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Biomedical subjects

J H Lawson

Publications and source records attributed to J H Lawson.

At least 19 recordsLinked to original sources
PubMed1992

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.

Chemical Phenomena↗
PubMed1992

The evaluation of complex-dependent alterations in human factor VIIa.

Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.

Calcium↗
PubMed1992

The role of the membrane in the expression of the vitamin K-dependent enzymes.

The hemostatic response to vascular damage results in the focal generation of thrombin to produce a fibrin/platelet clot at the site of vascular injury. This regulated hemostatic response derives from the assembly and activity of enzyme complexes that are localized to surfaces presented by the vascular damage. The product of each enzymatic complex provides the serine protease component required for the assembly and activity of each successive enzyme complex, ultimately leading to the formation of thrombin. When one limits attention to those complexes clearly associated with hemostatic or thrombotic risk, the significance of the vitamin K-dependent enzyme complexes becomes apparent. Each of these complexes involves a serine protease and a cofactor protein that assemble on a membrane surface in the presence of Ca++. The expression of an active complex involves, in addition to the activation of a zymogen to an enzyme, the presentation or activation of a cofactor protein and the provision of the appropriate membrane to support the reaction. The membrane plays an essential part in the formation and expression of vitamin K-dependent complexes; thus, its regulation is vital in the expression of procoagulant activity.

Blood Coagulation↗
PubMed1991

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation.

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.

Blood Coagulation↗
PubMed1990

Isolation and characterization of an acquired antithrombin antibody.

A 68-year-old man, following mitral valve replacement, presented with a low-grade chronic consumptive coagulopathy. Laboratory analysis showed mild fibrinolysis, minimal effect of coumadin therapy, and a prolonged thrombin time (greater than 150 seconds using bovine IIa). When purified human IIa was used the thrombin time normalized to within 17 seconds of controls, suggesting a possible inhibitor of bovine IIa. An anti-IIa antibody was isolated by protein A-Sepharose (Sigma, St Louis, MO) chromatography followed by affinity chromatography using a bovine IIa-Sepharose column. The effects of this purified anti-IIa antibody on both bovine and human IIa procoagulant and anticoagulant functions were studied. The isolated immunoglobulin G (IgG) was observed to inhibit bovine IIa in all assays tested. This IgG was also able to slightly prolong fibrinogen clotting by human IIa. Using an enzyme-linked immunosorbent assay it was observed that the IgG bound to bovine IIa, bovine II, human IIa, but not to human II. Further, binding was detectable at approximately 50-fold lower concentrations to bovine IIa (1 nmol/L IgG concentration) than to human IIa (50 nmol/L IgG concentration). The effect of the antibody on the reaction between IIa and AT III/heparin was investigated. Human IIa was found to be protected from AT III/heparin neutralization in the presence of this antibody. These results suggest that this patient developed an antibody that strongly binds to and inhibits the bovine IIa in all assays tested. However, the antibody only significantly affects human IIa neutralization by AT III/heparin, and has little effect on the human IIa procoagulant activity. These data suggest that the decreased effect of AT III/heparin on this patient's IIa may have been a contributing factor in his coagulopathy. The exact cause of this antibody development is unclear, but the role of bovine topical thrombin used during cardiac valve replacement surgery is suspect.

Aged↗
PubMed1979

Six-month survival of a calf with an artificial heart.

A pneumatically powered artificial heart, constructed primarily from a polyurethane, was implanted in the chest of a calf and supported the calf for more than 6 months. The heart, which was designed to fit in the chest of a 90 kilogram calf, was able to suppor the animal when it weighed 180 kilograms. During the first 105 days the calf remained strong and healthy. The animal grew progressively weaker after day 106, and by day 160 right heart failure became apparent. The principal cause of the right heart failure was an obstructive growth between the right atrium and the right ventricle. An attempt to correct the problem on day 184 with an artificial heart resulted in the animal's death.

Animals↗
PubMed1978

Legionnaires' Disease--the Benidorm episode.

The clinical pattern of illness in 2 fatal cases of Legionnaires' Disease is described. Common factors in the 2 patients were residence in a hotel in Benidorm, Spain, a severe and progressive pneumonia unaffected by wide-spectrum antibiotics and failure to incriminate an infecting organism. The similarities with the Philadelphia outbreak, in which a bacillus known as the Legionnaire agent was isolated, led to a retrospective diagnosis in the Benidorm episode. Subsequent serological surveys indicate that Legionnaires' Disease is widespread in nature; it is not a new disease.

Female↗
PubMed1978

The beat goes on: status of the artificial heart, 1977.

After two decades of continuous research on the artificial heart, survival times of experimental animals indicate that clinical application of such a device is definitely feasible. However, a number of problems remain to be solved. Durability of the device, infection, pannus formation at the interface between the device and natural tissue and thrombosis within the device appear to be the major problems. Development of an implantable power source is also an area of important research. Questions of efficiency, cosmetic and psychological acceptability and cost are just beginning to be considered.

Animals↗
PubMed1977

The effect of quinidine, propranolol and their combination on experimental atrial and ventricular arrhythmias.

The comparative antiarrhythmic activity of quinidine, propranolol and the combination of these two drugs was studied in experimental atrial and ventricular arrhythmias in the dog. Quinidine, but not propranolol, suppressed atrial arrhythmias produced by topical application of aconitine to the atrium, as well as the ventricular arrhythmias that developed approximately 20 hr following coronary artery ligation. When the two drugs were coadministered, synergism occurred in the atrial arrhthmias but not in the ventricular arrhythmias. Some possible mechanisms of action of these drugs are discussed. The change in response to quinidine by coadministration with propranolol may have resulted in part from suppression of atrial automaticity in the aconitine experiments and from further slowing of ventricular conduction in the coronary artery ligation experiments. Both effects might have occurred as a result of myocardial beta adrenergic receptor blockade.

Animals↗