Gel filtered human platelets contaminated with less than 0.02% of plasma protein S contained 490 ng of protein S antigen per 3 X 10(8) platelets, equivalent to 2.5% of protein S in whole blood. Three patients with heterozygous plasma protein S deficiency, a congenital disorder associated with venous thrombotic disease, had platelet protein S antigen levels that were 40% of the mean platelet level in ten normal volunteers. In immunoblotting analysis, platelet protein S was indistinguishable from plasma protein S. Thrombin stimulation of platelets caused release of 63% of total protein S antigen and this release was abolished when platelets were preincubated with metabolic inhibitors. Thrombin effected limited proteolysis of platelet protein S and this reaction was inhibited by calcium ions. Immunofluorescent staining of platelets using protein S antibodies demonstrated that protein S colocalized with fibrinogen, an established alpha-granule protein. Thus, human platelets contain protein S in alpha granules that can be released by thrombin stimulation. The released protein S may bind to stimulated platelets and thereby promote and localize the anticoagulant activity of activated protein C on the platelet surface.

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.

The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.