Background Hemostatic clots have a P‐selectin positive platelet core covered with a shell of P‐selectin negative platelets. Objective To develop a new human blood microfluidic assay to interrogate core/shell mechanics. Methods A 2‐stage assay perfused whole blood over collagen/± tissue factor (TF) for 180?seconds at 100?s ?1 wall shear rate, followed by buffer perfusion at either 100?s ?1 (venous) or 1000?s ?1 (arterial). This microfluidic assay used an extended channel height (120?μm), allowing buffer perfusion well before occlusion. Results Clot growth on collagen stopped immediately with buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100?s ?1 ) and ~30% (at 1000?s ?1 ) by 1200?seconds. Thrombin generation (on collagen/TF) reduced erosion at either buffer flow rate. P‐selectin–positive platelets were stable (no erosion) against 1000?s ?1 , in contrast to P‐selectin negative platelets. Thrombin inhibition (with D‐Phe‐Pro‐Arg‐CMK) reduced the number of P‐selectin‐positive platelets and lowered thrombus stability through the reduction of P‐selectin–positive platelets. Interestingly, fibrin inhibition (with H‐Gly‐Pro‐Arg‐Pro‐OH acetate salt) increased the number of P‐selectin–positive platelets but did not lower stability, suggesting that fibrin was only in the core region. Thromboxane inhibition reduced P‐selectin–positive platelets and caused a nearly 60% reduction of the clot at arterial buffer flow. P2Y1 antagonism reduced clot size and the number of P‐selectin–positive platelets and reduced the stability of P‐selectin–negative platelets. Conclusion The 2‐stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P‐selectin–positive platelets never left the clot.
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