Proteolytic inactivation of ADAMTS13 by thrombin and plasmin

Abstract

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...

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

“…Firstly, it is secreted and circulates as an active enzyme 19, 20. Secondly, ADAMTS‐13 has a long plasma half‐life (2–3 days) and has no known physiological inhibitors 21.…”

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

“…The expression vector for WT ADAMTS13 and its mutagenesis have been described elsewhere (25). WT ADAMTS13 and ADAMTS13 variants were transiently expressed in HEK293T cells (23).…”

Von Willebrand Factor: Multimeric Structure and Functional Activity in Patients With Atrial Fibrillation With and Without Oral Anticoagulation

“…4,32 Patients with stroke were not on OAC as the diagnosis of AF was established at admission. As it is well known, OAC inhibits thrombin formation and thrombin inactivates ADAMTS13, 45 and in consequence, formation of HMWMs; larger multimeric forms of vWF are more active in hemostasis, 4,34 promoting a prothrombotic state. 35 Nevertheless, this could be due to the acute response to stroke, so further studies are needed to confirm whether vWF parameters are related to an increased risk of stroke.…”