Summary. Microparticles have been prepared from human and animal plateletfree plasma by dilution of the plasma to lower the specific gravity followed by high‐speed ultracentrifugation. The protein, cholesterol, and phospholipid content (and cholesterol‐phospholipid ratios) of these microparticles have been determined and compared with values obtained with whole platelet homogenates and various isolated platelet subcellular fractions. Immunological investigations on the microparticles and on the density gradient fractions of platelet homogenates suggest that the particles may have their origin either in the surface membrane or within the intracellular membrane structures of the blood platelets. The microparticles have an associated ATP‐ase activity which, though lower in specific activity than isolated platelet ‘contractile protein’ (thrombosthenin), has similar characteristics in response to divalent cations, mersalyl and ouabain. Thrombosthenin antisera and antisera to the microparticles both inhibit clot retraction in vitro at high dilution and also inhibit the Mg2+ ATP‐ase activity of platelet homogenates and platelet thrombosthenin. When tested against platelet subcellular fractions from sucrose density gradients both these antisera showed precipitating antibodies to the low density membrane fraction. This fraction consists almost entirely of small vesiculated and larger sheet membrane fragments. In ultrastructure, the microparticles resemble the fine granular material present in the interior of thin‐walled sacs which may often be seen in electron‐micrographs prepared from fresh platelets. These sacs are believed to be the result of either budding of the pseudopodia or herniation of the cytoplasmic contents through the platelet wall.

Evidence has accumulated in support of a role for intracellularly generated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in raising cytosol [Ca2+] when various hormones, neurotransmitters, growth factors and other stimulants act on cell surfaces. The increase in [Ca2+] that follows stimulant-receptor interaction is accompanied by rapid hydrolysis of phosphoinositides. One product, Ins(1,4,5)P3, arising from the breakdown of phosphatidylinositol 4,5-bisphosphate was shown to promote the release of Ca2+ from non-mitochondrial stores in a variety of cells. Although platelet intracellular membranes have been implicated in the control of cytosol [Ca2+] and we previously characterized a Ca2+-sequestering mechanism associated with them, we have as yet no knowledge of how this Ca2+ store is mobilized after a stimulus-receptor interaction at the platelet surface. Using free-flow electrophoresis, we isolated and purified human platelet intracellular membranes. They show high enrichment and exclusive localization of the endoplasmic-reticulum marker NADH:cytochrome c reductase, and they sequester Ca2+ by an ATP-dependent process, reaching steady-state values in 10-12 min. Saturation with Ca2+ occurs at around 10-30 microM external Ca2+. When Ins(1,4,5)P3 is added to the 45Ca-loaded vesicles, a rapid release of Ca2+ occurs (approx. 35% in 15-30s). The magnitude of the release depends upon external [Ca2+], being maximum in the range 0.3-0.8 microM and low at external [Ca2+] greater than 1 microM. After release there is a rapid re-uptake of Ca2+, with restoration of the former steady-state values within 1 min. Half-maximal release occurs at approx. 0.25 microM-Ins(1,4,5)P3. This release and re-uptake pattern is not observed with ionophore A23187 or arachidonic acid, both of which liberate Ca2+ irreversibly. Inositol 1,4-bisphosphate was ineffective in releasing Ca2+ from these intracellular membranes. The results support the role of Ins(1,4,5)P3 as a specific intracellular mediator, transducing the action of excitatory agonists acting on the platelet surface into metabolic, mechanochemical and other functional events, known to occur during platelet activation.

By using density-gradient fractionation and high-voltage free-flow electrophoresis, human platelet membranes were separated into highly purified subfractions of surface (SM) and intracellular (IM) origin. Associated exclusively with the IM fraction is an ATP-dependent Ca2 + uptake that, in the absence of oxalate, reaches steady-state levels in 5-10min. When Ca2 +-EGTA buffers were used to control the external Ca2 + concentrations (range 0.1-50 .sM) there was an increase in the intravesicle steady-state level of Ca2 + up to 1OpM external Ca2 + concentration.Above this level the intravesicle space becomes saturated at a concentration between 10 and 20nmol of Ca2 +. (mg of protein)-'. The ionophore A23187 promotes a rapid and almost total release of the sequestered Ca2 + (> 90%, ti 1-2 min). The presence of oxalate in the external medium greatly enhances the Ca2 + accumulation to levels as high as 200 nmol-(mg of protein)-', but the uptake process is more variable and rarely reaches steady-state level even after 2h incubation. Moreover, accumulation in the presence of oxalate effects ionophore release with less than 80% depletion in 45-60min. These findings, taken together with the known presence in the platelet of a wide variety of functional and metabolic processes triggered by this cation, suggest that the platelet IM has a key role in controlling cytosolic Ca2+ concentrations.In a previous report we presented some evidence for an ATP-dependent Ca2 +-sequestering process associated with human platelet intracellular membranes. The membranes had been isolated from freshly sonicated platelets and purified free from surface-membrane elements by high-voltage free-flow electrophoresis . In order to isolate well-sealed and functionally competent vesicles, capable of accumulating Ca2 , fresh platelets were required, and the membrane vesicles also needed a short exposure to slightly hyperosmolar conditions during the isolation. In addition to ATP-dependence, the uptake of Ca2 + into these intracellular membrane In the present paper we have further characterized this Ca2 +-uptake property, particularly with respect to the rate of uptake and the steadystate levels of intravesicular Ca2 + reached in the presence and absence of oxalate. We have also investigated the effect on the uptake process of a range of different concentrations of Ca2 + outside the vesicles and also the action of the calcium ionophore A23187 on the sequestered Ca2+ at steadystate levels.Other fairly recent papers from this group (Lagarde et al., 1981(Lagarde et al., , 1982Carey et al., 1982) reported studies that have established that platelet intracellular membranes are the predominant site for the phospholipid-modifying enzymes that liberate arachidonic acid and the synthetases for cyclic endoperoxide and thromboxane production. These enzymic data, together with the present report, support the concepts put forward by Gerrard and his colleagues (Gerrard et al., 1982) that the internal membrane complexes involved in

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

Objectives-To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation ofneutrophils. Methods-Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure ofoxidative activity using 10' mom N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10 moUl platelet activating factor (PAF) as the priming agent.