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Biomedical subjects

M J Griffith

Publications and source records attributed to M J Griffith.

At least 73 records · Page 4Linked to original sources
PubMed1986

Tissue thromboplastin induced reversible DIC and heparin-enhanced inhibitors in dogs.

Reversible acute disseminated intravascular coagulation (DIC) has been induced in dogs by intravenous injection of homologous tissue thromboplastin. There was no measurable consumption of antithrombin III and heparin cofactor II even if fibrinogen was reduced during DIC by more than 80% of its baseline. The prothrombin level remained practically constant. These data correspond to the generation of a few nanomoles of thrombin in vivo with subsequent pseudo-first order inactivation by the major thrombin inhibitors. An ex vivo measure of the pseudo-first order rate constant (dynamic thrombin inhibitory capacity, DTIC) was a sensitive probe of circulating heparin. There was no change of DTIC during DIC in the absence of exogenous heparin suggesting that heparin-like endogenous glycosaminoglycans were not released in substantial amounts. Pretreatment with heparin efficiently inhibited the development of tissue thromboplastin induced DIC. This animal model may serve as a tool for the study of glycosaminoglycan anticoagulants in vivo.

Animals↗
PubMed1986

Structure-function relationships in heparin cofactor II: chemical modification of arginine and tryptophan and demonstration of a two-domain structure.

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 M GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.

Amino Acids↗
PubMed1985

Comparison of lipid binding and kinetic properties of normal, variant, and gamma-carboxyglutamic acid modified human factor IX and factor IXa.

The abilities of normal and three abnormal factor IXa molecules to activate factor X and to bind to phospholipid membranes have been compared to define the contributions of protein-lipid interactions and factor IXa light chain-heavy chain interactions to the functioning of this protein. The abnormal proteins studied had altered amino acid residues in their light chains. The heavy-chain regions, containing the active site serine and histidine residues, were normal in the abnormal proteins on the basis of titration by antithrombin III. The binding constants (Kd) for normal (N), variant [Chapel Hill (CH) and Alabama (AL)], and gamma-carboxyglutamic acid (Gla) modified (MOD) factors IX and IXa to phosphatidylserine (PS)/phosphatidylcholine (PC) small, unilamellar vesicles (SUV) were measured by 90 degrees light scattering. The Kd values for factor IXN binding were quite sensitive to the PS content of the membrane but less sensitive to Ca2+ concentrations between 0.5 and 10 mM. The zymogen and activated forms of both normal and abnormal factor IX bound with similar affinities to PS/PC (30/70) SUV. In the cases of factor IXaN and factor IXaAL, but not factor IXaCH or factor IXaMOD, irreversible changes in scattering intensity suggested protein-induced vesicle fusion. Since the activation peptide is not released from factor IXaCH, the normal interaction of factor IXa with a membrane must require the release of the activation peptide and the presence of intact Gla residues. The rate of factor X activation by normal and abnormal factor IXa was obtained by using a chromogenic substrate for factor Xa in the presence of PS/PC (30/70) SUV and 5 mM Ca2+.

1-Carboxyglutamic Acid↗
PubMed1985

Structural evidence for leucine at the reactive site of heparin cofactor II.

The reaction products formed during the enzymatic inactivation of heparin cofactor II (HCII) by a proteinase isolated from Echis carinatus were analyzed by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and by reverse-phase high-performance liquid chromatography. By NaDodSO4-polyacrylamide gel electrophoresis, limited proteolysis of HCII was observed, which resulted in a decrease in the apparent molecular weight of the protein from approximately 68 000 to approximately 53 000. By reverse-phase high-performance liquid chromatography, at least 20 peptides were observed. Primary structure analysis of these peptides indicated that significant proteolysis had occurred in the NH2-terminal region of the protein. HCII inactivation, however, coincided with the appearance of a peptide from the COOH-terminal region of the protein. The peptide differed from the previously identified reactive site peptide [Griffith, M. J., Noyes, C. M., & Church, F. C. (1985) J. Biol. Chem. 260, 2218-2225] by only one residue: a leucyl residue at the NH2-terminal of the peptide. We conclude that leucine, as opposed to the expected arginine, is at the reactive site of HCII.

Antithrombins↗
PubMed1985

Enzymatic inactivation of heparin cofactor II by a proteinase (proteinase-1) isolated from Echis carinatus venom.

Heparin cofactor II was enzymatically inactivated by incubation with Echis carinatus venom in the presence of calcium. The initial rate of inactivation increased proportionately with the addition of heparin to a final concentration of 50 micrograms/ml. A proteinase, termed proteinase-1, was purified 17.5-fold from the venom which also enzymatically inactivated heparin cofactor II in the presence of calcium. The initial rate of heparin cofactor II inactivation by proteinase-1 was not increased by heparin at concentrations as high as 200 micrograms/ml. Heparin cofactor II was not inactivated by either unfractionated venom or proteinase-1 in the absence of calcium. The results indicate that heparin cofactor II, like antithrombin III, is susceptible to enzymatic inactivation by metalloproteinases in snake venoms.

Animals↗
PubMed1985

Activation of normal and abnormal human factor IX with trypsin.

Human factor IX is activated to factor IXa beta when factor XIa cleaves two peptide bonds, Arg 145-Ala 146 and Arg 180-Val 181, to release an activation peptide. In factor IX Chapel Hill (IXCH), isolated from a hemophilia B patient with a mild bleeding disorder, the arginine 145 residue has been replaced with a histidine. Thus factor IXCH is activated by factor XIa by cleaving only at the Arg 180-Val 181 bond, leaving the activation peptide attached, and resulting in an activated species, factor IXa alpha CH, that, like normal factor IXa alpha, is only 20% as active as factor IXa beta. It is reported that both factor IX and factor IXCH could be activated by trypsin to forms of factor IXa beta and factor IXa beta CH that had clotting activities identical to factor XIa-activated factor IX. Amino-terminal amino acid sequence analysis showed that trypsin cleaved factor IX at the same bonds as did factor XIa; factor IXCH was cleaved at the Arg 180-Val 181 bond, as normal, and was cleaved near the histidine 145, at the Lys 142-Leu 143 bond, releasing a slightly larger activation peptide than from normal factor IXa beta. Metal ions had no effect on the rate of activation of factor IX by trypsin; however, metal ions had a profound effect on the rate at which further incubation with trypsin inactivated factor IXa. Calcium and manganese protected factor IXa from inactivation by trypsin more effectively than magnesium, which was more effective than no metal ion. It is concluded that trypsin can activate normal factor IX and factor IXCH to fully active IXa beta forms.

Amino Acid Sequence↗
PubMed1985

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.

1-Carboxyglutamic Acid↗
PubMed1985

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III.

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.

Amino Acid Sequence↗
PubMed1985

Inhibition of chymotrypsin by heparin cofactor II.

Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin.

Binding Sites↗
PubMed1985

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.

Blood Coagulation↗
PubMed1985

Heparin-enhanced inhibitors during reversible disseminated intravascular coagulation.

Intravenous injection of homologous lung or brain tissue thromboplastin in dogs under general anesthesia induced changes of conventional hemostasis variables consistent with acute DIC (prolongation of prothrombin times, thrombin times, APTT, drop of fibrinogen and a transient reduction of the platelet count). The animals reacted with accelerated respiration and pulse rates. After recovery from anesthesia they resumed their normal activity as before. Fibrinogen reached a minimum within 40 min after the DIC trigger dose had been injected. Dependent on the size of the latter up to 80% of clottable fibrinogen was consumed. No consumption of antithrombin III and heparin cofactor II could be demonstrated by functional assays based on thrombin inhibition by diluted plasma in the presence of heparin or dermatan sulfate. Prothrombin measured amidolytically by an Echis Carinatus venom assay remained practically unchanged. These findings are consistent with free thrombin concentrations in the nanomolar range sufficient to clot fibrinogen rapidly without visibly affecting the up to 1,000 fold higher concentrations of inhibitors and prothrombin. Heparin administered before tissue thromboplastin virtually suppressed the evolution of DIC but its protective effect was overcome by higher trigger doses. Heparin injected after the induction of DIC had no protective effect. The reversible DIC model in dogs may be a promising tool to study activated coagulation in vivo at practically constant inhibitor concentrations. One dog can be used for several acute experiments with homologous tissue thromboplastin, thus the number of animals and their costs may remain within reasonable limits.

Animals↗
PubMed1985

The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation.

The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

Blood Cells↗
PubMed1984

Evidence for essential lysines in heparin cofactor II.

Covalent modification with pyridoxal 5'-phosphate was used to study the function of lysyl residues in heparin cofactor II, a heparin-dependent plasma protease inhibitor. Reduction of the Schiff base with sodium borohydride resulted in modification of 3-4 lysyl residues of heparin cofactor II at high concentrations of pyridoxal 5'-phosphate, one of which was protected in the presence of heparin. The antithrombin activity of modified heparin cofactor II was enhanced compared to the native protein. However, the heparin cofactor activity for thrombin inhibition was reduced significantly or completely eliminated in the modified protease inhibitor depending on the extent of phosphopyridoxylation. In contrast to native heparin cofactor II, the modified protease inhibitor did not bind to a heparin-agarose column. The results suggest that lysyl residues are essential for heparin cofactor activity during thrombin inhibition.

Chromatography, Affinity↗
PubMed1984

Inactivation of human blood coagulation factor X by chemical modification of gamma-carboxyglutamic acid residues.

The inactivation of human factor X by incubation with a reagent known to chemically modify gamma-carboxyglutamic acid to gamma-methylene glutamic acid was studied. Incubation of factor X at pH 5.0 with a preincubated formaldehyde/morpholine mixture (0.9 M/1.0 M) resulted in a progressive decrease in factor X coagulant activity. In the presence of calcium (20 mM) the rate of factor X inactivation was decreased -3-fold. By using [14]C-formaldehyde, modified-factor X (less than 5% residual activity) was found to contain 7 mols of [14]C per mol of protein. Modified-factor X was not activated by Russell's viper venom in the presence of calcium, suggesting that the loss of coagulant activity was related to the inability of modified-factor X to be activated.

1-Carboxyglutamic Acid↗
PubMed1984

Interaction of substrates with glutamine synthetase after limited proteolysis.

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1984

Utilization patterns of health maintenance organization disenrollees.

This article addresses several questions about the effect of disenrollment on the start-up phenomenon in health maintenance organizations that were raised in a recent exchange of articles and comments in this journal. The results show that disenrollees, like continuous enrollees, go through a start-up phase of utilization that decreases over duration of membership to a stable lower level. The study also differentiates between voluntary and involuntary disenrollees, both of which go through start-ups. The different characteristics of the start-ups of both groups of disenrollees are contrasted with those of continuous enrollees. The implications of these findings for planners and administrators are discussed, and a future research agenda is recommended.

Adult↗
PubMed1984

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.

Animals↗