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The profibrinolytic effect of activated protein C in clots formed from
plasma is TAFI-dependent
L Bajzar, ME Nesheim and PB Tracy
Department of Biochemistry, University of Vermont, Burlington, USA.
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.
Volume 88,
Issue 6,
pp. 2093-2100,
09/15/1996
Copyright © 1996 by The American Society of Hematology
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