Abstract
Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.
Highlights
Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity
The results indicate that a soluble form of thrombomodulin prolongs the lysis time of clots formed from both purified components and human plasma
Thrombomodulin Prolongs the Lysis Time of Clots Formed from Purified Plasma-derived Components or barium citrate-adsorbed plasma (BAP) in the Presence of TAFI—Fibrin clots were formed from a solution comprising purified components including fibrinogen, plasminogen, prothrombin, factor V, antithrombin III, and ␣2antiplasmin at approximately 1⁄3 their respective plasma concentrations
Summary
Proteins and Reagents—Human proteins including fibrinogen (330 kDa, ⑀1%, 280 ϭ 16.0), plasminogen, (91 kDa, ⑀1%, 280 ϭ 16.1), prothrombin, (72 kDa, ⑀1%, 280 ϭ 13.8), protein C, (62 kDa, ⑀1%, 280 ϭ 14.5), antithrombin III, (57 kDa, ⑀1%, 280 ϭ 7.0), recombinant ␣2-antiplasmin, (65 kDa, ⑀1%, 280 ϭ 7.0), TAFI (48 kDa, ⑀1%, 280 ϭ 26.4), and factor V (330 kDa, ⑀1%, 280 ϭ 9.6) were purified and activated, when required, as described previously [10]. After rinsing the wells of a 96-well microtiter plate containing the confluent monolayer of HUVECs with HBS, an aliquot (25 l) containing both thrombin (6 nM, final concentration) and CaCl2 (10 mM, final) in HBS was added to each well, immediately before the addition of 25 l of 2:3 diluted plasma. One hundred l of the mixture was pipetted into the well of a microtiter plate that contained separated, 2-l aliquots of thrombin, activated protein C, tPA, and CaCl2 Their respective final concentrations were 6.0 nM, 10 or 50 nM, 294 pM, and 5.0 mM. Activated TAFI was produced from purified TAFI (1.0 M) by incubating it at 22 °C for 15 min in a solution of 0.02 M HEPES, 0.15 M NaCl, 5.0 mM CaCl2, pH 7.4, containing thrombin (20 nM) and soluble thrombomodulin (50 nM). Two 100-l aliquots of these samples were added
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
