Thromboelastographic study of fibrinolytic agents.
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Fibrin↗
Biomedical subjects
L Summaria
Publications and source records attributed to L Summaria.
An anti-plasminogen antibody preparation that inhibits the activation of human plasminogen but enhances the activator activity of the B-chain-streptokinase complex.
An anti-Glu-plasminogen (GLU-PLG) polyclonal antibody antiserum was prepared in the goat, and specific IgG anti-GLU-PLG antibodies were purified by affinity chromatography using a sepharose-GLU-PLG column. In chromogenic assay studies, the anti-GLU-PLG antibody preparation was found to be an effective inhibitor of the activation of PLG, and it produced different inhibition curves with four different PLG activators. 80% inhibition of streptokinase (SK) activation of GLU-PLG occurred with an anti-GLU-PLG antibody/GLU-PLG molar ratio of 1:1, whereas at this ratio only 28% and 36% inhibition of the plasmin-streptokinase (PLN-SK) and the B-chain-streptokinase (B-SK) complexes occurred. At a 1:1 molar ratio of antibody to PLN, no inhibition of PLN activity occurred. When the anti-GLU-PLG antibody preparation was incubated with each PLG activator, an enhancement in the activator activity of the B-SK complex, but not the other activators was observed with mini-plasminogen (MINI-PLG). Enhancement occurred at a molar ratio of 1:1 and reached a peak of 97% enhancement at a molar ratio of 10:1. Enhanced activator activity of the B-SK complex of 49% occurred at a molar ratio of 1:1 when GLU-PLG was the substrate. At higher molar ratios inhibition of activator activity on GLU-PLG was observed, but not on MINI-PLG. These results indicate that there are multiple activator binding sites on PLG, that these four activators all bind differently to GLU-PLG and MINI-PLG, that some antibody populations that are specific for the PLN B-chain can stimulate activator activity, while other antibody populations that are specific for either the PLN A-chain or the B-chain are inhibitory.
Animals↗
Comparison of human normal, full-term, fetal and adult plasminogen by physical and chemical analyses.
Human fetal plasminogen was isolated from fetal cord blood obtained from full-term normal newborns. Two fetal plasminogen preparations were characterized by physical analyses and compared to adult human Glu-plasminogen. The protein concentration of plasminogen in each full-term fetal plasma was approximately 50% of the concentration found in adult plasma. The specific activity of the isolated plasminogen from both full-term fetal plasmas was 28.8 +/- 1.5 IU/mg protein, approximately the same as that of adult Glu-plasminogen. No significant difference was observed in the rate at which plasmin was generated from the normal fetal plasminogen and the adult Glu-plasminogen using streptokinase, urokinase, and tissue plasminogen activator. Electrophoretic analyses in an acrylamide gel/sodium dodecyl sulfate dissociating system showed that the fetal plasminogens and the adult Glu-plasminogen were the same molecular size. Analyses in an acrylamide gel isoelectric focusing system indicated that fetal and adult plasminogen both contained the same twelve isoelectric forms, however, there was a slight difference in the distribution of the isoelectric forms. The fetal and adult plasminogens both contained 792 +/- 1 amino acid residues, and there were no significant differences in amino acid composition between the fetal and adult preparations. These comparisons indicate that normal, full-term, fetal and adult Glu-plasminogen are identical.
Adult↗
Decreased platelet number and function and increased fibrinolysis contribute to postoperative bleeding in cardiopulmonary bypass patients.
We simultaneously evaluated platelet and fibrinolytic parameters to assess their individual and combined contributions to postoperative blood loss in cardiopulmonary (CP) bypass patients. Platelet count, platelet aggregability, hematocrit, plasminogen (PLG) concentration, alpha 2-antiplasmin (AP) concentration, free protease activity (fPA), and antithrombin-III (AT-III) were measured in nine patients undergoing surgery using cardiopulmonary bypass. Chest tube drainage was used as the measure of postoperative blood loss. Hematocrit, platelet count, PLG, AP, and AT-III all decreased during CP bypass, with PLG and AT-III decreasing much more than dilution. During CP bypass, platelet aggregability to ADP did not change significantly from pre-bypass, but aggregability to arachidonic acid (AA) decreased significantly. Following protamine administration there was a large increase (83%) in fPA, the platelet count showed a further drop (from 61% to 50% of pre-bypass levels), and platelet aggregability decreased significantly (from 95% to 34% of pre-bypass levels for ADP, and from 55% to 11.9% for AA). Chest tube drainage during the first four postoperative hours correlated positively (p less than 0.05) with the combination of increase in free protease activity and decrease in platelet count. The total chest tube drainage correlated significantly with the combination of decrease in platelet count and the decrease in platelet aggregability. These combinations of changes correlated significantly with postoperative blood loss whereas the individual changes did not.(ABSTRACT TRUNCATED AT 250 WORDS)
Antithrombin III↗
Relationship between postsurgical fibrinolytic parameters and deep vein thrombosis in surgical patients treated with compression devices.
This study consisted of 52 patients admitted for orthopedic surgery and 28 patients admitted for general surgery, who were treated with Sequential Compression Devices (SCD) and Thromboembolic Deterrent Stockings (TEDS) and monitored for the development of deep vein thrombosis (DVT). Coagulation and fibrinolytic profiles were carried out on these patients preoperatively, and on days one, three, and six postoperatively. All patients were followed by I-125-Fibrinogen scanning, Venous Doppler, and Impedance Plethysmography studies for clot detection. In the orthopedic surgery group, six (11.5%) developed DVT, and in the general surgery group, one (3.6%) developed DVT. No patients developed pulmonary embolism. The combined incidence of DVT was 8.8 per cent. A variety of parameters was measured in order to determine whether compression devices prevent a fibrinolytic shut-down commonly seen in the postsurgical patient. A combination of three assays was found to be significant in demonstrating a fibrinolytic response. These parameters were a post-surgical decrease in the plasminogen level, an increase in the level of free protease activity postoperatively, and an increase in the level of tissue plasminogen activator after surgery. 56.3 per cent of all patients treated with SCD and TEDS showed a fibrinolytic response on postoperative day one by a combination of all three of these parameters. In the group of patients that developed DVT none showed an increase in free protease activity, and five of seven showed no significant decrease in plasminogen and no increase in tissue plasminogen activator. Patients who developed thrombosis had measurable differences in their fibrinolytic system compared to those without postoperative thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Clothing↗
The isolation and characterization of a ternary human plasmin B-chain-streptokinase-plasminogen complex.
A ternary equimolar human plasmin B-chain-streptokinase-plasminogen complex was isolated from a mixture of the plasmin B-chain-streptokinase complex and human plasminogen at 0 degrees C and 37 degrees C. A ternary complex which was shown to be species specific, was identified and characterized by ultracentrifugal, acrylamide gel electrophoretic, and agarose double diffusion analyses. When mixed at a 1:1 molar ratio at 0 degrees C, 39.9% of the preparation existed as a plasmin B-chain-streptokinase-plasminogen complex; when mixed at 37 degrees C, 86.4% existed as a complex, which was identified by electrophoretic analyses to be a plasmin B-chain-streptokinase-plasmin complex. Sedimentation velocity analyses gave s degrees 20,w values of 3.79 for the plasmin B-chain-streptokinase complex, 4.10 for Lys-plasmin, and 6.23 for the plasmin B-chain-streptokinase-plasmin complex. Sedimentation equilibrium analyses gave molecular weights of 73,900 for the plasmin B-chain-streptokinase complex, 82,900 for Lys-plasmin, and 153,100 for the plasmin B-chain-streptokinase-plasmin complex. The diisopropylphosphorofluoridate (DFP)-inhibited and the p-nitrophenyl-p-guanidino-benzoate (NPGB)-inhibited plasmin B-chain-streptokinase complexes both retained their ability to form a ternary complex with human plasminogen, but this complex did not convert to a plasmin B-chain-streptokinase-plasmin complex. Thus, the active site serine residue is essential for the activator activity of the plasmin B-chain-streptokinase complex, but it is not necessary for the binding of the plasmin B-chain-streptokinase complex to plasminogen to form a ternary complex.
Binding Sites↗
In vitro comparison of fibrinolytic activity of plasminogen activators using a thrombelastographic method: in vivo evaluation of the B-chain-streptokinase complex in the dog model using pre-titered doses.
Thrombelastography was used to quantitatively compare the clot-lysing efficiency of 6 different plasminogen activators, using human whole blood, pooled normal plasma, and platelet rich plasma. The activators compared were the B-chain-streptokinase complex, the plasmin-streptokinase complex, the mini-plasminogen-streptokinase complex, tissue plasminogen activator, streptokinase, and urokinase. The most efficient activator found was the B-chain-streptokinase complex. This complex was 4.0 times more effective than streptokinase, 3.0 times more effective than the plasmin-streptokinase complex, 1.3 times more effective than the mini-plasminogen-streptokinase complex, 2.3 times more effective than tissue plasminogen activator, and 16.0 times more effective than urokinase. Although there were differences in both the coagulation and fibrinolysis thrombelastographic patterns between plasma and whole blood, the comparative efficiencies of each activator were the same with either plasma or blood. The B-chain-streptokinase complex was evaluated as a thrombolytic agent in clot-lysis experiments in the jugular vein in the dog model, using a thrombelastographic method to determine the minimum dose of activator necessary for clot-lysis. With 6 dogs infused locally with 0.25 mg (8000 I.U.) of the plasmin-streptokinase complex, the cumulative clot-lysis was 18.0 +/- 3.0% with the first dose, 33.0 +/- 2.1% with the second dose, and 55.2 +/- 8.6% with the third dose. With 6 dogs infused locally with 0.03 mg (2000 I.U.) of the B-chain-streptokinase complex, the cumulative clot-lysis was 30.6 +/- 6.4% with the first dose, 54.4 +/- 9.6% with the second dose, and 80.2 +/- 9.0% with the third dose.
Animals↗
Hydrodynamic studies on the streptokinase complexes of human plasminogen, Val442-plasminogen, plasmin, and the plasmin-derived light (B) chain.
Sedimentation velocity and sedimentation equilibrium studies have been carried out on the Glu- and Lys-plasminogen-streptokinase complexes as well as on the complexes formed by Val442-plasmin and the light (B) chain of plasmin. Sedimentation equilibrium molecular weights are consistent with a 1 to 1 molar complex in all cases and give values consistent with the differences in size of the plasminogen moieties. Sedimentation velocity determinations in the presence of protease inhibitors give values consistent with the conformational differences already reported for the Glu- and Lys-plasminogen molecules. However, unlike Glu-plasminogen, the addition of epsilon-aminocaproic acid or lysine does not alter the conformation of the Glu-plasminogen complex. The values of the sedimentation coefficient and the molecular weight of the plasmin and the Val442-plasmin-streptokinase complexes increase to those of a dimer when determined in the absence of active-site inhibitors but return to monomer values when these inhibitors are added. Thus, dimer formation requires the presence of an available active site in at least one of the two molecules involved and is reversible.
Fibrinolysin↗
The human plasmin-derived light (B) chain X streptokinase complex: a second-generation thrombolytic agent.
Specific assay methods for the human plasmin-derived light (B) chain X streptokinase (B X SK) complex, in terms of both streptokinase (SK) and urokinase (UK) International Units, are described. The kinetic properties of various SK activator complexes with plasminogen, Val442-plasmin, and the plasmin-derived light (B) chain were compared to SK in terms of their catalytic efficiencies and Lineweaver-Burk plots. Similar kinetic data, and Lineweaver-Burk plots, are described for both highly purified high-molecular weight UK and low-molecular weight UK, including different clinical UK preparations. The B X SK complex has the highest catalytic efficiency of all the activator species studied. The Lineweaver-Burk plots of each of the various activator species are "fingerprints" of the enzymatic character of the activator. The B X SK complex is more like UK than SK, as an activator, in activating non-human plasminogen species. The biological half-life of the B X SK complex, in a dog model, was determined to be about 4 hr which is longer than the biological half-life(s) of SK in the same animal model, namely 0.6 hr (47%) and 2.8 hr (53%). This new second-generation activator complex may prove to be a useful thrombolytic agent in the treatment of thromboembolic diseases.
Animals↗
Comparative activation kinetics of mammalian plasminogens.
Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.
Amino Acids↗
Human plasminogen variant Chicago III.
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Adult↗
A virgin enzyme derived from human plasminogen. Specific cleavage of the arginyl-560-valyl peptide bond in the diisopropoxyphosphinyl virgin enzyme by plasminogen activators.
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Binding Sites↗
Plasmin and plasminogen activators: kinetics, and kinetics of plasminogen activation.
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Fibrinolysin↗
Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C.
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Enzyme Activation↗
Physiological activation of the human fibrinolytic system. Isolation and characterization of human plasminogen variants, Chicago I and Chicago II.
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Enzyme Activation↗
Recombinant human Lys-plasmin and the Lys-plasmin . streptokinase complex.
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Binding Sites↗
Steady state kinetics of activation of human and bovine plasminogens by streptokinase and its equimolar complexes with various activated forms of human plasminogen.
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Animals↗