Minhua Ling

Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13.

Elevated plasma von Willebrand factor (VWF) and low ADAMTS13 activity have been reported in several inflammatory states, including sepsis and acute respiratory distress syndrome. One hallmark of inflammation is neutrophil activation and production of reactive oxygen species, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). HOCl is produced from hydrogen peroxide and chloride ions through the action of myeloperoxidase. HOCl can oxidize methionine to methionine sulfoxide and tyrosine to chlorotyrosine. This is of interest because the ADAMTS13 cleavage site in VWF, the Tyr(1605)-Met(1606) peptide bond, contains both oxidation-prone residues. We hypothesized that HOCl would oxidize either or both of these residues and possibly inhibit ADAMTS13-mediated cleavage. We therefore treated ADAMTS13 substrates with HOCl and examined their oxidative modification by mass spectrometry. Met(1606) was oxidized to the sulfoxide in a concentration-dependent manner, with complete oxidation at 75muM HOCl, whereas only a miniscule percentage of Tyr(1605) was converted to chlorotyrosine. The oxidized substrates were cleaved much more slowly by ADAMTS13 than the nonoxidized substrates. A similar result was obtained with multimeric VWF. Taken together, these findings indicate that reactive oxygen species released by activated neutrophils have a prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13.

High density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

The ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

Simultaneous Exposure of Sites in von Willebrand Factor for Glycoprotein Ib Binding and ADAMTS13 Cleavage: Studies With Ristocetin

Objective—Platelet-bound von Willebrand factor (VWF) was recently demonstrated to be a better substrate for ADAMTS13, suggesting that 1 conformational change exposes both the glycoprotein Ib&agr; binding site in the A1 domain and the ADAMTS13 cleavage site in the A2 domain. Because ristocetin induces VWF to bind glycoprotein Ib&agr; in the absence of shear stress, we evaluated whether it could also enhance ADAMTS13 proteolysis of VWF. Methods and Results—We used several VWF sources: plasma, purified plasma VWF, recombinant VWF fragments encompassing A1A2A3, A1A2, and 2 A2 domains, 1 containing a ristocetin-binding site (Asp1459–His1472) and the other lacking it. Ristocetin accelerated ADAMTS13 cleavage of multimeric VWF and of each of the recombinant VWF fragments except for the A2 domain lacking the ristocetin-binding site. We also examined the effect of ristocetin on the conformation of the A2 domain by assessing its effect on the susceptibility of Met1606 at the ADAMTS13 cleavage site to be oxidized by hypochlorous acid. Ristocetin markedly enhanced oxidation of Met1606 and Met1521 of the A2 domain. Conclusion—These data indicate that exposure of the sites for glycoprotein Ib&agr; and ADAMTS13 are coupled, explaining why platelet-bound VWF is a better ADAMTS13 substrate and why enhanced proteolysis is often observed in type 2B von Willebrand disease.

Structural Basis of Type 2A von Willebrand Disease Investigated by Molecular Dynamics Simulations and Experiments

The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered in silico the tensile force necessary to undock the C-terminal helix 6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix 6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.

Normal cleavage of von Willebrand factor by ADAMTS-13 in the absence of factor VIII in patients with severe hemophilia A

Von Willebrand factor (VWF) is required for hemostasis, recruiting platelets from rapidly flowing blood to sites of vessel injury and protecting factor VIII (FVIII) from degradation. The adhesive activity of VWF correlates with its size: large VWF multimers bind more avidly to platelets [1]. VWF multimer distribution is regulated by the metalloprotease ADAMTS13 in plasma [2,3]. In the absence of ADAMTS13 activity, ultra-large VWF (ULVWF) multimers accumulate and induce spontaneous platelet clumping [4] and cause the life-threatening disease thrombotic thrombocytopenia purpura [5]. Recent studies by Cao et al. [6] showed that under shear stress in a system using purified proteins, exogenous FVIII enhanced the cleavage of high-molecular-weight VWF multimers by ADAMTS13. Based on this result, the authors proposed that FVIII is a cofactor for ADAMTS13, suggesting that reduced VWF processing and increased platelet adhesion could represent a form of hemostatic compensation in patients with severe hemophilia A. This finding predicts that absence of FVIII in hemophilia A patients would reduce VWF proteolysis, which would be normalized by infusion of FVIII. Here, we assessed VWF multimer distribution, VWF antigen levels, and ADAMTS13 activity in the plasmas of seven patients with severe hemophilia A before recombinant FVIII infusion. In two patients, we also examined the VWF cleavage by endogenous ADAMTS13 before and after FVIII infusion.