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Subcellular fractionation of human platelets labeled with radioactive sodium chromate was utilized to study distribution and characterization of the 51Cr binding sites in these cells. The cytoplasmic fraction of the platelets contained the major portion of the radioactivity while microsomal and mitochondrial fractions played a minor role in the binding of chromate. The stromal sediment had approximately one third of the total radioactivity. Only about 20 per cent of the 51Cr taken up by the platelets was in a free ionic form and was in the cytoplasmic fraction. The major portion of the 51Cr in the platelets was bound to nucleotides in the platelet cytoplasm. Nucleotides of the α-granules had no radioactive chromate. Hypertonic media and platelet antiserum produced marked release of 51Cr from the platelets, whereas aggregating agents produced none. In physiologic conditions, elution of 51Cr from labeled platelets is very limited because there is only little 51Cr in free ionic form in the cells. Release of considerable amount of 51Cr from the platelets can occur only in case of cell damage with release of nucleotides from the cytoplasmic pool. This was seen with hypertonic solutions and with platelet antibody. Platelet degranulation, as it occurs with aggregating agents, is not associated with release of 51Cr since the granular pool of nucleotides is not labeled by this compound.

Summary. The lowest concentration of ADP and adrenaline which resulted in a secondary wave of platelet aggregation at 37°C was determined in 60 normal subjects. The range of such ‘threshold’ concentrations was 0.2–1.4μM for ADP and 0.1–1.0μM for adrenaline. The mean threshold concentration of each reagent was unrelated to age but was higher for men than for women; this difference disappeared when the results were corrected for the effect of differences in PCV on the citrate concentration in platelet-rich plasma. The variation between separate determinations of threshold concentration in the same individual was of the same order as that between individuals; no evidence was found that the threshold concentration of either reagent was related to the phase of the menstrual cycle in four young women tested repeatedly. There was a highly significant correlation between the threshold concentrations of the two reagents in individual subjects. No correlation was found between the threshold concentration of either reagent and the total platelet ATP, ADP or ATP: ADP ratio. Concentrations of adrenaline at or slightly above the threshold caused the release of up to about 50% of the platelet ADP and 25% of the ATP during the second phase of aggregation, while lower concentrations produced no significant release. The ultrastructural changes during the first phase of aggregation were similar with all concentrations of ADP tested; threshold concentrations and above caused dense packing of platelets during the second phase, with the formation of giant pseudopodia and no tendency to disaggregation during the period of observation, while at lower concentrations disaggregation was accompanied by a retraction of pseudopodia. There was no clear evidence of platelet degranulation during either first or second phase aggregation.

Electron microscopy has confirmed previous studies and has provided much new information on the mechanism of endotoxin-platelet interaction. The Boivin lipopolysaccharide preparation is particulate, and on electron microscope examination appears as a three-layered structure, morphologically similar to bacterial cell wall. In vitro and in vivo experiments have demonstrated that these endotoxin particles adhere to platelets. In some species, particularly the rabbit, this is associated with loss of platelet contents, due to lysosomal and cell membrane lysis, resulting in platelet sphering and apparent cell death. Serial observation of degranulating platelets and metabolic studies indicate that although some platelet engulfment of endotoxin occurs, degranulation is not dependent upon phagocytosis. Several observations suggest that these endotoxin effects are mediated through immune mechanisms: (1) Inactivation of complement in the suspending plasma by heating to 56°C, anticoagulation with EDTA, a reaction temperature of 5°C, ammonium hydroxide incubation, and adsorption with either zymosan or washed antigen-antibody complexes, inhibits both endotoxin adherence and platelet degranulation. (2) The reaction requires a plasma cofactor, possibly antibody, which can be adsorbed out by endotoxin. (3) Endotoxin adheres selectively to nonprimate platelets and primate red cells, a pattern conforming to immune adherence, a phenomenon requiring antigen, antibody, and complement. It is suggested that endotoxin-induced platelet damage is dependent upon the intimate contact provided by immune adherence. We have not established whether degranulation is an endotoxin or complement effect. The species variation in susceptibility to endotoxin also merits further investigation.

The ultrastructural changes and the kinetics of 14C-serotonin release during the adhesion-aggregation reaction of human platelets induced by Thrombofax® (Ortho Pharmaceuticals), a commercial cephalin prepared as an ether extract of acetone dried bovine brain, were analyzed. The initial phase of interaction of Thrombofax with platelets is characterized by the development of adhesion sites on normally shaped cells; thereafter, loss of discoid shape, pseudopod formation and inward movement of granules, surrounded by a circumferential bundle of microtubules, become apparent. Measurements of 14C-serotonin release show that these ultrastructural changes precede the release reaction. The later stages which are detectable turbidometrically and which only occur in stirred samples and in the absence of release inhibitors, are characterized mainly by large aggregate formation, platelet degranulation and strong release of 14-C-serotonin.

Compound 48/80 degranulates mast cells and release histamine and serotonin. Since platelet degranulation is an important aspect of platelet function, the effect of this compound on platelets and platelet aggregation were investigated by transmission and scanning electron microscopy. Low concentrations of 48/80 did not alter platelet ultrastructure but high concentrations suppressed platelet pseudopod formation and altered the surface connecting system. Low concentrations of 48/80 interfered with platelet aggregation by thrombin and collagen but not by ADP; platelets incubated with high concentrations of 48/80 did not aggregate when exposed to ADP or collagen but the addition of large quantities of thrombin overcame this inhibition and caused platelet aggregation as well as plasma clotting. The effect of thrombin on platelets exposed to 48/80 was not related to the presence of fibrin. The observations provide evidence that the platelet membrane plays an essential role in aggregation and that the mechanisms leading to platelet release and aggregation by various agents may be different.