von Willebrand factor binding to myosin assists in coagulation

Abstract

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 factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

Graphical abstract

Figure 1.

Myosin binds to VWF. VWF:Ag is shown for plasma-derived VWF and recombinant VWF. (A) Myosin binding in plasma samples from healthy donors (“control”) and donors with VWF (types 3, 1, and 2A). No significant difference was found between the control myosin binding and type 1 myosin binding, but control myosin binding and type 1 myosin binding were significantly different compared with type 2 myosin binding. (B) Myosin binding is dependent on presence of high molecular weight multimers using recombinant VWF separated into fractions based on the molecular weight of the multimers contained therein (ultra high, high, medium, and low). A significant difference was noted for the medium and low molecular weight multimers compared with the high and ultrahigh molecular weight multimers. (C) Myosin binding to recombinant VWF, including WT recombinant VWF, constructs containing VWF variants that affect multimerization (87S and 2773R), the VWF A3 domain (1786D), which affects collagen III binding, a C-terminal domain RGD site variant (2509E), a D′ variant causing type 2N VWD (791M), and a construct without any VWF sequence as a negative control (mock). (D) A1 domain specificity of myosin binding to recombinant VWF constructs. n ≥ 3. Error bars denote 1 standard deviation. *P < .05, **P < .005.

Figure 2.

VWF binding to myosin inhibited by anti-A1 domain antibodies and collagen. VWF binding to myosin was measured with or without the addition of anti-VWF antibodies or collagen. The y-axis shows the percentage of VWF bound to myosin compared with a sample with VWF and no inhibitor. AVW3 is an anti-VWF A1 domain antibody. AVW5 is also an anti-VWF antibody, but it is located outside of the A1 domain. DAKO is a polyclonal anti-VWF antibody. AP2 is an anti-platelet αIIbβ3 antibody added as a negative control. Collagen IV (COL4) and collagen III (COL3) were also added as potential inhibitors. Error bars denote 1 standard deviation. n ≥ 3 for each experiment. **P < .005 vs control plasma (Plasma).

Figure 3.

FVIII binding to myosin detected only in presence of VWF. FVIII activity was measured with a chromogenic FVIII activity kit. Recombinant FVIII or plasma-derived combination VWF/FVIII concentrate was added to a plate coated with myosin. Bound FVIII was detected using a chromogenic FVIII activity kit. No activity was seen when only FVIII was added (preparation 1, full-length FVIII; preparation 2, B-domain–deleted FVIII), but FVIII activity was detected in the presence of VWF.

Figure 4.

Myosin enhances thrombin generation in the absence of platelets. Thrombin generation was performed for platelet-rich plasma (PRP) and platelet-poor plasma (PPP), with or without the addition of 200 nM myosin. All samples had tissue factor added to initiate thrombin generation. The graph shows total thrombin generated (area under the curve). No difference was seen for PRP, but addition of myosin increased thrombin generation for PPP. Error bars denote 1 SD. N ≥ 3 for each experiment. NS, not significant.