91: 473-479.
• 47 Leurs J, Wissing BM, Nerme V. et al. Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma. Thromb Haemost 2003; 89: 264-271.
• 48 Miah MF, Boffa MB. Functional analysis of mutant variants of thrombin-activat- able fibrinolysis inhibitor resistant to activation by thrombin or plasmin. J Thromb Haemost 2009; 7: 665-672.
• 49 Vercauteren E, Emmerechts J, Peeters M. et al. Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model. Blood 2011; 117: 4615-4622.
• 50 Wu C, Kim PY, Manuel R. et al. The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex. J Biol Chem 2009; 284: 7059-7067.
• 51 Marx PF, Brondijk TH, Plug T, Romijn RA, Hemrika W, Meijers JC, Huizinga EG. Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation. Blood 2008; 112: 2803-2809.