Human Platelet Plasma Membrane Research Articles

Phosphoinositides take a central stage in regulating blood platelet production and function

Blood platelets are produced by megakaryocytes through a complex program of differentiation and play a critical role in hemostasis and thrombosis. These anucleate cells are the target of antithrombotic drugs that prevent them from clumping in cardiovascular disease conditions. Platelets also significantly contribute to various aspects of physiopathology, including interorgan communications, healing, inflammation, and thromboinflammation. Their production and activation are strictly regulated by highly elaborated mechanisms. Among them, those involving inositol lipids have drawn the attention of researchers. Phosphoinositides represent the seven combinatorially phosphorylated forms of the inositol head group of inositol lipids. They play a crucial role in regulating intracellular mechanisms, such as signal transduction, actin cytoskeleton rearrangements, and membrane trafficking, either by generating second messengers or by directly binding to specific domains of effector proteins. In this review, we will explore how phosphoinositides are implicated in controlling platelet production by megakaryocytes and in platelet activation processes. We will also discuss the diversity of phosphoinositides in platelets, their role in granule biogenesis and maintenance, as well as in integrin signaling. Finally, we will address the discovery of a novel pool of phosphatidylinositol 3-monophosphate in the outerleaflet of the plasma membrane of human and mouse platelets.

Clinical Application of Epithelial Sodium Channel (ENaC) as a Biomarker for Arterial Hypertension.

Arterial hypertension (HTN) is a global public health concern and an important risk factor for cardiovascular diseases and renal failure. We previously reported overexpression of ENaC on the plasma membrane of human platelets is a hallmark of HTN. In this double-blinded study of an open population (n = 167), we evaluated the sensitivity and specificity of a diagnostic assay based on gold nanoparticles (AuNPs) conjugated to an antibody against epithelial sodium channel (ENaC) expressed on platelets, which is detected using a fluorescent anti-ENaC secondary antibody and spectrofluorometry. Using the cutoff value for the AuNP-anti-ENaC assay, we confirmed the diagnosis for 62.1% of patients with clinical HTN and detected 59.7% of patients had previously undiagnosed HTN. Although some shortcomings in terms of accurately discriminating healthy individuals and patients with HTN still need to be resolved, we propose this AuNP-anti-ENaC assay could be used for initial screening and early diagnosis to critically improve opportune clinical management of HTN.

Biomimetic Platelet-Camouflaged Nanorobots for Binding and Isolation of Biological Threats.

One emerging and exciting topic in robotics research is the design of micro-/nanoscale robots for biomedical operations. Unlike industrial robots that are developed primarily to automate routine and dangerous tasks, biomedical nanorobots are designed for complex, physiologically relevant environments, and tasks that involve unanticipated biological events. Here, a biologically interfaced nanorobot is reported, made of magnetic helical nanomotors cloaked with the plasma membrane of human platelets. The resulting biomimetic nanorobots possess a biological membrane coating consisting of diverse functional proteins associated with human platelets. Compared to uncoated nanomotors which experience severe biofouling effects and hence hindered propulsion in whole blood, the platelet-membrane-cloaked nanomotors disguise as human platelets and display efficient propulsion in blood over long time periods. The biointerfaced nanorobots display platelet-mimicking properties, including adhesion and binding to toxins and platelet-adhering pathogens, such as Shiga toxin and Staphylococcus aureus bacteria. The locomotion capacity and platelet-mimicking biological function of the biomimetic nanomotors offer efficient binding and isolation of these biological threats. The dynamic biointerfacing platform enabled by platelet-membrane cloaked nanorobots thus holds considerable promise for diverse biomedical and biodefense applications.

Nanoparticle biointerfacing by platelet membrane cloaking.

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can negatively impact nanoparticle effectiveness in complex, physiologically relevant systems1–3. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates4–7. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. As compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and are absent of particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

Concurrent and separate inside‐out transition of platelet apoptosis and activation markers to the platelet surface

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P-selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9±2·8% of total cells), (ii) CD62 but not PS (86·2±1·3%), (iii) both PS and CD62 (89·6±1·0%) or (iv) neither PS nor CD62 (87·9-97·5%), when platelets were treated at optimal conditions with pro-apoptotic BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT-737 is a slow temperature-dependent process requiring 90min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin-induced CD62 exposure and A23187-induced PS and CD62 exposure showed fast temperature-independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance.

PLA2 Type IIA Increases Platelet Plasma Membrane Rigidity During Cold Induced Activation

There is a wide discussion regarding the origin and function of lipid domains in human cells. An example in which lipid domains may play a functional role is the plasma membrane of human platelets. Platelets are susceptible to chilling, activating when the temperature falls below 20 °C. This limitation represents a crucial issue in terms of storage of platelets. It has been previously shown that cold-induced platelet activation is correlated with the formation of macroscopic lipid domains during cooling. This is a result of the fact that platelets present low cholesterol content (15 mol%), which results in the presence of a cooperative lipid melting transition centered at 15°C. This transition is responsible for the formation of the macroscopic lipid domains during cooling. Human secretory phopholipase A2 type IIA (sPLA2-IIA) catalyses the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. Recently we have shown that its activity is triggered by the local enrichment of anionic lipids in fluid domains during phase coexistence. Since human platelets expose their anionic lipid content during chilling, we propose that PLA2-IIA is triggered during this process, and that the activity of the enzyme on the fluid domains induces an overall increase in rigidity in the platelet plasma membrane due to an increase in the proportion of ordered lipids. We present a combined FTIR and Laurdan fluorescence spectroscopy study on the thermotropic phase behavior of human platelets, and show that during cold-induced activation membrane rigidity increases. We present results on how the activity of sPLA2-IIA is regulated by the thermotropic phase behavior of the platelet plasma membrane after platelets have been artificially activated through sonication. We show that PLA2-IIA activity increases the rigidity of these activated platelet plasma membranes.

Synthesis and structure-activity relationships of 5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine derivatives: novel arginine vasopressin antagonists.

A variety of novel heterocyclic compounds having thienoazepine, pyrroloazepine, furoazepine, and thienodiazepine skeletons were synthesized, most of which exhibited potent antagonism of [(3)H]-AVP specific binding in assays using rat liver (V1), rat kidney (V2), human platelet plasma membranes, and recombinant human CHO cells (V2), as well as antagonizing AVP-induced hypertension in rats (V1, intravenous) and showing a diuretic effect in rats (V2, oral). By detailed studies of the structure-activity relationships of these compounds, the thienoazepine derivative 1 was found to be a very potent combined V1 and V2 antagonist. After further pharmacological and toxicological evaluation as well as physical properties, the hydrochloride 2 (JTV-605) of compound 1 was selected for clinical studies as a potent AVP antagonist with a long duration of action.

Platelet glycoprotein IIb/IIIa receptors and Glanzmann's thrombasthenia.

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Isoforms of purified methyltransferase from human blood platelets

A membrane-bound protein with N-methyltransferase activity, associated with phospholipid metabolism, has been isolated from purified human blood platelet plasma membranes. The activity of this enzyme has been detected in crude platelet preparations. However, the nature and properties of this enzyme and its purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation and solubilized in buffer containing 0.5% Triton-X 100. The partially purified extracts were further purified using a combination of ion-exchange chromatographic procedures, preparative IEF and preparative SDS-PAGE. The preparative rotorfor electrofocusing, using pH 3-10 ampholyte at 40EC, yielded activities at both acidic and basic pHs of approximately 3.5 and 8.5, respectively. The pH 8.5 activity yielded predominantly a single 67 KDa band on SDS-PAGE gel and the pH 3.5 activity also gave a similar band and a few lighter bands at 35 KDa and 50 KDa. The acidic and basic fractions catalysed the transfer of methyl groups from S-adenosyl inethionine to PE with a predominant formation of PME (ca 90%). The fraction purified by preparative SDS-PAGE also yielded a major methylating activity that eluted between 60-70 KDa. This latter protein was washed free of SDS and its activity measured; it, too yielded predominaly the monomethylated PE. These data are consistent with the hypothesis that several SAM-dependent isozymes also exist in human blood platelets that convert PE to PC. JOURNAL OF THE GHANA SCIENCE ASSOCIATION Volume 2 No. 3 (2000) pp. 98-102

T-antigen (Gal β3 GaINAc α-) containing glycoproteins of human reace

Desialation of cell surface glycoconjugates due to bacterial or viral infection can expose epitopes like T-antigenic structure which can also occur during oncological transformations. Human platelet plasma membrane glycoproteins were isolated by jacalin affinity chromatography. Potential T-antigen containing glycoproteins which were not reported before could be identified on the Western blot using peanut agglutinin-horse radish peroxidase (PNA-HRP) after neuraminidase treatment. Alpha-galactosyl epitopes recognized by anti-gal were found to be absent in human platelet plasma membrane glycoproteins. Under the experimental conditions employed, the Gp IIbα was identified most rich in T-antigenic structures. Probable role of exposed T-antigenic structures and α-galactosyl epitopes in pathological conditions is discussed. The identity of major glycoprotein bands was confirmed by differential lectin-binding studies with Concanavalin A on the Western blot. The higher binding affinity of jacalin for T-antigenic structures when compared to PNA enabled the isolation and detection of the antigen containing platelet surface glycoproteins which were not reported before.

Distinct cholesterol and phospholipid incorporation at the platelet plasma membrane of hyperlipidemic subjects: structural order and function

Plasma lipid composition, platelet aggregation, cholesterol (Ch)/glycoprotein IIb-IIIa (GP) and phospholipid (Ph)/GP molar ratios, fatty acid composition and structural order (1, 6-diphenyl-1, 3, 5-hexatriene (DPH) fluorescence anisotropy at 35 C (r) of human platelet plasma membranes (HPPM) were measured in four DPH,35 groups of hyperlipidemic patients (II: plasma Ch < 250 mg/dl and TG (triglycerides) <220 mg/dl, n = 21; III: Ch > 250 mg/dl and TG < 220 mg/dl, n = 23; IV: Ch < 250 mg/dl and TG > 220 mg/dl, n = 18; and V: Ch > 250 mg/dl and TG > 220 mg/dl, n = 12) and compared with those of the control group (I). Our results were: (i) in groups III, IV and V the HPPM (Ch + Ph)/GP molar ratio increased 7.0 7.7% (mean SD); (ii) the Ph/GP molar ratio increased significantly in groups III, IV and V, but most in IV and V, while the Ch/GP molar ratio increased only in groups III and V; (iii) the mean relative increase of Ch with respect to Ph in the HPPM of groups III, IV and V was 140% 21% and 54%, respectively; (iv) the Ch/GP molar ratio was correlated with LDL-Ch (0.41 0.16, P < 0.002, n = 55, for all the subjects and 0.60 0.11, P < 2.10-4, n = 33, for subjects with TG < 220 mg/dl), however, it was totally uncorrelated with HDL-Ch; (v) the HPPM Ch/Ph molar ratio was positively correlated with plasma Ch (r = 0.51 0.08, P < 1.10-6, n = 83) and with (LDL + HDL) Ch (r = 0.64 0.07, P < 1.10-6, n = 73), the former correlation increased significantly ( r = 0.67 0.07, P < 1.10-6, n = 53) when done only for subjects with TG < 220 mg/dl; (vi) the Ch/Ph molar ratio was only increased in group III (0.70 0.03, P < 3.10-5, n = 23) and decreased in group IV (0.62 0.02, P < 0.001, n = 18); (vii) the fatty acid/GP molar ratio was significantly increased in groups IV and V, however, a significant absolute and relative increase of C16:0 and C18:1 was observed only in severe hypertriglyceridemia (> 500 mg/dl), together with a relative decrease of C18:0 and C20:4 ( n - 6); (viii) the HPPM structural order, as probed by r , was negatively correlated with DPH,35 plasma TG (r =- 0.61 0.10, P < 4.10-5, n = 39), the Ph/GP molar ratio (r =-0.58 0.10, P < 2.10-4, n = 39) and the the (C18:1 + C18:2))/GP molar ratio (r =- 0.80 0.05, P < 1.10-6, n = 39), however, it was independent of plasma and HPPM Ch; (ix) the higher HPPM Ch/Ph molar ratio in group III was associated (r = 0.58 0.12, P < 0.005, n = 22) with a moderately higher platelet reactivity to collagen. We conclude that Ch and Ph were distinctly incorporated to HPPM in the different groups of hyperlipidemia and, therefore, that the absolute increase of Ch and Ph was more informative to understand the structural and functional modifications of the HPPM in hyperlipidemias, than the Ch/Ph molar ratio. On the other hand, the r was sensitive to the DPH,35 increase in the content of HPPM Ph and C18:1 + C18:2 and it was insensitive to the increase in the Ch content.

Labeling of human platelet plasma membrane thromboxane A 2/prostaglandin H 2 receptors using SQB, a novel biotinylated receptor probe

This study reports the synthesis, biological evaluation, and application of a new biotinylated derivative 1-[[1 S-[1α,2α( Z),3α,4α]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino]methyl]-7-oxabi-cyclo [2.2.1]hept-2-yl]-5-heptenoyl]-2-[hexahydro-2′-oxo-1 H-thieno[3′,4′ d][imidazole-4′-pentanoyl]hydrazine (SQB) of the thromboxane A 2/prostaglandin H 2 (TXA 2/PGH 2) receptor antagonist [1 S-[1α,2α( Z),3α, 4α]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31.491 with biotin hydrazide, and the product was purified by flash chromatography. It was found that SQB specifically inhibited platelet aggregation in response to U46619 with an ic 50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregation. Competition binding studies revealed that SQB produced a concentration-dependent inhibition of [ 3H]-[1S-[1α,2β(5Z),3β,4α]]-7-[3-[[2[( phenylamino)carbonyl]hydrazino]methyl]-7- oxabicyclo[2.2.1] hept-2- yl]-5- heptenoic acid ([ 3H]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized platelet membranes, with a K i of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA 2/PGH 2 receptor antagonist BM13.177. Finally, incubation of gel-filtered platelets or platelet-rich plasma with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated fluorescent labeling of platelet plasma membrane TXA 2/PGH 2 receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA 2/PGH 2 antagonist SQ29,548. Based on the ability of SQB-FITC-avidin to label intact platelets, it can be concluded: (1) that a pool of platelet TXA 2/PGH 2 receptors resides in the plasma membrane; and (2) that the binding domains for these receptors are oriented at or near the external membrane surface. Collectively, these data demonstrate that SQB is a highly specific probe for TXA 2/PGH 2 receptors, which should be of significant value for receptor localization studies in platelets and other tissues.

Effect of cetirizine on plasma membrane of human eosinophils, neutrophils and platelets: a dose response study

The effect of different concentrations of cetirizine (0.1, 0.3, 0.5, 0.7, 0.9 ug/ml) on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils and platelets were investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results demonstrate that cetirizine induced a significant increase in the lipid order in the exterior part of the membrane and a decrease in membrane heterogeneity of eosinophils at concentrations of 0.7 ug/ml and higher and that at a concentration of 0.9 ug/ml it does likewise in neutrophils and platelets. Moreover, cetirizine abolished the PAF induced changes in membrane fluidity in these cells. These data suggest that cetirizine interacts with the plasma membrane of eosinophils, neutrophils and platelets inducing functional changes.

A new look at the lipid composition of the plasma membrane of human blood platelets relative to the GPIIb/IIIa (integrin cxIIβ3) content

The total lipids of the human platelet plasma membrane (HPPM) from 50 ml of blood of healthy subjects were extracted, quantified and related to the mass content of the major intrinsic membrane protein, the glycoprotein IIb/IIIa (GPIIb/IIIa). The HPPM total lipid/GPIIb/IIIa weight ratio determined was 5.40 ± 0.20, independently of the membrane washing procedure used, with the cholesterol/GPIIb/IIIa and phospholipid/GPIIb/IIIa molar ratios of 800 ± 50 and 1200 ± 40, respectively. If the distribution of lipids around each intrinsic protein were proportional to its mass, the lipids around a molecule of GPIIb/IIIa will occupy about 120 nm(2) of the membrane plane, which is about one and a half times the cross-sectional area of the extracellular head of GPIIb/IIIa, as estimated by electron microscopy. The lipid extracts were further subjected to thin-layer chromatography to separate and quantify the different phospholipid fractions, the free fatty acids and the neutral lipid fraction and the distribution of fatty acids in each fraction was determined by gas chromatography after methanolysis. The phospholipid molar distribution was SPM(22.3 ± 0.9%), PC(36.2 ± 1.0%), PE(24.9 ± 0.9%), PS(12.1 ± 0.6%) and PI(4.5 ± 0.4%) and the free fatty acid fraction represented 2.9 ± 0.4% of the total fatty acids in HPPM. The fatty acid chain length ranged from 14 to 24 carbons, comprising unsaturated fatty acids (47.3% molar per cent of the total) of which 40.7 ± 2.0% were monosaturated and 40.7 ± 0.9% tetraunsaturated. Palmitic, stearic, oleic and arachidonic acids represent 66% of the total fatty acids of HPPM, being: 68.9 ± 5.3% of palmitic acid and 63.3 ± 6.9% of oleic acid in PC; 50.9 ± 3.8% of arachidonic acid in PE; and 30.5 ± 2.4% of stearic acid in PS. We discuss the methodological modifications and the new data in relation with the major differences in HPPM lipid composition found in the literature. The data obtained provides a comprehensive and accurate description of the lipid composition of HPPM on which to rely as a reference for basic and medical studies.

Distribution and Activation of cAMP- and cGMP-Dependent Protein Kinases in Highly Purified Human Platelet Plasma and Intracellular Membranes

Previously cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK) have been found predominantly associated with the particulate fraction in human platelets. We now report the distribution and activation of cAMP-PK and cGMP-PK in highly purified fractions of human platelet plasma (PM) and intracellular membranes (IM) prepared using high voltage free flow electrophoresis. Two non-hydrolysable analogues of cAMP and cGMP namely Sp-5,6-DCI-cBiMPS and 8-p-CPT-cGMP have been used to activate cAMP-PK and cGMP-PK respectively. Addition of either agonist with [gamma 32P]ATP stimulated the endogenous activity of cAMP-PK or cGMP-PK in PM but not in IM. With PM Sp-5,6-DCI-cBiMPS stimulated the phosphorylation of protein substrates of Mr 16, 22, 24, 46-50, 66, 90, 160 and 250 kDa. A specific peptide inhibitor of cAMP-PK inhibited the phosphorylation of all of the substrates by Sp-5,6-DCI-cBiMPS. 8-pCPT-cGMP also induced the phosphorylation of a number of substrates particularly 16, 22, 46-50, 90 and 250 kDa proteins. Inclusion of the cAMP-PK inhibitor peptide totally blocked the phosphorylation of the 16 and 22 kDa proteins, partially inhibited phosphorylation of 46-50 and 90 kDa proteins and had no effect on the 250 kDa protein indicating the 46-50, 90 and 250 kDa proteins were also cGMP-PK substrates. Western blotting with antibodies to cGMP-PK and the catalytic subunit of cAMP-PK revealed the presence of the kinases to be exclusively associated with PM with no detection in IM. The presence of cAMP-PK substrates in IM was investigated by exogenous addition of catalytic subunit of cAMP-PK. Phosphoproteins of Mr 16, 22, 27, 30, 45, 75, 116 and 250 kDa were detected. A range of antibodies to cAMP-PK substrates were used to identify and localise the substrates. These antibodies revealed GPIb and VASP to be exclusively associated with PM fractions. Rap IB was also predominantly associated with PM with a small level detected in IM. Antibodies to the IP3 receptor (18A 10 and 4C11) revealed the protein to be predominantly associated with IM. Additionally the antibody 4C11 recognised a 230 kDa protein band in PM that was not seen in IM. From the known specificity of these antibodies the results confirm the presence of a type 1 IP3 receptor in IM and a distinct (possible type III) IP3 receptor with the PM. The 16, 22, 27, 30, 75 and 116 kDa proteins in IM represent newly detected substrates for cAMP-PK of presently unknown identity.

Cholesterol effect on the physical state of lipid multibilayers from the platelet plasma membrane by time-resolved fluorescence

There are indications that the plasma membrane lipid composition and, in particular, the cholesterol/phospholipid (C/PL) ratio, affects platelet function. As a first approximation to the molecular characterization of the effect of cholesterol on the order, fluidity and lateral heterogeneity of the platelet plasma membrane, the steady-state and time-resolved fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trans-parinaric acid (tPnA) has been studied in multibilayer vesicles of phospholipids extracted from human platelet plasma membrane with different cholesterol/phospholipid molar ratios modified in vitro from 0.07 to 0.9. The DPH studies show that the increased presence of cholesterol has a stronger effect on the order than on the fluidity of the bilayer, as has been previously observed in other lipid membranes. On the other hand, from the analysis of the fluorescence kinetics of tPnA we conclude that a higher cholesterol content gives rise to an increase of the heterogeneity of the bilayer, due to a larger fraction of solid-like lipid domains. These domains contain a cholesterol concentration much higher than the macroscopic average value.

Localization and identification of Ca2+ATPases in highly purified human platelet plasma and intracellular membranes. Evidence that the monoclonal antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in human platelets.

The Ca2+ATPase activities of highly purified human platelet membranes prepared by high-voltage free-flow electrophoresis have been analysed by using [gamma-32P]ATP hydrolysis, recognition by antibodies and phosphoenzyme-complex formation. The Ca2+ATPase activity present in mixed membranes was found to be predominantly associated with intracellular membranes after subfractionation, with only a low level of activity associated with plasma membranes. The intracellular-membrane Ca2+ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca2+ATPase was not significantly affected, suggesting that the latter does not belong to the SERCA (sarco-endoplasmic-reticulum Ca2+ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca2+ATPase [Cheng, Magocsi, Cooper, Penniston and Borke (1993) Cell Physiol. Biochem. 4, 31-43] recognized two bands at 135 and 150 kDa in mixed membranes and plasma membranes, and the corresponding bands in red-blood-cell membranes, confirming the Ca2+ATPase to be of the PMCA (plasma-membrane Ca2+ATPase) type. No recognition of any band was detected in intracellular membranes. Identification of the intracellular-membrane Ca2+ATPase activity was carried out with polyclonal antibodies with known specificity towards SERCA 2b (S.2b) and SERCA 3 (N89), and a monoclonal antibody, PL/IM 430, raised against platelet intracellular membranes. All of these antibodies recognized the 100 kDa Ca2+ATPase in mixed membranes and intracellular membranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and antiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca2+ATPase recognized by PL/IM 430 was immunoprecipitated, and the immunoprecipitated protein was specifically recognized by the antiserum N89, but not by S.2b. Analysis of the phosphoenzyme-complex formation revealed potent phosphorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 430 and N89. These studies report the presence of a PMCA in a purified plasma-membrane fraction from human platelets, and that the antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in intracellular membranes.

Distribution of plasma membrane Ca 2+-ATPase and inositol 1,4,5-trisphosphate receptor in human platelet membranes

Human platelet plasma membranes were prepared by the glycerol lysis method of Harmon et al. [Harmon JT. Greco NJ. Jamieson GA. (1992) Isolation of human platelet plasma membranes by glycerol lysis. Meth. Enzymol., 215, 32–36]. The membranes were observed to contain a Ca 2+-ATPase with different properties than those of internal membranes. The specific activity of Ca 2+-ATPase was lower in plasma membranes (10–40 nmol ATP hydrolyzed/min/mg), but the ATPase was less sensitive to thapsigargin (41% inhibition at 500 nM) and more sensitive to vanadate (50% inhibition at 4 μM) than the Ca 2+-ATPase in internal platelet membranes. The plasma membranes contained a Ca 2+-ATPase detectable by monoclonal and polyclonal antibodies against erythrocyte Ca 2+-ATPase that had a molecular mass of 144 kD. However, an anti-peptide antibody against an N-terminal sequence of the inositol 1,4,5-trisphosphate receptor recognized this protein in internal membranes, but not plasma membranes.

Calcium-induced transbilayer scrambling of fluorescent phospholipid analogs in platelets and erythrocytes

The non-random distribution of phospholipids in the plasma membrane of human platelets and erythrocytes is at least partially maintained by the ATP-dependent aminophospholipid translocase, but can be disturbed by a calcium-induced scrambling of lipids. Using fluorescent NBD-phospholipid analogs, we demonstrate that in both cells the aminophospholipid translocase has a slightly higher preference for the naturally occurring l-isomer of the polar headgroup of phosphatidylserine as compared to the d-isomer. Calcium-induced outward movement of internalized phosphatidylserine probe, however, is not affected by the stereochemical configuration of the serine headgroup and is virtually identical to both the inward and outward movement of the phosphatidylcholine probe. The data also indicate that both in platelets and red blood cells the calcium-induced transbilayer movement is bidirectional and involves all major phospholipid classes, with reorientation rates of sphingomyelin being appreciably lower than that of the other phospholipid classes. While our results largely support earlier observations on red cells, they clearly differ from a recent study on platelets which suggested that calcium-induced scrambling is restricted to aminophospholipids and would not involve cholinephospholipids. The present results indicate that the same mechanism is responsible for calcium-induced lipid scrambling in red blood cells and platelets.

Loss of phospholipid asymmetry in human platelet plasma membrane after 1-12 days of storage. An ESR study.

We used paramagnetic analogs of endogenous phospholipids to study modification of phospholipid distribution in platelet plasma membranes during aging. Asymmetrical distributions and translocation kinetics were very different for spin-labeled phosphatidylserine and spin-labeled phosphatidylcholine in fresh platelet plasma membranes. In freshly prepared platelets and up to day 7, spin-labeled phosphatidylserine very rapidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin-labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin-labeled phospholipids at equilibrium. Inhibition of translocase activity and modification of membrane stability accounted for these transformations. The rapid re-exposition of spin-labeled phosphatidylserine after stimulation by the calcium ionophore A23187, measured in fresh platelet concentrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppression of beta-N-acetyl glucosaminidase secretion and vesicle formation after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these results showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet activation. In addition, all of the observed events occurred simultaneously between day 7 and day 9. These results highlight the importance of maintaining plasma membrane asymmetry to increase the hemostatic effectiveness of transfused platelet concentrates.