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1.
江浙蝮蛇毒酸性磷脂酶A2抑制制血小板作用的机理 总被引:7,自引:3,他引:4
江浙蝮蛇毒酸性磷脂酶A2(APLA2)对多种致聚剂引起的血小析具有抑制作用,这种抑制并非是由于APLA2与血小板膜结合,引起膜流动性降低造成。APLA2不裂解血小板,电镜观察表明它能抑制血小析部颗粒的中心化,使之不易被激活。进一步研究显示APLA2是作用在骨架上,使致聚剂引肌动蛋白单体聚合难以进行,APLA2导致的cAMP浓度的升高正说明了这点。结果提示APLA2首先作用于轿小板细胞膜,引起cAM 相似文献
2.
尖吻蝮蛇酸性磷脂酶A2的纯化和初步晶体学 总被引:5,自引:0,他引:5
为进一步揭示影响血小板聚集活性的结构基础,纯化了江西尖吻蝮蛇(Agkistradun acutus)酸性磷脂酶A2。江西省吻蝮蛇蛇毒经CM-Sepharose离子交换柱层析,两步DEAE-Sepharose离子交换柱层析以及Mono)GFPL离子交换柱层析,得到了SDS聚丙烯酰胺凝胶电泳和等和集电泳均一的磷脂酶A2(PLA2),其分子量为16.5KD,等电点为4.3,经测定,该酶具有抑制ADP诱地 相似文献
3.
人血小板生成素受体c—MPL膜内部分的聚合作用 总被引:3,自引:0,他引:3
血小板生成素(TPO)是调节血小板生成最主要的细胞因子,其生物学效应由其受体c-MPL介导。利用酵母双杂合系统研究c-MPL膜内部分在TPO信号转导途径中的功能。首先用反转录PCR(RT-PCR)方法从人红白血病HEL细胞系总RNA中扩增并克隆P型c-MPL(MPLP)膜内部分cDNA,经测序验证后克隆至双杂合载体pAS2和pGAD424中,重组质粒命名为pASMM和pGADMM。将pASMM与p 相似文献
4.
PKC,PKA和TPK在血小板激活中的作用 总被引:3,自引:0,他引:3
利用^32P-NaH2PO4标记猪血小板,然后,以PMA,凝血酶,PGE1腺苷等处理,结果表明,随着PMA激活PKC,血小板发生聚集,35μmol/LPGE1或1mmol/LdbcAMP不能抑制50nmol/LPMA诱导的血小板聚集,腺苷却能抑制PMA诱导的血小板聚集(EC50=0.1mmol/L,db-cAMP,腺苷都不能抑制100nmol/LPMA诱导的40kD蛋白磷酸化,PKA激活不能抑制P 相似文献
5.
本工作采用分离培养家兔肺内小动脉平滑肌细胞(PASMCs),观察了外源性血小板活化因子(plateletactivatingfactor,PAF)、BN52021(PAF受体拮抗剂)、吲哚美辛、维拉帕米对PASMCs产生血栓素A_2(TxA_2)、前列环素(PGI_2)及对细胞膜Ca~(2+)-ATPase活力的影响。结果表明:(1)基础状态下PASMCs存在花生四烯酸(AA)代谢。(2)外源性PAF通过受体后途径激活环加氧酶促进AA代谢致TXA_2及PGI-2增加,TXA_2/PGI_2比值无明显变化。(3)外源性PAF能直接抑制Ca~(2+)-ATPase活力。(4)维拉帕米可逆转PAF抑制PASMCs膜Ca~(2+)-ATPase活力的效应。 相似文献
6.
PKC、PKA和TPK在血小板激活中的作用 总被引:1,自引:0,他引:1
利用~(32)P-NaH_2PO_4标记猪血小板,然后以PMA、凝血酶、PGE_1、腺苷等处理,结果表明,随着PMA激活PKC,血小板发生聚集。35μmol/LPGE_1或1mmol/LdbcAMP不能抑制50nmol/LPMA诱导的血小板聚集,腺苷却能抑制PMA诱导的血小板聚集(EC_(50)=0.1mmol/L),db-cAMP、腺苷都不能抑制100nmol/LPMA诱导的40kD蛋白磷酸化。PKA激活不能抑制PMA激活的PKC。在PMA、凝血酶激活的血小板中,PKC、TPK都发生激活,40kD底物既是PKC的底物又是TPK的底物,PKC和TPK在血小板聚集中起着重要的调节作用。 相似文献
7.
钙调素拮抗剂对人肺癌细胞增殖抑制及与cAMP信号系统相关性之初探 总被引:1,自引:0,他引:1
对钙调素(CaM)拮抗剂—三氟拉嗪(trifluoperazine,TFP)在人肺癌细胞PLA801的增殖抑制中的作用和CaM与cAMP信号系统水平的变化进行了研究.用5、10、15和20μmol/LTFP处理人肺癌细胞时观察到TFP在抑制细胞内CaM活性的同时,抑制了细胞的增殖.药物处理的细胞在软琼脂中形成的集落数减少且明显小于对照组细胞.使用流式细胞光度术分析细胞周期的结果表明:10μmol/LTFP处理抑制了G1期细胞向S期的转移.当用10μmol/LTFP作用细胞5min时,细胞内cAMP水平达到正常水平的1.8倍,直到3h仍明显高于正常水平.同时,cAMP依赖的PKA的活性在加药后15min上升到正常水平的2.8倍,直到加药3h.活性仍保持较高水平,结果表明:钙调素功能的抑制,提高了PLA-801细胞内cAMP系统的水平,Ca2+-CaM和cAMP-PKA两个信号系统的协调作用,抑制了细胞的增殖 相似文献
8.
用钙离子沉淀和柱层析的方法从猪血小板中分离纯化到了一个分子量为54kD、具有Annexin样生物学作用的钙结合蛋白,并用对钙离子有高度特异亲和力的偶氮胂试剂确定了该蛋白质具有钙结合能力。为了研究54kD钙结合蛋白对猪血小板膜磷脂酶A2的作用,我们用密度梯度离心方法制备了猪血小板膜,并用其作为酶和底物的材料,用游离脂肪酸的微量显色法建立了测定猪血小板膜结合的PLA2活性的方法。我们的实验结果表明:54kD钙结合蛋白在反应体系钙离子浓度低于1mmol/L时,对膜结合的PLA2具有激活作用;在钙离子大于1mmol/L时;对膜结合的PLA2具有抑制作用。并且抑制作用不随底物浓度的升高而降低. 相似文献
9.
精-甘-天冬-丝氨酸四肽对二磷酸腺苷诱导大鼠血小板活化的信号转导的影响 总被引:3,自引:0,他引:3
本工作在二磷酸腺苷(ADP)活化的大鼠血小板上,观察精-甘-天冬-丝上肽(RGDS肽)对血小板聚集、蛋白磷酸化、蛋白激酶C和丝裂素活化蛋白激酶活性的影响。结果发现,50μmol/LADP引起血小板聚集时,蛋白激酶C(PKC0及丝裂经蛋白激酶(MAPK)活性增加,并引起95和66kD蛋白磷酸化。应用50,100和200μmol/LRGDS肽与基共同孵育,呈浓度依赖地抑制ADP引起的血小板聚集和对PK 相似文献
10.
将尖吻蝮蛇毒酸性磷脂酶 A2 I( A.a A P L A2 I) 的基因克隆至表达载体p B L M V L2 , 在大肠杆菌 R R1 中成功表达。表达产物 A.a A P L A2 I约占细菌蛋白质总量的30 % , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 F P L C Superose T M12 纯化, 产物经过 S D S P A G E 检测只有单一条带。对表达的 A.a A P L A2 I进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 A.a A P L A2 I的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 A2( A P L A2) 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 A2( P L A2) 的结构与这些活性的关系进行了讨论 相似文献
11.
R N Puri F X Zhou R F Colman R W Colman 《Biochemical and biophysical research communications》1989,162(3):1017-1024
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. 相似文献
12.
Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena. 相似文献
13.
J C Yun K P Ohman J R Gill H Keiser 《Canadian journal of physiology and pharmacology》1991,69(5):599-604
The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP. 相似文献
14.
《The International journal of biochemistry》1976,7(11):535-540
- 1.1. Cyclic AMP (cAMP), an inhibitor of platelet aggregation, is pointed out to exert its effect on platelet function solely by stimulating platelet protein kinase.
- 2.2. Data from our laboratory and others indicate that in the presence of physiological concentrations of cAMP, ATP and Mg+, platelet protein kinase(s) phosphorylates and alters the activity of key platelet membrane and cytoplasmic enzymes. These enzymes include those involved in platelet cAMP synthesis and degradation, ATP→ADP hydrolysis coupled with either monovalent ion transport or contractile function, glycogen synthesis and degradation, prostaglandins and endoperoxides production, and other protein substrates such as microtubules whose function in platelets is yet to be determined.
- 3.3. A scheme is presented in which an increase in platelet cAMP concentration and phosphorylation correlates with an inhibition of platelet aggregation.
15.
Edema toxin is a key virulence determinant in anthrax pathogenesis that causes augmentation of cAMP inside host cells. This exotoxin has been implicated in facilitating bacterial invasion by impairing host defenses. Here, we report for the first time that edema toxin plays an important role in suppression of platelet aggregation; an effect that could be of vital significance in anthrax afflicted subjects. It was found that edema toxin induces a dose dependent and time dependent increase in cAMP inside rabbit platelets. Elevation of cAMP led to suppression of platelet aggregation as demonstrated by in vitro aggregation assays. A 95% suppression of platelet aggregation in response to thrombin and a complete suppression in response to ADP, at toxin concentrations of 7 and 2.2 nM, respectively, were observed. Antibody neutralized wild type edema factor and non-toxic mutants of this binary toxin failed to show any alteration in the normal aggregation pattern. Edema toxin caused the activation of cAMP dependent protein kinase A inside platelets, a phenomenon that could be speculated to initiate the cascade of events responsible for suppressing platelet aggregation. Furthermore, in vivo bleeding time registered a sharp increase in response to edema toxin. These findings can explicate the systemic occurrence of hemorrhage, which is a prominent symptom of anthrax. This study exemplifies how Bacillus anthracis has evolved the ability to use host's physiological processes by mimicking the eukaryotic signal transduction machinery, thus inflicting persistent infection. 相似文献
16.
G.I. Johnston S. Heptinstall W. Lösche 《Biochemical and biophysical research communications》1985,127(3):822-827
We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins. 相似文献
17.
The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation. 相似文献
18.
K Kitamura K Kangawa M Kawamoto Y Ichiki H Matsuo T Eto 《Biochemical and biophysical research communications》1992,185(1):134-141
An increase in the cellular concentration of cAMP leads to the inhibition of platelet aggregation. We have been investigating endogenous peptides which inhibit platelet function, using an assay which detects increase in platelet cAMP. Compared with the human adrenal medulla, a pheochromocytoma (PC) contained abundant peptides that elevate platelet cAMP. About 90% of the activity was found in the SP-III fraction which contained strongly basic peptides. From the SP-III fraction, peptides P-1, P-2 and P-3 were purified to homogeneity as endogenous peptides which elevated platelet cAMP. A gas phase sequencer was used to identify these peptides as follows: P-1 = vasoactive intestinal peptide (VIP); P-2 = calcitonin gene related peptide-I (CGRP-I); P-3 = CGRP-II. It is well known these peptides are potent vasorelaxants. VIP and CGRP-I significantly increased platelet cAMP levels 15- and 6-fold, respectively. These results suggest that VIP and CGRP-I and -II act upon platelets as well as upon vascular tissue. 相似文献
19.
Torti M Bertoni A Sinigaglia F Balduini C Payrastre B Plantavid M Chap H Mauco G 《FEBS letters》2000,466(2-3):355-358
Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2. 相似文献
20.
Several studies have shown that platelets are a major source of circulating neuropeptide Y (NPY) immunoreactivity in rats, but the effects of this vasoconstrictor peptide on platelets are not well-defined. Recently, it was reported that porcine NPY was an inhibitor of in vitro human platelet aggregation induced by epinephrine, an observation which would have important implications regarding platelet-vascular interactions during states involving platelet activation and thrombosis. Thus, we undertook the present studies, in an attempt to confirm the earlier report, and to extend those observations to human NPY. In contrast to the recent report, we found no inhibitory effect of either human or porcine NPY on epinephrine- or collagen-induced aggregation of human platelets from normal subjects. Likewise, specific NPY Y-1 and Y-2 agonists had no direct or indirect action on platelet aggregation. Finally, the effect of human NPY on intraplatelet cAMP was measured. The peptide had no effect on either basal or iloprost-stimulated cAMP levels. We hypothesize that the role of NPY in the platelet-vascular interaction is in promoting vasoconstriction associated with platelet aggregation, and does not include inhibition of further thrombosis. 相似文献