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T Mather

Publications and source records attributed to T Mather.

17 recordsLinked to original sources
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

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5.

Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly(12) and Gly(13) of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala(30) residue (the equivalent of Gly(13) of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly(12) of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution surprisingly stimulated the GTPase activity by 5-fold. Molecular modelling suggests that this built-in arginine mimics the catalytic arginine residues found in trimeric GTPases and GTPase-activating proteins in providing the positive charge to facilitate the GTP hydrolysis reaction. We investigated further the biological activity of the Rab5 mutants in relation to stimulating endocytosis. When expressed in cultured baby hamster kidney cells, both arginine and proline mutants, like wild-type Rab5, stimulated endocytosis. However, the arginine mutant was a more potent stimulator than the proline mutant (3-fold stimulation as against 1.7-fold). The tryptophan mutant, on the other hand, was completely deficient in activity in terms of the stimulation of endocytosis, demonstrating the importance of the phosphate-binding loop in Rab GTPase function.

Alanine↗
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↗
PubMed1999

The elongin B ubiquitin homology domain. Identification of Elongin B sequences important for interaction with Elongin C.

Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.

Amino Acid Sequence↗
PubMed1999

Area x-ray beam equalization for digital angiography.

An area beam equalization technique has been investigated in order to generate patient-specific compensating filters for digital angiography. An initial image was used to generate the compensating filter, which was fabricated using a deformable compensating material, containing CeO2, and an array of square pistons. The CeO2 attenuator thicknesses were calculated using the gray level information from the initial unequalized image. The array of pistons was pressed against a uniform thickness of attenuating material to generate a filter for x-ray beam equalization. The filter was subsequently inserted into the x-ray beam for the final equalized radiograph. It was positioned close to the focal spot (magnification of 8.0) in order to minimize edge artifacts from the filter. The equalization of x-ray transmission across the field exiting from the object significantly improved the image quality by preserving local contrast throughout the image. The contrast-to-noise ratio (CNR) in the equalized images was increased-by up to fivefold. Phantom studies indicate that equalized images using a relatively small array of pistons (e.g., 8 x 8) produce significant improvement in image quality with negligible perceptible artifacts. Animal studies showed that beam equalization significantly improved fluoroscopic and angiographic image quality. X-ray beam equalization produced an image with a relatively uniform scatter-glare intensity and it reduced the scatter-glare fraction in the previously underpenetrated region of the image from 0.65 to 0.50. Also, x-ray tube loading due to the mask assembly itself was negligible. In conclusion, area beam equalization reduces the scatter-glare fraction and significantly improves CNR in the previously underpenetrated region of the image.

Angiography, Digital Subtraction↗
PubMed1999

Inflammation, sepsis, and coagulation.

The molecular links between inflammation and coagulation are unquestioned. Inflammation promotes coagulation by leading to intravascular tissue factor expression, eliciting the expression of leukocyte adhesion molecules on the intravascular cell surfaces, and down regulating the fibrinolytic and protein C anticoagulant pathways. Thrombin, in turn, can promote inflammatory responses. This creates a cycle that logically progresses to vascular injury as occurs in septic shock. Most complex systems are regulated by product inhibition. This inflammation-coagulation cycle seems to follow this same principle with the protein C pathway serving as the regulatory mechanism. The molecular basis by which the protein C pathway functions as an anticoagulant is relatively well established compared to the mechanisms involved in regulating inflammation. As one approach to identifying the mechanisms involved in regulating inflammation, we set out to identify novel receptors that could modulate the specificity of APC in a manner analogous to the mechanisms by which thrombomodulin modulates thrombin specificity. This approach led to the identification of an endothelial cell protein C receptor (EPCR). To understand the mechanism, we obtained a crystal structure of APC (lacking the Gla domain). The crystal structure reveals a deep groove in a location analogous to anion binding exosite 1 of thrombin, the location of interaction for thrombomodulin, platelet thrombin receptor and fibrinogen. Thrombomodulin blocks the activation of platelets and fibrinogen without blocking reactivity with chromogenic substrates or inhibitors. Similarly, in solution, EPCR blocks factor Va inactivation without modulating reactivity with protease inhibitors. Thus, these endothelial cell receptors for the protein C system share many properties in common including the ability to be modulated by inflammatory cytokines. Current studies seek to identify the substrate for the APC-EPCR complex as the next step in elucidating the mechanisms by which the protein C pathway modulates the response to injury and inflammation.

Amino Acid Chloromethyl Ketones↗
PubMed1998

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Amino Acid Sequence↗
PubMed1997

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three-dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 A resolution using Patterson's search technique and refmed to an R-factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (alpha3)2 into alpha3'alpha3". The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re-arrangements for pyrophosphatase function is discussed.

Ammonium Sulfate↗
PubMed1997

Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 A resolution. Mechanism of hydrolysis.

Crystalline holo inorganic pyrophosphatase from Escherichia coli was grown in the presence of 250 mM MgCl2. The crystal structure has been solved by Patterson search techniques and refined to an R-factor of 17.6% at 1.9 A resolution. The upper estimate of the root-mean-square error in atomic positions is 0.26 A. These crystals belong to space group P3(2)21 with unit cell dimensions a = b = 110.27 A and c = 78.17 A. The asymmetric unit contains a trimer of subunits, i.e., half of the hexameric molecule. In the central cavity of the enzyme molecule, three Mg2+ ions, each shared by two subunits of the hexamer, are found. In the active sites of two crystallographically independent subunits, two Mg2+ ions are bound. The second active site Mg2+ ion is missing in the third subunit. A mechanism of catalysis is proposed whereby a water molecule activated by a Mg2+ ion and Tyr 55 play essential roles.

Binding Sites↗
PubMed1996

The 2.8 A crystal structure of Gla-domainless activated protein C.

The structure of the Gla-domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)-like domain modified by a large insert containing an additional disulfide, followed by a typical EGF-like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin-like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin-like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non-primed side.

Binding Sites↗
PubMed1994

Mutation of Glu-80-->Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex.

Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X. In thrombin, which does not bind Ca2+, Glu-70 is replaced by Lys, creating an internal salt bridge with Glu-80. We constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant is accelerated by thrombomodulin in a Ca(2+)-independent fashion. Unlike wild type Gla-domainless protein C, Ca2+ no longer inhibits activation of the mutant by free thrombin, and Ca2+ stimulation of chromogenic activity is also absent. The characteristic Ca(2+)-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca(2+)-binding site in protein C critical for zymogen activation involves Glu-80. The Glu-80 to Lys mutation probably results in a salt bridge with Glu-70 that stabilizes protein C zymogen in a conformation similar, if not identical, to the Ca(2+)-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.

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↗
PubMedDate not supplied

Effect of fibrin on endothelial cell production of prostacyclin and tissue plasminogen activator.

Fibrin formed on endothelial cells has previously been shown to have deleterious effects on the cells. Additionally, substances that cause endothelial cell damage have been reported to induce cultured endothelial cells to synthesize prostacyclin and tissue plasminogen activator (t-PA). The present studies were undertaken to determine whether fibrin formed on cultured human umbilical vein endothelial cells would alter synthesis of prostacyclin and t-PA by the cells. Fibrin was found to increase synthesis of both prostacyclin and t-PA in a dose and time dependent manner. Stimulation of prostacyclin synthesis was completely inhibited by indomethacin; partially inhibited by actinomycin D, cycloheximide, and trifluoperazine; and not affected by cytochalasin D or vinblastine. In contrast, stimulation of t-PA synthesis was completely inhibited by actinomycin D and cycloheximide; partially inhibited by cytochalasin D, vinblastine, and trifluoperazine; and not affected by indomethacin. Fibrin I, formed with Reptilase, caused only slight stimulation of t-PA production, but virtually no stimulation of prostacyclin synthesis. Neither collagen polymerization on the cells nor thrombin added in concentrations that did not induce fibrin polymer formation stimulated production of either substance. Furthermore, soluble fibrin II generated in the presence of the fibrin polymerization inhibitor gly-pro-arg-pro also failed to stimulate either prostacyclin or t-PA production. The presence of platelets in the plasma from which the fibrin was formed did not affect the amount of stimulation of the cells. Fibrin-induced stimulation of endothelial cell production of prostacyclin and t-PA could act to limit vascular occlusion in vivo by inhibiting platelet function and by stimulating fibrinolysis via t-PA.

6-Ketoprostaglandin F1 alpha↗