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Partial purification and properties of a thrombin activated fibrinolysis inhibitor (TAFI)
94
ORAL COMMUNICATATIONS l-7: Interrelation
Between Coagulation
261 PARTIAL PURIFICATION AND PROPERTIES OF A THROMBIN ACTIVATED FIBRINOLYSIS INHIBITOR (TAFT). Baizar. L. and Nesheim. M.E. Queen’s University, Kingston Ont. CANADA Previous studies in our lab (Bajzar, L. and Nesheim, M.E. J. Biol. Chem. 268 8608-8616, 1993) both demonstrated that
activation of prothrombin during tPA-induced fibrinolysis of fibrin clots inhibits lysis and suggested the existence of a thrombin activated fibrinolysis inhibitor (TAFI). Thus an assay was devised based on the prolongation of fibrinolysis upon prothrombin activation in a system comprising purified fibrinogen, plasminogen (Pgn), a,-antiplasmin (AP), antithrombin III (AT-III) and components of prothrombinase. TAFI activity was indicated by prolonged lysis times when prothrombin activation was initiated by Factar Xa (FXa) relative to the lysis time observed in the absence of FXa. The assay was used to monitor activity upon fractionation of barium citrate adsorbed human plasma. Partial purification (approx. 500 fold) was accomplished by (NH&SO,
262 REQUIREMENT OF Zn*+ FOR BINDING OF HISTIDINERICH GLYCOPROTEIN TO PVC-ADSORBED FIBRINOGEN AND FIBRIN. P.A. van Boheemen ,uS L G SlaoDendel, B.C. Hennis, G. Dooiiewaard and C. Kluft. Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. Histidine-rich glycoprotein (HRG) is an cY,-glycoprotein present in human plasma in a concentration of 100 pglml. It has strong affinity for divalent metal ions such as Zn*+. The function of HRG in haemostasis is ambiguous. By binding to plasminogen (Kd of 1 PM), HRG reduces the amount of plasminogen available for binding to fibrin. However, it has also been reported that HRG itself binds to fibrinogen and fibrin. We demonstrate that HRG binds in a Zn’+-dependent manner to fibrinogen adsorbed to PVC microtiter wells. Optimal binding was obtained at a ZnCl, concentration of 50 PM. The binding of HRG in the presence of 50 FM Zn*+ to adsorbed fibrinogen is concentration-dependent and
263 STREPTOKINASE STIMULATES THROMBIN GENERATION INDEPENDENT OF ITS FIBRINOLYTIC EFFECT PLASMINOGEN. Meiier P 7-7 Kluft C Brommer EJP. Gaubius Laboratory, TNO-PG, L&den, The Netherlands.
VIA
Reports on coagulation activation during thrombolytic treatment prompted us to evaluate the direct effects of thrombolytic agents on thrombin generation in traditional activation systems. We preincubated platelet-poor titrated plasma with either thromboplastin or ellagic acid reagent to start activation. During preincubation the following thrombolytics were included: urokinase, streptokinase, recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA). The formed thrombin was measured with a chromogenic substrate (S-2238) at several time intervals after recalcification of the preincubation mixture. With both activation procedures, thrombin generation, expressed as the lag time before explosive thrombin formation occurred, was greatly accelerated in the presence of streptokinase. The lag time was reduced from about 7 to 2 minutes. The effects of the
and Fibrinolysis:
2
fractionation (4570%), ionic exchange on Q-Sepharose fastflow, gel filtration on a tandem column of AcA-44/AcA-54 and ion exchange on DEAE-Sepharose fast-flow. Gel filtration suggests a mass of between 40-50 KDa. The activity is heat labile and can be stably stored in 50% glycerol/H,0 at -20°C. TAFI gives a dose dependent relative prolongation of lysis time in both the presence and absence of
the inhibitors AT-III and AP. The maximum relative increase in lysis time was 2.5 fold in the presence of the inhibitors and 1.7 fold in the absence of the inhibitors. The relative prolongation is independent of the initial concentration of Glu-Pgn between 0.2 and 0.8 PM Glu-Pgn. When Glu-Pgn was replaced with Lys-Pgn, however, no TAFI-dependent prolongation was observed, suggesting that TAFI exerts its effect early in the fibrinolytic process.
saturable, with an apparent Kd of 12 nM. Without Zn*+ or in the presence of 1 mM CaCl,, no binding of HRG to fibrinogen was found. Conversion of adsorbed fibrinogen to fibrin by thrombin did not alter the affinity for HRG. Only high concentrations of fluid-phase fibrinogen were able to compete for HRG binding to adsorbed fibrinogen with 50% inhibition occurring at 1 PM. This suggests that a conformational change induced by adsorption of fibrinogen is essential for the strong binding between HRG and fibrinogen. The interaction of HRG with adsorbed fibrinogen in the presence of 50 PM Zn*+ was shown to be pHdependent, with an optimum at pH 6.0. This finding and the fact that Zn*+ is essential, suggests that the histidine-rich region of HRG is involved in the interaction between HRG and fibrinogen. The Zn*+-dependent interaction of HRG with fibrinogen and fibrin might have an impact on physiological processes like plasminogen activation at a fibrin surface.
other thrombolytics were clearly smaller. A dose dependent effect was observed with a maximum acceleration obtained with streptokinase concentrations of 100 IUlml or more. Addition of hirudin quenched the conversion of the chromogenic substrate completely, confirming that thrombin was the measured enzyme. Both calcium and phospholipids were necessary for the acceleration of the thrombin generation by streptokinase. The streptokinase effect was not abolished by the addition of aprotinin (300 KIU/ml) to the preincubation, and the effect remained in plasminogen-depleted plasma. Addition of anti Lp(a) IgG did not alter the streptokinase effect. These findings indicate an effect of streptokinase on thrombin generation independent of plasminogen, plasmin formation and the plasminogen related molecule Lp(a). Experiments with plasma samples deficient for coagulation factors II, V. VIII, IX or X showed that streptokinase cannot suhstitute for any of these coagulation factors. It suggests a direct effect of streptokinase on the prothrombinase complex independent of its tihrinolytic effect via plasminogen.
ORAL COMMUNICATATIONS l-7: Interrelation
Between Coagulation
261 PARTIAL PURIFICATION AND PROPERTIES OF A THROMBIN ACTIVATED FIBRINOLYSIS INHIBITOR (TAFT). Baizar. L. and Nesheim. M.E. Queen’s University, Kingston Ont. CANADA Previous studies in our lab (Bajzar, L. and Nesheim, M.E. J. Biol. Chem. 268 8608-8616, 1993) both demonstrated that
activation of prothrombin during tPA-induced fibrinolysis of fibrin clots inhibits lysis and suggested the existence of a thrombin activated fibrinolysis inhibitor (TAFI). Thus an assay was devised based on the prolongation of fibrinolysis upon prothrombin activation in a system comprising purified fibrinogen, plasminogen (Pgn), a,-antiplasmin (AP), antithrombin III (AT-III) and components of prothrombinase. TAFI activity was indicated by prolonged lysis times when prothrombin activation was initiated by Factar Xa (FXa) relative to the lysis time observed in the absence of FXa. The assay was used to monitor activity upon fractionation of barium citrate adsorbed human plasma. Partial purification (approx. 500 fold) was accomplished by (NH&SO,
262 REQUIREMENT OF Zn*+ FOR BINDING OF HISTIDINERICH GLYCOPROTEIN TO PVC-ADSORBED FIBRINOGEN AND FIBRIN. P.A. van Boheemen ,uS L G SlaoDendel, B.C. Hennis, G. Dooiiewaard and C. Kluft. Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. Histidine-rich glycoprotein (HRG) is an cY,-glycoprotein present in human plasma in a concentration of 100 pglml. It has strong affinity for divalent metal ions such as Zn*+. The function of HRG in haemostasis is ambiguous. By binding to plasminogen (Kd of 1 PM), HRG reduces the amount of plasminogen available for binding to fibrin. However, it has also been reported that HRG itself binds to fibrinogen and fibrin. We demonstrate that HRG binds in a Zn’+-dependent manner to fibrinogen adsorbed to PVC microtiter wells. Optimal binding was obtained at a ZnCl, concentration of 50 PM. The binding of HRG in the presence of 50 FM Zn*+ to adsorbed fibrinogen is concentration-dependent and
263 STREPTOKINASE STIMULATES THROMBIN GENERATION INDEPENDENT OF ITS FIBRINOLYTIC EFFECT PLASMINOGEN. Meiier P 7-7 Kluft C Brommer EJP. Gaubius Laboratory, TNO-PG, L&den, The Netherlands.
VIA
Reports on coagulation activation during thrombolytic treatment prompted us to evaluate the direct effects of thrombolytic agents on thrombin generation in traditional activation systems. We preincubated platelet-poor titrated plasma with either thromboplastin or ellagic acid reagent to start activation. During preincubation the following thrombolytics were included: urokinase, streptokinase, recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA). The formed thrombin was measured with a chromogenic substrate (S-2238) at several time intervals after recalcification of the preincubation mixture. With both activation procedures, thrombin generation, expressed as the lag time before explosive thrombin formation occurred, was greatly accelerated in the presence of streptokinase. The lag time was reduced from about 7 to 2 minutes. The effects of the
and Fibrinolysis:
2
fractionation (4570%), ionic exchange on Q-Sepharose fastflow, gel filtration on a tandem column of AcA-44/AcA-54 and ion exchange on DEAE-Sepharose fast-flow. Gel filtration suggests a mass of between 40-50 KDa. The activity is heat labile and can be stably stored in 50% glycerol/H,0 at -20°C. TAFI gives a dose dependent relative prolongation of lysis time in both the presence and absence of
the inhibitors AT-III and AP. The maximum relative increase in lysis time was 2.5 fold in the presence of the inhibitors and 1.7 fold in the absence of the inhibitors. The relative prolongation is independent of the initial concentration of Glu-Pgn between 0.2 and 0.8 PM Glu-Pgn. When Glu-Pgn was replaced with Lys-Pgn, however, no TAFI-dependent prolongation was observed, suggesting that TAFI exerts its effect early in the fibrinolytic process.
saturable, with an apparent Kd of 12 nM. Without Zn*+ or in the presence of 1 mM CaCl,, no binding of HRG to fibrinogen was found. Conversion of adsorbed fibrinogen to fibrin by thrombin did not alter the affinity for HRG. Only high concentrations of fluid-phase fibrinogen were able to compete for HRG binding to adsorbed fibrinogen with 50% inhibition occurring at 1 PM. This suggests that a conformational change induced by adsorption of fibrinogen is essential for the strong binding between HRG and fibrinogen. The interaction of HRG with adsorbed fibrinogen in the presence of 50 PM Zn*+ was shown to be pHdependent, with an optimum at pH 6.0. This finding and the fact that Zn*+ is essential, suggests that the histidine-rich region of HRG is involved in the interaction between HRG and fibrinogen. The Zn*+-dependent interaction of HRG with fibrinogen and fibrin might have an impact on physiological processes like plasminogen activation at a fibrin surface.
other thrombolytics were clearly smaller. A dose dependent effect was observed with a maximum acceleration obtained with streptokinase concentrations of 100 IUlml or more. Addition of hirudin quenched the conversion of the chromogenic substrate completely, confirming that thrombin was the measured enzyme. Both calcium and phospholipids were necessary for the acceleration of the thrombin generation by streptokinase. The streptokinase effect was not abolished by the addition of aprotinin (300 KIU/ml) to the preincubation, and the effect remained in plasminogen-depleted plasma. Addition of anti Lp(a) IgG did not alter the streptokinase effect. These findings indicate an effect of streptokinase on thrombin generation independent of plasminogen, plasmin formation and the plasminogen related molecule Lp(a). Experiments with plasma samples deficient for coagulation factors II, V. VIII, IX or X showed that streptokinase cannot suhstitute for any of these coagulation factors. It suggests a direct effect of streptokinase on the prothrombinase complex independent of its tihrinolytic effect via plasminogen.