长角血蜱唾液腺Apyrase的生物学特性和功能的研究

长角血蜱在吸血时 ,其唾液腺中的 apyrase可明显地抑制由 A DP所诱导的血小板聚集反应 ,并呈剂量反应关系 ,1/ 2对唾液腺可以完全抑制血小板的聚集反应。通过 apyrase的动力学、 HPLC分子筛、等电聚焦电泳和温度敏感性实验证明 ,水解 ATP和 ADP并不是 ATP酶、 ADP酶或几种酶共同作用的结果 ,而是一种酶即 apyrase的作用结果。Apyrase对 Ca2 具有依赖性。而 Mg2 、Zn2 、Mn2 对之影响较小 ,Hg2 和EDTA是 apyrase的抑制剂。吸血对蜱唾液腺 apyrase活性影响较大 ,吸血 6天时其活性最大 ,吸血后活力明显下降。A pyrase与哺乳动物的 5′-核苷酸酶相似 ,它是一种糖基 -磷脂酰肌醇 ( GPI)膜锚结构蛋白。     

Edema-inducing activity of phospholipase A2 purified from human synovial fluid and inhibition by aristolochic acid

A neutral-active, Ca²⁺-dependent phospholipase A₂ (PLA₂) purified 11,000-fold from human synovial fluid (HSF) induced edema when injected into the mouse foot pad. The edema produced by HSF-PLA₂ was dose-dependent and was positively correlated with the dose-dependent in vitro expression of PLA₂ activity. Maximum edema was achieved within 45 min after the injection and persisted for atleast 6 h. Aristolochic acid [8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid], a major chemical component derived from various species ofAristolochia plant, produced a dose-dependent inhibition of in vitro phospholipid hydrolysis by HSF-PLA₂, porcine pancreatic PLA₂, snake venom (Naja naja) PLA₂, and PLA₂ isolated from human platelet. The sensitivity of these PLA₂s to inhibition by aristolochic acid varied markedly: HSF-PLA₂>N.naja PLA₂>human platelet PLA₂>porcine pancreatic PLA₂. The inhibition of HSF-PLA₂ by aristolochic acid was independent of substrate concentration (18–144M and Ca²⁺ concentration (0.1–4.0 mM). These observations indicate that inhibition of HSF-PLA₂ by aristolochic acid may result from direct interaction with the enzyme. When aristolochic acid was mixed with HSF-PLA₂ and then injected into the mouse foot pad, edema was inhibited in a dose-dependent manner and was positively correlated with in vitro inhibition of PLA₂ activity. Alkylation of HSF-PLA₂ withp-bromophenacyl bromide concomitantly inhibited both enzyme and edema-inducing activity. These results clearly demonstrate that the neutral-active, Ca²⁺-dependent PLA₂ isolated from human synovial fluid is proinflammatory and that catalytic activity is positively correlated with in vivo proinflammatory effects.     

芪丹通脉片对家兔血小板聚集的影响

目的 :观察芪丹通脉片对实验性家兔血小板聚集的影响。方法 :连续 7天灌胃给药后 ,采用Bom氏比浊法测量其血小板聚集率。结果 :实验结果表明 ,与对照组比较 ,芪丹通脉片明显降低二磷酸腺甘(ADP)和花生四烯酸 (AA)诱导的家兔血小板聚集率 (P <0 .0 5～ 0 .0 1) ,对胶原诱导的家兔血小板聚集率有降低趋势。结论 :芪丹通脉片具有抑制血小板聚集的作用。     

Substances that increase the cyclic AMP content prevent platelet aggregation and the concurrent release of pharmacologically active substances evoked by arachidonic acid

Arachidonic acid-induced platelet aggregation was inhibited by prostaglandins E₁ and F_2α (PGE₁ and PGF_2α), papaverine and dibutyryl cyclic AMP. Prostaglandin E₂ displayed a biphasic effect, as concentrations below 2 μM potentiated aggregation, whereas concentrations above it were inhibitory. Isoproterenol (up to 10 mM) failed to block aggregation but inhibition was uncovered in presence of adrenergic α-blocking agents. Isoproterenol potentiated aggregation due to sub-threshold amounts of arachidonic acid, and this effect, but not that due to PGE₂, was suppressed by the α-blocking agents. Isoproterenol and PGE₂ appear thus to enhance arachidonic acid-induced platelet aggregation after interacting with different receptor sites. The yield of rabbit aorta contracting activity formed during AA-induced aggregation was markedly reduced by PGE₁, dibutyryl cyclic AMP and high concentrations of PGE₂, and was increased by low concentrations of the latter. PG-like activity was not significantly reduced when aggregation and generation of rabbit aorta contracting activity were inhibited by bibutyryl cyclic AMP. It is hypothesized that interaction of human platelets and arachidonic acid results in formation of different pharmacologically active materials, possibly bearing similar lipoperoxide structures. Generation of one portion of these materials is controlled by the adenyl cyclase-cyclic AMP system, whereas another portion, that comprises the natural PG, is cyclic AMP-independent. Prostaglandins formed during platelet aggregation have a regulatory role and modulate the platelet response, rather than constitute a trigger stimulus for aggregation.     

Inhibition of platelet phospholipase-A2 as a mechanism for the anti-aggregating effect of linoleic acid

Linoleic acid was incorporated into platelet phospholipids and then released after activation of phospholipase A₂ (PL-A₂) with thrombin or ionophore A23187. The rate of this release was tenfold lower for linoleic than for arachidonic acid. This observation strongly suggests that incorporation of linoleic acid in platelet phospholipids might inhibit platelet PL-A₂ and might explain the anti-aggregating effect of linoleic acid. In fact it has also been shown that linoleic acid inhibits PL-A₂ activity at concentrations which antagonize platelet aggregation. Moreover, in experimental conditions where platelet cyclo-oxygenase is totally inhibited, aggregation does occur and can still be blocked by linoleic acid. This latter observation led to the conclusion that the anti-aggregating effect of linoleic acid is independent from prostaglandin pathway and is probably related to the phospholipid metabolism.     

中性粒细胞对洗涤血小板聚集功能的影响

目的:观察非激活或激活的中性粒细胞(PMN)对洗涤血小板聚集功能的影响。方法:采用Born方法测定血小板聚集功能。结果:非激活的PMN(5×10⁹cells/L)上清液对ADP和花生四烯酸(AA)诱导的血小板聚集具有显著抑制作用,阿司匹林可增强这种抑制作用;肉豆寇佛波醇(fMLP)及血小板活化因子(PAF)激活的PMN悬液或其上清液均能活化血小板聚集,且PMN悬液的诱聚作用比其上清液更强;阿司匹林对fMLP或PAF激活的PMN悬液或其上清液均无明显抑制作用。结论:不同状态(非激活或激活状态)的PMN对正常血小板的反应性表现出完全相反的作用,即非激活的PMN抑制血小板反应性,而激活的PMN则具有促血小板活化聚集作用。     

葡萄籽原花青素体外抗血小板聚集的机制研究

目的: 研究葡萄籽原花青素(PC)体外抗血小板聚集的可能机制。方法: 应用血小板聚集仪研究PC、二亚苯基碘鎓(DPI,非特异性NADPH氧化抑制剂)和夹竹桃麻素(apocynin,特异性NADPH氧化酶抑制剂)对胶原诱导的健康志愿者血小板最大聚集率的影响。用化学发光仪检测PC对血小板NADPH氧化酶活性、一氧化氮(NO)含量和超氧阴离子(O₂^-·)水平的影响。用流式细胞术观察PC对血小板活化标志物PAC-1和CD62P表达率的影响。结果: PC(100 μmol/L)、apocynin(10 μmol/L)和DPI(100 μmol/L )均可显著抑制胶原蛋白诱导的血小板最大聚集率(P<0.01)。胶原蛋白激活的血小板NO含量显著降低,O₂^-·含量显著增加,100 μmol/L PC可使二者明显恢复(P<0.05)。添加了100 μmol/L PC的样本中,血小板NADPH氧化酶活性受到明显的抑制(P<0.01),血小板PAC-1 和CD62P表达率也显著降低(P<0.05)。结论: 葡萄籽原花青素可能通过抑制NADPH氧化酶的活性,进而影响血小板NO和O₂^-·含量,在一定程度上阻断血小板活化过程,最终达到抗血小板聚集的作用。     

The role of phospholipids in platelet function

Platelet phospholipids undergo significant alterations during aggregation induced by thrombin or other agents. There is an early increase in phosphatidic acid, with a decrease in phosphatidyl inositol. De novo synthesis of most phospholipids from 14C-glycerol is decreased. Thrombin stimulates 32P-phosphate incorporation into di- and triphosphoinositides, suggesting increased phosphorylation of phosphatidyl inositol during aggregation. Arachidonic acid for prostaglandin synthesis is released from platelet phospholipids. Thrombin induced aggregation results in release of arachidonic acid primarily from phosphatidyl choline and phosphatidyl inositol. The availability of free arachidonic acid may be regulated by platelet phospholipase A2 activity. The latter activity is stimulated by thrombin, requires calcium ions, and is inhibited by agents which elevate cyclic adenosine monophosphate. Phospholipids are probably an essential component of the platelet surface lipoprotein procoagulant activity known as platelet factor 3. There is evidence that calcium ions may mediate binding between gamma carboxyglutamic acid residues on the amino terminal portion of prothrombin and negatively charged phosphate groups on phospholipid micelles. Binding of prothrombin to phospholipid on the platelet surface may orient the former such as to facilitate the prothrombinase activity of Factor Xa. Platelet phospholipids and platelet factor 3 activity are decreased in some congenital and myeloproliferative disorders. Increases in these factors may be associated with thrombotic and arterial occlusive disorders.     

Tea polyphenols inhibit acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and platelet-activating factor-induced platelet aggregation

BACKGROUND: Tea extracts have antiallergic and anti-inflammatory actions in rats and mice. However the mechanism through which tea polyphenols act in vivo are still incompletely understood. We found inhibitory effects of black tea extracts on an fMLP-induced aggregating response in a rabbit platelet-polymorphonuclear leukocyte (PMN) system. METHOD: To elucidate whether 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) production in PMNs and/or PAF-stimulated platelet activation were inhibited, the effects of tea polyphenols were investigated on the enzyme activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), PAF biosynthesis in A23187-activated rabbit PMNs, and rabbit platelet aggregation. By comparing the inhibitory effects of 31 galloyl esters and gallic acid, the structure-inhibitory activity relationship was characterized. RESULTS: Theaflavin and its galloyl esters and pentagalloyl glucose were found to be potent inhibitors of the acetyltransferase (IC(50) = 28-58 microM) and the PAF biosynthesis as well as (-)-epicatechin-3-O-gallate (IC(50) = 72 +/- 13 microM) and (-)-epigallocatechin-3-O-gallate (IC(50) = 46 +/- 6 microM). On the other hand, flavan-3-ols without galloyl group at C-3 and gallic acid did not show significant enzyme inhibition. In addition, theaflavin and its galloyl esters (IC(50) = 32-77 microM) and geranyl gallate, farnesyl gallate and geranylgeranyl gallate (IC(50) = 6.4-7.6 microM) were found to be potent inhibitors of PAF- and TPA-induced rabbit platelet aggregation but not A23187-induced aggregation. CONCLUSIONS: Theaflavin and its galloyl esters in black tea extract, and isoprenyl gallates were potent inhibitors of PAF synthesis and platelet aggregation and these activities may be relevant to the claimed therapeutic effects of tea extracts.     

Profile analysis and immunoglobulin E reactivity of wheat protein hydrolysates

BACKGROUND: Wheat protein hydrolysates have been traditionally used as food additives and are now being used in cooking worldwide. There have been a few studies on the relationship between the molecular mass distribution and the immunoglobulin E (IgE) reactivity of the wheat protein hydrolysates. METHOD: We analyzed the peptide profile of commercial wheat protein hydrolysate samples from enzymatic or acid hydrolysis of wheat protein using size exclusion chromatography. We further investigated the IgE reactivity of the wheat protein hydrolysates using the inhibition ELISA method and sera of 5 patients sensitive to wheat. RESULTS: The wheat protein enzymatic hydrolysate samples showed high concentrations of peptides with molecular masses greater than 1,050 Da, whereas in contrast, the wheat protein acid hydrolysates showed extremely low concentrations of peptides with molecular masses greater than 1,050 Da. Tested wheat protein acid hydrolysates hardly inhibited the patient IgE binding ability to wheat proteins in the five patient sera. On the contrary, some tested wheat protein enzymatic hydrolysate samples inhibited the IgE binding ability to wheat proteins. CONCLUSION: These results suggested that the uptake of wheat protein enzymatic hydrolysates might still have the possibility of causing food allergic reactions in patients allergic to wheat and the processed foods containing them.     

Effect of fibrinogen degradation products on platelet aggregation

The digestion of fibrinogen with various concentrations of trypsin results in the formation of a variety of degradation products. Degradation products formed in this way have been purified by DEAE cellulose column chromatography and their effects on platelet aggregation investigated.TWO METHODS HAVE BEEN USED TO STUDY PLATELET AGGREGATION: a turbidimetric method which assesses platelet aggregation by the ability of adenosine diphosphate (ADP) to clump platelets and a method which assesses platelet adhesiveness by their ability to adhere to glass and to each other (modified Hellem technique, 1960).Three breakdown products produced by trypsin-digested fibrinogen were studied and all showed ;antithrombin' activity: two inhibited platelet aggregation, but one accelerated aggregation in both systems. Another product prepared by digestion of fibrinogen with urokinase-activated plasminogen has been shown to possess the ability to enhance ADP-induced platelet aggregation.     

Regulation of canine platelet function II. Catecholamines

The action of epinephrine (E) on canine platelet aggregation is described. Although E did not induce a change in platelet shape or aggregation, potentiation of aggregation induced by the following agents was observed at physiological E concentrations (that is, less than 10 nM/1): arachidonic acid; the dense granule agonists, ADP and serotonin (5-HT); and collagen. Epinephrine-induced potentiation was in part independent of formation of arachidonic acid metabolites, and E potentiated the aggregating action of the bivalent cationophore A23187. Potentiation was inhibited by alpha-adrenergic receptor antagonists phenoxybenzamine, phentolamine, and ergotamine, and mimicked by alpha-adrenergic receptor agonists norepinephrine, clonidine, and in some cases, phenylephrine. The beta-adrenergic receptor agonists isoproterenol and dobutamine inhibited ADP-induced aggregation, and this action was presented by pretreating the platelets with propranolol and dichloroisoproterenol. An augmentation of the aggregation response of platelets to arachidonic acid was observed in blood samples withdrawn when circulating catecholamines were elevated. The physiological implication of epinephrine acting as a gain controller that alters the relationship between actuating signal and the platelet response to an agonist is discussed.     

急性心肌梗塞患者血小板聚集性改变及药物的影响

急性心肌梗塞患者肾上腺素诱导血小板聚集性降低,与病情有一定关系,一般两周内恢复;5-羟色胺诱导聚集阳性率及不解聚率均明显高于正常对照组;每日口服100mg阿斯匹林明显抑制5-羟色胺及肾上腺素诱导聚集,但不能完全抑制尿激酶引起的血小板聚集增加;蝮蛇抗栓酶对5-羟色胺诱导聚集有明显抑制作用。     

Relationship of inhibition of prostaglandin synthesis in platelets to anti-aggregatory and anti-inflammatory activity of some benzoic acid derivatives

The relationships between inhibition of platelet prostaglandin (PG) synthesis and aggregation, and suppression of inflammation were investigated with a number of benzoic acid (aspirin-like) chemicals. The compounds studied were 2-acetylbenzoic acid (ABA), 3-methylphthalide (3-MP), 3-propionyloxybenzoic acid (3-PBA) and 2-propionyloxybenzoic acid (2-PBA). At 0.5–0.6 mM, 3-MP inhibited the second phase of ADP-induced aggregation in human platelets, and reduced collagen-induced aggregation by 50%. Previous studies have shown 2-PBA to inhibit aggregation by 50%. Previous studies have shown 2-PBA to inhibit aggregation at similar concentrations. In contrast, ABA required 10 times higher concentrations, and low concentrations actually potentiated aggregation. Inhibition of PG synthesis from¹⁴C-arachidonic acid (AA) by human platelets was shown for 2-PBA, but not for 3-PBA, or ABA. At high concentration (1 mM), 3-MP showed modest inhibitory activity. Significant inhibition of AA aggregation was produced by ASA (83%), 2-PBA (76%) and 3-MP (69%), an order reflecting their inhibition of PG synthesis, where ABA and 3-PBA did not inhibit AA aggregation. Carrageenin-induced edema of the rat paw was suppressed by 3-MP, ABA and 2-PBA; all being roughly equipotent with aspirin. In contrast, 3-PBA did not suppress edema. Following oral administration of the drugs to rats, PG synthesis from labeled AA by rat platelets showed similar profiles to effects of the drugs on PG synthesis in human platelets. This suggests that biotransformation or species differences are not explanations for the observed differences in activity in the various test systems. The results indicate that, in a related series of chemicals there is not a good correlation between ability to inhibit platelet PG synthesis, anti-aggregatory activity and anti-inflammatory activity. Multiple mechanisms of action, differing sensitivities of various tissue PG synthetases, or unidentified factors could be involved.Reprint requests should be sent to R.B. Philp.     

柚皮素通过PI3K/Akt通路拮抗血小板聚集的体外研究

目的:探讨柚皮素拮抗二磷酸腺苷(ADP)诱导的血小板聚集的作用及机制。方法:采用血小板聚集仪观察不同浓度柚皮素对ADP诱导的大鼠血小板聚集的影响。采用荧光显微镜观察柚皮素对血小板在固化纤维蛋白原上扩展功能的影响。采用Western blot检测磷脂酰肌醇3-激酶(PI3K)表达及蛋白激酶B(Akt)磷酸化水平。结果:柚皮素能剂量依赖性地抑制ADP诱导的体外血小板聚集,其半数抑制浓度(IC_(50))值为(132.1±31.7)μmol/L。柚皮素灌胃给药,对ADP诱导的大鼠体内血小板聚集也有明显的抑制作用。柚皮素还能显著抑制血小板在固化纤维蛋白原上的扩展。Western blot结果显示,柚皮素能明显抑制ADP刺激的PI3K表达和Akt磷酸化同时,PI3K广谱抑制剂LY294002能增强柚皮素对Akt磷酸化的抑制作用。结论:柚皮素可能通过抑制血小板PI3K/Akt信号通路发挥抗血小板聚集的作用。     

Effects of sodium periodate on platelet functions.

Removal of N-acetylneuraminic acid from the platelet surface causes rapid removal of platelets from the circulation but causes little change in other platelet functions. We have now investigated the effects of sodium periodate which is thought to oxidize the sialic acid of glycoproteins on cell surfaces and has been shown to affect the functions of other cells. NaIO4 (1 to 10 mm) caused aggregation of stirred suspensions of washed platelets from rabbits. Calcium was required in the suspending medium for NaIO4-induced aggregation. Aggregation was not accompanied by the release of amine storage granule contents nor by cell lysis. Aggregation induced by NaIO4 was not inhibited by creatine phosphate-creatine phosphokinase, by platelet inhibitors that raise platelet cyclic AMP levels such as prostaglandin E1 or methylxanthines, by agents that modify platelet surface--SH groups (N-ethylmaleimide, p-chloromercuribenzene sulfonate), nor by cytochalasin B and/or colchicine which interfere with platelet contractile processes. Drugs such as acetylsalicyclic acid, penicillin G, or cephalothin had no effect on NaIO4-induced aggregation. NaIO4-induced aggregation was practically independent of platelet metabolism since it was not affected by low temperatures and was only slightly inhibited by a combination of antimycin and iodoacetate. Periodate treatment enhanced CO2 production by platelets. When rabbit platelets were pretreated, without stirring, with NaIO4 (0.01 to 1 mm), they did not aggregate. They retained their disc shape and granule contents. However, this pretreatment with NaIO4 inhibited aggregation induced by ADP and inhibited both aggregation and release induced by collagen, thrombin, arachidonic acid, and the ionophore A23,187. The extent of inhibition corresponded to the concentration of NaIO4 used to pretreat the platelets. In contrast, concanavalin A-induced aggregation was unchanged by NaIO4 pretreatment. When NaIO4 oxidation was followed by sodium borohydride (NaBH4) reduction, the effects caused by NaIO4 pretreatment on ADP-induced aggregation and collagen- or thrombin-induced aggregation and release were partially reversed. Pretreatment with NaIO4 also diminished the rate of serotonin uptake and decreased the ability of platelets to adhere to collagen-coated surfaces or to the subendothelial structures of the rabbit aorta. Platelets which had been treated with NaIO4 and then reinfused into rabbits did not survive, and in this way were similar to platelets from which surface sialic acid had been removed by neuraminidase treatment. Since NaIO4 has been shown to oxidize sialic acid on red cell membranes, it seems probably that alteration of surface sialic acid resulted in recognition of the periodate-treated platelets as "foreign" by the reticuloendothelial system. When NaIO4 oxidation was followed by NaBH4 reduction, platelet survival returned toward normal values.     

The importance of aspartate aminotransferase for platelet aggregation

In bovine platelets aspartate aminotransferase has a high activity. The enzyme in vitro is inhibited in a dose dependent manner by aminooxyacetate (IC50 = 10(-4) M), hydroxylamine (IC50 = 10(-4) M), and cycloserine (IC50 = 5 X 10(-3). The inhibitory effect of all the three compounds is strongest at low substrate (aspartate) concentration. Blocking of aspartate aminotransferase activity by these compounds in intact platelets is accompanied by the inhibition of ADP and collagen-induced aggregation. Among the three compounds the strongest inhibitor of platelet aggregation was hydroxylamine, which was also the most effective inhibitor of aspartate aminotransferase. Other metabolic blockers, i.e. dinitrophenol (DNP), rotenone and antimycin also inhibited the aggregation of platelets, and a synergism has been demonstrated between DNP, rotenone and antimycin A action on platelet aggregation and blockade of aspartate aminotransferase activity. The results are interpreted to mean that transamination is of importance in the energy production in the activated platelet, probably through its participation in reducing equivalents transport from the cytosol to the mitosol via the malate: oxaloacetate: aspartate shuttle.     

Pyridoxal phosphate inhibition of platelet function

The effect of pyridoxal phosphate (PLP) on human platelet function in vitro was studied. PLP inhibited adenosine diphosphate (ADP)-induced shape change, aggregation, and the potentiation by ADP of arachidonic acid-induced aggregation. This inhibition could easily be reversed by increasing concentrations of ADP or by removing PLP. The addition of sodium borohydride to PLP-treated platelets produced an irreversible inhibition of ADP aggregation. Thus it is possible that PLP inhibited ADP-induced platelet function by forming a Schiff base with platelet-surface amino groups. PLP also produced a partial inhibition of platelet aggregation to epinephrine, arachidonic acid, A23187, and a dose-dependent inhibition of [14C]serotonin release to epinephrine and arachidonic acid. PLP did not inhibit [14C]serotonin release to A23187, nor did it suppress arachidonic acid-induced malondialdehyde production. The conclusion is drawn that the partial inhibition by PLP of platelet aggregation observed to epinephrine, arachidonic acid, and A23187 resulted from PLP's inhibition of the effect of released ADP.     

2型糖尿病患者血小板聚集功能与肾微血管病变的关系

目的 :探讨 2型糖尿病 (DiabetesMellitus,DM )患者血小板聚集活性与肾微血管病变的关系。方法 :测定 15 6例 2型DM患者血小板功能指标 ,如血小板聚集率、血小板平均体积和肾微血管功能指标 ,如 2 4h尿白蛋白 (Alb)、2 4h尿β2 微球蛋白 ( β2 Microglobulin ,β2 MG)排泄量 ,分析二类指标的相关性。结果 :随着DM患者病情进展 ,尿A1b和尿β2 MG排泄不断增加 ,血小板聚集率逐渐上升 ,血小板平均体积逐渐增大 ,两者成明显正相关。结论 :血小板功能活性升高可能是糖尿病肾脏微血管损伤的促进因素之一 ,抑制血小板过度活化 ,是糖尿病治疗中的重要环节     

抗血小板糖蛋白VI单克隆抗体的制备及其功能研究

目的:制备抗血小板糖蛋白VI(GPVI)单克隆抗体,观察其在体外抗血小板黏附和聚集功能。方法:采用基因重组技术体外表达血小板糖蛋白VI胞外区重组蛋白(rGPVI)。以rGPVI免疫小鼠,经细胞融合及筛选后制备抗GPVI单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convu lxin及ADP诱导的血小板聚集的影响;利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果:正确构建了rGPVI表达载体pET-20b(+)-GPVI,rGPVI在原核细胞中有效表达。rGPVI能够被抗Penta-H is单抗和抗GPVI多抗识别。制备的抗GPVI单克隆抗体SZ118能够识别rGPVI,并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convu lxin诱导的血小板聚集,呈抗体剂量依赖性;对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明,SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论:成功制备抗GPVI单克隆抗体SZ118,该抗体与血小板有良好的结合能力,显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。