Flow Cytometric Detection of Activated Platelets: Comparison of Determining Shape Change, Fibrinogen Binding, and P-Selectin Expression

Abstract

Platelet activation plays an important role in the pathomechanisms of arterial vascular disorders including stroke, peripheral arterial disease (PAD), and myocardial infarction. Circulating activated platelets may be useful markers of local thrombotic events occurring in these diseases. Using flow cytometry circulating activated platelets can be detected by determining: 1. the platelets' shape change on the basis of the different light scatter properties of discocytes and spherocytes, 2. the expression of platelet bound fibrinogen or conformation specific neoantigens on fibrinogen and on its platelet receptor, and 3. the exposure of granule membrane proteins such as P-selectin as a result of platelet secretion. The concentration-effect relationships were determined for the ADP and U46619 induced shape change, fibrinogen binding, and expression of P-selectin. The EC50 for the shape change was 4 times lower than the EC50 for the fibrinogen binding and 29 times lower than the EC50 for the expression of P-selectin. These data clearly demonstrate that the shape change is a more sensitive indicator of platelet activation in vitro than fibrinogen binding or P-selectin expression. Both the shape change and fibrinogen binding were reversible, whereas the expression of P-selectin was irreversible upon stimulation. Reversibility of the shape change may be responsible for the fact that in patients with stroke or PAD the fraction of discocytes did not differ from controls, whereas more than 80% of them revealed a significantly higher fraction of P-selectin positive platelets. Thus the determination of the P-selectin expression reveals a higher diagnostic sensitivity for detecting a platelet activation in vivo than the determination of the shape change.

Course of Platelet Activation Markers After Ischemic Stroke

“…Peripheral venous blood was collected into an aldehyde-based fixation solution that blocks metabolic processes within milliseconds. 5,16 This fixation process was shown to preserve CD62p and CD63 for at least 24 hours at room temperature. The fraction of circulating platelets that express the activation markers p-selectin and CD63 was determined by flow cytometry as described previously.…”

“…The fraction of circulating platelets that express the activation markers p-selectin and CD63 was determined by flow cytometry as described previously. 5,16 Briefly, the samples were incubated with saturating concentrations of fluorescein isothiocyanate conjugated (FITC)-labeled antibodies (Immunotech) against p-selectin (Clone CLBThromb6) or CD63 (Clone CLBGran12) and with R-phycoethrin (PE)-labeled antibodies against CD41a (clone P2) for 15 minutes at room temperature in the dark (double immunolabeling). The antibody clone P2 binds to the fibrinogen receptor (glycoprotein IIb/IIIa; CD41/CD61) of platelets independently of the activation state of the receptor.…”

The plaque lipid lysophosphatidic acid stimulates platelet activation and platelet-monocyte aggregate formation in whole blood: involvement of P2Y1 and P2Y12 receptors

“…Platelet shape change was determined by flow cytometry collecting 10 000 platelet events and measuring the ratio forward scatter/side scatter. 17 Values in control samples (0.18-0.19) were set to 100%.…”

Platelet P-Selectin Levels in Relation to Plasma Soluble P-Selectin and β-Thromboglobulin Levels in Atrial Fibrillation

“…[5][6][7] Although P-selectin has been identified on endothelial cells, it is becoming clear that the majority of, if not all, soluble P-selectin in the plasma (sP-selectin) arises from the platelet. 8 -10 Expression of P-selectin on the platelet surface on stimulation is an irreversible phenomenon, 11 with the main function being to mediate adhesion of activated platelets to monocytes and neutrophils. 12,13 While sP-selectin levels are quantified by enzyme-linked immunosorbent assay (ELISA), platelet membrane P-selectin requires detection and quantification with flow cytometry or whole platelet ELISA.…”