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M C Guillin

Publications and source records attributed to M C Guillin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Functional consequences of the Ser334-->Pro mutation in a human factor X variant (factor XMarseille).

A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T-->C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334-->Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser-->Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.

Animals↗
PubMed1995

Role of the thrombin insertion loop 144-155. Study of thrombin mutations W148G, K154E and a thrombin-based synthetic peptide.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-D-Lys-(epsilon-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide ([K154E]thrombin) or tosyl-Gly-Pro-Arg-NH-nitroanilide ([W148G]thrombin), and in the second-order rate constants of inhibition by antithrombin III ([K154E]thrombin) and ([W148G]thrombin) compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.

Adult↗
PubMed1995

Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

Amides↗
PubMed1995

Limited proteolysis of human alpha-thrombin by urokinase yields a non-clotting enzyme.

Limited proteolysis of human alpha-thrombin by various proteases has been efficiently used to demonstrate the importance of two insertion loops located on the surface of this molecule. In the present study, we demonstrate that two-chain urokinase (tcu-PA) specifically cleaves the B chain of alpha-thrombin giving rise to a transient derivative, consisting of two non-covalently linked subunits. Although the thrombin derivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 microM and kcat = 145 s-1 versus respectively 4.5 microM and 149 s-1 for alpha-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platelets is considerably reduced (threshold for full platelet aggregation 2.5 nM versus 0.25 nM). The thrombin fragments were separated by HPLC and after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded to B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, indicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 74 in the B chain. The proteolytic derivative obtained, designated beta u-thrombin, is therefore identical to the transient proteolytic derivative, beta 1-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to gamma t-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Thrombin specificity.

A model of thrombin interaction with distinct substrates or ligands has been derived from the crystallographic studies of thrombin-inhibitors complexes, and buttressed by functional studies with mutant thrombins, thrombin proteolytic derivatives or antibodies against thrombin. The unique specificity of thrombin for its substrates and ligands may be ascribed to multiple interactions with both the active site cleft and exosite(s) distinct from the active site. Two prominent insertion loops around Trp 50 and Trp 148 project over the active site cleft and play an important role in the substrates selection. Several substrates (fibrinogen, thrombin receptor, heparin cofactor II) or ligands (thrombomodulin, glycoprotein Ib) interact with a large exosite located on the surface of the loop segment 65-76, mainly constituted of basic amino acids, designated anion binding exosite 1. Interaction with these various macromolecules appears to involve a limited number of residues within the large exosite 1. It is conceivable that exosite 1 contains distinct subsites, although most of them may overlap. A second basic exosite (anion binding exosite 2) is located close to the carboxy-terminal B chain helix. Exosite 2 interacts with heparin, the chondroitin sulfate moiety of thrombomodulin and prothrombin activation fragment 2. Interaction of ligands with either exosite 1 or exosite 2 leads to conformational changes of the thrombin molecule, that may be important determinants of thrombin specificity. Whether exosite 2 cooperates with exosite 1 for thrombin interaction with fibrin(ogen) or the thrombin receptor remains to be determined.

Amino Acid Sequence↗
PubMed1994

Mechanism of the abnormal vitamin K-dependent gamma-carboxylation process in human hepatocellular carcinomas.

BACKGROUND: An important marker for hepatocellular carcinoma is the presence of des-gamma-carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown. METHODS: Two groups of patients were defined according to the absence (Group I, n = 7) or presence (Group II, n = 8) of des-gamma-carboxy prothrombin. The enzymatic activity of gamma-carboxylase and the total microsomal prothrombin concentration were determined in all tumors. The kinetic parameters for the synthetic peptide Phe-Leu-Glu-Glu-Leu (FLEEL) were measured in eight tumors. The gamma-carboxylase mRNA expression was evaluated by Northern blot analysis in 12 of 15 tumors. In addition, the total vitamin K content (K1, K1 epoxide, and menaquinones 4-10) in 10 tumors was investigated by high performance liquid chromatography. RESULTS: Concentrations of menaquinones 4-10 were normal in the nontumorous part of the liver but significantly decreased (P = 0.02) in all the tumors (Groups I and II). This decrease was more severe in Group II (P = 0.02). The tumors in Group I had normal or increased gamma-carboxylase activity and increased mRNA expression (P < 0.02) as compared with their nontumorous counterparts. The tumors in Group II were heterogeneous. Five tumors displayed low gamma-carboxylase activity, associated with low mRNA expression in two, whereas two others had high gamma-carboxylase activity and mRNA expression. The concentration of FLEEL at half-maximal velocity was normal in all the tumors examined (Groups I and II), and a relation was found between the level of expression of gamma-carboxylase and the maximal velocity for FLEEL carboxylation in the tumors in Group II (r = 0.98; P < 0.01). The microsomal content of normal prothrombin was within normal limits in all tumors (Groups I and II). CONCLUSIONS: Tumor vitamin K content has a critical role in the synthesis of des-gamma-carboxy prothrombin. Furthermore, the gamma-carboxylase defect, which is observed in some secreting tumors, is the result of the defective gene expression of a normal enzyme and not the consequence of the presence of a competitive inhibitor. It is possible that a 75% reduction in gamma-carboxylase gene expression could take a part in the secretion of des-gamma-carboxy prothrombin, but this mechanism is not predominant.

Biomarkers↗
PubMed1993

Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition.

A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.

Amino Acid Sequence↗
PubMed1993

Late-fibrin(ogen) fragment E modulates human alpha-thrombin specificity.

Fibrinogen contains at least two independent sites having demonstrable affinity for alpha-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of alpha-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on alpha-thrombin specificity. We show that fragment E modulates alpha-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin-thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of alpha-thrombin-catalyzed fibrinopeptide-A cleavage (Ki = 5.2 +/- 1.3 microM), indicating that alpha-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited alpha-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC50 = 10 microM) and alpha-thrombin binding to GPIb. Fragment E competitively inhibited alpha-thrombin binding to thrombomodulin (Ki = 18.3 +/- 0.8 microM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on alpha-thrombin.

Amino Acid Sequence↗
PubMed1993

Paris I dysfibrinogenemia: a point mutation in intron 8 results in insertion of a 15 amino acid sequence in the fibrinogen gamma-chain.

Paris I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal gamma-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the gamma-chain region of the Paris I subject's genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A-->G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I gamma-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature gamma Paris I chain mRNA, and encodes a 15 amino acid insert after gamma 350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I gamma-chain mRNA also results after translation into a substitution of S for G at position gamma 351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the gamma Paris I chain. We conclude that the insertion of this amino acid sequence leads to a conformationally-altered, and dysfunctional gamma-chain in Paris I fibrinogen.

Afibrinogenemia↗
PubMed1992

Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.

We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

Amino Acid Sequence↗
PubMed1992

Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.

Alanine↗
PubMed1991

Effect of the hirudin carboxy-terminal peptide 54-65 on the interaction of thrombin with platelets.

The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed beta cleavage site, we have analyzed the consequences of alpha to beta-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The beta cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the beta cleavage site at Arg 73 on the thrombin B chain.

Amino Acid Sequence↗
PubMed1991

Effect of phosphopyridoxylation on thrombin interaction with platelet glycoprotein Ib.

The purpose of this study was to determine the effect of chemical modification of lysyl residues on thrombin interaction with platelet membrane proteins. Modification of lysyl residues by pyridoxal-5'-phosphate affected two different sites on thrombin and resulted in a greatly decreased binding to platelets. Using a crosslinking bifunctional reagent [bis(sulphosuccinimidyl) suberate (BS3)], we show that modified thrombin retained the ability to form high molecular mass (greater than or equal to 400 kDa) complexes with yet unidentified platelet proteins and to bind to platelet protease nexin I, but had lost the ability to bind to platelet glycoprotein Ib (GPIb). As previously reported by others, heparin protected one of the two sites from phosphopyridoxylation. In contrast modified thrombin, heparin-protected modified thrombin retained the ability to bind to GPIb, indicating that the lysyl residue(s) protected by heparin from the modification are essential for GPIb binding. While unprotected modified thrombin failed to bind hirudin, heparin-protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide H54-65. Tritium-labelling of the modified lysyl residues and degradation of modified thrombins by CNBr or trypsin confirmed that the lysyl residue(s) protected by heparin and essential for GPIb binding are located in the thrombin binding domain for the carboxyl-terminal tail of hirudin, within the sequence 18-73 of the thrombin B chain.

Binding Sites↗