P2Y(12) or P2Y(1) inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions

摘要

Determination of the patient-specific response to antiplatelet agents facilitates proper dosing for both acute and chronic prophylaxis. "Closed'' systems (with or without flow) may fail to predict pharmacological potency in situations where platelets rapidly accumulate under flow conditions at a site of thrombosis ("Open'' systems). Using an 8-channel microfluidic flow assay of human whole blood with corn trypsin inhibitor (perpendicular to PPACK) perfused over focal zones of collagen, dose-response curves were measured for pharmacological agents at a wall shear rate of 210 s(-1). The P2Y(1) inhibitor MRS 2179 (IC50 = 0.233 +/- 0.132 mu M) and P2Y(12) inhibitor 2-MeSAMP (IC50 = 2.558 +/- 0.799 mu M) were potent blockers of secondary platelet accumulation under flow, while the P2X(1) inhibitor (NF 449) and apyrase failed to reduce platelet accumulation. MRS 2179 and 2-MeSAMP had undetectable effects on initial platelet adhesion to collagen. Numerical simulation of convective-diffusive transport and apyrase-mediated catalytic degradation of ADP indicated that ultra-high concentrations of apyrase (B2000 U mL(-1)) would be required to have the same effect under flow as much lower concentrations (1 U mL(-1)) currently used in closed systems (aggregometry or cone-and-plate viscometer). This is the first evaluation of IC50 values for P2Y(12) and P2Y(1) antagonists under controlled flow conditions. Evaluation of antiplatelet agents in open flow systems demonstrates that inhibition of either ADP by apyrase or antagonism of P2X(1) signaling had no inhibitory effect on platelet accumulation. This technique provides a platform for rapidly investigating effects of antithrombotic therapies simultaneously in a model injury system.