Core and shell platelets of a thrombus: A new microfluidic assay to study mechanics and biochemistry

Abstract

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.