thromboplastin and calpeptin

Vesicles shed by U87-MG cells contain coxsackievirus and adenovirus receptor (CAR) protein that has been posttranslationally modified. Relative to full-length CAR, migration of the vesicle-associated soluble CAR antigen (CARd6) on SDS-polyacrylamide gels indicated a loss of approximately 6 kDa. HeLa and END-HHV6 cells also shed a similar vesicle-associated CAR protein. Vesicles shed by U87-MG cells following stimulation with calcium and A23187 contained CARd6 similar to that present in vesicles shed constitutively. RD cells transfected to express full-length CAR produced CARd6, but cells that expressed CAR with a truncated cytoplasmic domain produced no equivalent to CARd6. In U87-MG cells, calpain activity was required for release of CARd6 with shed vesicles, and accumulation of CARd6 in cells that rounded up and released from the plastic substrate in response to A23187 treatment was blocked by N-ethylmaleimide. These experiments show that CAR, posttranslationally modified in the cytoplasmic domain, can be released with vesicles shed by cells. Posttranslational modification of the CAR cytoplasmic domain occurs during cell rounding and release from the culture substrate. This modified, vesicle-associated CAR was the principal form of soluble CAR released by the cells.

Topics: Adenoviridae; Calcimycin; Cell Adhesion; Cell Line, Tumor; Culture Media; Cytoplasmic Vesicles; Dipeptides; Endothelial Cells; Enterovirus; Glioblastoma; HeLa Cells; Humans; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptors, Virus; Thromboplastin; Umbilical Veins

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Topics: Blood Coagulation; Blood Platelets; Calcimycin; Calpain; Cell Membrane; Collagen; Dipeptides; Flow Cytometry; Humans; Microscopy, Electron, Scanning; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Thrombin; Thromboplastin; von Willebrand Factor