Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Abstract

BACKGROUND Microfluidic devices recreate the hemodynamic conditions of thrombosis. METHODS Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate = 200 s−1) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 and P2Y12 inhibitors. RESULTS High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM),or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p < 0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. RCOX and RP2Y (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had RCOX < 1 or RP2Y < 1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had RP2Y < 1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual 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.

Dynamic platelet adhesion in patients with an acute coronary syndrome: The effect of antiplatelet therapy

“…A role of the α2β1 receptor in the inhibition by clopidogrel and ticagrelor of platelet adhesion and further activation is less possible since this receptor must be initially activated by inside-out signaling [34], and in our adhesion experiments we did not use any platelet agonist. Finally, platelet inhibition by aspirin may not be involved in the above effects of clopidogrel and ticagrelor, since aspirin has no effect on the initial stage of platelet adhesion [3,37], consistent with our findings. Therefore, we can assume that our findings are only the result of the effect of P2Y 12 antagonists.…”

“…However, the delicate balance between preventing excessive clotting and increasing bleeding risks requires careful use and in some cases, monitoring of antiplatelet therapies. The evaluation of the effect of antiplatelet agents on platelet function often relies on tests with poorly defined fluid mechanics and flow fields (e.g., aggregometry) that fail to replicate platelet adhesive mechanisms under flow conditions, in which the efficacy of antiplatelets greatly depend on granule release, platelet-platelet contacts, and convective removal of autocrinic agonists from the injury site [8]. Microfluidic devices can create hemodynamic conditions very similar to those occurring in vivo and allow the actual study of platelet-surface interaction and the detection of different phases of thrombus formation.…”

Microfludic platforms for the evaluation of anti-platelet agent efficacy under hyper-shear conditions associated with ventricular assist devices

“…While previous studies have evaluated efficacy of AP drugs in vitro [41,43–46] few have examined efficacy under shear flow conditions [33,47]. The present study provides an advance in both device and method, affording the possibility of testing platelet responsiveness following AP drug treatment in vitro under realistic and VAD-specific shear stress conditions.…”

Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation

“…Such designs may include obstructions to simulate a stenosis (66,67), microarrays of protein spots to evaluate adhesion and concentration thresholds (83,84) or coupled parallel channels for simplified dose-response and inhibitor evaluations (85,86).…”

“…At a low, venous shear rate, the overall pressure, in the flow chamber, during thrombus formation is low, a higher pressure can cause instabilities and embolization. The use of higher shear rates are clearly favourable from a physiological perspective but requires techniques that can adjust the pressure as it builds up in the channel, this issue has been discussed and studied by others (85,86,88).…”