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C T Esmon

Publications and source records attributed to C T Esmon.

At least 37 records · Page 2Linked to original sources
PubMed2001

A 23bp insertion in the endothelial protein C receptor (EPCR) gene impairs EPCR function.

EPCR is a type I transmembrane protein, highly expressed on the endothelium of large vessels, that binds protein C and augments its activation. In this study, a 23bp insertion in the EPCR gene was found in 4/198 survivors of myocardial infarction and 3/194 patients with deep vein thrombosis. The EPCR gene with the insertion predicts a protein that lacks part of the extracellular domain, the transmembrane domain and the cytoplasmic tail. Expression studies showed that the truncated protein is not localized on the cell surface, cannot be secreted in the culture medium, and does not bind activated protein C. Since protein C activation depends on the concentration of EPCR, patients with the EPCR insertion could have a diminished protein C activation capacity. Further clinical studies of adequate samples size are necessary to establish whether or not the EPCR insertion predisposes to the development of thrombotic events.

Adult↗
PubMed2000

Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor.

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.

Amino Acid Sequence↗
PubMed2000

Lipid oxidation enhances the function of activated protein C.

Although lipid oxidation products are usually associated with tissue injury, it is now recognized that they can also contribute to cell activation and elicit anti-inflammatory lipid mediators. In this study, we report that membrane phospholipid oxidation can modulate the hemostatic balance. Oxidation of natural phospholipids results in an increased ability of the membrane surface to support the function of the natural anticoagulant, activated protein C (APC), without significantly altering the ability to support thrombin generation. Lipid oxidation also potentiated the ability of protein S to enhance APC-mediated factor Va inactivation. Phosphatidylethanolamine, phosphatidylserine, and polyunsaturation of the fatty acids were all required for the oxidation-dependent enhancement of APC function. A subgroup of thrombotic patients with anti-phospholipid antibodies specifically blocked the oxidation-dependent enhancement of APC function. Since leukocytes are recruited and activated at the thrombus or sites of vessel injury, our findings suggest that after the initial thrombus formation, lipid oxidation can remodel the membrane surface resulting in increased anticoagulant function, thereby reducing the thrombogenicity of the thrombus or injured vessel surface. Anti-phospholipid antibodies that block this process would therefore be expected to contribute to thrombus growth and disease.

Humans↗
PubMed2000

The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18.

The protein C pathway is a primary regulator of blood coagulation and a critical component of the host response to inflammatory stimuli. The most recent member of this pathway is the endothelial protein C receptor (EPCR), a type I transmembrane protein with homology to CD1d/MHC class I proteins. EPCR accelerates formation of activated protein C, a potent anticoagulant and antiinflammatory agent. The current study demonstrates that soluble EPCR binds to PMA-activated neutrophils. Using affinity chromatography, binding studies with purified components, and/or blockade with specific Abs, it was found that soluble EPCR binds to proteinase-3 (PR3), a neutrophil granule proteinase. Furthermore, soluble EPCR binding to neutrophils was partially dependent on Mac-1 (CD11b/CD18), a beta(2) integrin involved in neutrophil signaling, and cell-cell adhesion events. PR3 is involved in multiple diverse processes, including hemopoietic proliferation, antibacterial activity, and autoimmune-mediated vasculitis. The observation that soluble EPCR binds to activated neutrophils via PR3 and a beta(2) integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.

Blood Coagulation Factors↗
PubMed2000

Characterization and regulation of the 5'-flanking region of the murine endothelial protein C receptor gene.

The protein C pathway plays a critical role in the negative regulation of blood coagulation. The nucleotide sequence of the murine endothelial protein C receptor (mEPCR) gene was determined for 8.8 kilobase pairs of the genomic structure and 3.4 kilobase pairs of the 5'-flanking region. RNase protection assay revealed six major transcription start sites clustered at -100 to -109 upstream of the translation initiation site. A series of 5'-promoter deletion fragments were fused to a luciferase reporter gene and transiently transfected into bovine aortic endothelium. Deletion of the sequence from -220 to -180 dramatically reduced luciferase expression in bovine aortic endothelial cells. This region of the murine endothelial protein C receptor gene contains one AP4 site and one SP1 site. Mutations in the core sequence of the AP4 and SP1 sites impaired both nuclear protein binding and luciferase expression. These results suggest important roles for AP4 and SP1 in the constitutive expression of mEPCR. A thrombin response element (CCCACCCC) was found to mediate the induction of mEPCR by thrombin in cell culture. Transgenic mice were developed expressing green fluorescent protein driven by the -350 to -1 or -1080 to -1 promoter. Thrombin up-regulated mEPCR and the transgene in vivo.

Animals↗
PubMed2000

Regulation of blood coagulation.

The protein C anticoagulant pathway converts the coagulation signal generated by thrombin into an anticoagulant response through the activation of protein C by the thrombin-thrombomodulin (TM) complex. The activated protein C (APC) thus formed interacts with protein S to inactivate two critical coagulation cofactors, factors Va and VIIIa, thereby dampening further thrombin generation. The proposed mechanisms by which TM switches the specificity of thrombin include conformational changes in thrombin, blocking access of normal substrates to thrombin and providing a binding site for protein C. The function of protein S appears to be to alter the cleavage site preferences of APC in factor Va, probably by changing the distance of the active site of APC relative to the membrane surface. The clinical relevance of this pathway is now established through the identification of deficient individuals with severe thrombotic complications and through the analysis of families with partial deficiencies in these components and an increased thrombotic tendency. One possible reason that even partial deficiencies are a thrombotic risk is that the function of the pathway can be down-regulated by inflammatory mediators. For instance, clinical studies have shown that the extent to which protein C levels decrease in patients with septic shock is predictive of a negative outcome. Initial clinical studies suggest that supplementation with protein C may be useful in the treatment of acute inflammatory diseases such as sepsis.

Animals↗
PubMed2000

The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis.

The influence of the endothelial protein C receptor (EPCR) on the host response to Escherichia coli was studied. Animals were treated with 4 separate protocols for survival studies and analysis of physiologic and biochemical parameters: (1) monoclonal antibody (mAb) that blocks protein C/activated protein C binding to EPCR plus sublethal numbers of E coli (SLEC) (n = 4); (2) mAb to EPCR that does not block binding plus SLEC (n = 3); (3) SLEC alone (n = 4); and (4) blocking mAB alone (n = 1). Those animals receiving blocking mAb to EPCR plus sublethal E coli died 7 to 54 hours after challenge, whereas all animals treated with the other protocols were permanent survivors. Histopathologic studies of tissues from animals receiving blocking mAb plus SLEC removed at postmortem were compared with those animals receiving SLEC alone killed at T+24 hours. The animals receiving the blocking mAb exhibited consumption of fibrinogen, microvascular thrombosis with hemorrhage of both the adrenal and renal cortex, and an intense influx of neutrophils into the adrenal, renal, and hepatic microvasculature, whereas the tissues from animals receiving only sublethal E coli exhibited none of these abnormal histopathologic changes. Compared with the control animals, the animals receiving the blocking mAb exhibited significantly elevated serum glutamic pyruvic transaminase, anion gap, thrombin-antithrombin complex, IL-6, IL-8, and soluble thrombomodulin. The levels of circulating activated protein C varied too widely to allow a clear determination of whether the extent of protein C activation was altered in vivo by blocking protein C binding to EPCR. We conclude that protein C/activated protein C binding to EPCR contributes to the negative regulation of the coagulopathic and inflammatory response to E coli and that EPCR provides an additional critical step in the host defense against E coli. (Blood. 2000;95:1680-1686)

Animals↗
PubMed2000

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo.

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor-alpha (TNF-alpha) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF-alpha failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo. (Blood. 2000;95:1687-1693)

Animals↗
PubMed2000

Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin.

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Antibodies, Monoclonal↗
PubMed2000

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Binding Sites↗
PubMed2000

Metalloproteolytic release of endothelial cell protein C receptor.

Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increase in inflammatory diseases. In this study we examine the potential mechanisms involved in release of EPCR from cells. We find that EPCR is released from the surface of endothelium and transfected 293 cells by a metalloprotease in a constitutive fashion. The mass of soluble EPCR is 4 kDa less than intact EPCR. Release is blocked by either the hydroxamic acid based inhibitor, KD-IX-73-4 or by 1,10-phenanthroline, but not by matrix metalloprotease inhibitors. Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1beta, and hydrogen peroxide. Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein C activation to a limited extent. The influence of phorbol 12-myristate 13-acetate on both EPCR release and inhibition of protein C activation are enhanced by microtubule disruption with nocodazole. EPCR release is augmented by transfection of EPCR expressing 293 cells with caveolin, suggesting that release is caveolae dependent. These studies indicate that metalloproteolytic release of EPCR is a highly regulated process that is sensitive to both coagulation factors and inflammatory mediators.

Blood Coagulation Factors↗
PubMed2000

The anticoagulant and anti-inflammatory roles of the protein C anticoagulant pathway.

Recent research has revealed a number of links between inflammation and coagulation. The protein C anticoagulant pathway appears to be the major pathway involved in the cross-talk between inflammation and coagulation. Studies indicate that inflammatory mediators can downregulate key components of the pathway through transcriptional control, proteolytic inactivation and oxidant damage. In turn, in vivo and in vitro studies have revealed mechanisms by which the components of the pathway may inhibit inflammatory responses. These include inhibition of cytokine responses to endotoxin, inhibition of leukocyte attachment to the activated endothelium and inhibition of thrombin and factor Xa generation in the microcirculation where both enzymes can lead to endothelial cell activation, further potentiating the inflammatory response. The ability of the protein C system to modulate both inflammation and coagulation may explain i part why specific defects in the pathway appear to be associated with both arterial and venus thrombosis.

Activated Protein C Resistance↗
PubMed2000

Antiphospholipid antibodies and the protein C pathway.

Among the mechanisms suggested for the prothrombotic activity of lupus anticoagulant and antiphospholipid antibodies is the direct inhibition of the anticoagulant activated protein C (APC) pathway. Although some pathological antibodies may be directed towards the proteins involved, we hypothesize that populations exist which selectively inhibit the APC complex as a result of differences in the phospholipid requirements of this complex as compared to those of the procoagulant complexes. The most prominent feature is the requirement for the presence of phosphatidylethanolamine in the membrane for APC anticoagulant function. This mimics the requirements for inhibitory activity of at least a subset of autoantibodies associated with thrombosis. The role of oxidation of the phospholipid in APC function and antibody reactivity is also discussed.

Antibodies, Antiphospholipid↗
PubMed2000

Modeling zymogen protein C.

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.

1-Carboxyglutamic Acid↗
PubMed2000

Does inflammation contribute to thrombotic events?

Recent studies have focused on a myriad of mechanisms by which inflammation can potentiate blood clotting. Inflammatory mediators like endotoxin and tissue necrosis factor (TNF)-alpha can cause the expression of tissue factor on monocytes and, possibly, endothelium, thereby initiating the coagulation cascade. Activation of the complement system can lead to exposure of membrane surfaces capable of amplifying the initial tissue factor stimulus by facilitating the assembly of the factor VIIIa-factor IXa and the factor Xa-factor Va complexes. Inflammatory mediators, particularly interleukin-6, can also increase the levels of fibrinogen, an acute-phase reactant. In addition, the inflammatory mediators can elevate the levels of plasminogen activator inhibitor, thus suppressing the fibrinolytic system. These studies alone, however, do not prove that inflammation can trigger clinically relevant thrombus formation in vivo. For instance, TNF-alpha has been studied in cancer patients as a potential cure for cancer, and even though these patients are hypercoaguable, thrombosis was not commonly observed as a side effect of the near-lethal doses of TNF-alpha that were administered. Based on primate studies, inflammatory mediators like TNF-alpha can promote clot deposition effectively only if there is reduced flow and inhibition of the natural anticoagulant pathways. The requirement for multiple simultaneous injurious events probably explains why inflammation alone is not observed as a major cause of thrombosis.

Animals↗
PubMed2000

Antibodies to thrombomodulin are found in patients with lupus anticoagulant and unexplained thrombosis.

OBJECTIVE: To test the hypothesis that thrombomodulin (TM) may be a target for lupus anticoagulant (LAC) antibodies. METHODS: A recombinant soluble form of TM was produced and used as an antigen for an ELISA to detect antibodies to TM (TMAB). Sixty-one samples from 58 patients identified by the coagulation laboratory as having a LAC and 200 patients with unexplained thrombosis were evaluated along with 201 healthy controls. RESULTS: Eighteen (30%) of the 58 patients with a LAC and 20 (10%) of 200 patients with unexplained thrombosis had antibodies to TM. Similar antibodies were found in only 4 (2%) of 201 normal controls. TMAB show selectivity for TM lacking chondroitin sulfate, but do not otherwise have an immunodominant region. The IgG from 6 patients with TMAB was purified, and it bound TM in our ELISA. Three of the 6 IgG fractions inhibited protein C activation 40% to 70% compared to no inhibition in 7 healthy controls. CONCLUSION: Some patients with LAC and unexplained thrombosis have antibodies to TM that may arise in response to TM that has been altered and lost its chondroitin sulfate attachment. Antibodies to TM may be an important risk factor for inflammation and thrombosis in these patients.

Adult↗