WHOLE BLOOD AGGREGATION AND PLASMA LYSO-PAF RELATED TO SMOKING AND ATHEROSCLEROSIS

1. Aggregation of diluted whole blood (impedance method) and thromboxane B2 production during aggregation were measured in cigarette smokers and non-smokers, aged 41-68 years, with (n = 14) and without (n = 15) major symptomatic peripheral vascular disease. The plasma level of the lyso derivative of platelet activating factor (lyso-PAF) was also measured using a bioassay with 14C-serotonin labelled rabbit platelets, after extraction and acetylation to active PAF. 2. Aggregation to ADP and collagen was significantly less in non-smokers without vascular disease (n = 8) than in the other three groups (P less than 0.01; ANOVA). Thromboxane B2 production was not significantly different between the groups. There was no significant difference in plasma lyso-PAF between groups. No change was found in any variable after smokers smoked two cigarettes. 3. In these older age subjects, both vascular disease and the smoking habit were associated with greater whole blood aggregation. However, current smoking and the smoking of two cigarettes did not affect aggregation in subjects with vascular disease and plasma lyso-PAF levels were not consistently related to either smoking or vascular disease.     

Effect of cefaclor on guinea pig platelet aggregation in vitro

Effects of cefaclor (3-chloro-7-D-(2-phenyl-glycinamido)-3-cephem-4-carboxylic acid) on PAF, ADP, collagen, endotoxin, and thrombin-induced platelet aggregation were examined in vitro with the use of guinea pig platelet-rich plasma and washed platelets. PAF, even at concentrations lower than its minimum effective concentration, enhanced ADP- or endotoxin-induced platelet aggregation and prolonged the time to attain the maximum aggregation. PAF also enhanced collagen-induced platelet aggregation and shortened the lag time. Cefaclor (CCL) inhibited the PAF, ADP or thrombin induced platelet aggregation and shortened their maximum aggregation times at higher concentrations such as 300 micrograms/ml or more. CCL also inhibited the collagen-induced platelet aggregation and prolonged the lag time, but showed no effect on endotoxin-induced platelet aggregation. The effect of CCL was almost the same as that of latamoxef (LMOX). CCL and LMOX, however, showed no effect on cellular Ca2+ increase produced by PAF, ADP, or thrombin, suggesting that the inhibitory effect of CCL and LMOX on platelet aggregation is caused by the inhibition of fibrinogen binding to the glycoprotein IIb/IIIa complex.     

Effects of aspirin,dipyridamole, nifedipine and cavinton which act on platelet aggregation induced by different aggregating agents alone and in combination

Summary The effects of aspirin, nifedipine, dipyridamole and cavinton on platelet aggregability in patients with atherosclerosis has been studied using various agents to induce aggregation.The drugs reduced platelet aggregability when aggregation was induced by ADP, adrenaline, or collagen alone. However, if platelet aggregation were induced by combinations of the agonists (including combinations of ADP with either adrenaline or platelet-activating factor (PAF), adrenaline with PAF, and collagen with ADP), the anti-aggregant effects of aspirin, dipyridamole, and cavinton were significantly reduced. The effect of nifedipine was less markedly reduced, especially by combinations which included adrenaline.The data suggest that positive agonist interactions may lead to a reduction in the therapeutic activity of antiplatelet drugs.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Contribution of ADP to the amplification of primary platelet aggregation by platelet activating factor (PAF): modulatory role of aspirin

Platelet Activating Factor (PAF)-induced human platelet aggregation in citrated plasma is accompanied by activation of the cyclo-oxygenase pathway and release of intracellular constituents including Adenosine-5'-diphosphate (ADP). Inhibition of the cyclo-oxygenase pathway by aspirin prevented the amplification of primary platelet aggregation induced by threshold concentrations of PAF. Removal of ADP by enzymatic systems had little or no effect on PAF-induced full aggregation, but reversed the aggregating effect of PAF (at 10 times threshold concentrations) on 'aspirinated' platelets. Aspirin also prevented the synergism between PAF and ADP when subthreshold concentrations of both compounds were combined. Similar results were obtained in heparinized platelet-rich plasma. Thus, ADP may amplify the primary response to PAF but its role is modulated by the availability of the cyclo-oxygenase pathway products.     

Platelet aggregation inhibitors from Agkistrodon acutus snake venom

Among all the purified components from A. acutus venom, including ADPase, 5'-nucleotidase, phospholipase A2 and fibrinogenases, only the venom ADPase (50-100 micrograms/ml) shows marked inhibitory action on ADP (10 microM)-, collagen (10 micrograms/ml)- and sodium arachidonate (100 microM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5'-nucleotidase (100 micrograms/ml) inhibited ADP-induced platelet aggregation by 31 +/- 4% (n = 4, P less than 0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 micrograms/ml) did not significantly inhibit platelet aggregation induced by ADP (10 microM), collagen (10 micrograms/ml) or sodium arachidonate (100 microM). However, when the fibrinogenase (fraction IX, 100 micrograms/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen (20 micrograms/ml)- and ADP (10 microM)-induced platelet aggregations by 34 +/- 9% (n = 4, P less than 0.05) and 35 +/- 6% (n = 4, P less than 0.05), respectively. The phospholipase A2 (100 micrograms/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 mu moles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5'-nucleotidase activities.     

SHORT TERM VITAMIN E SUPPLEMENTATION HAS NO EFFECT ON PLATELET FUNCTION, PLASMA PHOSPHOLIPASE A2 AND LYSO-PAF IN MALE VOLUNTEERS

1. Based largely upon in vitro studies, vitamin E has been reported to inhibit phospholipase A2 activity, to alter phospholipid metabolism and reduce platelet aggregation. 2. The effect of dietary supplementation with D-alpha-tocopherol (1500 iu/day for 14 days) was studied in nine males, 41-63 years old, comparing active treatment with a preceding placebo period. 3. Despite an increase from 2.6 +/- 0.8 (s.d.) x 10(-5) mol/L to 6.0 +/- 1.8 10(-5) mol/L in plasma vitamin E there were no significant changes in the aggregation of diluted whole blood or platelet rich plasma to adenosine diphosphate (ADP) or collagen, in plasma phospholipase A2 activity or plasma lyso-platelet-activating factor (lyso-PAF) (bioassay after in vitro acetylation to PAF). 4. High dose vitamin E dietary supplementation had no effect on these phospholipid and platelet parameters.     

Pharmacokinetic and pharmacodynamic profiles of vapiprost, a selective, long-lasting thromboxane receptor antagonist, after single and multiple oral administration to healthy volunteers.

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time-profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet-rich plasma (PRP) induced by a stable TXA2 receptor agonist U-46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U-46619 ex vivo were simultaneously examined and compared. In the single-dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one-compartment open model with a first-order absorption. The area under plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives (t1/2) remained approximately constant within the range of 0.99-1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U-46619 and the secondary aggregation by ADP were inhibited; that induced by U-46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time-profile of inhibiting whole blood platelet aggregation that was induced by U-46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nifedipine Reduces Thromboxane A2 Production by Platelets Without Changing Aggregation in Hypertensive Pregnancy

Abstract: Platelet aggregation and thromboxane A₂ generation were studied in hypertensive pregnant women using normotensive non-pregnant and pregnant controls. In hypertensive pregnancy, adrenaline- and adenosine diphosphate-induced platelet aggregation was at the non-pregnant level and lower than in normotensive pregnancy. Collagen-induced aggregation was at a lower level in hypertensive pregnancy than in both control groups. Thromboxane generation during spontaneous clotting and in platelet-rich plasma did not differ between the three groups. However, thromboxane generation was low during aggregation induced by small collagen concentrations in hypertensive pregnancy, but at higher collagen concentrations the difference between the groups disappeared. During nifedipine treatment (10 mg t.i.d.), aggregation and thromboxane production in platelet-rich plasma induced by the three stimuli remained unaltered in hypertensive pregnancy, while thromboxane synthesis during spontaneous clotting was reduced. In nifedipine-treated non-pregnant controls, only EC₈₀ for adrenaline-induced aggregation decreased. In vitro, pharmacological concentrations of nifedipine inhibited platelet aggregation and thromboxane production. In conclusion, nifedipine reduces thromboxane generation in spontaneous clotting, without inhibiting platelet aggregation and thromboxane production in platelet-rich plasma in hypertensive pregnancy. Reduced aggregability of platelet ex vivo may reflect their continuous activation and desensitiza-tion in vivo in hypertensive pregnancy.     

Inhibition of platelet aggregation following chronic in vivo treatment of rats with nicotine: prevention by simultaneous application of propranolol

Platelet aggregability is known to be enhanced and platelet-survival time shortened in smokers when compared with nonsmokers. Up to now it is unknown which of the substances in tobacco smoke are responsible for these effects. To evaluate a possible role of nicotine, rats were chronically treated with the alkaloid (10 mg/kg/day), continuously released from subcutaneously implanted osmotic minipumps. Surprisingly, after 8 weeks, platelet sensitivity toward the aggregating stimulus adenosine 5'-diphosphate (ADP) was markedly reduced. The mean ADP concentration required to induce half the maximum rate of aggregation (EC50) was 0.88 mumol/L in nicotine-treated animals, as compared with 0.67 mumol/L in controls (p less than 0.002). Platelet aggregability remained normal when the rats were treated simultaneously with nicotine and the beta blocker propranolol (3.5 mg/kg/day); for these animals, the mean EC50 for ADP was 0.73 mumol/L. These results are suggestive of a catecholamine-mediated action of nicotine. However, neither the basal levels of cAMP in platelet-rich plasma, nor the cAMP levels attained after stimulation of platelet adenylate cyclase with prostaglandin E1 (PGE1), were affected by 8 weeks of treatment with nicotine or nicotine plus propranolol. No effect on platelet aggregation was observed when the rats were treated with nicotine for only 2 weeks, or when nicotine or nicotine plus cotinine were added to platelet-rich plasma in vitro in concentrations equal to those attained in vivo after 8 weeks. Thus, prolonged application of nicotine in vivo caused an inhibition of ADP-induced rat platelet aggregation presumably mediated by beta-catecholaminergic stimulation of platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

Captopril augments both basal and frusemide-induced natriuresis in normal man by suppression of circulating angiotensin II.

1. The effect of increasing doses of orally administered aspirin (30-900 mg) on platelet aggregation and ATP release induced by arachidonic acid (AA), collagen and platelet activating factor (PAF) was assessed in 12 normal volunteers. 2. Aspirin ingestion was associated with a significant increase in EC50 for AA (P less than 0.0001) and collagen (P less than 0.0001) but not for PAF (P greater than 0.495) although the normal biphasic aggregation response for the latter was abolished. Maximum ATP release was reduced by aspirin for all three agonists. 3. The mean maximum degrees of inhibition of platelet aggregation induced by aspirin for AA, collagen and PAF were 100%, 48% and 21% of baseline, respectively. The corresponding mean maximum inhibition of ATP release was 100%, 63% and 57%. The minimum cumulative doses of aspirin producing these effects were 240, 240 and 90 mg for AA, collagen and PAF respectively. For collagen alone, there was a significant decrease in the degree of inhibition of aggregation between the last dose on day 1 (150 mg) and the baseline measurement on day 2. 4. Platelets from female subjects were more sensitive to collagen (P less than 0.05) and AA (P less than 0.01) stimulation compared with males. However, prior to aspirin ingestion, PAF produced a greater maximum response in platelets from females (P less than 0.02) while following aspirin ingestion PAF-induced activation was inhibited to a greater degree in females (P less than 0.02). 5. These results indicate that collagen- and PAF-induced platelet activation are only partially dependent on cyclo-oxygenase and for PAF this seems related only to the second phase of aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of platelet inhibitors in normal and in hypercholesterolaemic subjects.

Dibutyryl cyclic AMP, forskolin, dipyridamole and butyl imidazole inhibited platelet aggregation (induced by ADP or collagen) in washed platelets more than in platelet-rich plasma preparations. Aspirin, indomethacin and epoprostenol (prostacyclin, PGI2) showed no preferential inhibition of these platelet preparations. When platelet-rich plasma from either normal or familial hypercholesterolaemic (FH) subjects was used, aspirin, indomethacin and dipyridamole (but not forskolin) inhibited platelet aggregation in normal subjects more than in FH patients. When low doses of aspirin (75 mg daily for 7 days) or dipyridamole (250 mg, single dose) were administered in vivo, platelet aggregation was inhibited more in the normal subjects in comparison to the patient group.     

Regulation of human platelet activation--analysis of cyclooxygenase and cyclic AMP-dependent pathways

We have studied the regulation of human platelet activation by cyclic AMP (cAMP), and the cyclooxygenase products by examining the effect of prostacyclin (PGI2) and indomethacin on platelet aggregation, release reaction and thromboxane B2 (TxB2) generation induced by the full dose range of common platelet agonists in both platelet-rich plasma and washed platelets. Platelet aggregation, [14C]-5HT and TxB2 release induced by "threshold" and "supramaximal" concentrations of ADP, adrenaline, platelet-activating factor (PAF) and U46619 were totally abolished by low concentrations of PGI2 (3-6 nM). In contrast, platelet activation induced by submaximal concentrations of collagen, thrombin and the calcium ionophore A23187 was only partially inhibited by PGI2 (3-3000 nM). PAF-induced release reaction like that induced by ADP and adrenaline was totally dependent on the cyclooxygenase products and aggregation, while U46619-induced release reaction was only partially dependent on aggregation and the cyclooxygenase products. While both PGI2 (18-3000 nM) and indomethacin (10 microM) abolished collagen-induced aggregation and the aggregation-mediated release reaction, neither inhibitor significantly inhibited platelet adhesion or the adhesion-mediated release reaction. Maximal thrombin-induced aggregation and release reaction was also not significantly inhibited by PGI2 (300 nM) or indomethacin (10 microM). Thromboxane (TxB2) generation induced by sub-maximal to maximal concentrations of collagen, thrombin and A23187 was, although significantly inhibited, not abolished by PGI2. These results demonstrate that PAF is a "weak" agonist similar to ADP and adrenaline, U46619 is an agonist intermediate between weak and strong which induces a release reaction that is only partially dependent on aggregation, but unlike the strong agonists, is totally susceptible to inhibition by PGI2, PGI2 is an indirect inhibitor of phospholipase activation, which does not significantly inhibit non-aggregation-mediated arachidonate mobilization, induced by the strong agonists, and the so-called third pathway in the collagen and thrombin-induced release reaction, which is insensitive to indomethacin, is also insensitive to elevators of cAMP such as PGI2.     

Derivatives of 2-methylenepropane-1,3-diol as new antagonists of platelet activating factor

Two new achiral platelet activating factor (PAF) antagonists, N-[5-[[2-methylene-3- [[(octadecylamino)carbonyl]oxy]propoxy]carbonyl]pentyl]pyridinium bromide and 3-[6-[[2-methylene-3- [[(octadecylamino)carbonyl]oxy]propoxy]carbonyl]hexyl]thiazolium bromide were synthesized from 2-methylenepropane-1,3-diol. Platelet aggregation in platelet-rich plasma from rabbits, induced by racemic C16-PAF, was competitively antagonized by 9 or 10. At concentrations less than or equal to 10(-4) M, neither compound 9 nor compound 10 caused platelet aggregation, nor did they inhibit platelet aggregation induced by collagen or adenosine diphosphate. Bronchoconstriction in the guinea pig and hypotension in the rat, induced by racemic C16-PAF, were also effectively antagonized by 9 and 10. Both appear to be more potent as PAF antagonists than Takeda's CV-3988.     

新型血小板GPⅡb/Ⅲa受体拮抗剂Z4A5抗血小板作用的研究

目的研究血小板GPⅡb/Ⅲa受体拮抗剂Z4A5抑制血小板聚集的活性、稳定性以及对已聚集血小板的解聚作用。方法采用比浊法测定Z4A5对体外二磷酸腺苷(aden-osine diphosphate,ADP)诱导血小板聚集的抑制作用和稳定性及对ADP诱导聚集血小板的解聚作用。结果 Z4A5浓度依赖性的抑制ADP诱导的血小板聚集,半数抑制浓度(50%concentration of inhibition,IC50)为(0.46±0.05)μmol.L-1(n=10,P<0.05);Z4A5在血浆中孵育3 h仍保持85.55%(P<0.05)的活性;10μmol.L-1 Z4A5在加入ADP诱导聚集的血小板3 min及7 min后的解聚率为分别为22.66%(P<0.05)及30.93%(P<0.01)。结论 Z4A5在体外有较强的、稳定的抑制血小板聚集作用,而且对聚集血小板有一定的解聚作用。     

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.     

Some properties of rat platelet aggregation and effects of butylated hydroxytoluene, warfarin and aspirin

The platelet aggregation characteristics of male Sprague-Dawley (Jcl:SD) rats were investigated. Epinephrine, ristocetin, serotonin and platelet-activating factor were ineffective in rat platelets. Heparinized platelet-rich plasma (PRP) was more sensitive than citrated PRP to three aggregating agents, ADP, collagen and arachidonic acid. Butylated hydroxytoluene (BHT) and BHT quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) inhibited ADP- and collagen-induced aggregation at concentrations over 10(-3) M in vitro. The ADP-, collagen- and arachidonic acid (0.5-2.0 mM)-induced aggregations of PRP obtained from rats given 1.20% BHT in the diet for 7 days were normal, while arachidonic acid (3.9 mM)-induced aggregation of PRP from BHT-fed rats was significantly lower than control. PRP from rats given aspirin and warfarin also aggregated normally with ADP or collagen addition. These results suggest that heparinized PRP may be preferable in platelet aggregation analyses in rats and reaffirmed that effects on platelet aggregation may not play a key role in BHT-induced bleeding. Platelet aggregation capacity also does not necessarily reduce in haemorrhages induced by aspirin or warfarin.     

Amplification of platelet response during acute inflammation in rats

Enhanced aggregation of platelets was observed in platelet-rich plasma, but not in washed platelet suspension (WPS), during acute inflammation in rats. Incubation of WPS with inflammatory plasma increased the aggregatory response to ADP, but the plasma itself did not cause aggregation of platelets. It potentiated the aggregatory response of normal platelets, when platelets were stimulated with arachidonic acid, thrombin, calcium ionophore A23187 or phorbol-12-myristate-13-acetate. Pretreatment of rats with indomethacin (10 mg/kg) did not prevent the increased aggregation response of platelets due to inflammation. This response was not due to any of the biogenic amines nor was it due to platelet factor 4. The activity of the inflammatory plasma was reduced when it was incubated with phospholipase A2, indicating the involvement of platelet-activating factor (PAF). The activity was present both in the lipid and the protein fraction of the inflammatory plasma. The results indicate that a substance(s) released in the circulation during inflammation renders the platelets hyperactive. This substance appears to be a protein which is present in the inflammatory plasma and acts together with PAF to cause increased aggregation of platelets.     

眼镜蛇毒金属蛋白酶atrase A抗血小板聚集作用及机制

目的研究中华眼镜蛇毒金属蛋白酶atrase A对血小板聚集的影响及其相关的机制。方法测定atrase A对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸、瑞斯托霉素、凝血酶诱导血小板聚集的影响情况,并通过蛋白质免疫印迹检测atrase A对血小板膜糖蛋白和血管假血友病因子的酶切情况。结果中华眼镜蛇毒金属蛋白酶atrase A能明显抑制由瑞斯托霉素和凝血酶诱导的血小板聚集,这种抑制作用呈量效、时效关系。而atrase A和血小板预孵5min后对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸诱导的血小板聚集有微弱的抑制作用,预孵时间延长至30min对血小板聚集有明显的抑制作用。蛋白质免疫印迹结果显示atrase A能特异性酶切血小板膜糖蛋白GPIb,但对vWF几乎没有酶切作用。结论中华眼镜蛇毒金属蛋白酶atrase A对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸、瑞斯托霉素、凝血酶诱导的血小板聚集均有抑制作用,其中对瑞斯托霉素和凝血酶诱导的血小板聚集具有明显的抑制作用,其机制是通过酶切血小板膜糖蛋白GPIb。