Jennie Le

The rate of hemolysis in sickle cell disease correlates with the quantity of active von Willebrand factor in the plasma

Vaso-occlusion, hemolysis, and oxidative stress are hallmarks of sickle cell disease (SCD). This pathology is accompanied by systemic endothelial activation, rendering the endothelium more adhesive for blood cells, including sickle erythrocytes. Activated endothelial cells display or secrete several adhesive molecules, including von Willebrand factor (VWF). We assessed several VWF parameters in SCD patients at baseline: multimer pattern, antigen concentration (VWF:Ag), activation factor (VWF:AF), and total active VWF (VWF:TA). VWF:AF was determined using a llama nanobody (AU/VWFa-11) that detects a platelet-binding conformation of the A1 domain; VWF:TA was calculated by multiplying VWF:Ag by VWF:AF. SCD plasma contained elevated VWF:Ag and ultralarge VWF multimers. VWF:TA, a measure of total VWF reactivity, correlated closely with hemolysis, as determined by serum lactate dehydrogenase. ADAMTS13 activity and antigen were normal in all patients. These findings suggest an important role for hyperreactive VWF in SCD pathology and connect SCD to other microangiopathies, particularly thrombotic thrombocytopenic purpura.

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.

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.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D′D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1–GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1–GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.