Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of alpha IIb beta 3 engagement.
Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and alphaIIbbeta3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s-1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with alphaIIbbeta3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of alphaIIbbeta3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% +/- 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti-phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s-1 but not at 200 s-1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block alphaIIbbeta3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of alphaIIbbeta3, is sufficient to induce SIPA but does not lead to stable thrombus formation.
Mekrache
Médina
M
Institut National de la Santé et de la Recherche Médicale (INSERM), Le Kremlin-Bicetre, France.
Bachelot-Loza
Christilla
C
Ajzenberg
Nadine
N
Saci
Abdelhafid
A
Legendre
Paulette
P
Baruch
Dominique
D
D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D010766:Phosphorylation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013314:Stress, Mechanical; D014842:von Willebrand Diseases / Q000097:blood*; D014841:von Willebrand Factor / Q000378:metabolism*
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human