Comparison of two platelet activation markers using flow cytometry after in vitro shear stress exposure of whole human blood.

Platelet activation is the initiating step to thromboembolic complications in blood-contacting medical devices. Currently, there are no widely accepted testing protocols or relevant metrics to assess platelet activation during the in vitro evaluation of new medical devices. In this article, two commonly used platelet activation marker antibodies, CD62P (platelet surface P-selectin) and PAC1 (activated GP IIb/IIIa), were evaluated using flow cytometry. Anticoagulant citrate dextrose solution A (ACDA) and heparin anticoagulated human blood from healthy donors were separately exposed to shear stresses of 0, 10, 15, and 20 Pa for 120 s using a cone-plate rheometer model, and immediately mixed with the platelet marker antibodies for analysis. To monitor for changes in platelet reactivity between donors and over time, blood samples were also evaluated after exposure to 0, 2, and 20 µM of adenosine diphosphate (ADP). Following ADP stimulation, the percentage of both CD62P and PAC1 positive platelets increased in a dose dependent fashion, even 8 h after the blood was collected. After shear stress stimulation, both CD62P and PAC1 positive platelets increased significantly at shear stress levels of 15 and 20 Pa when ACDA was used as the anticoagulant. However, for heparinized blood, the PAC1 positive platelets decreased with increasing shear stress, while the CD62P positive platelets increased. Besides the anticoagulant effect, the platelet staining buffer also impacted PAC1 response, but had little effect on CD62P positive platelets. These data suggest that CD62P is a more reliable marker compared with PAC1 for measuring shear-dependent platelet activation and it has the potential for use during in vitro medical device testing.