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The utility of a methods section of a research paper is often tempered by the brevity demanded by manuscript word limitations. Whilst word limits help streamline a paper, a Methods section often bears the brunt of the editorial scalpel, resulting in only brief sketches of experimental protocols and consignment of methodology to online supplementary information files. To retain a place for important detailed methodology, and to encapsulate and highlight new and existing important techniques for platelet and megakaryocyte biology, the Platelets Journal Editorial board now accept Methods manuscripts.

Although the female gender is generally less represented in cardiovascular studies, observational and randomized investigations suggest that-compared with men-women may obtain different benefits from antiplatelet therapy. Multiple factors, including hormonal mechanisms and differences in platelet biology, might contribute to such apparent gender peculiarities. The thrombotic and bleeding risks, as well as outcomes after a cardiovascular event, appear to differ between genders, partly in relation to differences in age, comorbidities and body size. Equally, the benefits of antiplatelet therapy may differ in women compared with men in different vascular beds, during primary or secondary prevention and according to the type of an antiplatelet agent used. This document is an attempt to bring together current evidence, clinical practices and gaps of knowledge on gender-specific platelet function and antiplatelet therapy. On the basis of the available data, we provide suggestions on current indications of antiplatelet therapy for cardiovascular prevention in women with different clinical features; no strong recommendation may be given because the available data derive from observational studies or post hoc/subgroup analyses of randomized studies without systematic adjustments for baseline risk profiles., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Background: Optimal treatment of acute ST-elevation myocardial infarction (STEMI) involves rapid diagnosis, and transfer to a cardiac centre capable of percutaneous coronary intervention (PCI) for immediate mechanical revascularisation. Successful treatment requires rapid return of perfusion to the myocardium achieved by thromboaspiration, passivation of the culprit lesion with stent scaffolding and systemic inhibition of thrombosis and platelet activation. A delicate balance exists between thrombosis and bleeding and consequently anti-thrombotic and antiplatelet treatment regimens continue to evolve. The desire to achieve reperfusion as soon as possible, in the setting of high platelet reactivity, requires potent and fast-acting anti-thrombotic/anti-platelet therapies. The associated bleeding risk may be minimised by use of short-acting anti-thrombotic intravenous agents. However, effective oral platelet inhibition is required to prevent recurrent thrombosis. The interaction between baseline platelet reactivity, timing of revascularisation and effective inhibition of thrombosis is yet to be formally investigated., Methods/design: We present a protocol for a prospective observational study in patients presenting with acute STEMI treated with primary PCI (PPCI) and receiving bolus/infusion bivalirudin and prasugrel therapy. The objective of this study is to describe variation in platelet reactivity, as measured by the multiplate platelet function analyser, at presentation, the end of the PPCI procedure and 1, 2, & 24 hours post-procedure. We intend to assess the prevalence of high residual platelet reactivity within 24 hours of PPCI in acute STEMI patients receiving prasugrel and bivalirudin. Additionally, we will investigate the association between high platelet reactivity before and after PPCI and the door-to-procedure completion time.This is a single centre study with a target sample size of 108 participants., Discussion: The baseline platelet reactivity on presentation with a STEMI may impact on the effect of acute anti-thrombotic and anti-platelet therapy and expose patients to a heightened risk of bleeding or ongoing thrombosis. This study will define the baseline variation in platelet reactivity in a population of patients experiencing acute STEMI and assess the pharmacodynamic response to combined treatment with bivalirudin and prasugrel. The data obtained from this trial will be hypothesis generating for future trials testing alternative pharmacotherapies in the acute phase of treatment for STEMI., Trial Registration: This study has approval from Wiltshire research ethics committee (10/H0106/87) and is registered with current controlled trials (http://www.controlled-trials.com/ISRCTN82257414).

The last decade has seen increasing use of advanced imaging techniques in platelet research. However, there has been a lag in the development of image analysis methods, leaving much of the information trapped in images. Herein, we present a robust analytical pipeline for finding and following individual platelets over time in growing thrombi. Our pipeline covers four steps: detection, tracking, estimation of tracking accuracy, and quantification of platelet metrics. We detect platelets using a deep learning network for image segmentation, which we validated with proofreading by multiple experts. We then track platelets using a standard particle tracking algorithm and validate the tracks with custom image sampling - essential when following platelets within a dense thrombus. We show that our pipeline is more accurate than previously described methods. To demonstrate the utility of our analytical platform, we use it to show that in vivo thrombus formation is much faster than that ex vivo . Furthermore, platelets in vivo exhibit less passive movement in the direction of blood flow. Our tools are free and open source and written in the popular and user-friendly Python programming language. They empower researchers to accurately find and follow platelets in fluorescence microscopy experiments.

Human platelets can be fractionated into three main subcellular components: granules, membranes, and a soluble fraction. In this study we determined the phospholipid and neutral lipid content of the granules and membranes. Quantitative relationships between lipids and protein were examined. The fatty acid and aldehyde composition of individual phospholipids and neutral lipids was also determined. Whole platelets had a lower lipid to protein ratio than did the subcellular particles, but the basic lipid composition of the granules, membranes, and platelets was similar. The phospholipid composition of platelets and subcellular fractions was found to differ only in that granules had a lower percentage of lecithin. Each of the phospholipid classes displayed a distinctive fatty acid pattern which was the same in all fractions and in whole platelets. The major neutral lipid was free cholesterol. Cholesteryl esters, triglycerides, and free fatty acids were minor components. The molar ratio of cholesterol to phospholipid in the platelet membranes was lower than that of brain myelin and erythrocyte ghosts. Some differences in fatty acid composition of the neutral lipids of platelet fractions were found. A special lipid composition or constituent that would correlate with platelet function has not been found.

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.

Background: Determining the required daily number of platelet units in hospitals is a challenging task due to the high uncertainty in daily usage and short shelf life of platelets., Study Design and Methods: We developed a linear prediction model to guide the daily ordering quantity of platelet units at a hospital that orders the required units from a central supplier. The predictive model relies on historical demand data and other information from the hospital's information system. The ordering strategy is to place an order at the end of each day to bring the platelet inventory to the predicted demand for the next day. Unlike typical prediction models, the quality of the predictions is measured with respect to the resulting inventory costs of wastage and shortage. We used data from two hospitals in Hamilton, Ontario from 2015 to 2016 to train our model and evaluated its performance based on the resulting wastage and shortage rates in 2017., Results: In 2017, respectively 1915 and 4305 platelet units were transfused at the two hospitals, with daily average (SD) usage of 5.2 (3.7) and 11.8 (4.4). The expiry (estimated shortage) rates were 8.67% (13.86%), and 2.28% (8.48%) at the two hospitals, respectively. Our baseline model would have reduced the expiry (shortage) rates to 2.54% (4.01%) and 0.05% (0.44%) for the two hospitals, respectively., Discussion: Guiding daily ordering decisions for platelets using our proposed model could lead to a significant reduction of wastage and shortage rates at hospitals., (© 2022 AABB.)

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases and the nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A 2  leading to the release of thromboxane A 2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade., (© 2021 Wiley Periodicals LLC.)

Plain language summary: What is it?: Fetal neonatal alloimmune thrombocytopenia (FNAIT), also known as neonatal alloimmune thrombocytopenia (NAIT) or fetomaternal alloimmune thrombocytopenia (FMAIT), is a rare condition which affects a baby's platelets. This can put them at risk of problems with bleeding, particularly into the brain. One baby per week in the UK may be seriously affected and milder forms can affect one in every 1000 births. How is it caused?: Platelets are blood cells that are very important in helping blood to clot. All platelets have natural proteins on their surface called human platelet antigens (HPAs). In babies, half of these antigens are inherited from the mother and half from the father. During pregnancy, some of the baby's platelets can cross into the mother's bloodstream. In most cases, this does not cause a problem. But in cases of FNAIT, the mother's immune system does not recognise the baby's HPAs that were inherited from the father and develops antibodies, which can cross the placenta and attack the baby's platelets. These antibodies are called anti‐HPAs, and the commonest antibody implicated is anti‐HPA‐1a, but there are other rarer antibody types. If this happens, the baby's platelets may be destroyed causing their platelet count to fall dangerously low. If the platelet count is very low there is a risk to the baby of bleeding into their brain before they are born. This is very rare but if it happens it can have serious effects on the baby's health. How is it inherited?: A baby inherits half of their HPAs from its mother and half from its father. Consequently, a baby may have different HPAs from its mother. As the condition is very rare, and even if the baby is at risk of the condition we have no way of knowing how severely they will be affected, routine screening is not currently recommended. What can be done?: FNAIT is usually diagnosed if a previous baby has had a low platelet count. The parents are offered blood tests and the condition can be confirmed or ruled out. There are many other causes of low platelets in babies, which may also need to be tested for. As the condition is so rare, expertise is limited to specialist centres and normally a haematologist and fetal medicine doctor will perform and interpret the tests together. Fortunately, there is an effective treatment for the vast majority of cases called immunoglobulin, or IVIg. This 'blood product' is given intravenously through a drip every week to women at risk of the condition. It may be started from as early as 16 weeks in the next pregnancy, until birth, which would be offered at around 36–37 weeks. Less common treatments that may be considered depending on individual circumstances include steroid tablets or injections, or giving platelet transfusions to the baby. What does this paper tell you?: This paper considers the latest evidence in relation to treatment options in the management of pregnancies at risk of FNAIT. Specifically, we discuss the role of screening, when IVIg should be started, what dose should be used, and what evidence there is for maternal steroids. We also consider in very rare selected cases, the use of fetal blood sampling and giving platelet transfusions to the baby before birth. Finally, we consider the approaches to blood testing mothers to tell if babies are at risk, which is offered in some countries, and development of new treatments to reduce the risk of FNAIT. [ABSTRACT FROM AUTHOR]

This article is a commentary celebrating the 50th anniversary of Seminars in Thrombosis and Hemostasis (STH), a journal that first published in 1974. The commentary focuses on the historical significance of a paper titled "Description of an in vitro platelet function analyzer–PFA-100," which was published in a supplementary issue of STH in 1995 and is one of the most highly cited papers in the journal's history. The paper describes the PFA-100, an instrument that has since evolved into the PFA-200 and has been widely used for various purposes in the field of hemostasis. The commentary also mentions the impact of the PFA-100 on research and its potential applications in different areas, such as screening for von Willebrand disease and monitoring platelet function in animals. The article concludes by acknowledging the 30th anniversary of the PFA-100 in 2024 and its significant contributions to the hemostasis community. [Extracted from the article]

Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory and other cognitive functions. Protein Kinase Cε (PKCε) is an isoform that most effectively suppresses Amyloid Beta (Aβ) production and synaptic loss. Methods: In this study, spatial learning and memory for treated rats were evaluated by the Morris water maze test. The activity (total PKC), mRNA expression, and protein level of PKCε in the platelet and hippocampal tissue were evaluated using immunosorbent assay, real-time qPCR, and western blotting analysis, respectively. Results: The traveled distance was significantly prolonged, and escape latency significantly increased in Aβ-treated groups. PKC activity assay showed that there was a remarkable difference between the Aβ-treated and sham-operated groups on days 10 and 30 in the hippocampus and also day 30 in platelet after the injection of Aβ. A significant effect in PKC activity was observed between days 0 and 10, days 0 and 30, as well as days 5 and 30. Aβ significantly downregulated the PKCε mRNA expression in the hippocampus of rats on day 30; however, no significant difference was observed in platelet. Western blot analysis demonstrated that Aβ significantly reduced PKCε protein expression in the hippocampus of treated groups on day 30. Conclusion: The expression level of PKCε was downregulated following the injection of Aβ in the hippocampus, but no significant difference was observed between the AD and sham groups in platelet that may be due to the low concentration of PKCε or duration of Aβ exposure in the rat brain. [ABSTRACT FROM AUTHOR]

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.

When platelets take part in the formation of hemostatic plugs and thrombi in vivo, electron microscopic evidence indicates that some of the platelets not only release their granule contents but also undergo lysis. In the present study we have examined, in vitro, the relation between the release reaction of platelets and platelet lysis in response to the release-inducing agents thrombin and collagen. Release was measured by determining the amounts of 14C-serotonin and adenine nucleotides that appeared in the ambient fluid of prelabeled platelets. Lysis was measured by determining the amount of either lactate dehydrogenase or 14C-labeled cytoplasmic ATP from platelets incubated with -14C-adenosine. Washed platelets prepared from rabbit, pig, or human blood lost some lactate dehydrogenase and 14C-ATP upon exposure to thrombin, but the amounts of lactate dehydrogenase and 14C-ATP lost from rabbit platelets were much greater than from pig or human platelets. The reason for this species difference is not aparent. The platelet release reaction appeared to be necessary for lysis to occur. Reduction of the extent of the release reaction by preincubation of rabbit platelets with metabolic inhibitors to deplete metabolic ATP reduced the extent of lysis. In addition, it was apparent that the fall in platelet metabolic pool ATP caused by thrombin was not responsible for platelet lysis. Lack of calcium, addition of prostaglandin E(1), OR Increasing the albumin concentration of the suspending medium of rabbit platelets inhibited platelet lysis. These conditions may prevent the loss of material that causes lysis, inhibit the action of this lost material, or inhibit the lytic reaction. Release and lysis may occur together and release can occur without detectable lysis, but lysis in response to a release-inducing agent does not take place unless the release reaction occurs.

Platelets in patients with storage pool disease are markedly deficient in a nonmetabolic (storage) pool of ADP that is important in platelet aggregation. They are also deficient in ATP, although to a lesser degree. In seven patients with this disorder, including one with albinism, platelet 5-hydroxytryptamine (5-HT) levels were reduced in proportion to the reduction in ATP (r = 0.94). Their platelets show diminished capacity to absorb [(14)C]5-HT, and the type of defect was similar to that produced in normal platelets by reserpine, a drug known to inhibit the uptake of 5-HT by the platelet dense granules. Storage pool-deficient platelets also converted more [(3)H]5-HT to [(3)H]5-hydroxyindoleacetic acid than did normal platelets, and the platelets in one of two patients studied contained increased amounts of 5-HT metabolites. The above findings, together with those reported previously, support the conclusion that the capacity of the dense granules (which may be either diminished or functionally abnormal) for storing 5-HT is decreased in storage pool disease; as a result, the 5-HT that enters the platelet may be more exposed to monoamine oxidases present on mitochondrial membranes. This diminished storage capacity (for 5-HT) may also explain why preincubating platelet-rich plasma with 5-HT for 45 min without stirring inhibits subsequent platelet aggregation by 5-HT to a greater degree in patients with storage pool disease than in normal subjects. The latter finding is also consistent with the theory that the aggregation of platelets by 5-HT is mediated by the same receptors on the plasma membrane that are involved in its uptake. The diminished release of platelet-bound [(14)C]5-HT by collagen that we found in these patients, as well as findings in previous studies, suggests that the release reaction may also be abnormal in storage pool disease.

Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1-4 and 10-30 pmol/10(8) cells, respectively.

The ability of sodium fluoride (NaF) and thrombin to stimulate aggregation and protein phosphorylation in intact human platelets was measured and compared. When platelets were stimulated by NaF, phosphorylation of the 20 KDa protein was transient and after 5-10 min returned to the same level as that of unstimulated cells. On the other hand, 47 KDa protein was slowly phosphorylated without obvious dephosphorylation. The slow activation of the 47 KDa protein phosphorylation correlated well with the time required for the aggregation and secretion. Phosphoamino acid analysis showed that the phosphorylated amino acids of the 47 KDa protein from platelets activated by NaF and thrombin were slightly different. These results suggest that different stimuli may lead to the same protein phosphorylation by different biochemical mechanisms of action.

A low molecular weight antiplasmin has been detected in human blood platelets. The antiplasmin is dialysable and a similar material is present in normal plasma. A simple method for rapid isolation of the antiplasmin based on ultrafiltration is described. The inhibition of plasmin by these materials under different conditions has been studied. In the ultracentrifuge, both antiplasmins showed a single broad peak with an approximate sedimentation coefficient of 0.5S. Paper chromatography and paper electrophoresis indicated the isolated material to be heterogeneous. The individual components were isolated by paper chromatography and the antiplasmin activity was measured by the fibrin plate method. Preliminary studies on the fraction with maximal antiplasmin activity suggest that the inhibitory effect might be due to enzyme-inhibitor complex formation. Based on the present data, it is concluded that the platelet antiplasmin and the serum antiplasmin are very similar. This antiplasmin material may be useful in fibrinolytic therapy.

The primary structure of human platelet profilin was determined by aligning the sequences of its tryptic peptides to the previously determined calf spleen profilin sequence [(1979) FEBS Lett. 101, 161-165]. Comparison of the peptide fingerprints of the two proteins suggested a higher homology than that found by direct sequence comparison. We therefore reinvestigated the sequences of the peptides from calf spleen profilin. We identified four incorrect charge assignments and a deletion of three residues. The similarity between the two vertebrate profilins amounts to 95%.

The influence of freshly purified ATP on the effects of aggregating agents on human platelets was studied. ATP inhibited aggregation induced by ADP competitively (Ki = 20 muM) and immediately without need for prior incubation. ATP had no effect on primary aggregation induced by adrenaline, thrombin, vasopressin, or 5-hydroxytryptamine (5HT). ATP inhibited the shape change and the consumption of metabolic ATP induced by ADP but did not inhibit these effects when induced by thrombin, vasopressin, or 5HT. ATP counteracted the inhibition by ADP of PGE1-stimulated cyclic AMP production in platelets but did not reduce inhibition by adrenaline. It is concluded that adrenaline, thrombin, 5HT, and vasopressin each can induce primary aggregation of human platelets by a mechanism independent of extracellular ADP.

The addition of thrombin to human platelets prelabeled with 32Pi led to significant loss of radioactivity in phosphatidylinositol 4,5-bisphosphate within 5 s, followed by recovery or even increase by 2 min. Loss of label from phosphatidylinositol phosphate was much less marked. Stimulated loss of label from phosphatidylinositol was not seen, while labeled phosphatidate increased severalfold. The principal labeled water-soluble phosphates observed, in addition to 32Pi and [32P] ATP, co-migrated with inositol diphosphate and inositol triphosphate. This suggests that a pool of polyphosphoinositides is constantly undergoing phosphodiesteratic cleavage and resynthesis. Thrombin addition led to rapid increase in radioactivity in inositol triphosphate, but not in inositol diphosphate. We conclude that this early consequence of the thrombin-platelet interaction is the result of an increase in the phosphodiesteratic cleavage of phosphatidylinositol bisphosphate.

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.

Platelets are a rich source for the study of inositol lipids in man. The substitution of an EDTA-KCl solution for the water component of the Bligh and Dyer procedure permitted quantitative extraction of polyphosphoinositides. The latter, with monophosphoinositide, were found to comprise, on a molar basis, 6.7% of total platelet phospholipids. Study of the incorporation of orthophosphate-(32)P into platelet phospholipids was further simplified by separating eight (32)P-labeled lipids, including the inositides, with a single chromatographic development on formaldehyde-treated paper. Particular attention was paid to the influence of ionic environment on the pattern and degree of labeling. In 300 mOsm media major phospholipids other than the inositides were not labeled. Small amounts of label appeared in certain trace phospholipids, notably phosphatidic acid. In 150 mOsm media, labeling of inositides was moderately increased, that of trace phospholipids enormously so. The increased labeling was not solely due to thrombocytolysis since (a) platelet disruption by sonication or freeze-thawing abolished (32)P incorporation into phospholipids and (b) in timed studies, restoration of osmolarity to 300 mOsm by addition of hypertonic sorbitol blunted the enhancement effect of previous 150 mOsm exposure. Lowering K and compensatorily increasing Na concentration of 300 mOsm media also stimulated (32)P labeling of inositides and, to a lesser extent, the trace phospholipids. However, the pattern and degree of stimulation were not as strikingly altered as in the osmolarity studies. These data show that drastic alterations of ionic environment can sharply influence the platelet's ability to incorporate orthophosphate-(32)P into its phospholipids.

Human platelets contain diglyceride kinase, an enzyme that catalyzes the phosphorylation of diacylglycerol by adenosine 5'-triphosphate to yield phosphatidic acid. The majority of the platelet enzyme is particulate-bound, and membrane fractions of platelet homogenates have a higher specific activity than granule fractions. Both deoxycholate and magnesium are necessary for optimal enzyme activity. The K(m) of the enzyme for adenosine 5'-triphosphate is 1.3 mm, and the apparent K(m) for diacylglycerol is 0.4 mm. The pH optimum is 6.6-6.8 in imidazole-HCl or maleate-NaOH buffer. The enzyme activity of platelets from normal subjects was similar to the activity from patients with renal and hepatic failure.

Radioactive ATP and ADP were found in platelets after incubation of human platelet-rich plasma with either [8-(14)C]adenosine or [8-(14)C]ADP. Treatment of the labelled and washed platelets with thrombin indicated that, though considerable amounts of ATP and ADP were released to the supernatant, radioactive ATP and ADP remained predominantly in the cellular fraction. Breakdown of radioactive ATP took place to form mainly IMP and hypoxanthine, the latter compound appearing in the supernatant. The results indicate the presence of at least two pools of nucleotide in platelets. Evidence is given that the two pools contain approximately the same amounts of ATP plus ADP, and that the ratio of ATP to ADP in the pool released to the supernatant by the action of thrombin is about 0.7-0.8.