Thrombomodulin in Human Plasma Contributes to Inhibit Fibrinolysis through Acceleration of Thrombin-dependent Activation of Plasma Procarboxypeptidase B

Abstract

SummaryThrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.

Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not

“…However, several recent reports suggest that excess activation of proCPR may exacerbate a state of disseminated intravascular coagulation (DIC) resulting from a Gram-negative bacterial infection (14,15,(22)(23)(24)(25)(26)(27). Following activation by thrombin, proCPR, otherwise known as TAFI, exerts an antifibrinolytic effect by removing carboxyl-terminal lysines on fibrin, preventing plasminogen binding and activation (15).…”

“…Following activation by thrombin, proCPR, otherwise known as TAFI, exerts an antifibrinolytic effect by removing carboxyl-terminal lysines on fibrin, preventing plasminogen binding and activation (15). On the other hand, it has been established that CPN does not remove carboxyl-terminal lysine residues from the clot surface despite the preference for lysine residues rather than arginine residues (15,26). Furthermore, according to the present study, LPS stimulation did not essentially alter CPN expression or consumption of CPN compared with proCPR following LPS injection (Fig.…”

“…Thrombomodulin (TM) is expressed on the surface of endothelial cells, and soluble functional proteolytic fragments of TM are also present in circulating plasma. It has been reported that the thrombin/TM complex activates TAFI (proCPR) 1250 times more rapidly than does free thrombin (14) and that plasma TM, including soluble TM fragments, inhibits fibrinolysis in a dosedependent manner via activation of plasma proCPB (ϭproCPR) (26). Plasma levels of soluble TM fragments are elevated in patients with DIC (27).…”

A murine model of myocardial microvascular thrombosis

“…These data suggest that in uPA -/-mice, a potential TM/thrombin complex may affect other non-APC-dependent targets. Previous studies have demonstrated that the TM/thrombin complex regulates the rate of plasmin generation through the effects of thrombin-activated fibrinolysis inhibitor (TAFI) on both tPA and uPA (36)(37)(38)(39). In addition, the TM/thrombin interaction has been shown to directly modulate the uPA receptor (40).…”

Preferential Detection of Pro‐Carboxypeptidase R by Enzyme‐Linked Immunosorbent Assay

“…For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (T-TM) can be used (2,14). Therefore, we used ELISA to evaluate the amount of antigens detected in plasma before and after activation by T-TM.…”