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Biomedical subjects

C T Esmon

Publications and source records attributed to C T Esmon.

At least 163 records · Page 9Linked to original sources
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

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex.

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.

Animals↗
PubMed1987

The regulation of natural anticoagulant pathways.

Vascular endothelium plays an active role in preventing blood clot formation in vivo. One mechanism by which prevention is achieved involves a cell surface thrombin-binding protein, thrombomodulin, which converts thrombin into a protein C activator. Activated protein C then functions as an anticoagulant by inactivating two regulatory proteins of the coagulation system, factors Va and VIIIa. The physiological relevance of the protein C anticoagulant pathway is demonstrated by the identification of homozygous protein C--deficient infants with severe thrombotic complications. Recent studies suggest that this pathway provides a link between inflammation and coagulation.

Animals↗
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

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents.

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adamantane↗
PubMed1987

Anticoagulation proteins C and S.

Proteins C and S are two vitamin K-dependent plasma proteins that work in concert as a natural anticoagulant system. Activated protein C is the proteolytic component of the complex and protein S serves as an activated protein C binding protein that is essential for assembly of the anticoagulant complex on cell surfaces. The anticoagulant activity is expressed through the selective inactivation of Factors Va and VIIIa. Many patients deficient in proteins C and S have been described and have an associated thrombotic tendency, but not all heterozygous protein C and S deficient individuals experience thrombotic complications. Multiple mechanisms and/or drugs can lead to acquired deficiencies of these proteins: oral anticoagulation, liver disease, DIC and in the case of protein S, lupus erythematosus, nephrotic syndrome, pregnancy and certain hormones. The anticoagulant activity of protein C decreases rapidly after administration of warfarin (i.e., with a time course similar to Factor VII). This rapid decrease may lead to a transient imbalance and contribute to coumarin induced skin necrosis. Protein S antigen levels do not decrease as rapidly, but protein S functional levels are often low in patients with an acute thrombus. The discrepancy between antigen and function results from elevations in C4b-binding protein, which complexes reversibly with protein S. Unlike free protein S, the complex does not function in the anticoagulant pathway. The available information all suggest that deficiency of protein C and protein S should be considered a risk factor contributing to recurrent thrombotic disease and that the function of these proteins is altered by many common clinical conditions which have associated an increased risk of thrombosis.

Anticoagulants↗
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↗
PubMed1987

Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon.

Gram-negative septicemia elicits multiple abnormalities of the coagulation system. Although products of coagulation can lead to clot formation, thereby potentiating organ damage, recent work has shown that low concentrations of thrombin can protect animals from the shock state. Because these amounts of thrombin also lead to formation in vivo of the anticoagulant enzyme, activated protein C, we examined the role of protein C in modulation of Escherichia coli shock in baboons. First, we infused activated protein C and lethal concentrations of E. coli organisms, which prevented the coagulopathic, hepatotoxic, and lethal effects of E. coli. Second, using an antibody to protein C we blocked protein C activation in vivo to determine if this influenced the response to lethal and sublethal concentrations of E. coli organisms. Under these conditions the response to lethal concentrations of E. coli organisms was made more severe and the response to sublethal concentrations of E. coli was made lethal. The coagulopathic, hepatotoxic, and lethal responses in this latter case were prevented by infusion of exogenous protein C.

Alanine Transaminase↗
PubMed1987

Protein S deficiency occurs in the nephrotic syndrome.

Protein S activity may be compromised in patients with the nephrotic syndrome and contribute to a thrombotic diathesis. Protein S is found in two forms in plasma as free and functionally active protein S, and complexed to C4b-binding protein. When compared with controls, patients with nephrotic syndrome had reduced functional levels of protein S (69% +/- 27% [SD], p less than 0.001) despite having elevated levels of total protein S antigen (139% +/- 42%, p less than 0.001). Decreased protein S activity was caused by significant reductions in free (active) protein S levels (90% +/- 38%, p less than 0.05) due to the selective urinary loss of free protein S and elevation of C4b-binding protein levels (170% +/- 52%, p less than 0.001) that favors complex formation; and in the specific activity of the circulating free protein S (0.76; p less than 0.001). Along with this reduction in specific activity, we noted the abnormal electrophoretic mobility of the protein S in the presence of calcium ions. We conclude that acquired protein S deficiency occurs in the nephrotic syndrome and may be a risk factor for the development of the thromboembolic complications.

Blood Coagulation↗
PubMed1987

Oral contraceptives and gender affect protein S status.

Protein S is a plasma protein that serves as a cofactor for the anticoagulant effects of activated protein C. Congenital protein S deficiency is often associated with thromboembolic disease. During pregnancy a decrease in the functional and antigenic levels of protein S occurs; this change in protein S status may contribute to the thromboembolic complications that sometimes occur during pregnancy. In certain patients, oral contraceptive use has also been associated with thrombotic complications. In this study, protein S status was determined in 21 women taking oral contraceptives and compared with that of 21 women not taking oral contraceptives and that of 21 men. The results show that women taking oral contraceptives have significantly lower total protein S (24.3 +/- 3.6 micrograms/mL; mean +/- SD) than women not taking oral contraceptives (28.6 +/- 3.9 micrograms/mL) (P less than .005). Men had significantly higher protein S levels (30.9 +/- 3.9 micrograms/mL, P less than .01) than age-matched women not taking oral contraceptives. In plasma, an equilibrium exists between free (functionally active) protein S and protein S complexed to C4b-binding protein, which is functionally inactive. The mean levels of C4b-binding protein were essentially the same among the three groups, but the levels of free protein S were significantly different and reflected different total protein S antigen levels. Additionally, we found that inflammation significantly elevated C4b-binding protein levels and could result in a further significant decrease in free protein S levels. These data indicate that plasma protein S levels are significantly affected by hormonal status and inflammation.

Adolescent↗
PubMed1987

Analysis of protein C and protein S in disease states.

Protein C and protein S are two vitamin K dependent plasma proteins which function in concert to serve as a natural anticoagulant. Deficiencies of both proteins are associated with thrombotic disease. Assay procedures employing monoclonal antibodies are discussed and compared to previous assay procedures. The assays are effective in patients undergoing heparin or oral anticoagulant therapy and are not influenced by inhibitory factors generated during intravascular coagulation. These assays should aid in our understanding of the role of protein C and protein S in thrombotic disease.

Antibodies, Monoclonal↗
PubMed1987

Protein C, isolation and potential use in prevention of thrombosis.

Protein C and protein S serve as natural anticoagulants. Deficiencies of these proteins are often associated with recurrent deep vein thrombosis and coumarin induced skin necrosis. These two proteins function by selectively inactivating factors Va and VIIIa, two of the "cofactors" of blood coagulation. Hence, inhibition of coagulation by this pathway complements the better known inhibition mediated by the antithrombin III-heparin system. These observations suggest that protein C and/or activated protein C may prove useful in controlling thrombosis and/or DIC. We have developed a Ca2+ dependent monoclonal antibody which allows the rapid isolation of human protein C. This rapid isolation has allowed us to demonstrate that activated protein C can protect baboons from the lethal effects of E. coli/endotoxin and that protein C supplementation can minimize fibrinogen consumption following tissue factor infusion into dogs.

Animals↗
PubMed1986

On the mechanism by which complement proteins C5b-9 increase platelet prothrombinase activity.

Membrane assembly of complement proteins C5b-9 on human platelets results in a dose-dependent increase in the binding of coagulation factors Va and Xa to the plasma membrane, concomitant with a marked increase in platelet prothrombinase activity. Factor Va binding increased by 6-15-fold in platelets treated with the C5b-9 proteins as compared to controls. In the presence of near-saturating concentrations of factor Xa, factor Va binding to C5b-9-treated platelets approximately doubled. In the absence of added factor Va, C5b-9-treated platelets bound 1700 molecules of factor Xa versus 50 molecules/cell bound to controls, suggesting that C5b-9 assembly on the platelet surface initiates the release of platelet factor V from the alpha-granules. The capacity of the C5b-9 proteins to initiate the nonlytic release of the platelet alpha-granule storage pool was confirmed by assay for platelet factor 4. When measured in the presence of exogenous factor Va (2 micrograms/ml), factor Xa uptake by C5b-9 platelets increased to approximately 5500 molecules/cell (versus 330 molecules/cell for controls). Removal of external Ca2+ inhibited the C5b-9-initiated release of the alpha-granule storage pool and reduced by approximately 50% the expression of new factor Va binding sites, suggesting that these two events contributing to increased platelet prothrombinase activity are mediated in part by the influx of Ca2+ across the C5b-9 pore.

Animals↗
PubMed1986

A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Cultured bovine aortic endothelial cells promote activated protein C-protein S-mediated inactivation of factor Va.

Previous studies have demonstrated that protein S is required for optimal activated protein C-mediated inactivation of Factor Va on the surface of either the platelet or phospholipid vesicles. In this report we demonstrate assembly of the activated protein C-protein S complex on the surface of cultured bovine aortic endothelial cells. Endothelial cell surface acceleration of Factor Va inactivation by activated protein C required the presence of protein S. Kinetic studies indicated that the rate of Factor Va inactivation was half-maximal at a protein S concentration of 0.2 nM and an activated protein C concentration of 0.05 nM. Binding of 125I-activated protein C to endothelial cell monolayers was absolutely dependent on the presence of protein S. At saturating levels of protein S, activated protein C binding was saturable with Kd = 0.04 nM. In contrast, specific, time-dependent, and saturable binding of 125I-protein S to endothelium occurred in the absence of activated protein C. Addition of activated protein C increased the affinity of protein S from Kd = 11 nM to 0.2 nM, but did not change the number of molecules bound per cell at saturation (85,000 molecules/cell). These studies suggest that activated protein C increases the affinity of protein S for pre-existing sites on the endothelial cell surface. The close correlation between the parameters of protein S-activated protein C binding to endothelium and Factor Va inactivation supports the concept that it is bound protein S and activated protein C that are the active species. Formation of functional activated protein C-protein S complexes thus occurs effectively on the endothelial cell surface and represents a new addition to the list of vessel wall anticoagulant properties.

Animals↗
PubMed1986

Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity.

Previous studies demonstrated that endothelial cells participate actively in both anticoagulant and procoagulant reactions. Although anticoagulant mechanisms predominate on the surface of quiescent endothelial cells, perturbed endothelial cells can promote coagulation through the coordinated induction of procoagulant activity and suppression of anticoagulant mechanisms. Purified recombinant interleukin 1 was infused intravenously into rabbits and coagulant properties of the native aortic endothelium were subsequently studied. Interleukin 1 infusion resulted in a time- and dose-dependent induction of the procoagulant cofactor tissue factor, while concomitantly blocking the protein C anticoagulant pathway. Tissue factor activity increased greater than 10-fold by 3-5 hr after the infusion, while endothelial cell-dependent thrombin-mediated protein C activation decreased by 72% and assembly of functional activated protein C-protein S complex on the vessel surface was decreased by greater than 90%. Scanning electron microscopy of major arteries demonstrated fibrin strands closely associated with the luminal endothelial cell surface with a predilection for bifurcations. Interleukin 1, a mediator of the inflammatory response, can shift the balance of procoagulant and anticoagulant reactions on the endothelium unidirectionally favoring clot formation. The surface of perturbed endothelium can thus provide a template, facilitating the development of a prethrombotic state, and provides a model for the early stages of thrombosis.

Animals↗