We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.

We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.

Human platelets were incubated with gold particles coupled to fibrinogen to label the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor after initial activation of the cells by contact with formvar-coated grid and glass surfaces. Fibrinogen-gold (Fgn-Au) markers were absent on discoid platelets, but diffusely spread over the surface and extended pseudopods of early dendritic cells. Conversion to spread platelets resulted in movement of ligand-receptor complexes away from the cell margin toward cell centres. However, Fgn-Au gold did not concentrate in the central region. Rather, the Fgn-Au, GPIIb-IIIa complexes in the middle of spread platelets appeared to move toward a belt-like, intermediate zone, as did the ligand receptor complexes from the cell margin and pseudopods. The ultimate destination of the mobile receptor-ligand complexes, however, appeared to be channels of the surface-connected open canalicular system (OCS). Fgn-Au was concentrated in OCS channels of most dendritic and a small proportion of spread platelets. The decreased frequency of Fgn-Au filled channels in more transformed platelets may have been due to collapse or evagination of the OCS. Examination of platelets exposed to Fgn-Au after spreading on glass and then prepared for thin sections confirmed that the OCS was the final destination for mobile ligand receptor complexes on surface-activated platelets. Findings of this study are consistent with previous work showing clearance of mobile receptor-ligand complexes to the OCS of platelets activated in suspension.

The fine structure and ultrastructure of the anterior descending coronary artery were studied in a series of perinatal piglets at 1 week prior to birth, and at 8, 24, 72 and 168 h after birth. In the anterior descending coronary artery, at or just distal to the branch point of the left circumflex artery, early plaque-like intimal lesions were present in the majority of animals. These consisted of subendothelial edema, fragmentation and dissolution of the internal elastic lamella, and the appearance of intimal myoid cells known as modified smooth muscle cells (MSMCs). These changes were present in all piglets at and before 8 h of age. They persisted and progressed during the first week of life in about half of the piglets. Beginning at 72 h and continuing through 168 h, there was an increase in MSMCs and the appearance of fibroblasts. Both fibroblasts and MSMCs were associated with the elaboration of dense collagen fibrils. Foamy macrophages appeared within the subendothelial intima having the appearance of lipophages. While the prevalence of these changes at birth indicates that they may be part of normal development, their persistence in half the piglets and structural features suggest reaction to intimal injury beginning prenatally. The lesions may be precursive of coronary atherosclerosis later in life and may parallel the early stages of atherogenesis in humans.

Activated platelets rapidly internalize ligand-alpha(IIb)beta 3 complexes and particulates. The platelets sequester these primarily to the surface-connected canalicular system (SCCS), alpha-granules or evaginated surfaces. This occurs swiftly as activation initiates shape change, internal transformation, and secretion out of the platelet SCCS. The transmembrane signaling mechanism for internalization is through the platelet submembranous, detergent-resistant, cytochalasin B-resistant cytoskeletal filaments, not cytoplasmic actin filaments. This work proposes the model that platelet internalization responses contribute in the retraction of clots and participate in vivo in thrombus development at sites of coronary artery injury. In isometric clot retraction, the retracting platelets show internalization of fibrinogen-gold and fibrin while spreading on the polymerizing fibrin strands. The combination of platelet internalization (retracting) with evagination (spreading) on fibrin strands appear to approximate and organize nearby platelets into the developing thrombi. Ex vivo findings are similar under flowing conditions on denuded rabbit aortic endothelium with fibrinogen-gold markers. Early thrombi developing in vivo at sites of congenital coronary artery lesions in perinatal piglets show fibrin strands internalized into the SCCS of platelets. A thrombogenesis model is proposed that shows that platelet internalization responses anchor fibrin and approximate platelets, thus enhancing thrombus development and retraction.