Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro

Abstract

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Keywords: factor VIII; hemophilia A; polyphosphates; von Willebrand diseases; von Willebrand factor.

Figure 1

Dynamic interaction between von Willebrand Factor and polyphosphate is stronger in the absence of factor VIII. Surface plasmon resonance analysis of immobilized von Willebrand Factor (factor VIII-free) (VWF) (a) or von Willebrand factor co-purified with factor VIII (VWFFVIII) (b), confronted with increasing concentrations of isolated polyphosphate (polyP₆₅, 0.25–13.34 µM). The corresponding fitted curves are presented as solid lines. A representative experiment is shown (n = 3).

Figure 2

Characterization of the polyphosphate-von Willebrand Factor binding, in the absence or presence of factor VIII, using urea–polyacrylamide gel electrophoresis. (a) Purified von Willebrand factor (factor VIII-free, 2.3 µg) and/or purified factor VIII (0.02 to 2 µg) were incubated with 0.8 mM of isolated polyphosphate (polyP₆₅). Samples were loaded onto urea–polyacrylamide gels, separated by electrophoresis, and stained using a polyphosphate-specific staining (toluidine blue). Arrows show the mobility of von Willebrand factor on the gels. Representative experiments are shown (n = 3). (b,c) Increasing amounts of von Willebrand factor, either factor VIII-free in (b), or von Willebrand factor co-purified with factor VIII in (c), were incubated with 0.8 mM of isolated polyphosphate (polyP₆₅) as described in Methods. Samples were separated as before and stained using a sensitive polyphosphate-specific staining (DAPI negative). Similar experiments, but stained with toluidine blue, are shown in Supplementary Figure S1.

Figure 3

PolyP increases the von Willebrand Factor ristocetin cofactor activity in factor VIII-deficient plasmas. (a,b) Aggregation curves of fixed platelets in a ristocetin co-factor activity assay using factor VIII-deficient plasmas. Prior to the assay, 50 µL of plasmas (diluted 1:2) were incubated in the absence (control) or in the presence of (a) isolated polyphosphate (polyP₆₅, 2.5 µM, and 7.1µM) and (b) recombinant yeast exopolyphosphatase (PPX, 0.68 µg, and 1.7 µg). Representative experiments are shown (n = 3). (c,d) Quantification of the differences in von Willebrand factor ristocetin co-factor activity in normal plasmas (Normal) and plasmas depleted of factor VIII (FVIII-def.) after the addition of (c) 2.5 µM of isolated polyphosphate (polyP₆₅) or (d) 1.7µg of recombinant yeast exopolyphosphatase (PPX). For each sample, the change was calculated as “activity without addition of polyP or PPX” minus “activity with the addition of polyP or PPX”; therefore, positive values mean an increase above the basal activity and negative values mean a decrease. Results are presented in a box-and-whiskers plot. An asterisk indicates a statistical difference of p < 0.01, determined by Mann–Whitney test using plasmas of 3 normal individuals vs. factor VIII-depleted plasmas from three different lots. Measurements were performed using an automated aggregometer (Helena AggRAM).

Figure 4

PolyP addition increases the von Willebrand factor activity in plasmas from patients with severe hemophilia A. Experiments were performed as described in Figure 3 but using plasmas from healthy individuals (Normal) and patients with moderate and severe hemophilia A (Moderate, Severe). Quantification of the differences in von Willebrand factor ristocetin co-factor activity after the addition of 2.5 µM of isolated polyphosphate (polyP₆₅). For each sample, the change was calculated as “activity without addition of polyP” minus “activity with the addition of polyP”. For comparison, we also include data from experiments using factor VIII-deficient plasmas (FVIII-def.). Results are presented in a box-and-whiskers plot. Symbols indicate a statistical difference of p < 0.01, determined by Mann–Whitney test, related to “Normal” plasmas (*), or “Moderate hemophilia” (‡) (n = 3). An asterisk indicates a statistical difference of p < 0.01, determined by Mann–Whitney test. Measurements were performed using an automated aggregometer (Helena AggRAM).

Figure 5

PolyP depletion does not affect von Willebrand factor ristocetin co-factor activity in the presence of factor VIII. Aggregation curves of fixed platelets in a ristocetin co-factor activity assay of plasma from patients with von Willebrand disease type 1. Prior to the assay, 50 µL of plasmas (diluted 1:2) were incubated for 10 min at 37 °C in the absence (control) or in the presence of 3.5 µg of von Willebrand factor (factor VIII-free) (VWF) (a) or von Willebrand factor co-purified with factor VIII (VWF–FVIII) (b). In addition, we performed similar plasma incubations but including 6.4 µg of recombinant yeast exopolyphosphatase (+PPX) (a,b). Measurements were performed using an optical platelet aggregometer (Chronolog Aggro-meter model 400). A representative experiment is shown (n = 3).

Figure 6

PolyP addition increases the von Willebrand factor ristocetin co-factor activity in plasmas from patients with von Willebrand disease type 2 and 3. Experiments were performed as described in Figure 3 but using plasmas from healthy individuals (Normal) and patients with von Willebrand disease type 2 (Type 2 VWD) and type 3 (Type 3 VWD). Quantification of the differences in von Willebrand factor ristocetin co-factor activity after the addition of 2.5 µM of isolated polyphosphate (polyP₆₅). Results are presented in a box-and-whiskers plot. An asterisk indicates a statistical difference of p < 0.01, determined by Mann–Whitney test. Numbers of individuals in each category are indicated in parenthesis. Measurements were performed using an automated aggregometer (Helena AggRAM).

Figure 7

Predicted interaction of polyP with VWF. (a) The most recent structure of the FVIII–VWF complex (D′D3 domain), from reference [24] (PDB code 7KWO). The square indicates the area zoomed in on in the other panels of this figure. (b) Interaction between the FVIII-a3 acidic peptide and the VWF-highly basic groove. Arginines are labelled in blue. (c) VWF-highly basic groove without FVIII. (d) PolyP was superposed with the VWF highly basic groove. The model for polyP was extracted from the PDB (code 5LLF). Images were created using Mol* [25].