The central role of thrombin in hemostasis.

Following vascular injury, blood loss is controlled by the mechanisms of hemostasis. During this process, the serine proteinase, thrombin, is generated both locally and rapidly at sites of vessel damage. It plays a pivotal role in clot promotion and inhibition, and cell signaling, as well as additional processes that influence fibrinolysis and inflammation. These functions involve numerous cleavage reactions, which must be tightly coordinated. Failure to do so can lead to either bleeding or thrombosis. The crystal structures of thrombin, in combination with biochemical analyses of thrombin mutants, have provided insight into the ways in which thrombin functions, and how its different activities are modulated. Many of the interactions of thrombin are facilitated by exosites on its surface that bind to its substrates and/or cofactors. The use of cofactors not only extends the range of thrombin specificity, but also enhances its catalytic efficiency for different substrates. This explains a paradox (i.e. thrombin is a specific proteinase, and yet one that has multiple, and sometimes opposing, substrate reactions). In this review, we describe the context in which thrombin acts during hemostasis and explain the roles that its exosites and cofactors play in directing thrombin function. Thereafter, we develop the concept of cofactor competition as a means by which the activities of thrombin are controlled.

Further characterization of ADAMTS-13 inactivation by thrombin.

BACKGROUND: The multimeric size and platelet-tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. OBJECTIVES: In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance. METHODS AND RESULTS: By N-terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS-13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS-13. Proteolysis of ADAMTS-13 by thrombin caused an 8-fold reduction in its affinity for VWF that contributed to its loss of VWF-cleaving function. Intriguingly, thrombin-cleaved ADAMTS-13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS-13, but not added ADAMTS-13, appeared resistant to coagulation-induced fragmentation. An estimation of the K(m) for ADAMTS-13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS-13. This was corroborated by the comparatively low affinity of ADAMTS-13 for thrombin (K(D) 95 nM). CONCLUSIONS: Together, our data suggest that ADAMTS-13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.