P2Y12 or P2Y1 inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions.
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.
Maloney
S F
SF
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.
Brass
Lawrence F
LF
Diamond
S L
SL
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*
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
Microvasc Res. 1978 Sep;16(2):183-95
570240
Arterioscler Thromb Vasc Biol. 2008 Mar;28(3):s43-50
18174448
J Med Chem. 1998 Jan 15;41(2):183-90
9457242
Circulation. 2004 Jun 29;109(25):3064-7
15226220
Eur J Pharmacol. 2001 Feb 2;412(3):213-21
11166284
Annu Rev Med. 1979;30:119-34
233610
Science. 1989 Mar 17;243(4897):1483-5
2467379
J Thromb Haemost. 2007 May;5(5):907-9
17461925
Biophys J. 1986 Nov;50(5):937-45
3790695
Platelets. 2006 May;17(3):178-84
16702045
Blood. 2003 May 15;101(10):3969-76
12521992
Trends Pharmacol Sci. 1998 Oct;19(10):391-4
9803826
Blood. 1999 Oct 15;94(8):2704-15
10515874
Biochemistry. 1973 Jun 19;12(13):2462-8
4709942
Nat Rev Drug Discov. 2003 Oct;2(10):775-89
14526381
Thromb Haemost. 1986 Jun 30;55(3):415-35
3750272
J Clin Invest. 2004 May;113(10):1440-6
15146241
Circ Res. 2006 Dec 8;99(12):1293-304
17158345
Integr Biol (Camb). 2009 Dec;1(11-12):655-63
20027374
Thromb Haemost. 1999 Jan;81(1):111-7
10348701
Blood. 2008 Apr 1;111(7):3507-13
18203955
Lab Chip. 2008 Sep;8(9):1486-95
18818803
J Exp Med. 2003 Aug 18;198(4):661-7
12913094
J Intern Med. 1996 Jan;239(1):17-34
8551196
J Biol Chem. 2009 Jun 12;284(24):16108-16117
19346255
Biophys J. 2006 Nov 1;91(9):3474-81
16905604
Pharmacol Ther. 2005 Nov;108(2):180-92
15955565
Annu Rev Pharmacol Toxicol. 2006;46:277-300
16402906
Am J Physiol. 1995 Dec;269(6 Pt 2):H2155-61
8594927
Anal Chem. 2008 Oct 1;80(19):7543-8
18729474
Nat Rev Drug Discov. 2003 Jan;2(1):15-28
12509756
Lab Chip. 2008 May;8(5):701-9
18432339
Circulation. 2007 Oct 16;116(16):1784-94
17909107
Anal Chem. 1998 Dec 1;70(23):4974-84
21644679
Thromb Haemost. 1995 Aug;74(2):736-42
8585015
Platelets. 2004 May;15(3):131-44
15203715
Circulation. 1988 Dec;78(6):1431-42
3263902
Semin Thromb Hemost. 2005 Apr;31(2):150-61
15852218
J Thromb Haemost. 2008 Dec;6(12):2193-201
18983510
J Clin Invest. 2002 Apr;109(8):1031-40
11956240
Blood. 2003 Sep 1;102(5):1716-22
12738662
Biochem J. 1977 Dec 1;167(3):525-9
203267