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Von Willebrand factor self-association on platelet GpIbalpha under hydrodynamic shear: effect on shear-induced platelet activation.

ABSTRACT: The function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ib? (GpIb?) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488-conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ?A1-488. Although ?A1-488 alone did not bind platelets under shear, this protein bound GpIb? on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ? 60 to 70 dyne/cm(2). ?A1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ?A1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-?m beads bearing either immobilized VWF or anti-GpIb? mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm(2). Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIb? via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

SUBMITTER: Dayananda KM

PROVIDER: S-EPMC2981547 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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von Willebrand factor self-association on platelet GpIbalpha under hydrodynamic shear: effect on shear-induced platelet activation.

Dayananda Kannayakanahalli M KM Singh Indrajeet I Mondal Nandini N Neelamegham Sriram S

Blood 20100809 19

The function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488-conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produc ...[more]

PMID: 20696943

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Project description:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

Dataset Information

Von Willebrand factor self-association on platelet GpIbalpha under hydrodynamic shear: effect on shear-induced platelet activation.

Publications

von Willebrand factor self-association on platelet GpIbalpha under hydrodynamic shear: effect on shear-induced platelet activation.

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