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

Abstract

The function of the mechanosensitive, multi-meric 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 unla-beled 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 plate-lets 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 plate-lets 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. (Blood. 2010;116(19):3990-3998) Introduction von Willebrand factor (VWF) is a large, multidomain glycoprotein found in normal blood at concentrations of approximately 10 g/mL. 1 The protein plays an important role in hemostasis by both carrying the coagulation protein factor VIII (FVIII) in circulation and by regulating the adhesion of platelets to sites of vascular injury. Whereas the DD3 domain of VWF binds FVIII, the A1 and C1 domains engage platelet receptors glycoprotein Ib (GPIb) and IIb 3 (GPIIb-IIIa), respectively. Monomeric VWF has a molecular mass of approximately 250 kDa. This unit further polymerizes, via disulfide linkage formation in the endoplasmic reticulum and Golgi of endothelial cells and megakaryocytes. Multimeric VWF size ranges from 0.5 to 20 MDa. 2 Ultra/unusually-large VWF is secreted from the Weibel-Palade bodies of endothe-lial cells on stimulation with a variety of agonists associated with inflammation and thrombosis, including thrombin, histamine, and tumor necrosis factor-. The hemostatic potential of VWF increases with protein size and the magnitude of the applied hydrodynamic shear. 3,4 Ultra/ unusually-large VWF secreted from endothelial cells under shear is extended in the form of strings or bundles on the vessel wall. 5,6 Shear-mediated extension enhances cleavage of the cryptic Y 1605-M 1606 bond within the VWF-A2 domain by the constitutively active blood metalloprotease, ADAMTS13. In addition to cleavage when immobilized on the endothelium, VWF subjected to fluid shear in flowing blood 7 and on platelets 8 is also susceptible to proteolysis by ADAMTS13. Together, these mechanisms reduce a...

Migration distance-based platelet function analysis in a microfluidic system

“…As the shear rate increased, MR rapidly decreased until it reached an asymptotic value between 1800 and 3600 s -1 . These results were confirmed in the previous studies 31,32 that the percentage of platelet activation was proportional to the applied shear rates. In other words, the higher shear rate is applied, the more platelets are activated and aggregated.…”

“…31,32 Addition of viscous medium such as 1.5% dextran (molecular weight 2Â10 6 ) increases suspending medium viscosity (from 1.1 cP to 2.2 cP) and doubles the shear stress without changing the rotating shear rate in whole blood sample. Subsequently, this led significant increase (3.3 folds) in platelet activation.…”

Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13

“…These were expressed in mammalian HEK293T cells and HUVECs. The backbone vectors for multimeric VWF expression pCDNAD3-VWF 19 and fluorescence resonance energy transfer (FRET) 20 were described previously. All recombinant constructs contain the VWF signal peptide.…”

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

“…VWF self-association has been described on glass or collagen surfaces, 8 on the endothelial surface to form strands, 6,9 and on platelet surfaces. 10 VWF self-association is promoted by fluid shear stress. 9,11 We recently showed, in an in vitro system of endothelialized microvessels, that VWF secreted from the vessel wall is able to span the lumen of vessels with diameters up to 300 mm at sites of turns and bifurcations.…”