Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogs

Tsiang, Manuel; Lentz, Steven R.; Dittman, William A.; Wen, Duanzhi; Scarpati, Eleonora M.; Sadler, J. Evan

doi:10.1021/bi00499a005

Cited by 95 publications

(71 citation statements)

References 54 publications

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“…The D3 ES cells (provided by R. Hynes, Massachusetts Institute of Technology) were electroporated with a Not I-linearized targeting vector (see Fig. 1A) and selected in growth medium containing G418 and gancyclovir (gift of Syntex, Palo Alto, CA); doubly resistant colonies were isolated as described (5 (6). The immunoprecipitates were collected on protein G-Sepharose (Pharmacia) and bound proteins eluted with SDS buffer were electrophoresed under reducing conditions.…”

Section: Methodsmentioning

confidence: 99%

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

Healy

Rayburn

Rosenberg

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

224

159

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We have targeted the murine thrombomodulin (TM) gene in embryonic stem cells and generated embryos as well as mice with TM deficiency. The heterozygous TMdeficient (TM+/-) mice as compared to wild-type (TM'/') littermates exhibit 50% reductions in the levels of TM mRNA and TM protein. However, TM+/-mice appear normal and are free of thrombotic complications. The homozygous TMdeficient (TM-/-) embryos die before embryonic day 9.5. An overall retardation in growth and development of TM-/-embryos is first evident on embryonic day 8.5 (8-12 somite pairs). However, no specific pathologic abnormalities are observed. These initial changes take place at a time when TM is normally expressed in the parietal yolk sac. The removal of embryonic day 7.5 TM-/-embryos from maternal decidua and their subsequent culture in vitro allow development to proceed to stages not observed in vivo (13-20 somite pairs) with the appearance of normal blood vessels in the visceral yolk sac and embryo. The results of our studies suggest that the failure of TM-/-embryos to survive at mid-gestation is a consequence of dysfunctional maternal-embryonic interactions caused by the absence of TM in the parietal yolk sac and demonstrate that the receptor is necessary for normal embryonic development in utero.Thrombomodulin (TM) is a natural anticoagulant that forms a 1:1 complex with thrombin (1). This interaction product converts protein C (PC) to its activated form (APC), which proteolytically destroys activated cofactors V and VIII and thereby suppresses generation of thrombin. TM is expressed on vascular endothelial cells and also on synovial cells, meningial cells, mononuclear phagocytes, and platelets (1, 2). TM has also been detected in the rodent embryo on the parietal endoderm of yolk sac, in the aortic arch, embryonic heart, blood vessels, lung buds, and in the developing central nervous system (3, 4). The widespread distribution of TM in embryos and adult animals suggests that the receptor may have alternative functions beyond anticoagulation.In the present investigation, we have targeted the intronless TM gene in embryonal stem (ES) cells and generated mice that transmit the receptor deletion to their progeny. The results demonstrate that mice heterozygous for the TM deletion exhibit half-normal levels of receptor and should serve as a model for defining the contributions of TM to thrombogenesis. The data also show that embryos homozygous for the TM deletion die in utero by embryonic day 9.5 (E9.5) and suggest this lethal phenotype is due to an overall growth retardation secondary to a defect in the parietal yolk sac. MATERIALS AND METHODSTargeting the TM Gene in ES Cells and Generation of TM-Deficient Mice. The D3 ES cells (provided by R. Hynes, Massachusetts Institute of Technology) were electroporated with a Not I-linearized targeting vector (see Fig. 1A) and selected in growth medium containing G418 and gancyclovir (gift of Syntex, Palo Alto, CA); doubly resistant colonies were isolated as described (5). The correctly targeted E...

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Section: Methodsmentioning

confidence: 99%

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

Healy

Rayburn

Rosenberg

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

224

159

View full text Add to dashboard Cite

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“…In view of the numerous non-enzymic (hormonal) activities of thrombin (Fenton, 1988;Davie et al, 1991;Tsiang et al, 1990;Vu et al, 1991;Tapparelli et al, 1993), hirudin fragments can possibly be usefully employed for dissecting molecular aspects of both the dual-type binding of hirudin to thrombin, as well as of other biological functions of this enzyme in vivo. For example, the data of this study show that more than half of the free energy of binding of hirudin HM2 to thrombin is contributed by the N-terminal core domain, as also reported for hirudin HV1 (Betz et al, 1992).…”

Section: S Y T D C T E S G a N Y C L C V G S N V C G E G K N C Q L mentioning

confidence: 99%

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

Vindigni¹,

Filippis²,

Zanotti³

et al. 1994

European Journal of Biochemistry

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Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41 -49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a Nterminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.

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“…In addition, this is the only region of the B chain to contain an amino acid deletion. The precise location of the thrombomodulin-binding site(s) in human a-thrombin has yet to be resolved (5,6,38,39).…”

Section: -----F-vndt-------f-vndt--------s-eds--------s-eds__pnb -F--mentioning

confidence: 99%

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

Banfield

MacGillivray

1992

Proc. Natl. Acad. Sci. U.S.A.

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The cDNA sequence of the B chain of thrombin (EC 3.4.21.5) has been determined from nine vertebrate species (rat, mouse, rabbit, chicken, gecko, newt, rainbow trout, sturgeon, and hagfish). The amino acid sequence identities vary from 96.5% (rat vs. mouse) to 62.6% (newt vs. hagfish). Of the 240 amino acids spanned in all the species compared, there is identity at 110 (45.8%) positions. When conservative changes are included, the amino acid similarity increases to 75%. The most conserved portions of the B chain are the active-site residues and adjacent amino acids, the B loop, and the primary substrate-binding region. In addition, the Arg-Gly-Asp motif is conserved in 9 of the 11 species compared, and the chemotactic/growth factor domain is well conserved in all ofthe 11 species compared. The least conserved regions of the B chain correspond to surface loops, including the putative thrombomodulin-binding sites and one of the hirudin-binding regions. The extent of the amino acid sequence smilarity and the conservation of many of the functional/structural motifs suggests that, in addition to their role in blood coagulation, vertebrate thrombins may also play an important role in the general mechanisms of wound repair.The final reaction of the coagulation pathway is the conversion of fibrinogen to fibrin by the serine protease thrombin (EC 3.4.21.5) (1, 2). In mammals, thrombin is generated from its zymogen, prothrombin, by the limited proteolytic action of factor Xa in the presence of factor Va, calcium ions, and phospholipid (2). In addition, thrombin also activates/ inactivates other coagulation factors such as factor XIII, factors V and VIII, and protein C. The anticoagulant activity of thrombin is regulated through the interaction of thrombin with thrombomodulin (2, 3), an endothelial cell membrane protein related in structure to the low density lipoprotein receptor (4). While thrombomodulin likely binds to thrombin by an exposed surface loop, the precise binding site has yet to be resolved (5, 6). The thrombin/thrombomodulin complex activates protein C, which in the presence of protein S then degrades factors Va and VIlla (2).The enzymatic activity of thrombin is regulated by the endogenous protease inhibitor antithrombin III (2). Thrombin is also inhibited by protein inhibitors from nonplasma sources such as hirudin, from the European medicinal leech (7). The substrate specificity of thrombin is similar to that of trypsin, cleaving after basic amino acid residues (8). However, thrombin has a more restricted substrate range than trypsin. The molecular basis of this restricted substrate specificity is still unresolved but is thought to be the result of interactions of the substrates with secondary binding sites distant from the active site (9, 10).Thrombin is also a potent stimulator of tissue plasminogen activator release from endothelial cells (11), and various forms of thrombin are chemotactic for monocytes and neutrophils (12)(13)(14). This chemotactic activity is blocked by binding with hirudin...

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Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogs

Cited by 95 publications

References 54 publications

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

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