Flow Cytometric Detection of Platelet Activation

Flow cytometric measurement of P-selectin on platelet surfaces, indicating degranulation of α-granules, was used as an indicator of platelet activation, as we have previously described (11). In brief, after 10 min of exposure to virus or activation controls, platelets were incubated with an antibody against P-selectin conjugated to Alexa-647 or allophycocyanin (33.3 ng/mL final concentration, clone Psel.KO.2.7, Novus Biologicals, Littleton, CO, USA) for 10 min, followed by quench dilution (400 μL) in flow buffer. The samples were then analyzed with a flow cytometer (FACSCalibur™, BD Biosciences, Franklin Lakes, NJ, USA), gating platelets on their characteristic forward and side scatter. The percentage of P-selectin-positive platelets was then determined from histogram plots of the gated platelets (Figure ​(Figure1A),1A), using commercial software (FlowJo v10, FlowJo LLC, Ashland, OR, USA).

Unfractionated heparin (UFH) inhibits EHV-1-induced platelet activation, as measured by P-selectin expression with flow cytometry. (A) Platelets were gated (R1) in a forward versus side scatter plot (left panel) and the percentage of P-selectin-positive cells in the gated region was measured (marker or M1 region) using histogram plots of P-selectin fluorescence (right panel). In the absence of UFH, thrombin (0.15 U/mL) and both RacL11 and Ab4 strains of EHV-1 at 1 PFU/cell activated platelets. Activation was evident as lengthening and narrowing of the platelet event cloud, with some microvesiculation (arrows) and >90% platelets expressing P-selectin. (B) In the presence of 2 U/mL UFH, inhibition of platelet activation is seen in forward versus side scatter (left panel) and histogram plots of P-selectin expression (right panel) in gated platelets (R1) exposed to thrombin and both viruses. No changes are seen in the PBS control with or without UFH. Residual small events (<10¹ forward scatter units) in the virus-exposed samples in the presence of UFH likely represent aggregated virus particles. Images in (A,B) are representative of one horse. Note that EHV-1-induced activation in this horse was not completely abolished in the presence of heparin, possibly due to mild “preactivation” of platelets (4% P-selectin expression in PBS control). (C) Quantification of the median percentage of P-selectin-positive platelets (columns), with superimposed individual data points, in response to thrombin (light gray columns) or both strains of EHV-1 (RacL11, dark gray columns; Ab4, black columns) in the absence or presence of 2 U/mL UFH (n = 6–7). *Numbers shown are P values (Wilcoxon matched pairs sign rank). (D) A heparin dose titration curve showed consistent inhibition of thrombin (light gray columns) and EHV-1-induced platelet activation at 0.05 U/mL (RacL11, dark gray columns; Ab4, black columns; n = 3–10). Columns represent medians with superimposed individual data points.