Thrombosis Research 133 (2014) 203–210 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres Regular Article Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay Ruizhi Li, Scott L. Diamond ⁎ Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, 1024 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA article info abstract Article history: Background: Microfluidic devices recreate the hemodynamic conditions of thrombosis. Received 30 July 2013 Methods: Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for Received in revised form 28 October 2013 300 s (wall shear rate = 200 s-1)usingamicrofluidic flow assay. Platelet accumulation was measured in the pres- Accepted 29 October 2013 ence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 Available online 6 November 2013 and P2Y12 inhibitors. Results: High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM), or combined 2MeSAMP and MRS 2179 Keywords: decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p b 0.01), respectively. ASA reduced sec- platelet cyclooxygenase ondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to ADP 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 thromboxane and 105 s and secondary aggregation between 105 and 300 s. RCOX and RP2Y (defined as a ratio of secondary aggre- hemodynamic gation rate to primary deposition rate) demonstrated 9 of 10 subjects had RCOX b 1orRP2Y b 1followingASAor 2MeSAMP addition, while 6 of 10 subjects had RP2Y b 1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each indi- vidual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of RCOX and RP2Y to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). Conclusions: Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self- normalized metric of platelet function for a single blood sample. © 2013 Elsevier Ltd. All rights reserved. Introduction adenylate cyclase and stabilizes secondary platelet aggregation. Current therapies that target the P2Y12 receptor vary from prodrugs that irrevers- Antiplatelet therapies are used in a variety of clinical settings from ibly antagonize the P2Y12 receptor to direct, reversible antagonists [4,19]. management of unstable angina to risk reduction of myocardial infarction Thienopyridines clopidogrel and prasugrel are examples of the former, or stroke. Aspirin is used by over 50 million patients in the United States while ticagrelor is an example of the latter. Currently, no P2Y1 antagonists to reduce the risk of cardiovascular events [1]. Aspirin irreversibly acety- are on the market, however, combined P2Y1 and P2Y12 antagonists are in lates serine 529 of cyclooxygenase-1 (COX-1), blocking the enzyme active development [4,5]. To mimic the action of P2Y1 and P2Y12 antiplatelet site for arachidonic acid and inhibiting the generation of prostaglandin H2 therapies ex vivo, 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate and thus thromboxane A2 (TXA2) production from platelets [2]. Inhibition (MRS 2179) and 2-methylthioadenosine 5-monophosphate (2MeSAMP) of platelet TXA2 synthesis prevents platelet activation through the TXA2 are used in this study as highly selective P2Y1 and P2Y12 antagonists, receptor (TP), a receptor encoded by the TBXA2R gene. respectively. In addition to TXA2, adenosine disphosphate (ADP) receptors are an- Targeting signaling pathways such as TXA2 production and ADP/ other target of antiplatelet therapies. The platelet plasma membrane con- P2Y12 signaling reduces secondary platelet aggregation while not severe- tains two ADP receptors, P2Y1 and P2Y12, which are purinergic G protein ly altering primary haemostasis. However, the delicate balance between coupled receptors. P2Y1 is linked to Gq and ADP signaling through this preventing excessive clotting and increasing bleeding risks requires care- pathway results in rapid Ca2+ mobilization and platelet shape change ful monitoring of antiplatelet therapies. The evaluation of the effect of [3,4]. P2Y12 is linked to a Gi protein. ADP binding to P2Y12 inhibits pharmacological agents on platelet function often rely on tests with poorly defined fluid mechanics and flow fields (eg. aggregometry) that fail to replicate platelet adhesive mechanisms under realistic and defined Abbreviations: (ASA), Aspirin; (COX-1), cyclooxygenase 1; (TXA ), thromboxane A ; 2 2 hemodynamic conditions. Under flow conditions, the efficacy of pharma- (ADP), Adenosine diphosphate. ⁎ Corresponding author. Tel.: +1 215 573-5702; fax: +1 215 573-6815. cological agents greatly depend on granule release, platelet-platelet con- E-mail address: [email protected] (S.L. Diamond). tacts, and convective removal of autocrinic agonists from the injury site. 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.10.043 204 R. Li, S.L. Diamond / Thrombosis Research 133 (2014) 203–210 Microfluidic devices can recreate the hemodynamic conditions required patterned equine fibrillar collagen type 1 (Chronopar, Chronolog). Chan- to study anti-platelet agents. These devices offer spatially controlled focal nels were spaced in close proximity to allow for all channels to be im- injuries with collagen or collagen with tissue factor bearing surfaces aged simultaneously with a 2X objective lens using an inverted [6–8].Microfluidic devices have also been used to study clot contraction microscope (IX81, Olympus America Inc) equipped with a CCD camera and clot permeability with precise control of wall shear stress and (ORCA-ER, Hamamatsu). A custom stage insert held 3 microfluidic transthrombus pressure gradients [8,10]. In fact, the core-shell hierarchy of clots observed in vivo following laser injury [9] can be replicated in vitro with such devices [10]. Here we continue the development of microfluidic assay metrics found previously [11] and extend these metrics to examine two ADP an- tagonists and validate this assay for detection of anti-platelet therapies through Receiver-Operator Characteristic (ROC) analysis. For flow as- says to become a relevant clinical tool a large cohort of healthy donors must be tested with respect to response to antiplatelet agents. Toward that goal, we tested healthy subject platelet function with 38 donors and 66 independent blood draws (2 combined studies) after ex vivo ad- dition of ASA. While coagulation assays can rely on stable pooled plasma for calibration, live platelet function assays have no available standard to calibrate the assay. We sought to define a self-normalized parameter, the R-value, to score the accumulation of platelets on the surface for a single blood sample test without reference to a prior test value or cali- bration fluid. Materials and Methods Blood Collection, Labeling, and Antiplatelet Agents Blood was collected via venipuncture from 11 healthy subjects who self-reported as non-smoking, free of oral medication, and abstained from alcohol 48 hr prior to donation. All subjects were free from illness or bleeding disorders. Blood samples were drawn into H-D-Phe-Pro-Arg-chloromethylketone (100 μM PPACK final concentration, Haematologic Technologies). All volunteers provided informed consent in accordance with IRB approval and the Declaration of Helsinki. Whole blood was treated with Phycoerythrin (PE) Mouse Anti- Human CD61 (αIIbβ3) antibody (BD Biosciences) in a ratio of (1:50) 7 min prior to perfusion. This antibody has no effect on platelet αIIbβ3 function in the assay. PPACK-treated whole blood was perfused through the device within 25 min of phlebotomy. Blood was incubated with vehi- cle (0.1% DMSO final concentration) or indicated concentrations of ASA, 2MeSAMP, MRS 2179, or combined 2MeSAMP and MRS 2179 20 min prior to the assay. This time scale is consistent with previous studies done with these compounds [11–13]. Acetylsalicylic acid (ASA, Sigma Aldrich) was dissolved in DMSO at 500 mM, 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2MeSAMP, Sigma Aldrich) was dissolved in HEPES buffered saline (HBS, 20 mM HEPES, 160 mM NaCl, pH 7.5) at 1 mM/L, and 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate ammonium salt (MRS 2179, Tocris Bioscience) at 0.1 mM in HBS. A dilution of ASA was then made to the desired final concentration in HBS within 1 h of the test. Final concentrations of antiplatelet therapies used were: 500 μM ASA, 100 μM 2MeSAMP, and 10 μMMRS2179,wellinexcessoftheirIC50's in the assay of 10-20 μMASA,2.56μM 2MeSAMP, and 0.23 μM MRS 2179 [11,13]. Antiplatelet agent concentrations were used well in excess of their respective IC50 values to completely antagonize ADP receptors or abate COX-1 derived TXA2. Microfluidic Devices and Real Time Platelet Deposition Imaging Microfluidic devices were fabricated in polydimethylsiloxane (PDMS, Sylgard 184, Ellsworth Adhesives) according to previously Fig. 1. 8-channel microfluidic device, measured platelet fluorescence dynamics, and RCOX, fl described techniques [11,13].Themicrofluidic