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Aim: von Willebrand factor (VWF) plays an important role in the regulation of hemostasis and thrombosis formation, particularly under a high shear rate. However, the adhesive force due to the molecular interaction between VWF and glycoprotein Ibα (GPIbα) has not been fully explored. Thus, we employed atomic force microscopy to directly measure the adhesive force between VWF and GPIbα., Methods: We measured the adhesive force between VWF and GPIbα at the molecular level using an atomic force microscope (AFM). An AFM cantilever was coated with recombinant N-terminus VWF binding site of GPIbα, whereas a cover glass was coated with native VWF., Results: The adhesive force at the molecular level was measured using an AFM. In the presence of 1 μg/mL VWF, the adhesion force was nearly 200 pN. As per the Gaussian fit analysis, the adhesive force of a single bond could have been 54 or 107 pN., Conclusion: Our consideration with the Gaussian fit analysis proposed that the adhesive force of a single bond could be 54 pN, which is very close to that obtained by optical tweezers (50 pN).

Adsorption of plasma proteins such as von Willebrand factor (vWF) on thrombogenic surfaces can induce conformational changes in tertiary structure so that the prothrombotic functional epitopes are exposed for interactions with platelets, resulting in platelet adhesion and thrombus formation. Thus, understanding platelet binding following changes in the structure of vWF is critical in understanding the mechanisms of thrombogenesis. The present study examined the accessibility of platelet binding epitopes within vWF adsorbed on two different thrombogenic surfaces, a hydrophobic synthetic surface and collagen VI coated substrates, under physiological buffer conditions using atomic force microscopy (AFM) in combination with immunogold labeling. Our results demonstrated that the glycoprotein Ib (GPIb) binding domain in vWF undergoes changes when adsorbed on collagen VI compared to vWF on a hydrophobic synthetic surface. This study provides a basis for a novel approach to understand the molecular mechanisms of surface-induced thrombosis by directly examining the structure-function relationships of plasma proteins involved in the thrombus formation.

The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s(-1). At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.

Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ibalpha and von Willebrand factor (vWF) acted in synergy with alphaIIbbeta3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes.