Flow‐cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets

Abstract

Occlusive vascular diseases are promoted by a “prethrombotic state” with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow‐cytometric bio‐assay and the related change in the actin status by using the DNase‐I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F‐actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein “actin” and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow‐cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.

Effects of efonidipine on platelet and monocyte activation markers in hypertensive patients with and without type 2 diabetes mellitus

“…Circulating levels of CD62P-and CD63-positive platelets have recently been shown to be elevated in patients with coronary artery disease or diabetes, 43 and our findings agree with those reports. The present findings with respect to PDMPs, MDMPs, CD62P, CD63, PAC-1, and annexin V also suggested that enhanced platelet activity and related procoagulant activity may induce hypercoagulability in hypertension patients with diabetes.…”

Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus

“…Flow cytometric analysis of platelet CD62P and CD63 concentrations has been standardized [31,44]. With this technique, it has been shown that circulating concentrations of CD62P-and CD63-positive platelets are higher in patients with coronary artery disease and diabetes [44]. Our findings are consistent with those of these previous reports.…”

“…Quantitative increases in glycoproteins after platelet activation were not observed in GPIIb/IIIa or GPIb. Flow cytometric analysis of platelet CD62P and CD63 concentrations has been standardized [31,44]. With this technique, it has been shown that circulating concentrations of CD62P-and CD63-positive platelets are higher in patients with coronary artery disease and diabetes [44].…”

Significance of Platelet-Derived Microparticles and Activated Platelets in Diabetic Nephropathy

“…CD63 is a platelet lysosomal membrane protein, and is rapidly redistributed to the cell surface following platelet activation [34, 35, 36]. Flow-cytometric analysis of platelet CD62P and CD63 levels has already been standardized [12, 37]. With this technique, it has been directly demonstrated that elevated levels of CD62P- and CD63-positive platelets circulate in patients with coronary artery disease or diabetes patients [38].…”