The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is the precursor of an exopeptidase that is identical to plasma procarboxypeptidase B. Upon activation by thrombin, activated TAFI (TAFIa) attenuates fibrinolysis, presumably by catalyzing the removal of C-terminal lysines from partially degraded fibrin. Activated protein C (APC) proteolytically inactivates the essential cofactor in prothrombinase, factor Va, and limits both the formation of thrombin and subsequent activation of TAFI, thereby appearing profibrinolytic. TAFI is able to reconstitute an APC-dependent shortening of lysis time in a purified system; however, it remained to be determined the extent to which TAFI is involved in the profibrinolytic effect of APC in a plasma-based system. To aid in addressing this question, two monoclonal antibodies (MoAbTAFI#16 and #13) and a polyclonal antibody were produced against purified TAFI. MoAbTAFI#16 was shown to inhibit TAFI activation and thereby appears to stimulate fibrinolysis. Furthermore, an enzyme- linked immunosorbent assay was developed using MoAbTAFI#13 and the polyclonal antibody. Through its use, the plasma concentration of TAFI was determined to be 73 nmol/L. In addition, a turbidity assay was used to determine the effect of APC on tissue plasminogen activator-induced fibrinolysis of clots produced from normal human plasma (NHP), plasma immunodepleted of TAFI (TdP), and TdP reconstituted with purified TAFI. APC shortened lysis time of clots produced from NHP in a saturable and concentration-dependent manner. However, APC had no effect on lysis time of clots formed from either TdP or NHP in the presence of 80 nmol/L MoAbTAFI#16. The APC effect could be reconstituted in TdP by the addition of purified TAFI. The lysis time in TdP was increased from 50 to 180 minutes in a TAFI concentration-dependent manner. The EC50 was 15 nmol/L and saturation was approached at physiologically relevant concentrations (60 nmol/L). The profibrinolytic effect of APC was also compared with that of MoAbTAFI#16 and two competitive inhibitors, an inhibitor of the carboxypeptidase A and B family purified from potato tubers and 2-Guanidinoethylmercaptosuccinic acid (GEMSA). All were able to reduce lysis time of clots formed from normal human plasma by 90 minutes, yielding respective EC50 values of 5 nmol/L, 15 nmol/L, 50 nmol/L, and 90 mumol/L. Therefore, the majority of the profibrinolytic effect of APC, in an in vitro plasma system, is dependent on TAFI. Because TAFIa dramatically influences lysis time, inhibitors of TAFIa or TAFI activation may prove to be important adjuvants for thrombolytic therapy.