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Biomedical subjects

C T Esmon

Publications and source records attributed to C T Esmon.

At least 127 records · Page 7Linked to original sources
PubMed1992

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.

Amino Acid Sequence↗
PubMed1992

"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents.

Amino Acid Sequence↗
PubMed1991

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.

Amino Acid Sequence↗
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

The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity.

A thrombin receptor has recently been cloned and the sequence deduced. The sequence reveals a thrombin cleavage site that accounts for receptor activation. The receptor also has an acidic region with some similarities to the carboxyl-terminal region of the leech thrombin inhibitor, hirudin. Synthetic peptides corresponding to the receptor cleavage site (residues 38-45), the hirudin-like domain (residues 52-69), and the covalently associated domains (residues 38-64) were evaluated for their ability to bind to thrombin. Peptides 38-45 and 38-64 were competitive inhibitors of thrombin's chromogenic substrate activity (Ki = 0.96 mM and 0.6 microM, respectively. Residues 52-69 altered the chromogenic substrate specificity, resulting in accelerated cleavage of some substrates and inhibited cleavage of others. The same peptide binds to thrombin and alters the fluorescence emission intensity of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-thrombin in which the dansyl is attached directly to the active site serine (Kd = 32 +/- 7 microM). Residues 52-69 displace the carboxyl-terminal peptide of hirudin, indicating that they share a common binding site in the anion exosite of thrombin. These data suggest that the thrombin receptor has high affinity for thrombin due to the presence of the hirudin-like domain and that this domain alters the specificity of thrombin. This change in specificity may account for the ability of the receptor to serve as an excellent thrombin substrate despite the presence of an Asp residue in the P3 site, which is normally inhibitory to thrombin activity.

Amino Acid Sequence↗
PubMed1991

Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.

Amino Acid Sequence↗
PubMed1991

Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins.

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.

Animals↗
PubMed1991

Thrombin Glu-39 restricts the P'3 specificity to nonacidic residues.

Residue 39 of serine proteases neighbors positions P'2 to P'4 of the substrate. When Glu-39 of thrombin is replaced with Lys, the resultant enzyme (E39K) retains similar P1, P2, and P3 specificities but has altered P'3 and/or P'4 specificities. These conclusions are based on analysis of both p-nitroanilide and synthetic peptide hydrolysis. The activity of E39K is nearly normal toward 17 p-nitroanilide substrates. In peptide substrates, an acidic residue at either the P3 or P'3 position reduces the rate of cleavage by thrombin. A single substitution of Asp with Gly in either the P3 or P'3 position of a peptide corresponding to the P7-P'5 residues of protein C increases the rate of cleavage by thrombin 2-3-fold. Replacement of both Asp residues with Gly increases the rate of cleavage 30-fold. With E39K, the inhibitory effect of Asp in P3 remains unchanged, but Asp in the P'3 site is no longer inhibitory. Significant differences in the catalytic activity of E39K are also seen with respect to protein C activation. In the absence of thrombomodulin, E39K activates protein C 2.2 times faster than thrombin. In the presence of thrombomodulin, the rate of protein C activation is similar for E39K and thrombin. The second order rate constant of inhibition by antithrombin III, where P'4 is a Glu, is slightly increased (1.4-fold). The clotting activity is reduced 2.4-fold due to a lower rate of fibrinopeptides A and B release where P'3 is Arg. These data show that the P'3 position is a determinant of thrombin specificity and suggest that thrombomodulin may function in part by alleviating the inhibitory effects that may arise from the proximity of the Asp in P'3 of protein C with Glu-39 of thrombin.

Amino Acid Sequence↗
PubMed1991

C4b-binding protein exacerbates the host response to Escherichia coli.

Activated protein C is a plasma anticoagulant. For activated protein C to function as an anticoagulant, it must form a complex with protein S. Protein S anticoagulant activity is neutralized by formation of a reversible complex with C4b binding protein (C4bBP). C4bBP is an acute-phase plasma protein. When C4bBP levels increase, mass action forces the level of free protein S to decrease, giving rise to an acquired functional protein S deficiency. It has been proposed that these elevated C4bBP levels and the resultant acquired deficiency of protein S that occurs in inflammation could contribute to a hypercoagulable state. An experimental model to test this hypothesis was suggested by our previous studies that demonstrated that inhibition of protein C activation rendered baboons hypercoagulable in response to sublethal Escherichia coli infusion (J Clin Invest 79:918, 1987). We have extended these studies to examine the effect of inhibition of protein S activity with C4bBP in the host (baboon) response to infusion of sublethal concentrations of E coli organisms. Five sets of animals were studied: (1) those challenged with sublethal concentrations of E coli alone (0.4 x 10(10)/kg); (2) those supplemented only with C4bBP (20 mg/kg); (3) those challenged with the same level of E coli but supplemented with C4bBP (20 mg/kg); (4) those challenged with sublethal E coli and supplemented with C4bBP (20 mg/kg) and sufficient protein S (2.3 mg/kg) to fill the protein S binding sites on C4bBP; and (5) those challenged with lethal concentrations of E coli. Sublethal E coli infusion (group 1 animals) caused only an acute-phase response with no consumption of fibrinogen, detectable organ damage, or detectable tumor necrosis factor (TNF) in the plasma. C4bBP infusion (group 2 animals) resulted in no significant physiologic changes, no detectable plasma TNF, and little change in fibrinogen level. The group 3 animals, receiving both sublethal E coli and C4bBP, exhibited rapid consumption of fibrinogen, systemic organ damage, and detectable circulating TNF ultimately leading to death. The overall response of this group was very similar to the response of the group 5 animals receiving an LD100 dose of E coli. The group 4 animals, which were treated exactly as above except that C4bBP was supplemented with a slight excess of protein S, responded essentially like those that received sublethal E coli alone. These studies suggest that the elevation of C4bBP during an inflammatory response can contribute to fibrinogen consumption and vascular damage. This vascular damage may be associated with enhanced elaboration of cytokines like TNF.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage.

One of the aims of research in the area of thrombosis has been to design an effective anticoagulant that would function in a predictable and direct manner. In evaluating the role of coagulation in sepsis we used factor Xa blocked in the active center with [5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl+ ++ chloromethyl ketone (DEGR-Xa). We infused 1 mg/kg of DEGR-Xa together with LD100 concentrations of Escherichia coli (4 x 10(10) organisms/kg) into five baboons. As controls, we infused E coli alone into five baboons. The inflammatory, coagulant, and cell injury responses to E coli of both the treated and control groups were lethal and were similar in every respect except for the complete inhibition of the consumption of fibrinogen in the DEGR-Xa group. The half life of DEGR-Xa was approximately 10 hours and 2 hours, as determined by isotopic and enzyme-linked immunosorbent assays, respectively. These results for the first time demonstrate that, although coagulation occurs in E coli sepsis, fibrin formation per se did not influence the lethal outcome in this model. These results also show the effectiveness of DEGR-Xa as an anticoagulant and raise the possibility that it could serve as an alternative to anticoagulants currently in use.

Amino Acid Chloromethyl Ketones↗
PubMed1991

The natural anticoagulant protein S is decreased in male smokers.

Protein S is a natural anticoagulant plasma protein; familial deficiency is associated with thrombotic complications. Since smoking carries an increased risk of myocardial infarction and stroke, we investigated the protein S system of men who smoke. In plasma, free protein S is functionally active and protein S bound to C4b binding protein is inactive. Male smokers (n = 24) have lower total protein S than nonsmokers (n = 24) (16.8 +/- 4.3 micrograms/ml versus 19.8 +/- 5.1 micrograms/ml, p less than 0.02). C4b binding protein levels are higher (p less than 0.05) in smokers (212 +/- 47 micrograms/ml versus 186 +/- 52 micrograms/ml). Free protein S is significantly lower in smokers (6.9 +/- 2.3 micrograms/ml versus 8.0 +/- 1.9 micrograms/ml, p = 0.02). These alterations in protein S may contribute to the thrombotic complications associated with smoking.

Adult↗
PubMed1991

Deep venous thrombosis in the baboon: an experimental model.

Experimental models of deep venous thrombosis, heretofore, have not been available for laboratory studies. This investigation establishes a novel model of venous thrombosis by inhibiting the protein C system combined with venous stasis and subtle venous injury. Ten adolescent baboons were studied in pairs, with one animal receiving saline solution (B2, B4, B6, B8, B10) and one being exposed to thrombogenic reagents (B1, B3, B5, B7, B9). These reagents represented a combination of a monoclonal antibody (HPC4) to protein C, 1 to 4 mg/kg administered over 5 minutes, and tumor necrosis factor administered over 3 minutes at a dose of 150 micrograms/kg through a catheter placed into the left superficial femoral vein with distal ligation. To encourage stasis, a pediatric size blood pressure cuff was inflated to 40 mm Hg on the right thigh for 50 minutes of every hour during the first experimental day (day 1) in B5 to B10. The animals were observed for a 6-hour period on day 1 and then for an 11- to 15-day period until sacrifice. Hemodynamic and hematologic parameters were recorded along with duplex imaging of the iliac veins and inferior vena cava on a daily basis. Venography was performed on day 1, day 4, and the day of sacrifice. At sacrifice the entire iliac and vena caval system was carefully dissected, opened, and photographed. Experimental animals given the HPC4 and tumor necrosis factor developed left iliac vein thrombosis extending into the inferior vena cava. Duplex imaging, venography, and autopsy revealed that control animals receiving saline solution never developed comparable thrombus. Experimental subjects exhibited thrombus on duplex imaging by day 4 (B1), day 3 (B3), day 2 (B5), 120 minutes (B7), and 360 minutes (B9) after receiving HPC4 and tumor necrosis factor. Venograms performed on day 1 exhibited thrombus in B5, B7, and B9. The extent of thrombus, the timing of its occurrence, and its effect on the animals' left leg followed a dose-dependent relationship for the animals in which the occlusive blood pressure cuff was used. Significantly greater declines in blood pressure, white blood cell count, and platelet count were found in affected animals given HPC4 and tumor necrosis factor reagents as compared to control subjects. All affected animals demonstrated the appearance of fibrin split products and a markedly prolonged prothrombin time. This investigation, for the first time, establishes a reproducible model of deep venous thrombosis involving inhibition of protein C that will facilitate further laboratory studies on venous thrombosis.

Animals↗
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↗
PubMed1991

Protein C activation following coronary artery occlusion in the in situ porcine heart.

BACKGROUND: Activated protein C, the body's natural anticoagulant, may play a role in protecting the heart during and following an occlusion of the left anterior descending coronary artery (LAD) in the porcine heart. METHODS AND RESULTS: Infusion of 1 unit thrombin over 30 seconds into the LAD of juvenile pigs produced a prolongation of the Xa clotting time (153 +/- 14%) in blood drawn from the anterior interventricular vein (AIV). The action of the anticoagulant was blocked by a polyclonal immunoglobulin G antibody directed against porcine activated protein C. A brief (30 seconds) occlusion of the LAD induced a similar prolongation of the Xa clotting time (138 +/- 11%), which was also blocked by the polyclonal antibody. To determine whether activated protein C helps sustain the heart during and following a 2-minute occlusion, three groups of pigs were studied: 11 controls, six receiving activated porcine protein C, and nine receiving a monoclonal antibody (HPC4) that blocks protein C activation. Relative to the controls, HPC4-treated animals recovered function, as measured by the maximum time derivative of left ventricular pressure and segmental shortening, more slowly and were not able to sustain this recovery. Animals receiving activated protein C recovered more quickly and sustained this recovery. CONCLUSIONS: These data indicate that an ischemic insult induces rapid activation of protein C in the coronary microcirulation and that blocking this activation impairs recovery.

Animals↗
PubMed1990

The active site of membrane-bound meizothrombin. A fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va.

The distance between the phospholipid surface and the active site of membrane-bound meizothrombin, a derivative of prothrombin, was determined directly using fluorescence energy transfer. The active site of prothrombin was exposed after a single cleavage by Echis carinatus protease in the presence of [5-(dimethylamino)-1-naphthalenesulfonyl]glutamylglycylarginyl+ ++ (DEGR) chloromethyl ketone to yield DEGR-meizothrombin and thereby minimize secondary proteolysis. When DEGR-meizothrombin was titrated with 80% phosphatidylcholine, 20% phosphatidylserine vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes in the active sites of the membrane-bound proteins and the acceptor dyes at the outer surface of the phospholipid bilayer. This energy transfer required both Ca2+ and phosphatidylserine. Assuming k2 = 2/3, the dependence of the efficiency of energy transfer upon the acceptor density showed that the distance of closest approach between the active site probe and the bilayer surface was 71 +/- 2 A. In the presence of factor Va, the distance was 67 +/- 3 A. These direct measurements show that the active site of meizothrombin is located far above the membrane surface. Also, association of factor Va with meizothrombin on the phospholipid surface appears to cause a slight movement of the meizothrombin protease domain toward the membrane surface. The environment of the dansyl dye covalently attached to the active site of meizothrombin was particularly sensitive to the presence of calcium: addition of Ca2+ ions to metal-free DEGR-meizothrombin reduced the dansyl fluorescence lifetime from 11.7 to 9.0 ns and the dansyl emission intensity by 24%. Hence, the conformation of the active site changed when Ca2+ ions bound to meizothrombin. Since the intensity change was half-maximal at 0.2 mM and was also elicited by the binding of Mg2+ ions, this spectral change correlates with the calcium-dependent conformational change previously observed in fragment 1. We conclude, therefore, that the binding of Ca2+ ions to meizothrombin and, by extension, perhaps to prothrombin, elicits a conformational change that extends beyond the fragment 1 domains into the distant (cf. above) active site or protease domain. The association of factor Va with membrane-bound DEGR-meizothrombin increased both the dansyl emission intensity (by 7%) and polarization. This intensity change and the factor-Va dependent change in energy transfer indicate that the cofactor of the prothrombinase complex functions to modulate the conformation and orientation of both the substrate and the enzyme of the complex.

Animals↗