The Experts below are selected from a list of 219 Experts worldwide ranked by ideXlab platform
Victor Gurewich - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Highly Effective Fibrinolysis by a Sequential Synergistic Combination of Mini-Dose tPA plus Low-Dose Mutant proUK
2016Co-Authors: Ralph Pannell, Victor GurewichAbstract:Results of thrombolysis by monotherapy with either tPA or proUK have not lived up to ex-pectations. Since these natural activators are inherently complementary, this property can be utilized to a synergistic advantage; and yet, this has undergone little evaluation. ProUK is no longer available because at pharmacological concentrations it converts to UK in plas-ma. Therefore, a single site proUK mutant, M5, was developed to address this problem and was used in this study. Fibrinolysis was measured using preformed fluoresceinated 24 h old clots in a plasma milieu rather than by the standard automated method, because proUK/M5 is sensitive to inactivation by thrombin and activation by plasmin. The shortest 50 % clot lysis time that could be achieved by tPA or M5 alone was determined: mean times were 55 and 48 minutes respectively. These bench marks were matched by 6 % of the tPA mono-therapy dose combined with 40 % that of M5: mean lysis time 47 minutes with less associat-ed Fibrinogenolysis. Results showed that the tPA effect was limited to initiating fibrinolysis which was completed by M5 and then tcM5. Plasma C1-inhibitor inhibited Fibrinogenolysis by M5, providing protection from side effects not available for proUK. In conclusion, by utiliz-ing the complementary properties and sequential modes of action of each activator, more efficient fibrinolysis with less non-specific effects can be achieved than with traditional monotherapy. In vivo validation is needed, but in a previous clinical trial using a similar com-bination of tPA and proUK (5 % and 50%monotherapy doses) very promising results have already been obtained
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The effect of the synergistic dose combination in plasma alone:
2015Co-Authors: Ralph Pannell, Victor GurewichAbstract:The synergistic combination of the activators (0.2 μg/ml tPA and 6 μg/ml M5) was incubated (37°C) in plasma without a clot. No Fibrinogenolysis occurred for at least 3 h, showing that the plasminogen activation seen with the synergistic combination was fibrin-dependent and no non-specific effect was detected by the synergistic combination.
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Effect of C1-inibitor:
2015Co-Authors: Ralph Pannell, Victor GurewichAbstract:The experiment shown in Fig. 1B with M5 was repeated with prior addition of C1-inhibitor (750 μg/ml) to the plasma. The lysis rate was not inhibited, in fact was shortened, Fibrinogenolysis was prevented. The experiment was not repeated with tPA because C1-inhibitor inhibits lysis by tPA (28) and seen also in Fig. 3B.
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Recombinant human C1-inhibitor prevents non-specific proteolysis by mutant pro-urokinase during optimal fibrinolysis
Thrombosis and Haemostasis, 2009Co-Authors: Ralph Pannell, Victor GurewichAbstract:SummaryA single-site mutant of prouPA (M5) spared haemostatic fibrin during thrombolysis in dogs. Zymograms of plasma from these dogs showed an unusual inhibitor complex with C1-inhibitor (C1I). Purified C1I added to human plasma enhanced the fibrinspecificity of M5. In the present study, the effect of recombinant human C1I (recC1I) on high-dose M5 and tPA were compared using fluorescein-labeled standardised clots in a plasma milieu. The shortest time to complete clot lysis (maximum rate) was first determined. This was ∼65% per hour for both activators. By contrast, their top fibrin-specific lysis rate (<20% fibrinogen depletion) was less than half maximum (25–30% per hour). Adding recC1I (250–750 μg/ml) did not affect fibrinolysis, but prevented Fibrinogenolysis and plasminogen depletion by M5, raising its fibrin-specific lysis rate to the maximum. With tPA, the recC1I modestly attenuated Fibrinogenolysis, raising its fibrin-specific rate to about half the maximum. Consistent with this, the t½ inhibition by C1I was ∼90 min for tPA compared with ∼10 min for tcM5. The t½ of C1I for plasmin was ∼2 min. Zymograms of plasma after clot lysis indicated that recC1I prevented non-specific tcM5 generation from M5, as evidenced by suppression of tcM5:C1I complexes. In conclusion, recC1I raised the fibrin-specificity of M5 in plasma so that a maximum lysis rate could be achieved without Fibrinogenolysis. The inhibition by C1I of nonspecific but not fibrin-dependent plasminogen activation could not be duplicated by other serpins. The findings provide a potential means to optimize both the efficacy and safety of thrombolysis.
Jack Hirsh - One of the best experts on this subject based on the ideXlab platform.
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a2-Antiplasmin Supplementation Inhibits Tissue Plasminogen Activator-induced Fibrinogenolysis and Bleeding with Little Effect on Thrombolysis
2016Co-Authors: Jeffrey Weitz, Beverly Leslie, Jack Hirsh, P KlementAbstract:Tissue plasminogen activator (t-PA) causes fibrinogen proteo-lysis when a2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is pro-tected from a2-antiplasmin inhibition, we tested the possibility that a2-antiplasmin supplementation would block t-PA-in-duced Fibrinogenolysis and bleeding without affecting thrombol-ysis. When added to human or rabbit plasma, a2-antiplasmin inhibits t-PA-induced Fibrinogenolysis, but has little effect on the lysis of 1251-fibrin clots. To examine its effect in vivo, rab-bits with preformed 125I-labeled-jugular vein thrombi were ran-domized to receive t-PA, t-PA and a2-antiplasmin, or saline. a2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2 % to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87 % to 27 % (P = 0.0001), and decreased blood loss from standardized ear in-cisions from 5,594 to 656,ul (P < 0.0001). We hypothesize that a2-antiplasmin limits t-PA-induced hemorrhage by inhibit-ing Fibrinogenolysis and subsequent fragment X formation be-cause (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in a2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into he-mostatic plugs contributes to t-PA-induced bleeding. By block-ing t-PA-mediated Fibrinogenolysis, a2-antiplasmin supple-mentation may improve the safety of fibrin-specific plasmino
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Alpha 2-antiplasmin supplementation inhibits tissue plasminogen activator-induced Fibrinogenolysis and bleeding with little effect on thrombolysis.
The Journal of clinical investigation, 1993Co-Authors: J. I. Weitz, Beverly A. Leslie, Jack Hirsh, P KlementAbstract:Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced Fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced Fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting Fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated Fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.
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Standard and low molecular weight heparin have no effect on tissue plasminogen activator induced plasma clot lysis or Fibrinogenolysis.
Thrombosis and haemostasis, 1991Co-Authors: Jeffrey I. Weitz, Beverly A. Leslie, Jacob Kuint, Jack HirshAbstract:Although heparin is often given as an adjunct to tissue plasminogen activator (t-PA), the effect of heparin on t-PA induced fibrin(ogen)olysis is controversial. To address this controversy, we examined the effects of standard and low molecular weight heparin (enoxaparine) on both t-PA induced clot lysis and t-PA mediated Fibrinogenolysis in a human plasma system. Accordingly, 125I-labeled fibrin clots were incubated in t-PA containing citrated plasma in the presence or absence of these glycosaminoglycans, and the extent of thrombolysis was determined by measuring residual radioactivity of the clots, while B beta 1-42 levels were used as a specific index of Fibrinogenolysis. Over a wide range of t-PA concentrations (0.1 to 1.6 micrograms/ml), neither heparin nor enoxaparine influences either t-PA induced clot lysis or t-PA mediated B beta 1-42 generation. These findings suggest that either agent could be used as an adjunct to t-PA without compromising either the thrombolytic potential of t-PA or its clot-selectivity.
Ralph Pannell - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Highly Effective Fibrinolysis by a Sequential Synergistic Combination of Mini-Dose tPA plus Low-Dose Mutant proUK
2016Co-Authors: Ralph Pannell, Victor GurewichAbstract:Results of thrombolysis by monotherapy with either tPA or proUK have not lived up to ex-pectations. Since these natural activators are inherently complementary, this property can be utilized to a synergistic advantage; and yet, this has undergone little evaluation. ProUK is no longer available because at pharmacological concentrations it converts to UK in plas-ma. Therefore, a single site proUK mutant, M5, was developed to address this problem and was used in this study. Fibrinolysis was measured using preformed fluoresceinated 24 h old clots in a plasma milieu rather than by the standard automated method, because proUK/M5 is sensitive to inactivation by thrombin and activation by plasmin. The shortest 50 % clot lysis time that could be achieved by tPA or M5 alone was determined: mean times were 55 and 48 minutes respectively. These bench marks were matched by 6 % of the tPA mono-therapy dose combined with 40 % that of M5: mean lysis time 47 minutes with less associat-ed Fibrinogenolysis. Results showed that the tPA effect was limited to initiating fibrinolysis which was completed by M5 and then tcM5. Plasma C1-inhibitor inhibited Fibrinogenolysis by M5, providing protection from side effects not available for proUK. In conclusion, by utiliz-ing the complementary properties and sequential modes of action of each activator, more efficient fibrinolysis with less non-specific effects can be achieved than with traditional monotherapy. In vivo validation is needed, but in a previous clinical trial using a similar com-bination of tPA and proUK (5 % and 50%monotherapy doses) very promising results have already been obtained
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The effect of the synergistic dose combination in plasma alone:
2015Co-Authors: Ralph Pannell, Victor GurewichAbstract:The synergistic combination of the activators (0.2 μg/ml tPA and 6 μg/ml M5) was incubated (37°C) in plasma without a clot. No Fibrinogenolysis occurred for at least 3 h, showing that the plasminogen activation seen with the synergistic combination was fibrin-dependent and no non-specific effect was detected by the synergistic combination.
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Effect of C1-inibitor:
2015Co-Authors: Ralph Pannell, Victor GurewichAbstract:The experiment shown in Fig. 1B with M5 was repeated with prior addition of C1-inhibitor (750 μg/ml) to the plasma. The lysis rate was not inhibited, in fact was shortened, Fibrinogenolysis was prevented. The experiment was not repeated with tPA because C1-inhibitor inhibits lysis by tPA (28) and seen also in Fig. 3B.
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Recombinant human C1-inhibitor prevents non-specific proteolysis by mutant pro-urokinase during optimal fibrinolysis
Thrombosis and Haemostasis, 2009Co-Authors: Ralph Pannell, Victor GurewichAbstract:SummaryA single-site mutant of prouPA (M5) spared haemostatic fibrin during thrombolysis in dogs. Zymograms of plasma from these dogs showed an unusual inhibitor complex with C1-inhibitor (C1I). Purified C1I added to human plasma enhanced the fibrinspecificity of M5. In the present study, the effect of recombinant human C1I (recC1I) on high-dose M5 and tPA were compared using fluorescein-labeled standardised clots in a plasma milieu. The shortest time to complete clot lysis (maximum rate) was first determined. This was ∼65% per hour for both activators. By contrast, their top fibrin-specific lysis rate (<20% fibrinogen depletion) was less than half maximum (25–30% per hour). Adding recC1I (250–750 μg/ml) did not affect fibrinolysis, but prevented Fibrinogenolysis and plasminogen depletion by M5, raising its fibrin-specific lysis rate to the maximum. With tPA, the recC1I modestly attenuated Fibrinogenolysis, raising its fibrin-specific rate to about half the maximum. Consistent with this, the t½ inhibition by C1I was ∼90 min for tPA compared with ∼10 min for tcM5. The t½ of C1I for plasmin was ∼2 min. Zymograms of plasma after clot lysis indicated that recC1I prevented non-specific tcM5 generation from M5, as evidenced by suppression of tcM5:C1I complexes. In conclusion, recC1I raised the fibrin-specificity of M5 in plasma so that a maximum lysis rate could be achieved without Fibrinogenolysis. The inhibition by C1I of nonspecific but not fibrin-dependent plasminogen activation could not be duplicated by other serpins. The findings provide a potential means to optimize both the efficacy and safety of thrombolysis.
L B Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Human tryptase Fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.
Journal of Immunology, 1997Co-Authors: A E Lawson, Clive M. Baumgarten, M. Carr, L B SchwartzAbstract:Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of Fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase Fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.
Beverly A. Leslie - One of the best experts on this subject based on the ideXlab platform.
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Alpha 2-antiplasmin supplementation inhibits tissue plasminogen activator-induced Fibrinogenolysis and bleeding with little effect on thrombolysis.
The Journal of clinical investigation, 1993Co-Authors: J. I. Weitz, Beverly A. Leslie, Jack Hirsh, P KlementAbstract:Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced Fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced Fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting Fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated Fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.
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Standard and low molecular weight heparin have no effect on tissue plasminogen activator induced plasma clot lysis or Fibrinogenolysis.
Thrombosis and haemostasis, 1991Co-Authors: Jeffrey I. Weitz, Beverly A. Leslie, Jacob Kuint, Jack HirshAbstract:Although heparin is often given as an adjunct to tissue plasminogen activator (t-PA), the effect of heparin on t-PA induced fibrin(ogen)olysis is controversial. To address this controversy, we examined the effects of standard and low molecular weight heparin (enoxaparine) on both t-PA induced clot lysis and t-PA mediated Fibrinogenolysis in a human plasma system. Accordingly, 125I-labeled fibrin clots were incubated in t-PA containing citrated plasma in the presence or absence of these glycosaminoglycans, and the extent of thrombolysis was determined by measuring residual radioactivity of the clots, while B beta 1-42 levels were used as a specific index of Fibrinogenolysis. Over a wide range of t-PA concentrations (0.1 to 1.6 micrograms/ml), neither heparin nor enoxaparine influences either t-PA induced clot lysis or t-PA mediated B beta 1-42 generation. These findings suggest that either agent could be used as an adjunct to t-PA without compromising either the thrombolytic potential of t-PA or its clot-selectivity.
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Soluble fibrin degradation products potentiate tissue plasminogen activator-induced fibrinogen proteolysis.
The Journal of clinical investigation, 1991Co-Authors: Jeffrey I. Weitz, Beverly A. Leslie, Jeffrey S. GinsbergAbstract:Abstract Despite its affinity for fibrin, tissue plasminogen activator (t-PA) administration causes systemic Fibrinogenolysis. To investigate the mechanism, t-PA was incubated with plasma in the presence or absence of a fibrin clot, and the extent of Fibrinogenolysis was determined by measuring B beta 1-42. In the presence of fibrin, there is a 21-fold increase in B beta 1-42 levels. The potentiation of Fibrinogenolysis in the presence of fibrin is mediated by soluble fibrin degradation products because (a) the extent of t-PA induced Fibrinogenolysis and clot lysis are directly related, (b) once clot lysis has been initiated, Fibrinogenolysis continues even after the clot is removed, and (c) lysates of cross-linked fibrin clots potentiate t-PA-mediated Fibrinogenolysis. Fibrin degradation products stimulate Fibrinogenolysis by binding t-PA and plasminogen because approximately 70% of the labeled material in the clot lysates binds to both t-PA- and plasminogen-Sepharose, and only the bound fractions have potentiating activity. The binding site for t-PA and plasminogen is on the E domain because characterization of the potentiating fragments using gel filtration followed by PAGE and immunoblotting indicates that the major species is (DD)E complex, whereas minor components include high-molecular weight derivatives containing the (DD)E complex and fragment E. In contrast, D-dimer is the predominant species found in the fractions that do not bind to the adsorbants, and it has no potentiating activity. Thus, soluble products of t-PA-induced lysis of cross-linked fibrin potentiate t-PA-mediated Fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. The occurrence of this phenomenon after therapeutic thrombolysis may explain the limited clot selectivity of t-PA.