Von Willebrand Factor Binding to Myosin Assists in Coagulation

Introduction: Von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factors V and X (Deguchi et al, Blood 2016;128:1807-1878). We hypothesized that VWF could also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation.

Methods: Recombinant wild-type (WT) was transfected into HEK293T cells and supernatants collected for VWF to use in experiments. VWF variants containing point mutations at specific sites were constructed via site directed mutagenesis and expressed as above. Myosin from rabbit skeletal muscle was plated on maleic anhydride plates, recombinant VWF or plasma samples added and presence of VWF detected using anti-VWF monoclonal antibodies. Competition assays were performed with antibodies blocking various sites on VWF. FVIII activity was measured using a chromogenic substrate (Chromogenix Coatest). Thrombin generation was performed using a fluorogenic substrate from the Technothrombin TGA kit with either tissue factor +/- myosin or the RCL reagent +/- myosin added to platelet poor plasma from healthy controls.

Results: WT VWF and human plasma VWF from healthy controls bound myosin, while plasma lacking VWF failed to demonstrate binding. Binding was multimer dependent, with a dose dependent decrease in binding seen with increasing loss of high molecular weight multimers. When myosin binding to VWF:Ag ratios were compared, a solution of ultra high molecular weight multimers demonstrated a mean ratio of 1.6, a solution of high molecular weight multimers a ratio of 1.4, a solution of medium molecular weight multimers a ratio of 0.4 and binding was undetectable with only low molecular weight multimers. A polyclonal anti-VWF antibody (DAKO) completely blocked VWF binding to myosin as did an antibody directed against the VWF A1 domain (AVW-3). Antibodies directed against other VWF sites including the N and C terminal ends failed to affect VWF binding to myosin. Since collagen IV also binds in this region, collagen IV was added to the assay as competition and also completely blocked VWF binding to myosin. VWF variants p.R1395A and p.R1399A showed undetectable binding, while a variant in the A3 domain (that abrogates type III collagen binding) showed normal binding to myosin (95% of WT). However, additional residues that affect VWF-collagen IV interactions were tested and had minimal effect on myosin binding, including p.1392A and p.1402A. Taken together, these results suggest a binding site for myosin in the VWF A1 domain similar but not identical to that of collagen IV.

FVIII activity was detected when a VWF concentrate containing both VWF and FVIII was bound to myosin, with a dose-dependent increase in activity. However, no increase in FVIII activity was seen using recombinant FVIII alone in the absence of additional VWF. Because previous results from J. Griffin and coworkers showed a role for myosin in enhancing thrombin generation, we also looked at thrombin generation in platelet-poor plasma with and without myosin, but no difference was seen in the absence of additional phospholipids. Peak thrombin with myosin was 80 nM vs 62 nM without myosin (p=NS), and the area under the curve was 1022 vs 775 (p=NS). With additional phospholipids, there was a trend towards increased thrombin generation with myosin but the difference was again not statistically significant. Peak thrombin with myosin was 70 nM vs 64 nM without myosin (p=NS), and the area under the curve was 974 vs 867 (p=NS).

Discussion: Myosin can also serve as a surface for VWF binding. This may help facilitate delivery of FVIII to sites of injury and improve thrombin generation, although in our hands myosin did not substantially increase thrombin generation in the presence of additional phospholipid. Unlike factor V, FVIII does not intrinsically interact with myosin to accelerate thrombin generation. However, myosin, similar to collagen, can increase local accumulation of VWF-FVIII and indirectly accelerate thrombin generation and clot formation.