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Biomedical subjects

V Gurewich

Publications and source records attributed to V Gurewich.

At least 73 records · Page 4Linked to original sources
PubMed1987

Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments.

Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro-UK), purified human thrombin converted pro-UK (scu-PA) to a two-chain form that had no amidolytic activity. The two chains (Mr approximately 33,000 and 22,000) were disulfide linked and resistant to subsequent activation by plasmin. By contrast, thrombin did not inactivate tissue plasminogen activator or two-chain urokinase. The enzyme from snake venom Agkistrodon contortrix, relatively specific for fibrinopeptide B, had an effect similar to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod), specific for fibrinopeptide A, did not. When pro-UK (scu-PA) was present during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA) in the clot supernatant was seen, whereas virtually full recovery (95%) of radioactivity was found. A loss of latent amidolytic activity in the clot supernatant was also found, the extent of which could be correlated with the degree of degradation of the radiolabeled probe. It was concluded that thrombin inactivation of pro-UK (scu-PA) accounts for the loss of amidolytic activity in the clot supernatant, which has been attributed to fibrin binding. Further confirmation was obtained from experiments in which ancrod was used as the clotting agent. Full recovery of both radioactivity and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was obtained under these conditions. These findings indicate that thrombin may introduce an artifact in the results of certain experiments designed to study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).

Ancrod↗
PubMed1987

Lipids, lipoproteins, fibrinogen and fibrinolytic activity in angiographically assessed coronary heart disease.

Plasma lipids, lipoproteins, fibrinogen and fibrinolytic activity (FA) were measured in 202 consecutive patients undergoing coronary angiography. Twenty-one patients, 13 men and 8 women with a mean age of 52.8 years and 56.7 years respectively, were found to be angiographically free of coronary artery disease (CAD) and served as the principal control group. Since this group contained a disproportionate number of subjects with risk factors such as family history, hypertension and smoking, a second control group of clinically healthy subjects selected for age was also tested. Their laboratory results were not used in the statistical calculations. The group with angiographic CAD consisted of 130 men (mean age 57.6 years) and 51 women (mean age 61.5 years). Abnormal angiograms were graded according to the number of major vessels with more than 50% stenosis involved. The laboratory variables which were significantly (p less than .01-.001) associated with the presence of CAD were: High density lipoprotein (HDL) when determined by polyacrylamide gel electrophoresis (PAGE) and expressed as a percentage of total lipoproteins rather than concentration, presence of Intermediate Density Lipoprotein (IDL), percent of Very Low Density Lipoprotein (VLDL), fibrinogen concentration and FA. The HDL2 subfraction was significantly inversely correlated only in women. The total plasma cholesterol was normal and virtually identical in both groups. Within the CAD group, only two of the laboratory results were significantly correlated with the extent of disease. By univariate analysis, the FA showed the closest association with the score for severity of CAD (p less than .001) followed by the presence of IDL (p less than .01). In conclusion, lipoprotein analysis by a method which measures not only HDL, but also LDL, VLDL and IDL, together with the determination of fibrinogen and FA provides information useful in the identification of individuals at risk for CAD.

Adult↗
PubMed1987

Activation of plasminogen by single-chain urokinase or by two-chain urokinase--a demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase).

Single-chain urokinase (SC-UK) has an intrinsic amidolytic activity, as measured with synthetic substrate (Kabi S-2444; pyro-Glu-Gly-Arg-pNitroanalide), which was found to be 0.1% to 0.2% that of its plasmin-activated derivative, two-chain UK (TC-UK). A study of the reaction of SC-UK with plasminogen is complicated by the effect of the reaction product, plasmin, on both reactants. The resultant generation of TC-UK and Lys-plasminogen produces secondary reactions which greatly augment plasminogen activation. To confine enzymatic activity to the primary reaction, after pretreatment to eliminate trace TC-UK contaminants, SC-UK was incubated with Glu- or Lys-plasminogen in the presence of aprotinin (500 KIU/mL) to inhibit generated plasmin and dansyl-glutamyl-glycyl-arginyl-chloromethylketone (20 mumol/L), which irreversibly inhibited TC-UK but not SC-UK. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a plasminogen-activating activity for SC-UK that was approximately 0.4% that of TC-UK. Both SC-UK and TC-UK preferentially activated Lys-plasminogen over Glu-plasminogen. Similarly, Glu-plasminogen activation was augmented by lysine or soluble fibrin. The ratio of the reaction rates of SC-UK and TC-UK were comparable for Glu- and Lys-plasminogen. It is concluded that there is a major difference in the catalytic activities of SC-UK and TC-UK against plasminogen that is comparable to that against synthetic substrate.

Amino Acid Sequence↗
PubMed1986

A comparative study of the efficacy and specificity of tissue plasminogen activator and pro-urokinase: demonstration of synergism and of different thresholds of non-selectivity.

Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The fibrinolytic activity of pro-UK was expressed as latent units, i.e. measured after activation with plasmin on a fibrin plate against the reference standard. The t-PA unitage was assigned on a weight basis of a similar equivalence of 100,000 IU/mg. To simplify comparison, both activators were expressed in IU (1 IU = approximately 10 ng). At low concentration (1-50 IU/ml), t-PA induced more effective and more linear clot lysis, whereas pro-UK induced lysis was preceded by a lag phase. The two activators were equivalently effective at higher concentrations and saturated at the same lysis rate. Clots made from platelet rich plasma or whole blood were more responsive to lysis by pro-UK but not t-PA than corresponding platelet poor clots. At very low concentrations (2.5-5 IU/ml) of t-PA combined with moderate concentrations (25-50 IU/ml) of pro-UK, a synergistic effect on clot lysis, which was fibrin-specific, was observed. Plasminogen and fibrinogen and the appearance of plasmin-inhibitor complexes in plasma were measured after incubation with either activator with and without a clot present. Non-specific plasminogen activation occurred above a certain concentration of either activator but was found at lower concentrations of t-PA than pro-UK. In the absence of a clot, plasmin generation occurred with t-PA at about 30% of the concentration at which pro-UK induced a corresponding effect. It is concluded that there are important differences in the fibrinolytic and clot selective properties of t-PA and pro-UK, and that some of these properties may be complementary resulting in a fibrin specific, synergistic fibrinolytic effect.

Blood Platelets↗
PubMed1986

Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect.

Highly purified pro-urokinase (pro-UK) or single-chain urokinase-type plasminogen activator (scu-PA) was treated with diisopropylfluorophosphate (1 mmol/L) to eliminate traces of two-chain UK activity. This preparation was found to retain a low activity against a chromogenic substrate (S2444), equivalent to 0.1% to 0.5% of the activity of its plasmin-activated derivative. Evidence is presented that the intrinsic activity of pro-UK (scu-PA) was sufficient to activate plasminogen on a fibrin plate or in buffer and was far more reactive against Lys-plasminogen than against Glu-plasminogen. The relative resistance of Glu-plasminogen to activation was overcome by the addition of lysine (25 mmol/L) to the reaction mixture. By contrast, in plasma, pro-UK (scu-PA) was stable and nonreactive for greater than 72 hours when incubated (37 degrees C). Pro-UK (scu-PA) did not form sodium dodecyl sulfate-stable inhibitor complexes, whereas complexation occurred rapidly with UK. Only at high concentrations of pro-UK (scu-PA) (greater than or equal to 250 IU/mL) did plasminogen activation in plasma occur. The relative inertness of pro-UK (scu-PA) in plasma, in contrast to its low-grade enzymatic activity in buffer, was attributed to the effect of inhibitors. The addition of EDTA or the removal of divalent cations by dialysis was associated with a lower threshold for nonspecific plasminogen activation by pro-UK (scu-PA) in plasma. Replacement of Ca++ but not other cations restored baseline conditions. In the presence of a clot, fibrin-selective plasminogen activation and clot lysis were triggered. Lysis was accompanied by less than 10% conversion of pro-UK (scu-PA) to two-chain UK, suggesting that the intrinsic activity of pro-UK (scu-PA) itself may have been responsible for fibrinolysis, although a contribution by the small amount of UK generated could not be excluded. Similarly, pro-UK (scu-PA) supported clot lysis for several days in the same plasma before the effect dissipated as a result of degradation to UK. When Glu-plasminogen in plasma was replaced by Lys-plasminogen, or when lysine was added to normal plasma, nonselective plasminogen activation and fibrinogenolysis occurred. It was concluded that under the experimental conditions, the fibrin specificity of pro-UK (scu-PA) can be explained by its selective activation of fibrin-bound plasminogen and is due to the latter's Lys-plasminogen-like conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed1984

Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species.

A single-chain 55,000-mol wt form of urokinase (UK), similar to that previously isolated from urine, was purified from a transformed kidney cell culture medium and characterized; and its fibrinolytic properties were evaluated. The preparation immunoprecipitated with UK antiserum, had a low intrinsic amidolytic activity that was 0.1% of its active derivative, and resisted diisopropyl fluorophosphate treatment and inactivation by plasma inhibitors. The single-chain UK was therefore designated pro-UK. In the presence of plasmin and during clot lysis, activation by conversion to two-chain, 55,000-mol wt UK (TC-UK) was demonstrated. This did not occur during blood clotting nor on incubation with purified thrombin. Clot lysis in plasma consistently occurred in 2-5 h with 50-100 IU per ml of pro-UK, whereas comparable lysis was inconsistently achieved by 500-1,000 IU of UK. Pro-UK, in sharp contrast to UK, caused no fibrinogen degradation at fibrinolytic concentrations. In the absence of a clot, pro-UK in plasma was stable for more than 2 d. When a clot was added after incubation (37 degrees C) for 50 h, activation to full lytic activity took place. The findings in vivo were comparable but the rapid clearance of pro-UK required that it be given by a constant infusion despite its plasma stability. In rabbits, a UK-resistant species, pro-UK was significantly (P less than 0.001) more efficacious than TC-UK but neither induced significant fibrinogen degradation. In dogs, a more sensitive species, the high specificity of thrombolysis by pro-UK contrasted with the defibrinogenation and uncontrollable bleeding that accompanied thrombolysis by UK. It was concluded that clot lysis by pro-UK is more effective and specific than UK. The advantage of pro-UK is in the limitation of its activation to the site of a clot. This can be explained by an activation mechanism that is dependent, under physiological conditions, on fibrin-stabilized plasmin.

Animals↗
PubMed1983

The antithrombotic effect of aspirin and dipyridamole in relation to prostaglandin synthesis.

The antithrombotic effect of aspirin (ASA) and dipyridamole (DIP) was evaluated in rabbits in which platelet thromboxane A2 (TXA2) and arterial prostacyclin (PGI2) were measured. An intracarotid cannula thrombosis model previously shown to be sensitive to antiplatelet agents was used. Prostaglandins were determined by radioimmunoassays for thromboxane B2 (TXB2) and 6-keto-prostaglandin PGF1 alpha, the stable metabolites of TXA2 and PGI2. In the aspirin-treated animals, reduction in thrombosis was seen only in rabbits which received a low-dose (1-2 mg/kg), and was related to a selective suppression of platelet TXA2. In contrast, higher doses of ASA (10 or 100 mg/kg), which suppressed both TXA2 and PGI2, were not associated with thrombus inhibition. DIP alone had a lesser antithrombotic effect which was augmented by low-dose ASA but not by high-dose ASA. It is concluded that 1) the antithrombotic effect of ASA in this animal model is dependent on selective TXA2 suppression; 2) ASA has no antithrombotic properties beyond its inhibition of prostaglandin synthesis by platelets; 3) selective suppression of TXA2 in vivo can be achieved in rabbits by a single dose of ASA but only over a narrow dose-range; 4) DIP may have an antithrombotic effect additive to that of low-dose ASA; 5) measurement of serum TXB2 may be used to determine the minimal ASA dose necessary to suppress TXA2 and therefore be most likely to spare PGI2.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine.

Affinity chromatography on fibrin/celite, a previously described technique for isolating plasminogen activators with a high fibrin affinity (S. Husain, B. Lipinski, and V. Gurewich, 1981, Proc. Nat. Acad. Sci. USA 78, 4265-4269), was performed on freshly voided urine. About 10-25% of the plasminogen activator activity present was adsorbed whereas less than 1% was adsorbed when urine was stored at room temperature for 12-24 h. The adsorbed activator was isolated by elution with arginine (0.1 M) and further purified by gel filtration. This plasminogen activator was similar in molecular weight (56,000) to the known high-molecular-weight form of urokinase but differed from it by its binding affinity for fibrin/celite, single-chain subunit structure, lower specific activity (approximately 20,000 IU/mg), and slower reaction with diisopropylfluorophosphate. The enzymatic activity of the purified activator was quenched by anti-urokinase antibody and therefore established it to be a new form of urokinase. The relationship of this high affinity, single-chain urokinase to the previously described forms of urokinase is unclear.

Chemical Phenomena↗
PubMed1982

The value of measuring percent high-density lipoprotein in assessing risk of cardiovascular disease.

The measurement of high-density lipoprotein (HDL) for the purpose of assessing the risk of cardiovascular disease in the individual subject was evaluated. Three laboratory methods were compared, two electrophoretic and one heparin-manganese precipitation, and the HDL results were expressed both as a percent and as an absolute concentration. In phase 1 of the study, the optimal method and the best cut point were identified. In phase 2, these were applied to a larger population who were assigned, on the basis of clinical criteria, to a coronary heart disease and to a control group. The overall probability of correct classification of an individual by his HDL result was calculated. When HDL was expressed as a percent and determined by gel electrophoresis, 82.6% of control subjects and 83.0% of patients with coronary heart disease were classified correctly using the optimal cut point of 23.5%.

Adult↗
PubMed1980

Transfemoral temporary pacing and deep vein thrombosis.

The incidence of deep vein thrombosis following transfemoral temporary pacing was prospectively assessed in 20 consecutive patients using serial 125I-fibrinogen scanning, impedance plethysmography, and measurements of fibrin degradation products. Four patients underwent venography. Of the twenty patients, five (25%) had definite evidence of deep vein thrombosis, six demonstrated equivocal abnormalities, and nine had no evidence of thrombosis. Thrombosis was found only in the legs used for pacing. Of the five with deep vein thrombosis, one had positive findings in all the noninvasive tests. Venography confirmed the diagnosis in the other four; one had thrombosis of the femoral vein, one had thrombosis of several calf veins, and one had thrombosis of both the thigh and calf veins; the fourth patient had thrombosis of the entire deep venous system of the leg. This latter patient was the only one who had clinical evidence of venous thrombosis. The patients with deep vein thrombosis did not differ in clinical characteristics from those without thrombosis. There was no relationship between the occurrence of deep vein thrombosis and the number of venepunctures, the state of the electrode, the presence of the introducer sheath in the femoral vein, or the duration of pacing. In conclusion, deep vein thrombosis is a common complication of transfemoral temporary pacing and the high incidence of its occurrence should be considered before deciding upon the site of entry for a temporary pacemaker.

Adult↗
PubMed1980

Fibrin degradation products and impedance plethysmography. Measurements in the diagnosis of acute deep vein thrombosis.

Measurement of fibrin degradation products (FDP) was evaluated in 152 consecutive patients referred because diagnosis of deep vein thrombosis (DVT) was suspected. All patients underwent impedance plethysmography (IPG), and venograms were obtained in 59. Sensitivity and specificity of FDP measurement, using venography as the standard, was 88% and 66%, respectively. Sensitivity and specificity of IPG was 77% and 79%, respectively. Specificity of the two tests combined, when both yielded abnormal results, was 93%. Patients were classified as (1) normal, (2) abnormal, or (3) equivocal on the basis of IPG. A significant difference in mean FDP concentration was found between groups 1 and 2 and groups 2 and 3. An overall correlation was found between degree of abnormality on IPG and FDP concentration. A quantitative determination of FDP is a practical and reliable screening test for DVT and, when used in conjunction with IPG, improves both sensitivity and specificity of the latter.

Adult↗