The multimeric size and the function of circulating von Willebrand factor are modulated via its proteolytic cleavage by the plasma metalloproteinase, AD-AMTS13. It is unclear how ADAMTS13 activity is regulated within the vascular system. In the absence of a regulatory mechanism, ADAMTS13 activity might compromise platelet adhesion at sites of vascular injury. We hypothesized that at sites of vascular injury, ADAMTS13 activity could be regulated locally by coagulation proteinases. Initiation of coagulation in human plasma resulted in the disappearance of added full-length recombinant ADAMTS13. This loss was inhibited by hirudin. Using purified proteins, we showed that ADAMTS13 is proteolyzed at several cleavage sites by thrombin in a time-and concentration-dependent manner. Furthermore, this proteolysis ablated ADAMTS13 activity against purified von Willebrand factor. Preincubation of thrombin with soluble thrombomodulin, but not heparin, inhibited the proteolysis of AD-AMTS13, suggesting the involvement of IntroductionVon Willebrand factor (VWF) is a large (2050 amino acid/ϳ250 kDa) multidomain glycoprotein whose functions are critical for normal hemostasis. 1 VWF has 2 principal roles that influence the hemostatic process: (1) it acts as a carrier protein for coagulation factor VIII 2 and (2) it mediates rapid adhesion of platelets to sites of vascular perturbation. The latter is one of the first hemostatic events following endothelial disruption and occurs through the specific binding of VWF to exposed subendothelial matrix proteins (principally collagen). 3 Once immobilized, VWF affinity for the glycoprotein (GP) Ib-IX-V receptor complex on the surface of circulating platelets is significantly enhanced. 4 This results in the tethering of platelets at sites of vascular damage and in the formation of a primary platelet plug. Tethered platelets are subsequently activated and expose phosphatidylserine-rich surfaces that are critical for efficient thrombin generation to occur. 5 VWF is constitutively secreted into the blood by endothelial cells as multimers of varying size that differ predominantly in the number of component VWF units. A significant proportion is also stored within Weibel-Palade bodies, predominantly as "ultra-large" multimers (UL-VWF) 6 that may exceed 2 ϫ 10 4 kDa. 1 This pool is released on demand in response to endothelial cell activation. 7 The properties of circulating VWF are, in part, dependent on its molecular size. Larger VWF multimers not only bind circulating platelets more readily than smaller forms, but also undergo marked conformational changes in response to the rheologic forces exerted by the circulating blood. 8 Under normal flowing conditions, VWF multimers circulate in a globular form. However, when VWF is exposed to increased shear forces, these molecules unravel into a "stringlike" conformation. This increases the number of exposed platelet/matrix binding sites and thus enhances the platelet tethering potential of the VWF molecule.Thrombotic thrombocytopenic purpura (TTP) is...

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.Following initiation of coagulation, a series of carefully regulated serine proteinase reactions take place resulting in the generation of thrombin and the formation of an insoluble fibrin clot. Thrombin is a serine proteinase responsible for proteolytic cleavage of multiple substrates involved in the coagulation pathway (1, 2). One of the main roles of thrombin is the conversion of fibrinogen to an insoluble fibrin clot. This process is initiated by proteolytic cleavage of two pairs of fibrinopeptides, FPA 1 and FPB, from the A␣-and B␤-chains of fibrinogen, respectively. FPA is cleaved first, leading to spontaneous polymerization of the fibrin monomers. This is shortly followed by proteolysis of the B␤-chain, which is associated with lateral aggregation of the fibrin protofibrils to produce thicker fiber bundles (3, 4). To ensure a more stable clot structure, activated factor XIII (factor XIIIa), a transglutaminase, covalently crosslinks specific glutamine and lysine side chains of the protofibrils, resulting in increased resistance of the clot to chemical, physical, and proteolytic insults (5, 6).Factor XIII is a heterologous tetramer with a molecular mass of 324,000 Daltons. It consists of two A-subunits that contain the active site of the transglutaminase and two B-subunits that serve a carrier function for the hydrophobic A-subunit in the aqueous environment of human plasma (5, 6). Thrombin activates factor XIII by cleavage of a 37 amino acid activation peptide from the factor XIII A-subunits (7). Consequently, ...

The endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed ؊5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of ؊5.5HS coupled to either PP or the SV40 promoter were able to direct ␤-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The ؊5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell-specific transcriptional regulation that reflects the common origin of these cell types. IntroductionThe protein C (PC) anticoagulant pathway plays a crucial role in the regulation of blood coagulation and inflammation. 1 Activated PC (APC) generated in this pathway serves to confine the hemostatic plug to the site of vascular injury by inhibiting the cofactor function of clotting factors Va and VIIIa on intact endothelium. In addition, APC may also exert antiapoptotic and neuroprotective functions that influence inflammatory responses. 2,3 The important regulatory roles of APC in coagulation and inflammation are illustrated by the increased risk of thrombosis incurred by individuals with deficiencies in components of the PC pathway 4 and by the improved outcome of patients with severe sepsis treated with APC. 5 PC is activated by thrombin, but only when thrombin is bound to its receptor, thrombomodulin, present on endothelial cells (ECs). Activation is further enhanced by another EC receptor, the endothelial cell protein C receptor (EPCR). 6 By binding PC, EPCR helps present PC to the thrombin:thrombomodulin complex and so reduces the K m for PC activation. 7 In this way, EPCR enhances APC generation by at least 5-fold in vitro. 7,8 In vivo, blocking protein C-EPCR interactions results in an 88% decrease in circulating APC levels generated in response to thrombin infusion. 9 EPCR function is critical for embryo development because EPCR knockout mice die in midgestation. 10 The normal distribution of EPCR is highly tissue specific. During embryogenesis, EPCR is expressed by the embryonic giant trophoblast cells from approximately E7.5, and by certain embryonic EC from approximately E11.5. 11 In adults, EPCR is expressed almost exclusively by ECs, particularly those o...

The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demon-