Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus

This study describes the extraction and characterization of a platelet aggregation inhibitory peptide from Inonotus obliquus. Ethanol extract from I. obliquus ASI 74006 mycelia showed the highest platelet aggregation inhibitory activity (81.2%). The maximum platelet aggregation inhibitory activity was found when the mycelia of I. obliquus ASI 74006 was extracted with ethanol at 80 degrees C for 12 h. The platelet aggregation inhibitor was purified by systematic solvent fractionation, ultrafiltration, Sephadex G-10 column chromatography, and reverse-phase HPLC. The purified platelet aggregation inhibitor is a novel tripeptide with a molecular mass of 365 Da, having a sequence of Trp-Gly-Cys. The purified platelet aggregation inhibitor also showed high platelet aggregation inhibitory activity in Institute of Cancer Research (ICR) mice.     

Characteristic parameters of the vascular-platelet component of the hemostasis system in the immigrant and indigenous populations of the Far North

The platelet aggregation activity was studied in 79 healthy subjects, immigrant and indigenous, aged between 20 and 59 years, living in the Yamalo-Nenets Autonomous Area. The platelet count and spontaneous and induced platelet aggregation were assessed using the inducer adenosine diphosphate (ADP). It was established that the spontaneous platelet aggregation rate was higher (p < 0.01) in indigenous residents of the region than in the immigrant population, and the ADP-induced platelet aggregation values were higher in the immigrant population of the Far North (p < 0.01).     

Effect of homocysteine, cysteine, and their derivatives on the platelet link of hemostasis system

The influence of homocysteine, homocysteine thiolactone, cysteine and their derivatives on activation and aggregation of human platelets was investigated using the model systems in vitro. It was established that homocysteine and cysteine increased platelet aggregation induced by ADP, epinephrine, or collagen. Their action began in a range of concentrations such as their physiological blood levels (10 microM) and was increasing with the rise of their concentrations. Cysteine increased ADP-induced platelet aggregation, hardly any affect on epinephrine-induced platelet aggregation and depressed collagen-induced platelet aggregation in the highest concentration (1000 microM). Their disulfides and thioethers did not influence platelet aggregation.     

Signal transduction pathways involved in the platelet aggregation induced by a D-49 phospholipase A2 isolated from Bothrops jararacussu snake venom

Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A(2) (D-PLA(2)) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE(1)) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways.     

Improvement of platelet aggregation abnormalities in thrombocytosis after thrombocytopheresis

Platelet function tests were performed in three patients with thrombocytosis in myeloproliferative disorders before and after a swift reduction of platelet count by thrombopheresis. The decrease of platelet count obtained after the procedure was reversed in six days. In two patients with platelet aggregation defects, the normalization of aggregation abnormalities was observed after pheresis, followed by a progressive decrease of platelet response until the pre-pheresis values on 6th day. In the third patient with normal platelet aggregation, a progressive increase of platelet aggregation response was noted on the days following thrombopheresis with ischaemic symptoms of a foot toe. In all three patients, the changes of platelet aggregation were accompanied by a related increase of megathrombocytes. In the two patients with platelet aggregation abnormalities, plasma and platelet beta-thromboglobulin levels were related to changes in platelet count and aggregation.     

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.     

The Ratio of ADP- to TRAP-Induced Platelet Aggregation Quantifies P2Y12-Dependent Platelet Inhibition Independently of the Platelet Count

Objective

This study aimed to assess the association of clinical factors with P2Y₁₂-dependent platelet inhibition as monitored by the ratio of ADP- to TRAP-induced platelet aggregation and conventional ADP-induced aggregation, respectively.

Background

Controversial findings to identify and overcome high platelet reactivity (HPR) after coronary stent-implantation and to improve clinical outcome by tailored anti-platelet therapy exist. Monitoring anti-platelet therapy ex vivo underlies several confounding parameters causing that ex vivo platelet aggregation might not reflect in vivo platelet inhibition.

Methods

In a single centre observational study, multiple electrode aggregometry was performed in whole blood of patients after recent coronary stent-implantation. Relative ADP-induced aggregation (r-ADP-agg) was defined as the ratio of ADP- to TRAP- induced aggregation reflecting the individual degree of P2Y₁₂-mediated platelet reactivity.

Results

Platelet aggregation was assessed in 359 patients. Means (± SD) of TRAP-, ADP-induced aggregation and r-ADP-agg were 794 ± 239 AU*min, 297 ± 153 AU*min and 37 ± 14%, respectively. While ADP- and TRAP-induced platelet aggregation correlated significantly with platelet count (ADP: r = 0.302; p<0.001; TRAP: r = 0.509 p<0.001), r-ADP-agg values did not (r = -0.003; p = 0.960). These findings were unaltered in multivariate analyses adjusting for a range of factors potentially influencing platelet aggregation. The presence of an acute coronary syndrome and body weight were found to correlate with both ADP-induced platelet aggregation and r-ADP-agg.

Conclusion

The ratio of ADP- to TRAP-induced platelet aggregation quantifies P2Y₁₂-dependent platelet inhibition independently of the platelet count in contrast to conventional ADP-induced aggregation. Furthermore, r-ADP-agg was associated with the presence of an acute coronary syndrome and body weight as well as ADP-induced aggregation. Thus, the r-ADP-agg is a more valid reflecting platelet aggregation and potentially prognosis after coronary stent-implantation in P2Y₁₂-mediated HPR than conventional ADP-induced platelet aggregation.     

Structural-functional state of platelets at the initial stage of aggregation

ADP-induced changes of light transmission of platelet suspension were studied with a laser aggregometer at concentration variation of both cells and aggregation agents. It was shown that a decrease of light transmission of platelet suspension was due to structure-morphological changes of platelets and pseudopodia formation. An investigation method of platelet aggregation with a small content of cells in the solution permits to fix distinctly the stages of the platelet structural states in the processes of stimulation and aggregation.     

血小板反应性增高患者相关指标的检测与分析

目的:探讨反应性血小板增高患者凝血指标及血小板聚集功能指标变化规律。方法:143例血小板反应性增高患者和65例对照者同时检测血小板聚集功能、抗凝血酶活性(AT-Ⅲ)、凝血酶原时间(PT)和活化部分凝血活酶时间(APTT),并统计分析。结果:血小板反应性增高常见于肿瘤、肺炎、外伤患者;血小板反应性增高组患者AT-Ⅲ、PT和APTT与对照组无差异,但患者血小板聚集功能显著高于对照组。结论:血小板反应性增高患者血小板聚集功能增强,建议定期监测血小板聚集功能,必要时用抗血小板聚集药预防。     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Relationship of turbidity to the stages of platelet aggregation

The process of platelet aggregation as detected by turbidity changes in the platelet aggregometer was studied relative to light scattering by large particles. For latex beads a plot of light scattering intensity/unit mass versus particle size gave increased light scattering intensity for small particle sizes but decreased scattering at large particle size. This behavior is predicted by Rayleigh-Gans theory. These results were related to the platelet aggregometer, an optical instrument used to measure the association of small particles (monomeric platelets) to large particles (platelet aggregates). Formalin-fixed platelets do not show changes in light transmission due to energy-requiring processes, such as shape change, so that turbidity changes in the presence of aggregating agents could be attributed to a change in platelet aggregation state. Small platelet aggregates showed increased turbidity compared to a similar mass of monomeric platelets. In fact, very large platelet aggregates that were visible to the unaided eye were needed to produce a decrease in light scattering intensity. Thus, turbidity can either increase or decrease with platelet aggregation depending on the size of the aggregates. Studies of platelet aggregation that show no initial increase in turbidity must be characterized by dominance of large platelet aggregates and monomeric platelets.     

Relationships between Platelets and Blood Cells in Postprandial Glycemia in Healthy Individuals

The relationships between platelet aggregation and blood cell amount were studied in healthy subjects after fasting and after a standard carbohydrate breakfast. Positive relationships were revealed between certain parameters of platelet aggregation and the amounts of monocytes and eosinophils. Negative relationships were revealed between most parameters of platelet aggregation and the amount of platelets. Along with the well-known food leukocytosis and increased fibrinogen, postprandial conditions were characterized by an increase in certain parameters of platelet aggregation, destruction of the relationship between the fibrinogen concentration and platelet aggregation, impairment of the inverse relationship between the amount and aggregation of platelets, and moderate direct or inverse relationships between certain parameters of the first and the second stages of platelet aggregation and the erythrocyte parameters. As a disturbing factor, postprandial conditions activated platelet hemostasis by modulating the initial stages of aggregation and substantially changed the interrelations between blood cells.     

Characterization and platelet inhibitory activity of bitistatin, a potent arginine-glycine-aspartic acid-containing peptide from the venom of the viper Bitis arietans

A platelet aggregation inhibitory protein, bitistatin, was isolated from the venom of the puff adder Bitis arietans. This protein is a single-chain peptide containing 83 amino acids and 7 disulfide bonds. Bitistatin contains the sequence arginine-glycine-aspartic acid and shows considerable homology to two previously described snake venom platelet aggregation inhibitors, trigramin and echistatin. Bitistatin inhibited human and canine platelet aggregation initiated by 10 microM ADP in vitro with IC50 values of 237 +/- 13 and 28 +/- 3 nM, respectively. In order to assess the antithrombotic potential of bitistatin, a canine model of platelet-dependent coronary thrombus formation was utilized. Injection of bitistatin at 10-100 micrograms/kg (0.7-7 nmol/kg, intravenously (i.v.] resulted in dose-dependent inhibition of both platelet aggregation ex vivo and platelet-dependent cyclical flow reductions. The effective dose to inhibit cyclical flow reductions was 30 micrograms/kg, i.v. A higher dose of bitistatin (100 micrograms/kg, i.v.) inhibited cyclical flow reductions for 160 +/- 29 min as well as attenuated ex vivo platelet aggregation. Bitistatin at 100 micrograms/kg, i.v. prolonged the bleeding time 4 x normal at 15 min post-administration but returned to normal at 3 h. Thus, in a canine model of in vivo platelet aggregation, bitistatin is an effective antiplatelet agent to inhibit periodic cyclical flow reductions. Bitistatin also exhibits reversible effects of ex vivo platelet aggregation as well as on bleeding time.     

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.     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Influence of vitamin E on platelet aggregation and thrombocythemia in the rat.

Collagen-induced platelet aggregation was increased in 9-10 wk old vitamin E deficient rats although there was no difference in platelet count between deficient and control animals. With a more prolonged deficiency (at 15 wk) both platelet aggregation and platelet counts were elevated in the vitamin E deficient animals.     

Effect of aggregation inducers and inhibitors on the functional properties and the ultrastructure of platelets]

The influence of inductors and inhibitors of platelet aggregation on certain functional properties and structural peculiarities of platelets are investigated. Inductors of aggregation, such as ADP and thrombin, will cause the calcium binding to the platelet membrane to be diminished, whereas inhibitory substances of platelet aggregation will cause an increase of calcium binding. Inductors increase the number of partially degranulated platelets. Inhibitors, such as triphtazin and papaverin, inhibit these morphological changes.     

Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca2+-induced platelet aggregation

Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.     

Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation.

The effects of aqueous extracts of raw and boiled garlic and onions were studied in vitro on the collagen-induced platelet aggregation using rabbit and human platelet-rich plasma. A dose dependant inhibition of rabbit platelet aggregation was observed with garlic. Onion also showed dose-dependent inhibitory effects on the collagen-induced platelet aggregation but this inhibition was of a lesser magnitude compared to garlic when related to dose. The concentration required for 50% inhibition of the platelet aggregation for garlic was calculated to be approximately 6.6 mg ml(-1) plasma, whereas the concentration for onion was 90 mg ml(-1) plasma. Boiled garlic and onion extracts showed a reduced inhibitory effect on platelet aggregation. Garlic but not onion significantly inhibits human platelet aggregation in a dose-dependent fashion. The potency of garlic in inhibiting the collagen-induced platelet aggregation is approximately similar to that of rabbit platelets (8.8 mg ml(-1) produced 50% inhibition of platelet aggregation). The results of this study show that garlic is about 13 times more potent than onion in inhibiting platelet aggregation and suggest that garlic and onion could be more potent inhibitors of blood platelet aggregation if consumed in raw than in cooked or boiled form.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.