r-mVWF mediated platelet adhesion under physiologic high shear. (A) Impact of collagen-binding mutants on collagen I or III surface under high shear conditions. Whole blood from VWF knockout mice containing DiOC₆ (1 μM)–labeled platelets, anticoagulated with argatroban, was perfused over a type I or III collagen and recombinant mouse VWF-coated chamber under high shear rate (2500 S⁻¹). Representative images of collagen-binding mutants at 7 minutes after perfusion. p.L1757A showed accelerated platelet adhesion on both collagen I and III surfaces. p.S1731T and p.H1786D showed decreased platelet adhesion. These images were constructed from spinning disk confocal laser scanning microscopy–based data on successive horizontal slices. The images are representative of 4 flow experiments. Scale bar, 50 μm. (B) Flow-based analysis of platelet adhesion. Statistical analyses corresponding to the images in panel A. Error bars represent mean (± standard deviation) surface coverage and total thrombus volume in the flow experiments. Thrombus generation with the p.L1757A gain-of-function variant is accelerated and enhanced, whereas the loss-of-function mutants showed various degrees of functional deficit (n = 5).