Surfactin C inhibits platelet aggregation

This study was designed to investigate the effect of surfactin C, which is derived from Bacillus subtilis, on platelet aggregation and homotypic leucocyte aggregation. Surfactin C strongly and dose-dependently inhibited platelet aggregation, which was stimulated both by thrombin (0.1 U mL(-1)), a potent agonist that activates the G protein-coupled protease receptor, and by collagen (5 microg mL(-1)), a potent ligand that activates alpha(IIb)beta(3) with IC50 values (concentration inhibiting platelet aggregation by 50%) of 10.9 and 17.0 microM, respectively. Moreover, surfactin C significantly suppressed the intracellular Ca(2+) mobilization in thrombin-activated platelets. Surfactin C, however, did not affect various integrin-mediated U937 cell aggregation, implying that the anti-platelet activity of surfactin C was not due to its detergent effect but by its action on the downstream signalling pathway. Therefore, the results suggest that surfactin C may have a beneficial therapeutic effect on aberrant platelet aggregation-mediated cardiovascular diseases.     

Thrombin-induced platelet endostatin release is blocked by a proteinase activated receptor-4 (PAR4) antagonist

Endostatin is a potent endogenous inhibitor of angiogenesis that was recently shown to be stored in platelets and released in response to thrombin, but not ADP. In the present study, we have tested the hypothesis that thrombin-induced endostatin release from rat platelets is mediated via proteinase-activated receptor-4 (PAR4). Immunoprecipitation and Western blotting confirmed that endostatin is contained within rat platelets. Aggregation and release of endostatin could be elicited by thrombin (0.5 - 1.0 U ml(-1)) or by specific PAR4 agonist (AYPGKF-NH(2); AY-NH(2); 15 - 50 microM). Significant release of endostatin could be induced by a dose of thrombin below that necessary for induction of aggregation. An adenosine diphosphate (ADP) scavenger, apyrase, inhibited the platelet aggregation induced by thrombin, but not the release of endostatin. In contrast, a selective PAR4 antagonist (trans-cinnamoyl-YPGKF-NH(2); tcY-NH(2)) prevented endostatin release and aggregation induced by thrombin or by AY-NH(2). We conclude that thrombin-induced endostatin release from rat platelets is PAR4-mediated via an ADP-independent mechanism that can occur independently of platelet aggregation.     

Antiplatelet action of ilexoside D,a triterpenoid saponin fromIlex pubescens

The anti-platelet activity of ilexoside D isolated from the roots ofIlex pubescens Hook. et Arn. was investigated inin vitro andex vivo models of platelet aggregation induced by ADP, thrombin or collagen in rats.In vitro ilexoside D inhibited more effectively platelet aggregation induced by ADP and thrombin than by collagen as compared with aspirin.Ex vivo ilexoside D also inhibited platelet aggregation induced by ADP and collagen, but not by thrombin, and the inhibitory action of ilexoside D was more effective than that of aspirin. However,in vitro ilexoside D inhibited very poorly the generation of malonyldialdehyde, which is known to be concomitantly released with thromboxane A₂ during platelet aggregation. These results suggest that the anti-platelet activity of ilexoside D may not be responsible for prostaglandin synthesis in platelets.     

Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: critical roles of ERK2 and cAMP

Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti-platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti-platelet activity. Dihydroginsenoside Rg3 inhibited thrombin-induced platelet aggregation in a concentration-dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 +/- 0.4 microM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL(-1)) with an IC50 of 40.2 +/- 0.9 microM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand-induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen-induced platelet aggregation with an IC50 of 20.0 +/- 0.9 microM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 microg mL(-1)), but not of p38 mitogen-activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.     

The antiplatelet activity of PMC, a potent alpha-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase.

PMC, a potent alpha-tocopherol derivative, dose-dependently (5-25 microM) inhibited the ATP-release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose-dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25 microM) significantly inhibited collagen-stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 microM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 microM) did not affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. PMC (10 and 25 microM) markedly inhibited the exogenous arachidonic acid (100 microM)-induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 microM) in washed human platelets, indicating that PMC inhibits cyclo-oxygenase activity. We conclude that PMC may exert its anti-platelet aggregation activity by inhibiting cyclooxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP-release.     

Ginkgolide C inhibits platelet aggregation in cAMP- and cGMP-dependent manner by activating MMP-9

In this report, we investigated the effect of ginkgolide C (GC) from Ginkgo biloba leaves in collagen (10 mug/ml)-stimulated platelet aggregation. It has been known that matrix metalloproteinase-9 (MMP-9) is released from human platelets, and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GC to form an activated MMP-9 (86-kDa) on gelatinolytic activities. And then, GC dose-dependently inhibited platelet aggregation, intracellular Ca(2+) mobilization, and thromboxane A(2) (TXA(2)) formation in collagen-stimulated platelets. In addition, GC significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. Therefore, we demonstrate that the inhibitory effect of GC on platelet aggregation might be involved into the following pathways. GC may increase intracellular cAMP and cGMP production and MMP-9 activity, inhibit intracellular Ca(2+) mobilization and TXA(2) production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that GC is a potent inhibitor of collagen-stimulated platelet aggregation. It may be a suitable tool for a negative regulator during platelet activation.     

Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides

Xanthones and their glycosides were tested for their antiplatelet activities in washed rabbit platelets. Tripteroside acetate and norathyriol acetate were the most potent inhibitors. Tripteroside acetate inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, ionophore A23187 and thrombin. The IC50 values of tripteroside acetate toward arachidonic acid- (100 microM) and collagen- (10 micrograms/ml) induced platelet aggregation were 10 and 30 micrograms/ml respectively. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, thrombin and ionophore A23187 and also that caused by the incubation of lysed platelet homogenate with arachidonic acid. Tripteroside acetate decreased the formation of inositolphosphate caused by thrombin, collagen and PAF, whereas it had no direct effect on fibrinogen-platelet interaction. It is concluded that xanthone derivatives inhibited platelet aggregation and release reaction by diminishing thromboxane formation and phosphoinositide breakdown.     

Components of traditional Chinese medicine inhibit platelet aggregation by blocking ADP receptor subtypes P2Y_1 and P2Y_(12)

Platelets aggregation and thrombosis formation are major reasons of cardiovascular and cerebral vascular diseases.To develop new generative,potent and safe agents for inhibiting platelet aggregation and preventing above diseases are urgently required.Some traditional Chinese medicines of″Houxue Huayu″have been shown to inhibit platelet aggregation potently.In the present study the mechanisms and the molecular targets of puerarin,salvianolic acid B and the analogue of 3-n-butylphthalide,dl-PHPB were investigated and compared with ticlopidine.Four platelet aggregation inducers,ADP,arachidonic acid,collagen and thrombin were used in the study.It was found that puerarin and dl-PHPB specifically inhibited ADP induced platelet aggregation like ticlopidine did.However,salvianolic acid B inhibited both ADP and collagen induced platelet aggregations with similar potency.Due to existing two ADP receptor subtypes on platelets,P2Y1 and P2Y12,we studied the action of above compounds on the receptors and the signaling pathways.It was found that dl-PHPB decreased IP1 accumulation produced by ADP,but had no effect on IP1 level induced by m-3M3 FBS,an activator of PLC.M-3M3 FBS might attenuate the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation.In addition,dl-PHPB did not affect cyclic AMP formation in platelets by ADP,which is different from P2Y12 antagonist ticlopidine.Puerarin showed the similar effects of dl-PHPB.Therefore,the actions of dl-PHPB and puerarin might be through P2Y1receptor-PLC-βpathway.Salvianolic acid B did not reduce the IP1 accumulation stimulated by ADP.It might act on the receptor subtype P2Y12.Our results suggest that components of Chinese herb medicine might be a resource for development of novel anti-platelet drugs.     

Antiaggregation effect of alum on human platelets

OBJECTIVE: The purpose of this study was to evaluate the effect of alum on human platelet aggregation. METHODS: Platelet-rich plasma fractions were prepared from fresh blood drawn from a group of healthy male volunteers. Platelet aggregation was induced by various inducers. The percent aggregation was recorded in the absence and presence of various concentrations of alum. RESULTS: Alum at concentrations less than 1.5 mg/ml inhibited platelet aggregation induced by collagen, epinephrine. ADP and thrombin in a dose-dependent manner. The IC50s were 0.668, 0.324, 0.250 and 0.191 mg/ml of alum, for collagen-, epinephrine-, ADP- and thrombin-induced platelet aggregation, respectively. In contrast, alum at concentrations up to 1.5 mg/ml did not inhibit ristocetin-induced platelet aggregation. CONCLUSION: Alum has an anti-platelet action and should be used cautiously in the treatment of intractable intravesical hemorrhage. Alum is a cheap anti-platelet drug that needs further investigation.     

Genistein, a protein tyrosine kinase inhibitor, inhibits thromboxane A2-mediated human platelet responses

An isoflavone compound, genistein, which is known as a protein tyrosine kinase inhibitor, concentration-dependently (0.1-30 micrograms/ml) suppressed human platelet aggregation, serotonin secretion, and protein tyrosine phosphorylation induced by collagen or stable thromboxane A2 analogs [U46619 and 9,11-epithio-11,12-methano-thromboxane A2 (STA2)]. However, genistein did not inhibit these thrombin (0.1 unit/ml)-induced platelet responses. Although thrombin induced an increase in the platelet phosphotyrosine content, genistein at 100 micrograms/ml only slightly attenuated thrombin-induced protein tyrosine phosphorylation. Genistein competitively inhibited [3H]U46619 binding to washed platelets, in a concentration-dependent fashion. Daidzein (another isoflavone compound), which does not have a hydroxyl group at the 5-position of genistein and lacks inhibitory activity for protein tyrosine kinase, was found to suppress [3H]U46619 binding, leading to the inhibition of collagen- or STA2-induced platelet responses. These results indicate that the blockage by genistein of platelet responses induced by collagen or thromboxane A2 is due to its preventive action on thromboxane A2 binding to the receptor, rather than via inhibition of protein tyrosine phosphorylation, and that the drug does not appear to be a particularly good inhibitor of tyrosine phosphorylation in intact platelets.     

Effect of 2(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca⁺⁺, [¹²⁵I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca⁺⁺ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.     

Antiplatelet effects of piplartine,an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Objectives Piplartine (piperlongumine; 5,6‐dihydro‐1‐[1‐oxo‐3‐(3,4,5‐trimethoxyphenyl]‐2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in‐vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet‐rich plasma by the agonists collagen, adenosine 5′‐diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin‐ (37%), ADP‐ (71%) and collagen‐ (98%) induced aggregation. The inhibitory effect of piplartine on ADP‐induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l ‐arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y₁₂ purinoceptor antagonist). However, aspirin, a well‐known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic‐acid‐induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A₂ formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.     

Inhibition of collagen- and ADP-induced platelet aggregation by substance P in vivo: involvement of endothelium-derived relaxing factor.

We examined the effect of substance P, a potent stimulator of endothelium-derived relaxing factor (EDRF) release, on responses to collagen and adenosine 3',5'-diphosphate (ADP) in an in vivo model of platelet aggregation. Substance P inhibited platelet aggregation induced in vivo by both collagen and ADP. This anti-platelet effect was particularly pronounced against collagen-induced aggregation and was prevented by prior administration of haemoglobin (Hb), a known inhibitor of EDRF-mediated responses. Collagen-induced platelet aggregation in vitro was unaffected by a concentration of substance P equivalent to that achieved in plasma following in vivo administration. This study provides a clear demonstration of the anti-platelet activity of EDRF in vivo and an indication that its effectiveness may depend on the aggregating agent used.     

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.     

Effects of imidazoline and non-imidazoline alpha-adrenergic agents on canine platelet aggregation

Aggregatory and antiaggregatory effects of imidazol(in)e and non-imidazol(in)e alpha-adrenergic agents on canine platelets were examined turbidimetrically in citrated platelet-rich plasma or washed platelet solution. Each alpha-adrenoceptor agonist alone did not induce aggregation, but adrenaline and noradrenaline potentiated dose-dependently aggregation stimulated by ADP, collagen or thrombin. Small potentiation of ADP- or collagen-stimulated aggregation was also observed in response to oxymetazoline. The alpha2-adrenoceptor antagonists and/or imidazol(in)e alpha-adrenergic agents inhibit dose-dependently adrenaline-potentiated aggregation, whereas alpha1-adrenoceptor antagonists, a beta-adrenoceptor antagonist and non-imidazol(in)e alpha-adrenergic agents were no or less effective in inhibiting adrenaline-potentiated aggregation. The alpha2-adrenoceptor agonists did not reduce inhibitory effect of alpha2-adrenoceptor antagonists for adrenaline-potentiated aggregation. The alpha2-adrenoceptor antagonists and/or imidazol(in)es were no or less effective in inhibiting aggregation induced by ADP or thrombin alone. These results demonstrated that alpha2-adrenoceptor-blocking agents and/or imidazol(in)e alpha-adrenergic agents inhibit effectively the adrenaline-potentiated platelet aggregation.     

The novel and orally active thrombin receptor antagonist E5555 (Atopaxar) inhibits arterial thrombosis without affecting bleeding time in guinea pigs

Thrombin is a powerful agonist for platelets, the action of which is mediated by the thrombin receptor protease-activated receptor-1 (PAR-1). Recently, we discovered that E5555 (1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl) ethanone hydrobromide) is a potent thrombin receptor antagonist. We evaluated the anti-platelet and anti-thrombotic effects of E5555. E5555 inhibited the binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP) to PAR-1 with a half maximal inhibitory concentration (IC(50)) value of 0.019μM. E5555 showed potent inhibitory effects on human platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.064 and 0.031μM, respectively, but had no effect on platelet aggregation induced by either ADP or collagen. Similarly, E5555 showed potent and selective inhibitory effects on guinea pig platelet aggregation induced by thrombin and TRAP with IC(50) values of 0.13 and 0.097μM, respectively. The antithrombotic activity of E5555 in vivo was evaluated in a photochemically-induced thrombosis (PIT) model using guinea pigs. Oral administration of E5555 at 30 and 100mg/kg prolonged the time to occlusion by 1.8-fold and 2.4-fold, respectively, compared with controls. Furthermore, E5555 did not prolong bleeding time in guinea pigs at the highest tested dosage of 1000mg/kg. The drug interactions between E5555 and tissue plasminogen activator (tPA) were evaluated. Intravenous administration of 1mg/kg tPA significantly prolonged bleeding time, and its effects were not altered by the oral co-administration of 300mg/kg E5555. These results suggest that E5555 could be a therapeutic option for atherothrombotic disease.     

YD-3, a novel inhibitor of protease-induced platelet activation

1. In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole] were examined. 2. YD-3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC(50)=28.3 microM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet-activating factor (PAF) or U46619. YD-3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin-induced fibrin formation was not affected by YD-3, indicating YD-3 does not inhibit the proteolytic activity of thrombin. 3. In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (> or =0.1 u ml(-1)) of thrombin or SFLLRN, the protease-activated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC(50) values of 38.1 microM and 5.7 microM, respectively. 4. YD-3, at 100 microM, had no effect on ristocetin-induced glycoprotein Ib (GPIb)-dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5. In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml(-1)) could still elicit intracellular Ca(2+) mobilization, and the rise of [Ca(2+)](i) was prevented by either leupeptin or YD-3. 6. Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.     

Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation

Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis.     

尖吻蝮蛇毒小分子多肽的分离及抗血小板聚集作用

目的从尖吻蝮蛇毒中分离纯化一种抗血小板聚集小分子多肽,研究其理化性质以及对ADP、胶原、凝血酶诱导的血小板聚集反应的影响。方法经SephadexG-75凝胶过滤,超滤,DEAE-SepharoseCL-6B离子交换层析法分离纯化蛇毒组分,采用高效液相鉴定纯度,用SDS-凝胶电泳(SDS-PAGE)测定其分子量,用比浊法测定其抗血小板聚集活性。结果从尖吻蝮蛇毒中分离相对分子质量约为7862u等电点为4.29的组分,该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性。结论此法成功地从尖吻蝮蛇毒中纯化出抗血小板聚集组分。该组分与去整合素比较相似,可能属于去整合素家族。     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.