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Summary. Investigations of the haemostatic functions in three patients with the Wiskott‐Aldrich syndrome are presented. All patients had severe thrombocytopenia and prolonged bleeding times. The platelets had abnormal morphology with reduced size and variations of shape. Electron microscopy revealed ultrastructural abnormalities with a reduced number of organelles, and many of the platelets contained large numbers of tubules. Platelet electrophoretic mobility in citrated plasma was not reduced by collagen, and platelet aggregation by collagen and ADP was deficient. Biochemical studies revealed a lack of the storage pool of adenine nucleotides. Platelet adhesiveness in vitro in whole blood was reduced. Platelet factor‐3 release by kaolin, ADP and freezing and thawing was normal in one and reduced in another of the patients. Platelet survival studies showed a normal survival time of normal donor platelets in one patient, while autologous platelets had a markedly reduced survival time in two of the patients. The bone marrow contained normal numbers of megakaryocytes. By electron microscopy of the bone marrow, blood platelets were found to be phagocytosed by macrophages and reticulum cells. The main cause of the thrombocytopenia is most probably incrcased peripheral destruction of platelets. It is suggested that the qualitatively defective platelets are recognized in the reticulo‐endothelial system as foreign particles and phagocytosed.

Cat, cattle, dog, horse, human, mink, pig, and rabbit platelets were separated from plasma by gel filtration. The gel-filtered platelets (GFP) were treated with thrombin to induce maximal granule secretion and the potential dense granule constituents ATP, ADP, serotonin (5-HT), Ca2+, and Mg2+ were measured in GFP and in the control and thrombin-treated platelets and in the respective supernatants. The amount of Ca2+, Mg2+, 5-HT, ATP, and ADP within the nonreleasable pool for all species varied between 3.1 and 10.0 mumol/10(11) platelets for Ca2+ and Mg2+ was less than 1.5 mumol/10(11) platelets for ADP and 5-HT and was between 2.0 and 5.0 mumol/10(11) platelets for ATP. Marked differences were observed in the releasable fraction. Human platelets were characterized by the largest releasable Ca2+ pool (greater than 10 mumol/10(11) platelets), the smallest secretable 5-HT and Mg2+ pool (less than 0.5 mumol/10(11) platelets), and the lowest ATP-to-ADP ratio (greater than 1.0). Pig platelets had the highest amount of releasable Mg2+ (approximately 8.0 mumol/10(11) platelets). Rabbits platelets released the most 5-HT (greater than 3.0 mumol/10(11)) and had the highest ATP/ADP (greater than 5.0). The releasable pool of Ca2+, Mg2+, ATP, and ADP in the remaining species varied in mumol/10(11) platelets from approximately 1.5-4.0, approximately 1.0-3.0, 0.5-3.5, and approximately 0.5-1.5, respectively.

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine ␣-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMRmonitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent ␣-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of ␣-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.

SUMMARYWe earlier reported that neomycin blocked reversibly the binding of herpes simplex virus type 1 (HSV-1) to the receptor of BHK cells, while the binding of HSV-2 to the receptor was unaffected. We could not determine whether the effect was on the virus particle, the receptor, or both. We have now tested several other cationic substances, and report that polylysine (and polyarginine) block the binding of HSV-1 to the receptor by interfering with the cellular receptor function; higher molecular weight polylysines were more potent than those of lower molecular weight. Polylysine and neomycin showed additive effects. In vitro, polylysine showed the same strong binding to the plasma membrane phosphoinositides as did neomycin. Together these data suggest that the drugs may have a common target in the cell membrane. The HSV-1 and HSV-2 virus particles were unaffected by the drugs, as was the cellular HSV-2 receptor.

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About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 pmol/lO" cells of this F-actin-ADP complex compared to the total actin content of 0.8 pmol/1O1' cells, which is consistent with the view that actin is maintained in different pools (F-actin -ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 "C of 0.1 0.025 s-by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATPconsuming process that would account for up to 50% of total ATP consumption in resting platelets.Platelets contain two pools of adenine nucleotides: the cytosolic or metabolic pool (about 40% of total) and the granule pool (6O%), which can be secreted from the cells when they are properly stimulated; the cytosolic pool can be easily distinguished from the granule pool since it is specifically labelled when platelets are incubated with radioactive adenine or adenosine [l]. Extraction of platelet nucleotides with ethanol has demonstrated the presence of a third compartment: a small fraction of the total adenine nucleotides (about 3%) is retained by the insoluble pellet when platelets are extracted with ethanol; the nucleotides can be extracted from the pellet with HC104 and consist exclusively of metabolic ADP [2, 31. French and Wachowicz [4] extracted the material that contained platelet ethanol-insoluble ADP (ADP-protein complex precipitated by 43% ethanol) and demonstrated that this ADP was bound to a protein which eluted in the void volume of a Sephadex G-200 column. Based on this observation and the solubility properties of the material they suggested that the ethanol-insoluble ADP was bound to platelet actomyosin.We have previously shown that the ethanol-insoluble, protein-bound ADP has the same specific radioactivity as the ethanol-soluble (cytoplasmic) adenine nucleotides in platelets prelabelled with radioactive adenine [5]. These results suggested that exchange between the protein-bound and cytosolic pools reaches equilibrium in a period of less than 30 min. In this communication we have identified F-actin as Note. The term 'ethanol-insoluble ADP' is used here to refer to the ADP-protein complex that is precipitated by 43% ethanol. the protein that binds ethanol-insoluble ADP, and provide evidence that this actin-bound ADP exchanges very rapidly, thus accounting for upto 50% of the total ATP consumption in unstimulated platelets. MATERIALS AND METHODS MaterialsAll reagents were analytical grade or better, and deionized ...