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Biomedical subjects

M F Scully

Publications and source records attributed to M F Scully.

At least 55 records · Page 3Linked to original sources
PubMed1992

Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups.

The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.

Antithrombin III↗
PubMed1992

Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin.

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.

Binding Sites↗
PubMed1991

Influence of intrinsic and extrinsic plasminogen upon the lysis of thrombi in vitro.

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

Fibrin↗
PubMed1991

Influence of tryptophan modification upon digestion of antithrombin III by elastase.

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

Antithrombin III↗
PubMed1990

Properties of thrombolytic agents in vitro using a perfusion circuit attaining shear stress at physiological levels.

A perfusion circuit was designed to investigate in vitro some of the factors which may influence the success of thrombolytic treatment in vivo. The rate of lysis of clotted plasma and different types of artificial thrombi (fibrin thrombi or whole blood thrombi) was measured in citrated plasma or whole blood under static conditions or under shear stress equivalent to the arterial or venous circulation. With both streptokinase (SK) and tissue-type plasminogen activator (t-PA) the rate of lysis of fibrin thrombi and whole blood thrombi was reduced significantly, when compared to the conventional plasma gel clot model (25-fold and 8-fold, respectively). This occurred particularly with SK which showed a reduction (4-fold) in potency relative to t-PA under these conditions. Lysis of thrombi by both activators was observed to be faster in plasma than whole blood, and also faster with whole blood thrombi than fibrin thrombi. High shear stress, generally, caused a reduction in the rate of lysis of fibrin thrombi and an increase in the rate of lysis of whole blood thrombi compared to lysis rates under static conditions. Under all conditions of flow the lysis rate observed at 50 units t-PA per ml was much faster than that at 500 units per ml unlike the conventional plasma gel clot model.

Fibrin↗
PubMed1990

Influence of chemical modification of tryptophan residues on the properties of human antithrombin III.

According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.

Antithrombin III↗
PubMed1989

Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa.

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

Antithrombin III↗
PubMed1989

Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator.

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation Tests↗
PubMed1989

Influence of whole blood on standard curve for heparin measurement--possible heparin binding by red cells.

Measurement of heparin ex vivo is usually with reference to standard curve prepared with a "spiked" normal human plasma pool (NHP). When the calibration curve was prepared by addition of heparin to whole blood before plasma separation, although the linear relationship was maintained the slope was increased in comparison to the classical standard calibration curve. It is concluded that the preparation of the calibration curve by addition of heparin to NHP may give erroneously high heparin levels in treated patients' plasma, leading perhaps to inappropriate dosage. It was also observed that when heparin was added to blood of different haematocrit (prepared by addition of washed RBC to plasma) and plasma prepared, the subsequent APTT was decreased with the fall in haematocrit; suggesting that the laboratory monitoring of heparin treatment should take into account the patient's haematocrit.

Calibration↗
PubMed1989

Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes.

The binding of urokinase-type plasminogen activators (u-PA) to receptors on various cell types has been proposed to be an important feature of many cellular processes requiring extracellular proteolysis. We have investigated the effect of single-chain u-PA binding to the monocyte-like cell line U937 on plasminogen activation. A 16-fold acceleration of the activation of plasminogen was observed at optimal concentrations of single-chain u-PA. This potentiation was abolished by the addition of either 6-aminohexanoic acid or the amino-terminal fragment of u-PA, thus demonstrating the requirement for specific binding of both single-chain u-PA and plasminogen to the cells. The mechanism of the enhancement of plasmin generation appears to be due primarily to an increase in the rate of feedback activation of single-chain u-PA to the more active two-chain u-PA by cell-bound plasmin, initially generated by single-chain u-PA. This increased activity of the plasminogen activation system in the presence of U937 cells provides a mechanism whereby u-PAs may exert their influence in a variety of cell-associated proteolytic events.

Cell Line↗
PubMed1989

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity.

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.

Allosteric Regulation↗
PubMed1988

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis.

Heparan sulphate with no affinity for antithrombin III (ATIII) was observed to cause acceleration of the factor Xa:ATIII interaction by 1100-fold (k2, 7 X 10(7) M-1.min-1) and the prothrombinase:ATIII interaction by 2900-fold (k2, 2.5 X 10(7) M-1.min-1). Although high-affinity heparan sulphate catalyzed higher acceleration and at lower concentration, in natural mixtures of the two forms the activity of the no affinity form predominated. Heparan sulphate had no significant effect on the thrombin:ATIII interaction but inhibited its potentiation by heparin (Kd 0.3 microM). From the estimated concentration of heparan sulphate on the endothelial cell surface it is proposed that the non-thrombogenic property of blood vessels is due to the acceleration of the factor Xa or prothrombinase:ATIII interaction by the greater mass of surface-bound heparan sulphate rather than by the much smaller proportion of heparin-like molecules (with high affinity for antithrombin III) which may be present.

Angiotensin II↗