title: P2Y12 or P2Y1 inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions.
abstract: 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 (+/- 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 (IC(50) = 0.233 +/- 0.132 microM) and P2Y(12) inhibitor 2-MeSAMP (IC(50) = 2.558 +/- 0.799 microM) 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 ( approximately 2000 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 IC(50) 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Division of Hematology and Oncology, Institute for Medicine and Engineering, 1024 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Maloney
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Brass
authors.forename: S L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000008:administration & dosage*; D001081:Apyrase / Q000008:administration & dosage*; D002478:Cells, Cultured; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods; D010973:Platelet Adhesiveness / Q000187:drug effects*; D058919:Purinergic P2 Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D013927:Thrombosis / Q000473:pathology* / Q000503:physiopathology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; D058919:Purinergic P2 Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D001081:Apyrase; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone
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