Monitor Antiplatelet Therapy Among Patients with Ischemic Stroke By Platelet Function Assay-200 (PFA-200)

Aspirin (ASA) is an anti-inflammatory and anti-platelet agent that irreversibly inhibits the cyclooxygenase (COX) and, thus thromboxane (TX) generation. Insufficient response on antiplatelet medication has become an intensively discussed issue because of the risk factor of recurrent adverse cardiovascular events. However, the monitoring of antiplatelet therapy requires appropriate, robust and reliable test methods. For the measurement of thienopyridine effects, the manufacturer of the PFA-200 System provides the INNOVANCE PFA P2Y cartridge.

The aim of this study is to monitor the antiplatelet therapy among ischemic stroke patients with aspirin and/or clopidogrel by platelet function assay-200 (PFA-200).

Adult patients who developed ischemic stroke over the past six months in our hospital and received treatment either with Aspirin or Clopidogrel or both agents were enrolled. Compliance with therapy was ensured by hospital records. After daily aspirin 100 mg with or without clopidogrel 75 mg therapy for 7 to 10 days, citrated blood was obtained from patients for assay. Platelet functions were studied by platelet aggregometry induced by 0.5% Arachidonic acid (AA) and/or 20 μM adenosine diphosphate (ADP) and PFA-200 screen. The definitions of aspirin or clopidogrel resistance were defined as generally accepted criterion.

Of this 123 patients studied, 46 were on aspirin and 12 on clopidogrel monotherapy, 65 were on dual antiplatelet therapy, Maximum ADP-induced platelet aggregation (20 uM) by light transmittance aggregometry at 1 week (7 to 10 days) was 55.0 ± 27.4% (n = 77). Overall, 19 patients on aspirin (17.1%, 19/111) and 24 patients on clopidogrel (31.2%, 24/77) had resistance to anti-platelet effect. In 77 patients with ischemic stroke undergoing clopidogrel antiplatelet therapy, the new cartridge had a detection rate of 84.4% (65/77, closure times >106s as cut-off) for clopidogrel medication. In the 12 patients under clopidogrel monotherapy, the detection rate was 75.0% (9/12), while in the 65 patients on dual antiplatelet therapy, the detection rate was 86.1% (56/65).

103 healthy volunteers matched by sex and age were enrolled as control group. The PFA test is highly dependent on VWF levels because of the high-shear conditions within the cartridge capillary and aperture. The level of VWF: Ag in patients with ischemic stroke (median 176%, interquartile range [IQR] 114%-293%) was significantly higher than controls (105% [61%-145%], P < 0.001). However, the CTs of the conventional PFA collagen/ADP test cartridge in patients before therapy (median 85.5s, [IQR] 61%-145%) were no significant difference compared with controls (94.0s, [75.3-112.3s], P= 0.115).

24 patients with clopidogrel and/or aspirin resistance and 24 controls matched by sex and age were enrolled for further in vitro assay. The in vitro addition of the VWF antagonist aptarmer (AT-300) to citrated human whole blood resulted in a dose-dependent prolongation of closure times (CTs) of the conventional PFA collagen/ADP cartridge. Collagen/ADP induced platelet plug formation was inhibited by 50% (IC₅₀) in patients with ischemic stroke (median 0.277ug/ml, interquartile range [IQR] 0.207-0.481ug/ml) were significantly higher than controls (0.161 ug/ml, [0.114-0.222] ug/ml, P < 0.01).

In summary, resistance to the anti-platelet effect of clopidogrel (31.2%) appears to be more frequent than that due to aspirin (17.1%) in our patients. INNOVANCE PFA P2Y showed a high sensitivity for the detection of P2Y12 receptor blockade. Therefore, this new cartridge is a useful tool to rule out P2Y12 receptor inhibition. Its predictive value for risk assessment of thromboembolic events needs to be evaluated in clinical trials.

Funding

This work was supported by grants from the National Scientific Foundation of China (NSFC) (81472191), Jiangsu province Natural Science Foundation (BK20141203) , Jiangsu medical key talent (QNRC2016729) and Suzhou Natural Science Foundation (SYS201607) .