Biomedical subjects

N L Esmon

Publications and source records attributed to N L Esmon.

At least 19 recordsLinked to original sources

PubMed1992

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.

1-Carboxyglutamic Acid↗

PubMed1992

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation.

The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting of the six repeated growth factor-like domains of thrombomodulin (GF1-6) and the other containing only the fifth and sixth such domains (GF5-6). Both derivatives can bind to thrombin and block fibrinogen clotting activity, though only the larger GF1-6 can stimulate the activation of protein C. To ascertain whether the substrate specificity change from fibrinogen to protein C is accompanied by structural changes in the active site of the enzyme, fluorescent dyes were positioned at different locations within the active site. A 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached to the active site serine to form dansyl-thrombin, while either a fluorescein or an anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg linkage. The environment of the dansyl dye was altered in a similar fashion when either GF1-6 or GF5-6 bound to thrombin, since a similar reduction in dansyl emission intensity was elicited by these two thrombomodulin derivatives (25 and 32%, respectively). These spectral changes, and all others in this study, were saturable and reached a maximum when the ratio of thrombomodulin derivative to thrombin was close to 1. The environments of the fluorescein and ANS dyes were also altered when GF1-6 bound to thrombin because binding resulted in emission intensity changes of -13% and +18%, respectively. In contrast, no fluorescence changes were observed when the fluorescein and ANS thrombin derivatives were titrated with GF5-6. Thus, the structure of the active site was altered by thrombomodulin both immediately adjacent to the active site serine and also more than 15 A away from it. However, the structural change far from Ser-195 was only elicited by thrombomodulin species that stimulate thrombin-dependent activation of protein C.

Amino Acid Sequence↗

PubMed1991

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix.

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.

Antibodies, Monoclonal↗

PubMed1991

Initiation of the protein C pathway.

The protein C activation system provides an interesting model for the control of coagulation. Expression of the critical receptor appears to be under the control of inflammatory mediators. Open questions of considerable importance relate to the physiological significance of these observations. Preliminary evidence is emerging that thrombomodulin is down-regulated in patients and animals with inflammation, but it remains to be determined if the loss of thrombomodulin causes the thrombotic complications or occurs in response to these complications.

Animals↗

PubMed1990

Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions.

Cell Transformation, Viral↗

PubMed1990

Factor IXa and von Willebrand factor modify the inactivation of factor VIII by activated protein C.

Activated protein C inactivates factor VIII by proteolytic cleavage of the heavy chain of factor VIII. Protein S and calcium ions are cofactors in this reaction. We have examined the effects of several potential modulators of this reaction, including phospholipids, von Willebrand factor, and factor IXa, all of which bind factor VIII. Our results indicate that neither resting nor stimulated platelets nor phospholipid vesicles protect factor VIII from inactivation by activated protein C in either the presence or the absence of protein S. However, the addition of von Willebrand factor decreases the inactivation of factor VIII by activated protein C by 20% to 30%, and factor IXa, which is known to protect factor VIII from inactivation by activated protein C, confers additional protection with von Willebrand factor. The active site of factor IXa is necessary for the protective effect, because native factor IX and active site-inhibited factor IXa do not protect factor VIII from inactivation. Thus there is an additive protective effect when von Willebrand factor and factor IXa are present with factor VIII, leading to a decrease in the inactivation by activated protein C. These factors may be particularly important in stabilizing factor VIII in the circulation and during the early stages of coagulation.

Amino Acid Chloromethyl Ketones↗

PubMed1990

In vivo behavior of detergent-solubilized purified rabbit thrombomodulin on intravenous injection into rabbits.

Thrombomodulin is a thrombin endothelial cell membrane receptor. The thrombomodulin-thrombin complex rapidly activates protein C resulting in anticoagulant activity. We investigated the anticoagulant effects and pharmacokinetic behavior of detergent-solubilized purified rabbit thrombomodulin labeled with iodine 125 when intravenously injected into rabbits. Thrombomodulin half-life (t1/2) was determined by tracking the 125I-radiolabeled protein and the biologic activity as determined by the prolongation of the activated partial thromboplastin time (APTT) and thrombin clotting time (TCT). When 200 micrograms/kg 125I-thrombomodulin was injected into rabbits, the APTT and TCT were immediately prolonged, whereas no effect on the prothrombin time was seen. In vitro calibration curves enabled us to convert the prolongations of the clotting times into micrograms per milliliter thrombomodulin equivalents. The best fit (r greater than 0.99) for the disappearance curves was provided by a two-compartment model with mean t1/2 alpha (distribution phase) of 18 minutes for 125I, 12 minutes for APTT, and 20 minutes for TCT, and mean t1/2 beta (elimination phase) of 385 minutes for 125I, 460 for APTT, and 179 for TCT. The administration of two doses of endotoxin (50 micrograms/kg) 24 hours apart did not accelerate the turnover rate of 125I-thrombomodulin as measured by the disappearance of 125I from the circulation. Thus, detergent-solubilized purified thrombomodulin administered intravenously circulates in a biologically active form for appreciable time periods.

Animals↗

PubMed1989

The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface.

The location of the active site of the membrane-bound anticoagulant complex of thrombin and thrombomodulin has been determined relative to the membrane surface using fluorescence energy transfer. Thrombin was reacted with 5-(dimethylamino)-1-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (DEGR-CK) to yield DEGR-thrombin, an analogue of thrombin with a fluorescent dye covalently attached to its active site. When DEGR-thrombin was titrated with thrombomodulin that had been reconstituted into phospholipid vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes, each in an active site of a DEGR-thrombin bound to thrombomodulin, and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer reached a maximum when DEGR-thrombin and thrombomodulin were equimolar in the sample, as expected for the formation of a 1:1 complex between thrombin and thrombomodulin. This energy transfer was dependent upon the binding of DEGR-thrombin to thrombomodulin because no energy transfer was observed with vesicles that lacked thrombomodulin, and the extent of energy transfer was reduced greatly by the addition of excess unmodified nonfluorescent thrombin to compete with DEGR-thrombin for binding to the thrombomodulin. From the dependence of the energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between a dye in the active site of the thrombin-thrombomodulin complex and a dye at the membrane surface was determined to average 66 A (65 +/- 3 A for phosphatidylcholine vesicles without and 67 +/- 5 A for those with 20% phosphatidylserine). This distance was also insensitive to the presence or absence of Ca2+. These direct measurements indicate that the active site of the membrane-bound thrombin-thrombomodulin complex is located far above the phospholipid surface, that the peptide bond cleaved during the activation of protein C is situated about 66 A above the membrane, that the thrombin binding site on thrombomodulin is positioned more than 45 A above the membrane, ant that thrombin, with a diameter near 40 A, is not positioned alongside thrombomodulin near the membrane to form the thrombin-thrombomodulin complex but is instead bound "on top" of thrombomodulin.

Amino Acid Chloromethyl Ketones↗

PubMed1989

Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture.

Tumor necrosis factor (TNF), a mediator of the inflammatory response, induces tissue factor and decreases the expression of thrombomodulin (TM) on endothelial cells, thus shifting the hemostatic properties of the endothelium. To determine the mechanism of TM downregulation, bovine aortic endothelial cells in culture were treated with TNF (2 nmol/L) and the fate of TM followed. Both surface expressed TM (antigen and activity), and the total TM pool (measured by radioimmunoassay and activity in detergent extracts) dropped to less than or equal to 20% of control values within 12 hours of TNF treatment. TM was not found in an immunologically recognizable form in the supernatants of treated cultures. Chloroquine (greater than or equal to 100 mumol/L) was able to abrogate the TNF effect on the total TM pool but not the effect on surface-expressed TM activity. We conclude that TNF induces the internalization and subsequent degradation of the TM molecule. None of the components of the protein C anticoagulant pathway, either alone or in combination, prevented the TNF-dependent downregulation of TM antigen.

Animals↗

PubMed1989

Thrombomodulin.

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Amino Acid Sequence↗

PubMed1988

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody.

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.

Amino Acid Sequence↗

PubMed1988

Protein C and the endothelium.

With the continued accumulation of clinical and animal studies, it is becoming abundantly clear that the protein C anticoagulant pathway plays a critical role in the regulation of coagulation. Investigations also indicate that this pathway is intimately involved in the interaction of the coagulation and inflammatory systems. Although no direct information is presently available, the function of this pathway is likely depressed in the regions of atherosclerotic plaque. It is clear that monocytes accumulate in this region and release many growth factors and monokines that are capable of endothelial function perturbation. Perturbation of the protein C anticoagulant pathway is one viable mechanism for the hypercoagulable state in this disease. As indicated here, the endothelial cells of the vessel wall play a critical role in the initiation, and possibly expression, of this pathway. Any injury to these cells that affects the proper expression of thrombomodulin, synthesis of protein S, or Factor Va inactivation complex formation could potentially lead to a hypercoagulable state and thrombotic complications. As has been discussed, several inflammatory mediators are already known that fulfill the criteria of endothelial cell perturbants that may lead to such a state. What other entities might have similar effects, either directly or indirectly through induction of cytokines, is not known at this time. A more complete understanding of this critical pathway and the effects of vascular disease on it should lead to a better understanding of many diverse disease processes and potential therapeutic strategies in the future.

Animals↗

PubMed1988

Isolation of an abnormal protein C molecule from the plasma of a patient with thrombotic diathesis.

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin-thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.

Adult↗

PubMed1987

Reconstitution of rabbit thrombomodulin into phospholipid vesicles.

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.

Animals↗

PubMed1987

Proteolytic formation and properties of functional domains of thrombomodulin.

Thrombomodulin is an endothelial cell surface protein which complexes with thrombin to accelerate protein C activation. To gain insight into the mechanisms of thrombomodulin-membrane association, limited proteolytic digestions of thrombomodulin with trypsin and elastase were performed. Trypsin digestion resulted in two major fragments (Mr = 54,000 and 27,000), both of which bound to phosphatidylcholine/phosphatidylserine vesicles. Elastase digestion also yielded two major fragments (Mr = 50,000 and 25,000), but only the smaller fragment bound to the phospholipid vesicles. The larger fragment obtained from both enzymatic digestions retained the ability to accelerate protein C activation. The Mr = 54,000 fragment from the trypsin digest retained a high affinity for thrombin (Kd less than or equal to 0.5 nM), a Km for protein C of approximately equal to 8 microM, and a half-maximal Ca2+ dependence of 0.3 mM. The Mr = 50,000 fragment from elastase digestion had a lower affinity for thrombin (Kd approximately equal to 6 nM) than intact thrombomodulin, and the Km for protein C was decreased to 0.3 microM in the presence of 0.3 mM Ca2+. The Ca2+ dependence of protein C activation with the Mr = 50,000 fragment was distinctly different from that of thrombomodulin or the active tryptic fragment. The active elastase fragment exhibited a Ca2+ optimum at 0.3 mM and activity rapidly decreased with further increases in Ca2+. At the Ca2+ optimum, the Km for protein C was similar to that observed on endothelial cell surfaces or with thrombomodulin reconstituted into liposomes. Our data demonstrate that thrombomodulin has one or more membrane-binding domains and that an active soluble form with catalytic activity can be generated by limited proteolytic digestion. Digestion with elastase appears to expose a site on thrombomodulin capable of recognizing the gamma-carboxyglutamic acid domain of protein C (residues 1-44 of the light chain). Whether this is the same site which is exposed on thrombomodulin upon incorporation into phospholipid vesicles (see accompanying manuscript) remains to be determined.

Binding Sites↗

PubMed1987

Thrombomodulin.

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Animals↗

PubMed1987

Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro.

Endotoxemia is frequently associated clinically with disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in vivo is unclear. Modulation of tissue factor (TF) and thrombomodulin (TM) expression on the endothelial surface may be relevant pathophysiologic mechanisms. Stimulation of human umbilical vein endothelial cells with endotoxin (1 microgram/ml) increased surface TF activity from 1.52 +/- 0.84 to 11.89 +/- 8.12 mU/ml-10(6) cells at 6 h (n = 11) which returned to baseline by 24 h. Repeated stimulation at 24 h resulted in renewed TF expression. Endotoxin (1 microgram/ml) also caused a decrease in TM expression to 55.0 +/- 6.4% of control levels at 24 h (n = 10) that remained depressed at 48 h. Both effects were dose and serum dependent. A temporary rise in TF expression accompanied by a sustained fall in TM expression comprise a shift in the hemostatic properties of the endothelium that would favor intravascular coagulation and may contribute to the pathogenesis of DIC in gram-negative septicemia.

Cell Membrane↗