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神经病学与神经康复学杂志 2004年第1期1卷 58-61页

作者：冯智英苗玲上海第二医科大学附属仁济医院神经内科上海200001 上海第二医科大学附属仁济医院神经内科上海200001

自发性脑出血(ICH)是神经科常见的急症之一.最近的研究表明[1],脑血管疾病造成死亡中15%是由于自发性脑出血的缘故.自发性脑出血造成继发性神经损伤的主要原因有脑组织受损,脑水肿的形成等.目前,血肿周围的脑水肿可以加重脑出血后神经... 详细信息

自发性脑出血(ICH)是神经科常见的急症之一.最近的研究表明[1],脑血管疾病造成死亡中15%是由于自发性脑出血的缘故.自发性脑出血造成继发性神经损伤的主要原因有脑组织受损,脑水肿的形成等.目前,血肿周围的脑水肿可以加重脑出血后神经损伤的机制日益受到重视,而凝血酶(Thrombin)在其中起重要作用.

关键词：脑出血/酶学凝血酶/代谢综述人类

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Purification, crystallization and preliminary X-ray analysis of a Sco1-like protein from Bacillus subtilis, a copper-binding protein involved in the assembly of cytochrome c oxidase

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2003年第7期59卷 1299-1301页

作者： Imriskova-Sosova, I Ye, QL Hill, BC Jia, ZC Queens Univ Dept Biochem Kingston ON K7L 3N4 Canada

The putative copper-delivery protein BsSco from Bacillus subtilis is a member of the Sco family of cytochrome c oxidase assembly proteins. BsSco is a membrane protein and the soluble domain has been cloned and expressed in Escherichia coli as a fusion with glutathione-S-transferase. The fusion protein was isolated from the cell lysate using a glutathione-affinity column and the soluble domain of BsSco was released by treatment with thrombin. Sufficient amounts of the soluble domain have been obtained for crystallization. Crystals obtained by hanging-drop vapour diffusion diffract to a resolution of 2.3 Angstrom at a synchrotron source. The space group is P6 and the unit-cell parameters are a = 67.74, b = 67.74, c = 189.58 Angstrom.

关键词：枯草芽孢杆菌/化学细菌蛋白质类克隆分子铜结晶/方法电子传递复合物Ⅳ 谷胱甘肽转移酶膜蛋白质类/化学膜蛋白质类/遗传学膜蛋白质类/分离和提纯蛋白质结构三级重组融合蛋白质类溶解度凝血酶/代谢 X线衍射/方法

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Biotin derivatives of D-Phe-Pro-Arg-CH₂Cl for active-site-specific labeling of thrombin and other serine proteinases

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ANALYTICAL BIOCHEMISTRY 2001年第2期296卷 254-261页

作者： Anderson, PJ Bock, PE Vanderbilt Univ Sch Med Dept Pathol Med Ctr N C3321A Nashville TN 37232 USA

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor D-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91-96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88-1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89-1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a greater than or equal to7- to 14-atom spacer was needed for sensitive (similar to 10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizo-thrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin I that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity. (C) 2001 Academic Press.

关键词：亲和力标记物/化学亲和力标记物/代谢结合部位生物素/化学酶前体/代谢寡肽类/化学寡肽类/代谢凝血酶原/代谢丝氨酸内肽酶类/代谢凝血酶/代谢

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Platelet glycoprotein Ibα binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第9期276卷 6161-6168页

作者： Li, CQ Vindigni, A Sadler, JE Wardell, MR Washington Univ Sch Med Dept Biochem & Mol Biophys St Louis MO 63110 USA Washington Univ Sch Med Dept Med St Louis MO 63110 USA Washington Univ Sch Med Howard Hughes Med Inst St Louis MO 63110 USA

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha -thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate D-Phe-Pro-Arg-pNA. The K-d for the glycocalicin-thrombin interaction was 1 muM at physio logical ionic strength but was highly salt-dependent, decreasing to 0.19 muM at 100 mM NaCl (Gamma (salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha -binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K-d values for their interactions with GPIb alpha were similar to those of wild type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K-d values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, me demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteri

关键词：纤维蛋白肽A/分泌肝素低分子量/药理学水蛭素类/类似物和衍生物水蛭素类/药理学肽碎片/药理学血小板糖蛋白GPⅠb-Ⅸ复合物/代谢重组蛋白质类/代谢氯化钠/药理学凝血酶/化学凝血酶/代谢人类

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A high-throughput microtiter plate-based calcium assay for the study of protease-activated receptor 2 activation

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ANALYTICAL BIOCHEMISTRY 2001年第2期290卷 378-379页

作者： Hawthorne, SJ Macey, M Howells, G Walker, B Queens Univ Belfast Sch Pharm Ctr Med Biol Belfast BT9 7BL Antrim North Ireland Royal London Hosp Trust Dept Haematol London E1 2BB England St Bartholomews & Royal London Sch Med & Dent Dept Oral Pathol London E1 2AD England

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Role of arginine 129 in heparin binding and activation of antithrombin

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第25期275卷 18976-18984页

作者： Desai, U Swanson, R Bock, SC Björk, I Olson, ST Univ Illinois Ctr Mol Biol Oral Dis Chicago IL 60612 USA Virginia Commonwealth Univ Dept Med Chem Richmond VA 23298 USA Univ Utah Dept Med Salt Lake City UT 84132 USA Univ Utah Dept Bioengn Salt Lake City UT 84132 USA Swedish Univ Agr Sci Uppsala Biomed Ctr Dept Vet Med Chem SE-75123 Uppsala Sweden

The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of similar to 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state.

关键词：抗凝血酶类/化学抗凝血酶类/代谢精氨酸/代谢肝素/代谢动力学模型分子蛋白质结合蛋白质构象重组蛋白质类/化学重组蛋白质类/代谢凝血酶/代谢

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A binding site for the kringle II domain of prothrombin in the Apple 1 domain of factor XI

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第41期275卷 31954-31962页

作者： Baglia, FA Badellino, KO Ho, DH Dasari, VR Walsh, PN Temple Univ Sch Med Sol Sherry Thrombosis Res Ctr Philadelphia PA 19140 USA Temple Univ Sch Med Dept Med Philadelphia PA 19140 USA Temple Univ Sch Med Dept Biochem Philadelphia PA 19140 USA NCI Med Branch NIH Bethesda MD 20892 USA

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (Al) domain of factor XI (FM). Since prothrombin (and Ca2+) can bind FXI and can substitute for HK (and Zn2+) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant Al domain (rAl, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K-d similar to 71 nn) and the rAl domain (K-d similar to 239 nM) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit I-125-rAl (Glu(1)-Ser(90)) binding to prothrombin, I-125-prothrombin binding to FXI, and 125I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rAl binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the Al domain of FXI, acted synergistically to inhibit I-125-rAl binding to prothrombin. Mutant rAl peptides (V64A and I77A), which did not inhibit FXI binding to III, retained full capacity to inhibit rAl domain binding to prothrombin, and mutant rAl peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rAl domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the Al domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that th

关键词：结合部位结合竞争性因子Ⅺ/化学因子Ⅺ/遗传学因子Ⅺ/代谢碘放射性同位素动力学激肽原高分子量/代谢带双硫键三环蛋白结构区肽碎片/代谢肽碎片/药理学蛋白质结合/药物作用凝血酶原/化学凝血酶原/代谢重组蛋白质类/化学重组蛋白质类/遗传学重组蛋白质类/代谢表面等离激元凝血酶/代谢人类

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Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第30期275卷 22942-22947页

作者： Wang, W Nagashima, M Schneider, M Morser, J Nesheim, M Queens Univ Dept Biochem Kingston ON K7L 3N6 Canada Queens Univ Dept Med Kingston ON K7L 3N6 Canada Berlex BioSci Richmond CA 94804 USA

Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombin-activable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C;the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca2+-dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mM Ca2+. Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other.

关键词：氨基酸序列抗体单克隆/免疫学钙/代谢羧肽酶U 羧肽酶类/药理学纤维蛋白溶解/药物作用点突变蛋白质C/代谢凝血酶/代谢血栓调节蛋白/化学血栓调节蛋白/遗传学血栓调节蛋白/免疫学血栓调节蛋白/代谢

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α₁-Proteinase inhibitor, α₁-antichymotrypsin, or α₂-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第17期275卷 12799-12805页

作者： Ikari, Y Fujikawa, K Yee, KO Schwartz, SM Univ Washington Dept Pathol Seattle WA 98195 USA Univ Washington Dept Biochem Seattle WA 98195 USA Fred Hutchinson Canc Res Ctr Dept Mol & Cellular Biol Seattle WA 98109 USA

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be alpha(1)-proteinase Inhibitor, alpha(1)-antichymotrypsin, and alpha(2)-macroglobulin. The chemically inactivated alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only antiintegrin (alpha(nu)beta(3) and alpha(5)beta(1)) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.

关键词：主动脉/细胞学印迹法蛋白质细胞黏附细胞运动细胞培养的色谱法琼脂糖剂量效应关系药物纤维蛋白/代谢纤连蛋白类/代谢整合素类/代谢肌平滑血管/细胞学肌平滑血管/酶学沉淀素试验凝血酶/代谢时间因素 α1抗糜蛋白酶/生理学 α1抗胰蛋白酶/生理学 α巨球蛋白类/生理学动物牛人类

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Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第30期275卷 22756-22763页

作者： Bradford, HN Pixley, RA Colman, RW Temple Univ Sch Med Sol Sherry Thrombosis Res Ctr Philadelphia PA 19140 USA Temple Univ Sch Med Dept Med Philadelphia PA 19140 USA Temple Univ Sch Med Dept Physiol Philadelphia PA 19140 USA

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-M-V complex. Although factor XII will bind to the intact platelet through GP Ib alpha (glycocalicin) without activation, we now report that factor XIIa (0.37 mu M), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor Wa but not with factor XII zymogen or factor XIIa indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nM) binding to gel-filtered platelets and, at concentrations of 50 nM, was able to block 50% of the HX binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HX and factor XII bind to GP IB alpha. Factor XIII may serve to regulate thrombin binding to the GP Ib receptor by colocalizing with HK, to control the extent of platelet aggregation in vivo.

关键词：氨基酸序列抗体/免疫学结合竞争性印迹法蛋白质因子Ⅻ/代谢血小板聚集/药物作用血小板聚集抑制剂/药理学血小板糖蛋白GPⅠb-Ⅸ复合物/免疫学血小板糖蛋白GPⅠb-Ⅸ复合物/代谢蛋白质结合受体 PAR-1 受体凝血酶/代谢凝血酶/拮抗剂和抑制剂凝血酶/药理学人类

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