Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry.
Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D019007:P-Selectin; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D013861:Thiocyanates; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate