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C T Esmon

Publications and source records attributed to C T Esmon.

At least 73 records · Page 4Linked to original sources
PubMed1997

Influence of Arginines 93, 97, and 101 of thrombin to its functional specificity.

Mutation of three Arg residues, 93, 97, and 101, to Ala in thrombin (thrombin R93,97,101A) has previously been shown to eliminate most heparin acceleration of thrombin inhibition by antithrombin and most of the ability of chondroitin sulfate (CS) on thrombomodulin (TM) to enhance affinity for TM and to eliminate the characteristic high-affinity interaction with protein C observed with TM lacking CS. In this study we examined the relative impact of mutation of these Arg residues alone and in combination on the above reactions and, in addition, on the ability of rabbit TM to accelerate thrombin inhibition by antithrombin. The order of importance for heparin acceleration of inhibition by antithrombin was Arg 101, 93, and 97. In contrast, Arg 97 was the major residue required for TM-dependent acceleration of reactivity with antithrombin and for CS-dependent enhancement of TM affinity. Arg 101 and 93 were critical for TM-dependent, high-affinity protein C interaction at low Ca2+ concentrations, while Arg 97, which was critical for the other TM-dependent effects, played no detectable role in this metal dependence. These results illustrate that these Arg residues in anion binding exosite 2 contribute very differently to the diverse reactions dependent on that domain in thrombin.

Animals↗
PubMed1997

Identification of functional endothelial protein C receptor in human plasma.

The endothelial cell protein C receptor (EPCR) binds protein C and facilitates activation by the thrombin-thrombomodulin complex. EPCR also binds activated protein C (APC) and inhibits APC anticoagulant activity. In this study, we detected a soluble form of EPCR in normal human plasma. Plasma EPCR appears to be approximately 43, 000 D, and circulates at approximately 100 ng/ml (98.4+/-27.8 ng/ml, n = 22). Plasma EPCR was purified from human citrated plasma using ion exchange, immunoaffinity, and protein C affinity chromatography. Flow cytometry experiments demonstrated that plasma EPCR bound APC with an affinity similar to that previously determined for recombinant soluble EPCR (Kdapp = 30 nM). Furthermore, plasma EPCR inhibited both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage Factor Xa clotting assay. The physiological function of plasma EPCR is uncertain, but if the local concentrations are sufficiently high, particularly in disease states, the present data suggest that the soluble plasma EPCR could attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of APC.

Anticoagulants↗
PubMed1997

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.

Antithrombin III↗
PubMed1997

7E3 F(ab')2, a monoclonal antibody to the platelet GPIIb/IIIa receptor, protects against microangiopathic hemolytic anemia and microvascular thrombotic renal failure in baboons treated with C4b binding protein and a sublethal infusion of Escherichia coli.

We have used our previously described baboon model of infusion of both a sublethal dose of Escherichia coli and C4b binding protein to assess the impact of inhibiting platelet function with the F(ab')2 fragment of the monoclonal antibody 7E3, directed against the platelet glycoprotein (GP)IIb/IIIa receptor, on the characteristic microvascular changes. At a dose of 0.25 to 0.35 mg/kg bolus plus an infusion of 0.25 to 0.35 mg/kg over 6 hours, c7E3 F(ab')2 had only a minimal impact on fibrinogen consumption and delayed but did not prevent, the development of thrombocytopenia. Treatment with 7E3 F(ab')2, however, produced significant protection from the development of microangiopathic hemolysis and renal insufficiency. Histologic examination supported these observations, with treated animals having fewer schistocytes on blood smear and less evidence of ischemic renal changes. Treated animals also had more rapid recovery of peripheral white blood counts, suggesting a possible protective effect of treatment on ischemic damage to the bone marrow. These data indicate that potent inhibition of platelet function via GPIIb/IIIa receptor blockade can decrease ischemic organ damage in this animal model that has features similar to those found in diffuse intravascular coagulation, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.

Anemia, Hemolytic↗
PubMed1997

Endothelial cells and extracellular calmodulin inhibit monocyte tumor necrosis factor release and augment neutrophil elastase release.

Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-alpha release from whole blood or isolated mononuclear cells exposed to endotoxin. In contrast, the endothelial cells augmented neutrophil elastase release in the same blood. A protein with these functional properties was isolated from endothelial cell-conditioned media and, surprisingly, was identified as calmodulin. Authentic calmodulin mimicked the effect of endothelium. 125I-Calmodulin bound to a high affinity site on monocytic cell lines (Kd approximately 30 nM, in agreement with its functional activity). Cross-linking of 125I-calmodulin to monocytic cells identified a candidate calmodulin receptor. We conclude that calmodulin possesses an extracellular signaling role in addition to its intracellular regulatory functions. Calmodulin released at sites of tissue injury or possibly by specific mechanisms in the endothelium can bind to receptors, modulating the activities of inflammatory cells.

Amino Acid Sequence↗
PubMed1997

Thrombogenic mechanisms of antiphospholipid antibodies.

These studies indicate how immunoglobulin populations that react with phospholipid surfaces in the absence of other cofactor molecules can selectively inhibit anticoagulant pathways and lead to a prothrombotic state. These studies combined with those of others indicating the presence of a-PE antibodies (often in isolation) in thrombotic patients illustrate the need to better define the assays to determine patients at risk. Neither the LA assays nor the anti-cardiolipin assays presently in use may be testing for the population(s) of clinical importance. A better understanding of the biochemical requirements of the various reactions involved should help the rational design of such assays. The preliminary studies of Salmon, et al, also show that the genetic context of the patient may contribute to the thrombotic mechanism of any APA present. It is unlikely any single mechanism is responsible for the thrombogenic activity of all APAs associated with thrombosis and this will be a fertile field of investigation for a significant time to come.

Antibodies, Antiphospholipid↗
PubMed1996

Molecular basis of residue 192 participation in determination of coagulation protease specificity.

Residue 192 (chymotrypsin numbering system) in thrombin, activated protein C, and factor Xa contributes to the specificity of these enzymes toward their substrates and inhibitors. A Glu192-->Gln mutation in both thrombin and activated protein C yielded enzymes that reacted better with some, but not all, of their natural substrates and inhibitors. To determine whether the specificity change is due to productive interactions of Gln192 with substrates and inhibitors or elimination of repulsive electrostatic interactions, we prepared forms of thrombin, des-(1-45)-factor Xa and activated des-(1-45)-protein C with Glu, Gln, or Met at position 192 and compared their activities toward inhibitors and substrates. All mutants had nearly normal amidolytic activity. The Glu192-->Gln and Glu 192-->Met mutations of thrombin and activated des-(1-45)-protein C increased the second-order rate constant (k2) of inhibition by alpha 1-antitrypsin about 700-fold and 170-fold for thrombin, and 185-fold and 150-fold for activated des-(1-45)-protein C, respectively. [E192]faxtor Xa, but not [M192]factor Xa, was resistant to inhibition by alpha 1-antitrypsin. Glu-->Gln or Glu-->Met mutants of both thrombin and activated des-(1-45)-protein C were effectively inhibited by tissue factor pathway inhibitor (K1 < 200 nM) and, except for [M192]thrombin, by bovine pancreatic trypsin inhibitor (K1 < 60 nM). With respect to substrate cleavage, Glu192-->Gln and Glu192-->Met mutations of activated des-(1-45)-protein C both inactivated factor Va 2-3-fold faster than activated des-(1-45)-protein C. Thrombin and [M192]thrombin activated protein C at similar slow rates compared to rapid activation by [Q192]thrombin. The Gln192-->Met mutants of des-(1-45)-factor Xa activated prethrombin 1.8-11-fold slower than wild-type enzyme. With thrombomodulin or factor Va present, these differences in protein C and prethrombin 1 activation rates were decreased to about 2-fold. We conclude that residue 192 contribution to enzyme specificity is achieved by both productive and repulsive interactions and that the magnitude and nature of the participation varies among enzymes, substrates and inhibitors.

Animals↗
PubMed1996

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

Antibodies, Monoclonal↗
PubMed1996

The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor.

Expression of the endothelial cell protein C receptor (EPCR) gene in mammalian cells imparts the capacity to bind activated protein C (APC) or protein C. Immunochemical analysis of CCD41, apparently the murine homologue of EPCR, suggested centrosomal localization, raising questions about the location of the EPCR gene product and its role in protein C binding. In this study, we express a soluble form of EPCR, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. Affinity purified polyclonal and a monoclonal antibody against EPCR bound to the cell surface of EPCR-transfected cells but not to control cells. A 49-kDa protein, a mass similar to soluble EPCR, was immunoprecipitated from the cell surface of endothelium and cells transfected with human EPCR but not from control cells. The FLAGtrade mark antibody and APC bound to cells expressing an EPCR construct containing the FLAGtrade mark epitope located in a putative extracellular domain, whereas an EPCR construct truncated just before the putative transmembrane domain produced only soluble EPCR antigen. Soluble EPCR inhibited APC binding to EPCR expressing cells in a concentration-dependent fashion, Kd (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane protein that binds directly to APC.

Animals↗
PubMed1996

The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors.

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by alpha1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.

Amidohydrolases↗
PubMed1996

Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII.

The role of anion-binding exosites of thrombin in the activation of factor V and factor VIII was studied using thrombin Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala (thrombin RA), a recombinant exosite 2 defective mutant, and a synthetic N-acetylated dodecapeptide, Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-O-SO4Leu (hirugen), which competitively inhibits binding of macromolecules to exosite 1. The catalytic efficiency of the activation of factor VIII or of the first step of factor V activation by thrombin RA was approximately 10% that of wild-type thrombin. The overall rate of conversion to factor Va was not influenced by the mutation. In contrast to factor V, the slow activation of factor VIII by thrombin RA was associated with a decreased rate of cleavage at all three proteolytic sites (Arg372, Arg740, and Arg1689). Hirugen inhibited factor V and factor VIII activation. These results indicate that both anion-binding exosites of thrombin are involved in the recognition of factor V and factor VIII.

Animals↗
PubMed1996

Infusion of phospholipid vesicles amplifies the local thrombotic response to TNF and anti-protein C into a consumptive response.

Inflammation often is considered a contributing factor to both thrombosis and disseminated intravascular coagulation. The molecular mechanisms that dictate which of these clinical manifestations will result from the inflammatory stimulus remain obscure. Bacterial infection and certain tumors are common initiators of the disseminated intravascular coagulant response. Complement activation resulting from bacterial infection shares with selected tumors the capacity to generate or release membrane particles that lack functional adhesion receptors and hence could circulate to amplify a disseminated intravascular coagulant response. We developed a model of venous thrombosis that resulted in localized thrombus formation without disseminated intravascular coagulation. The model involves infusion of tumor necrosis factor, blockade of protein C and a partial decrease in venous flow caused by ligation of the superficial femoral vein without obstruction of the deep formal vein. Infusion of phospholipid vesicles into this model resulted in amplification of a localized thrombotic response into a consumptive response. Seven different groups of animals were studied. The first three groups established the conditions necessary to produce deep vein thrombosis. The second four groups established the conditions necessary to produce disseminated intravascular coagulation. The infusion of phospholipid vesicles plus tumor necrosis factor and anti-protein C antibody resulted in consumption of fibrinogen, the production of thrombin/antithrombin complexes, a fall in platelet count, and venous thrombosis. Without ligation and catheterization phospholipid vesicles failed to produce the consumptive response. We conclude, therefore, that phospholipid vesicles can amplify a local thrombotic response into a consumptive response, and that vesiculation accompanying inflammation is one means by which localized coagulant activity may be amplified to produce disseminated intravascular coagulation.

Animals↗
PubMed1995

Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex.

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI approximately equal to 140-fold (k2 = 2.4 x 10(6) in the presence of TM compared to 1.7 x 10(4) M-1 S-1 in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI approximately equal to 59-fold (k2 = 1.0 x 10(6) M-1 S-1). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe353 with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k2 = 6.3 x 10(5) M-1 S-1), but TM 4-6 enhances inhibition by this mutant approximately equal to 9-fold (k2 = 5.8 x 10(6) M-1 S-1) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

Animals↗
PubMed1995

Role of free protein S and C4b binding protein in regulating the coagulant response to Escherichia coli.

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S. The second objective was to examine the relevance of this model to the hemolytic uremic syndrome. Infusion of C4b-binding protein alone reduced free protein S and decreased platelet concentration to 20% of baseline, whereas infusion of the C4b-binding protein/protein S complex did not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a transient inflammatory response with no reduction of free protein S. However, coinfusion of C4b-binding protein with sublethal E coli reduced free protein S and produced a thrombocytopenia, anemia, and a microvascular thrombotic response, whereas infusion of the C4b-binding protein/protein S complex with sublethal E coli did not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing (S-145) antibodies with protein S coinfused with sublethal E coli produced similar contrasting results. Therefore, we concluded that neutralization of free protein S, and not some other property of C4b-binding protein influenced by protein S, accounted for this microvascular thrombotic response. This response is similar to the hemolytic uremic syndrome characterized by thrombocytopenia, anemia, shistocytosis, and renal glomerular thrombosis with uremia. Comparison of the respective renal histopathologic appearance supports this conclusion. This raises the possibility that inhibition of protein S activity (possibly by one of the forms of C4b-binding proteins) might be one of the factors contributing to microvascular thrombotic disorder, such as the hemolytic uremic syndrome.

Animals↗
PubMed1995

Tryptophans 231 and 234 in protein C report the Ca(2+)-dependent conformational change required for activation by the thrombin-thrombomodulin complex.

Human protein C circulates as both single- and two-chain zymogens. Activation by the physiological activation complex, thrombin-thrombomodulin, generates the anticoagulant enzyme, activated protein C. Ca2+ binding to the protease domain of protein C is accompanied by 5.5 +/- 0.2% quenching of intrinsic fluorescence that correlates with the conformational change required for the rapid activation by the thrombin-thrombomodulin complex. To map which Trp residues report this Ca2+ binding, candidate Trp residues at positions 84, 115, 145, 205, 231, and 234 were changed individually to Phe within a protein C deletion mutant lacking the Gla domain (GDPC). Of these, the Trp to Phe mutation at position 231 (W231F) eliminated the Ca(2+)-induced fluorescence quenching, and the Trp 234 to Phe mutation (W234F) increased the maximum quenching in protein C to 9.4 +/- 0.4%. Upon Ca2+ binding, the fluorescence emission intensity of the W231F mutant was increased 3.4% +/- 0.6%. The Kd for this site (84 +/- 20 microM) was similar to that of GDPC (Kd = 39 +/- 4 microM). To compare the properties of single- and two-chain protein C, we replaced the Lys156-Arg157 dipeptide cleavage site in protein C with Thr and Gln to form GDPCKR/TQ. GDPCKR/TQ and the two-chain form of protein C were activated at the same rate with the thrombin-thrombomodulin complex, they exhibited similar Ca2+ dependence for both activation and fluorescence quenching, and these enzymes had the same chromogenic activity. In contrast to the zymogen form, activated human Gla-domainless protein C did not undergo a Ca(2+)-induced fluorescence change. These results indicate that the environment of Trp 231 and 234 within the Ca2+ binding loop of the protein C zymogen are perturbed by Ca2+ binding to the zymogen.

Amino Acid Sequence↗
PubMed1995

Contribution of residue 192 in factor Xa to enzyme specificity and function.

Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln in thrombin and activated protein C has been shown to dramatically alter substrate and inhibitor specificity, in large part by allowing these enzymes to accept substrates with acidic residues in the P3 and/or P3' positions. In factor Xa, residue 192 is already a Gln. We now compare the properties of a Q192E mutant of Gla-domainless factor X (GDFX). Kinetic analysis of prothrombin activation indicates similar affinity of factor Va for GDFXa and GDFXa Q192E (Kd(app) = 3.6 and 3.7 microM, respectively). Prothrombin activation rates are similar for both enzymes with factor Va, but are approximately 10-fold slower for the Q192E mutant without factor Va. This defect is in the activation of prethrombin 2 and is corrected by factor Va only in the presence of fragment 2. Without factor Va, fragment 2 has no influence on bovine prethrombin 2 activation by GDFXa, but fragment 2 enhances prethrombin 2 activation by the Q192E mutant at least 10-fold. These results indicate that the fragment 2 domain of prothrombin probably alters the conformation of the prethrombin 2 domain, selectively improving its presentation to GDFXa Q192E. With respect to inhibition, tissue factor pathway inhibitor and bovine pancreatic trypsin inhibitor are > or = 30 times poorer inhibitors of GDFXa Q192E than of GDFXa, but these enzymes are inhibited at comparable rates by antithrombin. These results indicate that Gln-192 in factor Xa is a key determinant of substrate/inhibitor specificity.

Amino Acid Sequence↗