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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.

Adhesion of platelets from 15 patients with von Willebrand's disease was tested in an ex vivo human umbilical vein model. Experiments employed umbilical veins still in their umbilical cords taken from patients undergoing cesarean section and platelets (fetal, adult and vW) either apyrase-washed or used as platelet-rich plasma or whole blood. F VIII R:Ag, F VIII:Rcof, and F VIII:C were measured in initial fresh plasma and in effluents from the umbilical vein segments. F VIII:Rcof increased in most perfusates. Binding of latex-linked specific antihuman F VIII R:Ag demonstrated that F VIII R:Ag existed on subendothelium and on injured endothelial cells. Scanning electron microscopy three-dimensionally displayed vW platelet--vessel-wall interactions. Although vW platelets adhered to injured vein, both qualitative and quantitative differences existed in comparison with adhesion of normal platelets. The differences correlated best with the plasma F VIII:Rcof level. The best adhesion shown by vW platelets was only 51 percent of the adhesion of control fetal or adult platelets. vW platelets had less surface activity, fewer pseudopods, and little ability to spread and pave the exposed subendothelium. Pretreatment of the vein with F VIII R:Ag antibody partly blocked adhesion. Coperfusion of cryoprecipitate with vW platelets improved their adhesivity, state of activation on subendothelium, and ability to form aggregates. ABO differences in blood cell types of fetal material and platelet donors seemed without effect, which further establishes this model's validity for studies of platelet dysfunction and platelet or endothelial reactive agents.

A nine-year-old black girl with essential thrombocythemia developed hemoptysis. Only two other cases in the English literature have been described. Ultrastructure and functional characteristics of this patient's platelets were studied. Twenty-six percent of the patient's platelets were very large (megathrombocytes). Spontaneous aggregated from the patient's platelets were not compact, and the pseudopods did not interdigitate. Both qualitative and quantitative defects in platelet organelles were detected. The microtubular system was faulty in organization. Furthermore, the number of granules (especially alpha granules) was reduced. Platelet aggregation studies demonstrated subnormal aggregation in response to ADP, epinephrine, and collagen, but aggregation with ristocetin was normal. It is postulated that a platelet membrane abnormality may be the cause of their defective platelet aggregation.

Gel filtered human platelets were mixed with polylysine coated polyacrylamide beads. Platelet binding to individual beads within one preparation was variable, yet high and the bound platelets appeared to involve non-activated platelets. Some cells appeared to be in contact with more than one bead. When the attachment process was neutralized with 1 mg/ml of polyanionic dextran sulfate, significantly fewer platelets remained attached to the beads. Platelets which were recovered in the washes also were non-activated. Attached cells were capable of being activated and underwent release reactions when exposed to either ADP or thrombin. Release reactions were monitored by the liberation of either serotonin or beta-thromboglobulin. Platelets which were iodinated prior to attachment gave evidence that the failure to neutralize the bead before vortexing resulted in lower specific activities of bound iodine/protein.

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and beta-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and beta-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG1. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.

The effects of thrombin stimulation on megakaryocytopoiesis and pulmonary-platelet interactions were investigated before and after administration of the compound to 15 mongrel dogs. Each dog served as its own control. Thrombin was given to encourage the traffic of megakaryocytes into the lung and to study the thrombin-stimulated effects on megakaryocytopoiesis in the bone marrow. Our results showed that thrombin increased the numbers of bone marrow cells in general and megakaryocytes (MK) in particular. In addition, the maturation cycle of megakaryocytes was accelerated and the number of MK migrating into the central venous circulation was nearly doubled. Most of the circulating MK ultimately became sequestered in pulmonary capillaries, where platelets were shed into the arterial circulation. We conclude that thrombin has a major stimulatory effect on megakaryocytopoiesis in the bone marrow and that the lung plays an important role as a vascular filter and regulator of circulating platelet count.

This article summarizes recent ultrastructure findings from our laboratory and documents some of the information accumulated primarily since 1975 from many laboratories. Special attention is given to documentation by scanning electron microscopy which affords insight into platelet activation (adhesion, aggregation, release/secretion) and especially platelet-vessel wall interactions. Structural physiology of platelets is considered in some detail as a basis for understanding platelet disorders contributing to clinical problems of thrombosis and hemorrhage. The impaired ability of von Willebrand platelets to adhere to injured vessel wall is reported using the human umbilical vein perfusion model. Relationships between platelets and blood coagulation factors focus on the exquisite sensitivity of platelets to minute amounts of thrombin. Unmasking of platelet factor 3 sites is identified on activated platelets, after glutaraldehyde fixation, by their reaction ot latex bearing anti-platelet factor 3 markers. The basis for platelet-collagen interactions is reviewed. Conditions for and possible mechanisms behind platelet interaction with vessel wall are discussed. Ex vivo flowing blood-vessel wall models offer opportunities for improved understanding of the platelets role(s) in vascular diseases.