Destabilization of the A1 domain in von Willebrand factor dissociates the A1A2A3 tri-domain and provokes spontaneous binding to glycoprotein Ibalpha and platelet activation under shear stress

Abstract

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibalpha (GPIbalpha). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbalpha without ristocetin with an apparent K(D) of 85 +/- 34 nm, comparable with that of WT (36 +/- 12 nm) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s(-1) shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s(-1), platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbalpha and alphaIIbbeta3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

FIGURE 1.

Mutation R1450E impaired the interaction between A1 and A2 domain. A, increasing concentrations of recombinant A1 domain proteins were incubated with immobilized A2 domain. The bound A1 protein was determined by enzyme-linked immunosorbent assay as described under “Experimental Procedures” using monoclonal antibody 5D2. The graph is representative of two separated experiments. Each point represents the mean ± S.D. of values obtained from a triplicate assay. B, the binding of the biotinylated WT-A1 domain (250 nm) to immobilized A2 was measured in the presence of purified non-biotinylated WT-A1 or R1450E mutant at the indicated concentrations or the same volume of TBS in the control mixture. 100% is defined as the fraction of added A1 domain polypeptide bound with no A1 variants. Each point represents the mean ± S.E. of two independent sets of triplicate determinations.

FIGURE 2.

SDS-PAGE and gel filtration analyses of recombinant A1A2A3 proteins. Inset, a Coomassie Blue-stained SDS-PAGE (10%) verified the purity of the recombinant WT (1) and mutant (2) proteins under non-reducing conditions (MW, molecular mass). Both purified A1A2A3 proteins were subjected to gel filtration to analyze their native conformation. Using gel filtration column with a TBS mobile phase, the proteins were detected by their absorbance at 280 nm. The graph represents the elution profile for both A1A2A3 proteins (∼92 kDa), and the arrows above represent the elution volume for β-amylase (200 kDa) and human serum albumin (68 kDa). mAu, milliabsorbance units.

FIGURE 3.

Differential scanning calorimetry and urea denaturation of A1 and A3 single and A1A2A3 tri-domains. A, DSC of the WT and R1450E A1 domain and the A3 domain is shown. B, urea denaturation of WT and R1450E A1 domain at 25 °C via circular dichroism at 222 nm is shown. C and D, DSC of the WT and R1450E A1A2A3 tri-domains is shown. E and F, shown is activation energy of the single domains and of the first and second transition of the tri-domain corresponding to the A1 domain and A3 domain, respectively.

FIGURE 4.

Binding of recombinant A1A2A3 proteins to platelet GPIbα and antibody VP-1. Increasing concentrations of the recombinant A domain proteins were incubated with immobilized fixed platelets (A) or monoclonal antibody VP-1 (B). The bound protein was determined by enzyme-linked immunosorbent assay as described under “Experimental Procedures.” A, the mutant A1A2A3 (closed square) had a markedly different binding activity for GPIbα than WT A1A2A3 (closed diamond) in the absence of ristocetin. In the presence of ristocetin, the WT bound to GPIbα similarly to the mutant A1A2A3 without ristocetin. B, mutation R1450E also induced the exposure of the recognition site for the antibody in A2 domain, increasing the binding capacity for VP-1. The graphs are representative of two separated experiments. Each point represents the mean ± S.D. of values obtained from a triplicate assay.

FIGURE 5.

Effect of the A1A2A3 proteins in ristocetin-induced platelet agglutination and shear-induced platelet aggregation. A, the mutant (250 nm) or WT protein (1000 nm) was incubated with PRP for 2 min at 25 °C. The platelet agglutination was then initiated with the addition of ristocetin (1.0 mg/ml). The figure represents four separated experiments using different donors. B, the mutant (250 nm) was incubated with the same PRP used in ristocetin-induced platelet agglutination for 2 min at 25 °C. The mixture was then sheared at 500 or 5000s⁻¹ for 1 min, and platelet aggregation was determined by the reduction of single platelets. The graph depicts the average of percentage of inhibition of platelet aggregation from three separated experiments (mean ± S.D.).

FIGURE 6.

Effect of the mutant A1A2A3 in flow-dependent platelet adhesion to immobilized fibrin(ogen). A, whole blood mixed with WT (top panels) or mutant A1A2A3 (250 nm, bottom panels) was perfused over a surface coated with fibrinogen at different shear rates as indicated. After a 2-min perfusion, the plates were washed with TBS, and several frames of attached platelets were recorded. The photomicrographs represent three separated assays. B, the bar graph shows the percent of surface covered by firmly adhered platelets (mean ± S.E.) after a 2-min perfusion of the whole blood as described in A. The columns represent three separate experiments. C, whole blood containing the mutant A1A2A3 (250 nm) was incubated with either anti-αIIbβ3 (10E5), anti-GPIbα (6D1) (20 μg/ml), EDTA (5 mm), prostaglandin E1 (PGE1) (10 μm), or apyrase (10 units/ml). The blood was perfused over a surface coated with fibrinogen at shear rates of 1500 s⁻¹. After a 2-min perfusion, the plates were washed with TBS, and several frames of attached platelets were recorded and quantified.

FIGURE 7.

Effect of ristocetin on flow-dependent platelet adhesion to immobilized fibrin(ogen). Whole blood mixed with buffer (control, top panel) or ristocetin (0.5 mg/ml, middle panel) was perfused over a surface coated with fibrinogen at a shear rate of 1500 s⁻¹. After a 2-min perfusion, the plates were washed with TBS, and several frames of attached platelets were recorded, (control <1% surface coverage and with ristocetin, ∼68 ± 14% surface coverage). The effect of ristocetin was completely inhibited by the anti-αIIbβ3 antibody 10E5 (bottom panel, <1% surface coverage). The photomicrographs represent three separated assays.