Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation

Abstract

To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000- mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double- labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28- positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.

Multiple platelet defects identified by flow cytometry at diagnosis in acute myeloid leukaemia

“…The binding of PAC-1 antibody is competitively inhibited by Arg-Gly-Asp-Ser (RGDS peptide) (SIGMA), which was used to demonstrate specific PAC-1 binding (Shattil et al, 1985). Anti-CD63 PE (CLB-gran12; Immunotech) is directed against granulophysin (gp53), a membrane protein of lysosomes and dense granules exposed on the external membrane upon platelet stimulation and degranulation (Nieuwenhuis et al, 1987;Israels et al, 1992). Isotype-and fluorochrome-matched controls and RGDS were included in the analyses.…”

Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen‐activated and thrombin‐activated platelets

“…Exposure of the neo-epitopes CD62 (P-selectin) and CD63 (Glycoprotein 53). The thrombin-induced expression of the alpha-granule marker CD62 and the lysosomal/dense granule marker CD63 [33] increased in the presence of the autocrine inhibitors, particularly for CD62 (Table 1). We believe that this is not due to stimulation of the intracellular mechanisms governing neo-epitope exposure.…”

Large platelets continue to circulate in an activated state after myocardial infarction

“…2.17 reacts with the CD62-related antigen, a 140 kD component of the internal alpha granule membrane. These monoclonal antibodies react specifically with activated platelets [18,19,20].…”