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The anticoagulant mechanism of action of hirudin
THURSDAY
182 Annexln V (vascular antlcoagulant alpha), an antlcoagulant wlth a new mechanism of action REUIELINGSPERGER CPM, ?? ANDREE HAM, HEMKER HC Department of Biochemistry and *Research Institute of Cardiovascular Direases, Universiryof Limburg, Maastricht, 7&e Netherkmds
JUNE 28 1990
73
Binding studies demonstrate a high affinity (K&10%4) of annexin V for procoagulant phospholipid surfaces. It dislodges the coagulation factors of prothrombinase from the surface, which forms the basis of its anticoagulant action. Annexin V completes the set of anticoagulants (antithrombin IIUheparin, protein synthesis effecters, activated protein C) acting on the four component procoagulant enzyme complex of vitamin K-dependent coagulation factors, protein cofactors (V, and VIII,) and negatively charged phospholipid surfaces.
Negatively charged phospholipids play a mandatory role in coagulation by assembling the vitamin K-dependent factors into enzyme complexes with improved kinetic parameters. Annexin V (formerly, vascular anticoagulant alpha) was first discovered in the homogenates of vascularized tissue due to its strong anticoagulant activity. Molecular cloning of its cDNA identified this anticoagulant as a member of the Ca*+/phospholipid binding protein family the annexins.
The anticoagulant mechanism of action of hlrudln LINDHOUT T, BLEZER R, HEMKER HC Dept. of Biochemistv, Universityof Limburg, Maastricht, The Netherlands We studied the inhibitory action of the recombinant desulphatohirudin (CGP 39393) on thrombin generation in plasma. Plasma was activated either with thromboplastin or factor IXa. Hirudin delayed the onset of rapid thrombin generation, but it was unable to prevent the explosive appearance of thrombin. The dose-dependent prolongation of the lag phase of the intrinsic and extrinsic thrombin generation curve was not the result of titration of thrombin activity by hirudin but the result of a delayed formation of the prothrombin converting complex (prothrombinase). In case of extrinsic activation, hirudin did not affect factor Xa generation, but prolonged the lag phase of the factor Va
The comparative effects of recombinant hlrudln and standard heparln on lnhlbltlon of thrombus growth In rabbits AGNELLI G, PASCUCCI C, COSMI B, NENCI GG Institute di Semeiotka Medica, Universitddi Peru& Pen@, Itaiy Standard heparin (SH) is the accepted treatment for deep vein thrombosis for preventing thrombus extension. SH is a weak inhibitor of thrombin bound to fibrin. Recombinant hirudin (r-hirudin) (CGP 39393) is a selective inhibitor of thrombin, active on both free plasma thrombin and fibrin bound thrombin. The aim of this study was to compare the ability of SH and r-hirudin to inhibit fibrin accumulation on pre-existing thrombi in a rabbit jugular model. Doses of SH and r-hirudin equivalent in prolonging aPIT were first identified. 0.5 and 0.75 mg/kg of SH and 0.8 and 1.25 mg of r-hirudin infused over 3 hours produced a mean prolonga-
generation curve, causing its appearance when factor Xa generation was already in the decay phase. This provides hirudin with anti-prothrombinase activity. Because of its inhibitory action on the thrombin-mediated activation of factor VIII, hirudin prolonged the lag phase of the factor X converting complex that consists of factor IXa and factor VIIIa. Factor IXa was not inactivated by plasma proteinase inhibitors nor by hirudin. Our observations with hirudin are in keeping with the notion that inhibition of the thrombinmediated amplification reactions in blood coagulation is a very efficient way to delay or inhibit completely thrombin generation. Although extremely small amounts of hirudin neutralize stoichiomettic amounts of thrombin, the interaction between in situ generated thrombin and hirudin appears not to be fast enough to prevent trace amounts of thrombin to activate factors VIII and V. Consequently, an explosive thrombin generation is observed even when free hirudin is present.
tion of aPTT of 1.5 and 2, respectively. In saline treated rabbits 62 (27) pg of ‘zI-fibrinogen accumulation was observed. The lower doses of SH and r-hirudin produced an accumulation of 44*5 pg and 2524 pg of ‘2SI-fibrinogen, respectively (p
182 Annexln V (vascular antlcoagulant alpha), an antlcoagulant wlth a new mechanism of action REUIELINGSPERGER CPM, ?? ANDREE HAM, HEMKER HC Department of Biochemistry and *Research Institute of Cardiovascular Direases, Universiryof Limburg, Maastricht, 7&e Netherkmds
JUNE 28 1990
73
Binding studies demonstrate a high affinity (K&10%4) of annexin V for procoagulant phospholipid surfaces. It dislodges the coagulation factors of prothrombinase from the surface, which forms the basis of its anticoagulant action. Annexin V completes the set of anticoagulants (antithrombin IIUheparin, protein synthesis effecters, activated protein C) acting on the four component procoagulant enzyme complex of vitamin K-dependent coagulation factors, protein cofactors (V, and VIII,) and negatively charged phospholipid surfaces.
Negatively charged phospholipids play a mandatory role in coagulation by assembling the vitamin K-dependent factors into enzyme complexes with improved kinetic parameters. Annexin V (formerly, vascular anticoagulant alpha) was first discovered in the homogenates of vascularized tissue due to its strong anticoagulant activity. Molecular cloning of its cDNA identified this anticoagulant as a member of the Ca*+/phospholipid binding protein family the annexins.
The anticoagulant mechanism of action of hlrudln LINDHOUT T, BLEZER R, HEMKER HC Dept. of Biochemistv, Universityof Limburg, Maastricht, The Netherlands We studied the inhibitory action of the recombinant desulphatohirudin (CGP 39393) on thrombin generation in plasma. Plasma was activated either with thromboplastin or factor IXa. Hirudin delayed the onset of rapid thrombin generation, but it was unable to prevent the explosive appearance of thrombin. The dose-dependent prolongation of the lag phase of the intrinsic and extrinsic thrombin generation curve was not the result of titration of thrombin activity by hirudin but the result of a delayed formation of the prothrombin converting complex (prothrombinase). In case of extrinsic activation, hirudin did not affect factor Xa generation, but prolonged the lag phase of the factor Va
The comparative effects of recombinant hlrudln and standard heparln on lnhlbltlon of thrombus growth In rabbits AGNELLI G, PASCUCCI C, COSMI B, NENCI GG Institute di Semeiotka Medica, Universitddi Peru& Pen@, Itaiy Standard heparin (SH) is the accepted treatment for deep vein thrombosis for preventing thrombus extension. SH is a weak inhibitor of thrombin bound to fibrin. Recombinant hirudin (r-hirudin) (CGP 39393) is a selective inhibitor of thrombin, active on both free plasma thrombin and fibrin bound thrombin. The aim of this study was to compare the ability of SH and r-hirudin to inhibit fibrin accumulation on pre-existing thrombi in a rabbit jugular model. Doses of SH and r-hirudin equivalent in prolonging aPIT were first identified. 0.5 and 0.75 mg/kg of SH and 0.8 and 1.25 mg of r-hirudin infused over 3 hours produced a mean prolonga-
generation curve, causing its appearance when factor Xa generation was already in the decay phase. This provides hirudin with anti-prothrombinase activity. Because of its inhibitory action on the thrombin-mediated activation of factor VIII, hirudin prolonged the lag phase of the factor X converting complex that consists of factor IXa and factor VIIIa. Factor IXa was not inactivated by plasma proteinase inhibitors nor by hirudin. Our observations with hirudin are in keeping with the notion that inhibition of the thrombinmediated amplification reactions in blood coagulation is a very efficient way to delay or inhibit completely thrombin generation. Although extremely small amounts of hirudin neutralize stoichiomettic amounts of thrombin, the interaction between in situ generated thrombin and hirudin appears not to be fast enough to prevent trace amounts of thrombin to activate factors VIII and V. Consequently, an explosive thrombin generation is observed even when free hirudin is present.
tion of aPTT of 1.5 and 2, respectively. In saline treated rabbits 62 (27) pg of ‘zI-fibrinogen accumulation was observed. The lower doses of SH and r-hirudin produced an accumulation of 44*5 pg and 2524 pg of ‘2SI-fibrinogen, respectively (p