Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

广西眼镜蛇毒L-氨基酸氧化酶体内外抗血小板聚集实验研究

目的 研究广西眼镜蛇中提取的L-氨基酸氧化酶(L-amino acid oxidase)在人体外及家兔体内的抗血小板聚集作用.方法 用比浊法测定广西眼镜蛇毒L-氨基酸氧化酶对二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)在人体外及家兔体内引起的血小板聚集率的影响.结果 实验中,能明显抑制二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)引起的血小板聚集,并呈明显的正相关.结论 广西眼镜蛇毒L-氨基酸氧化酶在体内外均有较强的抗血小板聚集活性.     

Wortmannin对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸累积的影响

 Ｗ ｏｒｔｍ ａｎｎｉｎ 是肌醇磷脂 ３ 激酶的不可逆抑制剂．用比浊法分析血小板聚集;肌醇磷脂用３２ Ｐ 磷酸钠标记,用氯仿和甲醇抽提,用 Ｔ Ｌ Ｃ和放射自显影分析,研究了 Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸（ Ｐ Ｉ Ｐ３）累积的影响．结果显示, Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶（５００ Ｕ／ Ｌ）诱导的人血小板聚集有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（２０～８０μｍ ｏｌ／ Ｌ）．凝血酶（５００ Ｕ／ Ｌ）诱导人血小板 Ｐ Ｉ Ｐ３ 的累积, Ｗ ｏｒｔｍ ａｎｎｉｎ 对此累积有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（４０～１６０ μｍ ｏｌ／ Ｌ）．结果提示： Ｗ ｏｒｔｍ ａｎｎｉｎ 可能是潜在的抗血小板药物,抑制凝血酶诱导的人血小板聚集主要与其抑制 Ｐ Ｉ Ｐ３ 的累积有关．结果也提示,肌醇磷脂 ３ 激酶在血小板活化中起重要作用．     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Modulation of the platelet aggregation capacity by modified forms of thrombin

It was found that human platelets possess a high sensitivity towards alpha-thrombin (Km = 2 nM). Modified thrombin forms (beta/gamma-thrombin) with an impaired recognition site of high molecular weight substrates and DIP-alpha-thrombin and trypsin are incapable of inducing platelet aggregation when taken at concentrations corresponding to effective concentrations of alpha-thrombin. Beta/gamma-Thrombin and trypsin, unlike DIP-alpha-thrombin, cause platelet aggregation at concentrations of 100-200 nM. Studies on the modulating effects of modified thrombin forms, alpha-thrombin and trypsin, on platelet aggregation induced by alpha-thrombin revealed that beta/gamma-thrombin, alpha-thrombin and trypsin at concentrations causing no cell aggregation potentiate the platelet response after 2 min incubation and inhibit platelet aggregation upon prolonged (15 min) incubation. However, DIP-alpha-thrombin, irrespective of the incubation time (up to 30 min) increased the sensitivity of platelets to alpha-thrombin-induced aggregation. The activating effect of DIP-alpha-thrombin is characterized by an equilibrium constant (KA) of 17 nM. The experimental data confirm the hypothesis that the necessary prerequisite for an adequate physiological response of platelets to alpha-thrombin is the maintenance in the thrombin molecule of an intact active center and a recognition site for high molecular weight substrates. The specificity of thrombin as a potent platelet aggregation inducer is determined by the recognition site for high molecular weight substrates.     

Minimal sequence requirement of thrombin receptor agonist peptide.

A site-specific proteolytically generated neoamino terminus of the thrombin receptor having a sequence SFLLRNPNDKYEPF- has been reported to be a functional ligand of the receptor. This discovery raises question on the precise structural requirements of the "tethered ligand" responsible for receptor activation and signal transduction. By examining the agonist activity of a panel of synthetic sequence analogues of thrombin receptor agonist peptides (TRAP) on human platelet aggregation, we determined that the minimal sequence of the human platelet thrombin receptor ligand is SFLL-amide (TRAP1-4, EC50 = 300 uM). An extension of TRAP1-4 by an additional Arg-Asn segment yielded the most potent agonist among the series (TRAP1-6, EC50 = 1.3 microM). Based on the structure-activity relationships, we hypothesized a model of the ligand-binding site of the human platelet thrombin receptor that accommodates a hexapeptide structure. TRAP1-6, when administered intravenously, induced marked intravascular platelet aggregation in the anesthetized guinea pigs.     

A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

pH sensitivity of the thrombin-induced rise in fructose 2,6-bisphosphate content of human platelets

The stimulation of human platelets with thrombin results in a rapid and sustained increase in the fructose 2,6-bisphosphate content which may play an important role in the potentiation of glycolytic flux induced by the agonist. The investigation of the effect of pH on thrombin-induced rise in platelet fructose 2,6-bisphosphate content is reported here. The results indicate that the early intracellular alkalinization which follows platelet stimulation may contribute to mediate the positive effect of thrombin on the regulatory metabolite.     

Thrombosis Prevention by Acetylsalicylic Acid in Hyperlipemic Rats

In rats, administration of acetylsalicylic acid (ASA) by stomach tube two hours before blood removal, or addition of the drug to platelet-rich plasma in vitro, markedly inhibited platelet aggregation induced by thrombin, ADP and collagen. Addition of ASA in vitro to human platelet-rich plasma also inhibited platelet aggregation by thrombin, ADP and collagen. In hyperlipemic rats, ASA (100 to 200 mg./kg.), administered by stomach tube once or five times, markedly inhibited the production of thrombosis initiated by intravenous injection of S. typhosa endotoxin. In these experiments, thrombosis prevention by ASA was associated with both a decrease in platelet aggregation and an increase in the recalcification plasma clotting time.     

Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Influence of sodium conductances on platelet activation

The effects of extracellular Na+ and tetrodotoxin on resting membrane potential, cytosolic free Ca2+ levels and aggregation of human platelets have been studied. Neither the decrease in extracellular Na+-concentration (from 140 mmol/l to 0 mmol/l) nor the addition of tetrodotoxin (10(-7) to 10(-5) mol/l) modified the platelet membrane potential. Zero extracellular Na+ concentration or the presence of tetrodotoxin in the medium inhibited platelet aggregation; however, K+-depolarized platelets showed an unchanged aggregation induced by ADP or thrombin in media with zero or low extracellular Na+ concentrations or in the presence of tetrodotoxin. Moreover, zero extracellular Na+ concentration or tetrodotoxin inhibited calcium mobilization in platelets during activation induced by thrombin. Hence, voltage-dependent activation linked to Na+ influx appears to be necessary for ADP- and thrombin-induced platelet aggregation under control conditions. Mechanisms for the role of Na+ conductances in platelet function are discussed.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.     

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.     

Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors.

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.     

PDGF modifies phosphoinositide metabolism and inhibits aggregation and release in human platelets

While platelet derived growth factor (PDGF) did not induce any platelet aggregation nor secretion, it modified the polyphosphoinositide metabolism of human platelets prelabeled with 32P-orthophosphate. We found a decrease of 32P associated with phosphatidylinositol 4,5 bisphosphate after 3 min, with parallel increase of 32P-phosphatidylinositol 4 phosphate and 32P-phosphatidylinositol using 100 ng/ml of PDGF. This modification was PDGF concentration dependent. PDGF inhibited thrombin and collagen induced platelet aggregation and 14C-serotonin release in a dose dependent manner, but was without effect when arachidonic acid was used. These results suggest that PDGF (i) stimulated the hydrolysis of polyphosphoinositides (ii) and could exert a negative feedback control on platelet activation induced by thrombin or collagen.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.