Platelet endothelial aggregation receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.     

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.     

Modulating effects of adenosine on the process of human platelet activation]

The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.     

HSP27 phosphorylation is correlated with ADP-induced platelet granule secretion

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. ADP has been reported to induce heat-shock protein (HSP) 27 phosphorylation in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanisms and the roles of ADP-induced HSP27 phosphorylation in human platelets. We showed for the first time that both of decreased phosphorylation levels of HSP27 by PD98059, a MEK1/2 inhibitor and SB203580, a p38 MAPK inhibitor were correlated with the suppressed levels of platelet granule secretion but not with platelet aggregation. Furthermore, the inhibition of either the p44/p42 MAPK or p38 MAPK pathways had no effect on ADP-induced platelet aggregation. These results strongly suggest that the ADP-induced phosphorylation of HSP27 via p44/p42 MAPK and/or p38 MAPK is therefore sufficient for platelet granule secretion but not for platelet aggregation in humans.     

Novel factor Xa inhibitor,maslinic acid,with antiplatelet aggregation activity

As antithrombotic effects of maslinic acid (MA) have not yet been studied, MA-mediated downregulation of coagulation factor Xa (FXa) and platelet aggregation was studied. We show that MA inhibited the enzymatic activity of FXa and platelet aggregation, induced by adenosine diphosphate (ADP) and a thromboxane A₂ (TXA₂) analog, U46619 with a similar antithrombotic efficacy to rivaroxaban, a direct FXa inhibitor used as a positive control. Mechanistically, MA suppressed U46619- or ADP-induced phosphorylation of myristoylated alanine-rich C kinase substrate, and the expression of P-selectin, and activated PAC-1 in platelets. MA increased generation of nitric oxide, but downregulated excessive secretion of endothelin-1 in ADP- or U46619-treated human umbilical vein endothelial cells. In arterial and pulmonary thrombosis mouse model, MA showed prominent anticoagulant and antithrombotic effects. Our data suggest MA as a candidate molecule for a new class of drugs targeting anti-FXa and antiplatelet.     

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: Covalent modification of aggregin,a putative ADP receptor

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I₂). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [¹⁴C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [¹⁴C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.     

PLATELET PHAGOCYTOSIS AND AGGREGATION

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.     

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.     

Platelet count and function at high altitude and in high-altitude pulmonary edema.

Platelet aggregation is the key process in primary hemostasis. Certain conditions such as hypoxia may induce platelet aggregation and lead to platelet sequestration primarily in the pulmonary microcirculation. We investigated the influence of high-altitude exposure on platelet function as part of a larger study on 30 subjects with a history of high-altitude pulmonary edema (HAPE) and 10 healthy controls. All participants were studied in the evening and the next morning at low altitude (450 m) and after an ascent to high altitude (4,559 m). Platelet count, platelet aggregation (platelet function analyzer PFA100; using epinephrine and ADP as activators), plasma soluble P (sP)-selectin, and the coagulation parameters prothrombin fragments 1+2 and thrombin-antithrombin complex were measured. High-altitude exposure decreased the platelet count, shortened the platelet function analyzer closure time by approximately 20%, indicating increased platelet aggregation, increased sP-selectin levels to approximately 250%, but left plasma coagulation unaffected. The HAPE-susceptible subjects were prophylactically treated with either tadalafil (a phosphodiesterase 5 inhibitor), dexamethasone, or placebo in a double-blind way. Subgroup analyses between these different treatments and comparisons of the seven placebo-treated individuals developing HAPE and controls revealed no differences in platelet count, platelet aggregation, or sP-selectin values. We conclude that exposure to high altitude activates platelets, which leads to platelet aggregation, platelet consumption, and decreased platelet count. These effects are, however, not more pronounced in individuals with a history of HAPE or actually suffering from HAPE than in controls and therefore may not be a pathophysiological mechanism of HAPE.     

Aggregation of platelets by muscle actin. A multivalent interaction model of platelet aggregation by ADP

Filamentous muscle actin (F-actin) aggregated blood platelets while G-actin was ineffective. This aggregation could be blocked by ATP suggesting a possible role of actin-bound ADP in this process. Actin-bound ADP caused platelet aggregation at concentrations significantly lower than equivalent concentrations of free ADP. Thus, actin potentiates the aggregating action of ADP. An actin antibody or DNase I inhibited this aggregation showing the requirement of actin in this process. Like other physiological agents, Ca⁺⁺ was necessary for platelet aggregation by actin. Platelets fixed in formaldehyde were not aggregated by actin showing the need for viable platelets. Since F-actin contains 1 mole of bound ADP/mole protein, it is postulated that actin potentiates ADP-induced aggregation by providing multiple interaction sites for platelets.     

Alpha-adrenergic stimulation of phosphatidylinositol synthesis in human platelets as an alpha-2 effect secondary to platelet aggregation

Epinephrine and adenosine diphosphate (ADP) stimulated 3H-glycerol uptake into phosphatidylinositol of human platelets. Yohimbine, an alpha-2 adrenoceptor antagonist, markedly reduced epinephrine-stimulated 3H-glycerol uptake into phosphatidylinositol; while prazosin, an alpha-1 antagonist, was without effect. Likewise, yohimbine, but not prazosin, blocked epinephrine-induced platelet aggregation. Furthermore, clonidine, a specific agonist for alpha-2 adrenoceptors, stimulated incorporation of 3H-glycerol into phosphatidylinositol and promoted platelet aggregation in the presence of low concentrations of ADP. These studies indicate that the effects of epinephrine on platelet aggregation and phosphatidylinositol synthesis are mediated through alpha-2 adrenoceptors. Further, since the stimulation of phosphatidylinositol synthesis seen with epinephrine was also observed with ADP, this suggests that the increased 3H-glycerol labeling is an indirect result of platelet aggregation.     

Adenosine diphosphate receptors on blood platelets: potential new targets for antiplatelet therapy

Platelets play a key role not only in physiological haemostasis, but also under pathological conditions such as thrombosis. Platelet activation may be initiated by a variety of agonists including thrombin, collagen, thromboxane or adenosine diphosphate (ADP). Although ADP is regarded as a weak agonist of blood platelets, it remains an important mediator of platelet activation evoked by other agonists, which induce massive ADP release from dense granules, where it occurs in molar concentrations. Thus, ADP action underlies a positive feedback that facilitates further platelet aggregation and leads to platelet plug formation. Additionally, ADP acts synergistically to other, even weak, agonists such as serotonin, adrenaline or chemokines. Blood platelets express two types of P2Y ADP receptors: P2Y(1) and P2Y(12). ADP-dependent platelet aggregation is initiated by the P2Y1 receptor, whereas P2Y(12) receptor augments the activating signal and promotes platelet release reaction. Stimulation of P2Y(12) is also essential for ADP-mediated complete activation of GPIIb-IIIa and GPIa-IIa, and further stabilization of platelet aggregates. The crucial role in blood platelet biology makes P2(Y12) an ideal candidate for pharmacological approaches for anti-platelet therapy.     

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation.

There is broad agreement that platelet aggregation is generally dependent on fibrinogen (Fg) binding to the glycoprotein (GP) IIb-IIIa receptor expressed on the activated platelet surface. We therefore compared rates and extents of aggregation and of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted 10-fold) was stirred with adenosine diphosphate (ADP) for 10 s or 2 min to measure rates and extent of aggregation, respectively, determined from the decrease in the total number of particles. The number of fibrinogen receptors and bound Fg were measured from mean fluorescence values obtained with FITC-labeled IgM monoclonal antibody PAC1 and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry as presented in part I (Frojmovic et al., 1994). Because flow cytometric and aggregation measurements were routinely determined at room temperature and 37 degrees C, respectively, we also compared and found temperature-independent initial rates of aggregation. The fraction of platelets with fluorescence values above one critical threshold value, corresponding to maximally "activated" platelets (P*), increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r > 0.9). Aggregation was not rate-limited by fibrinogen receptor expression or by Fg binding. It appears that each platelet expresses its maximal Fg receptors at a critical ADP concentration, i.e., occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and capture of such "quantally activated" platelets into aggregates.     

精-甘-天冬-丝氨酸四肽对二磷酸腺苷诱导大鼠血小板活化的信号转导的影响

本工作在二磷酸腺苷（ＡＤＰ）活化的大鼠血小板上,观察精－甘－天冬－丝上肽（ＲＧＤＳ肽）对血小板聚集、蛋白磷酸化、蛋白激酶Ｃ和丝裂素活化蛋白激酶活性的影响。结果发现,５０μｍｏｌ／ＬＡＤＰ引起血小板聚集时,蛋白激酶Ｃ（ＰＫＣ０及丝裂经蛋白激酶（ＭＡＰＫ）活性增加,并引起９５和６６ｋＤ蛋白磷酸化。应用５０,１００和２００μｍｏｌ／ＬＲＧＤＳ肽与基共同孵育,呈浓度依赖地抑制ＡＤＰ引起的血小板聚集和对ＰＫ     

Effect of ethyl alcohol on selected parameters of the hemostasis system in vitro]

Alcohol produces several disorders in all components of hemostasis system. However, its mechanism of action is not clear. Therefore, an effect of ethyl alcohol on the selected parameters of both platelet and plasma hemostasis has been examined in vitro. Blood aggregation induced by ADP and PAF and platelet-leucocytic aggregates have been determined in vitro in the group of 45 healthy volunteers. Out of plasma parameters the selected factors of blood coagulation and fibrinolysis have been examined. Results suggest that the examined concentrations of ethyl alcohol mainly affect platelet function decreasing platelet aggregation. Alcohol does not affect significantly blood coagulation and fibrinolysis occurring in vitro.