Shear-Dependent Thrombus Formation Assay

Microspot-coated coverslips were mounted onto a transparent parallel-plate flow chamber (50 μm depth, 3 mm width and 20 mm length), and pre-rinsed with HEPES buffer pH 7.45 containing 0.1% BSA. Anticoagulated whole-blood samples (400–500 μl) were perfused through the flow chamber for a time period sufficient for full-thrombus formation on collagen I spots, that is, 6 min at 150 s⁻¹, 4 min at 1,000 s⁻¹ and 3.5 min at 1,600 s⁻¹. Where indicated, blood samples were preincubated for 5 min with DiOC₆ (0.5 μg ml⁻¹), and fluorescence images were recorded from the microspots during blood perfusion. In other cases, thrombi formed after blood flow were poststained by 2-min perfusion (1,000 s⁻¹) with colour-selected combinations of the following platelet activation markers: FITC-labelled anti-fibrinogen mAb (1:100), FITC-labelled anti-P-selectin mAb (1.25 μg ml⁻¹) and/or AF647-annexin A5 (0.25 μg ml⁻¹), all in HEPES buffer pH 7.45 supplemented with 0.1% BSA. After 2 min of staining (stasis), unbound label was removed by a short perfusion with the same HEPES buffer. No fixative was used.
To assess the role of thrombin on thrombus formation under high-shear flow conditions, tissue factor (500 pM) was immobilized together with a dual coating of vWF with fibronectin or collagen-I. Corn trypsin inhibitor (5 μg ml⁻¹) was present in the blood collecting tube to inhibit the contact activation pathway of coagulation. Blood was drawn on 0.32% trisodium citrate and Gly-Pro-Arg-Pro (5 mg ml⁻¹) was added to block fibrin polymerization and thus clot formation. During the flow perfusion, the blood was recalcified with 6.3 mM CaCl₂ and 3.2 mM MgCl₂ to allow thrombin to be formed via co-coated tissue factor. Platelet activation markers were determined as described for the noncoagulating conditions.