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Inhibition of fluid phase and clot-bound thrombin: Effects of heparin, hirudin and PEG-rm-hirudin
Vol. 70, Suppl. 1
ABSTRACTS
OF 12TH NATIONAL
CONGRESS
s17
c 017 INHIBITION OF FLUID PHASE AND CLOT-BOUND THROMBIN: EFFECTS OF HEPARIN, HIRUDIN AND PEGm-HIRUDIN. M. Leone, G. Agnelli, P. Melelli, C. Renga, G. G. Nenci. institute oflntemal and Vascfdar hfediche, Urive&y ol Pengia, ita&. Heparin and hirudin are antithrombotic agents able to inactivate thrombin through two different mechanisms: heparin inactivates thrombin through antithrombin III (ATIII); hirudin inhibits thrombin through a stoichiometric complex that does not require plasma cofactors. Recent studies have demonstrated that thrombin bound to fibrin is resistant to inactivation by the heparin-ATIII complex. This could explain the limited ability of heparin in preventing the extension of vein thrombi. The aim of this study was to compare the ability of unfractionated heparin, r-hi&n and PEG-rm-hirudin, a poly ethylene-glycol-coupled derivative of rm-hirudin with prolonged plasma half-life (manufactured by Knoll AG, Ludwigshafen, Germany), to inhibit thrombin, both in a fluid phase and bound to clot. The three agents were tested at concentrations able to produce an identical prolongation of the aPTT (x2). FPA generation (MA) was used as an index of thrombin activity. The results showed that r-hirudin and PEG-rm-hirudin at the concentration of 100 nM and heparin at the concentration of 0.25 U anti-Xa/ml produced an inhibition of thrombin in fluid phase by 100% for r-hirudin and PEG-rm-hirudin and by 90% for heparin (p=ns). The three inhibitors, at same concentrations, produced a different inhibition of FPA production induced by clot-bound thrombin: 88Ok8, 82%i7 and 42OM respectively for hirudin, PEG-rm-hirudin and heparin. In conclusion the 3 inhibitors are equally effective In inhibiting the fluid phase thrombin. r- and PEG-rm-hirudin maintain the same ability of inhibiting clot-bound thrombin. This is not the case of heparin, whose inhibiting action on bound thrombin is reduced by 50%.
c 018 UNFBACTIONATED HEPARIN AND LOW MOLECULAR WEIGHT HEPARINS (LMWH) EFFECTS ON BLEEDING TIME AND ANTI-Xa ACTIVITY IN HEALTHY VOLUNTEERS. EMPogliani, P.Bucciarelli. I.Baragetti. E.Bragani. lstituto di Scienze Biomediche, Universita di Milano, Div. Ematologia, Ospedale S.Gerardo.Monza Bleeding time and pharmacokinetlc parameters of Lh4WH heparins CY 216 SO75 Anti-Xa IU, Enoxaparin 2OOCland 4OW Anti-Xa IU and calcium heparin (Calciparina 5ooo IU t.i.d.) administered by subcutaneous route were Investigated In 12 healthy volunteers in a cross over study according to uncompleted balanced blocks design. The pharmacological effects were evaluated by measuring bleeding tiome (BT), chromogenic antlfactor Xa activii (S-2222, S-2732), activated partial thromboplastin time (APlT) and antithrombin Ill (AT Ill) after single and repeated administration (5 days). Calcium heparln did not prolong BT in a statlstically significant way after the first admlnlstratlon. while Lh4Wheparins CY 216 3075 AXa IU and enoxaparine 4OLXIAXa IU, significantly prolonged BT (p < 0.01) at Tmax After repeated doses, all heparins showed no statistical significant action on BT prolungation either at the basal or Tmax values in comparison with the same evaluation time at the fifth day of treatment. In fact, the increasing in ET, measured as the difference between Tmax and basal value, was not statistically significant different after repeated administrations, both for Lh4W heparins and calcium heparin. These results support the notion that heparins do not create an accumulation after repeated administrations of prophylactic dosages. No significant correlation has been found between the delta of BT and anti-Xa activity, APTT and anti-Xa/APTT ratio between the three different dosages of LMWH heparins expressed by IU. This observation confirms that LMW heparins are drugs with different biological and physical characteristics between each other.
ABSTRACTS
OF 12TH NATIONAL
CONGRESS
s17
c 017 INHIBITION OF FLUID PHASE AND CLOT-BOUND THROMBIN: EFFECTS OF HEPARIN, HIRUDIN AND PEGm-HIRUDIN. M. Leone, G. Agnelli, P. Melelli, C. Renga, G. G. Nenci. institute oflntemal and Vascfdar hfediche, Urive&y ol Pengia, ita&. Heparin and hirudin are antithrombotic agents able to inactivate thrombin through two different mechanisms: heparin inactivates thrombin through antithrombin III (ATIII); hirudin inhibits thrombin through a stoichiometric complex that does not require plasma cofactors. Recent studies have demonstrated that thrombin bound to fibrin is resistant to inactivation by the heparin-ATIII complex. This could explain the limited ability of heparin in preventing the extension of vein thrombi. The aim of this study was to compare the ability of unfractionated heparin, r-hi&n and PEG-rm-hirudin, a poly ethylene-glycol-coupled derivative of rm-hirudin with prolonged plasma half-life (manufactured by Knoll AG, Ludwigshafen, Germany), to inhibit thrombin, both in a fluid phase and bound to clot. The three agents were tested at concentrations able to produce an identical prolongation of the aPTT (x2). FPA generation (MA) was used as an index of thrombin activity. The results showed that r-hirudin and PEG-rm-hirudin at the concentration of 100 nM and heparin at the concentration of 0.25 U anti-Xa/ml produced an inhibition of thrombin in fluid phase by 100% for r-hirudin and PEG-rm-hirudin and by 90% for heparin (p=ns). The three inhibitors, at same concentrations, produced a different inhibition of FPA production induced by clot-bound thrombin: 88Ok8, 82%i7 and 42OM respectively for hirudin, PEG-rm-hirudin and heparin. In conclusion the 3 inhibitors are equally effective In inhibiting the fluid phase thrombin. r- and PEG-rm-hirudin maintain the same ability of inhibiting clot-bound thrombin. This is not the case of heparin, whose inhibiting action on bound thrombin is reduced by 50%.
c 018 UNFBACTIONATED HEPARIN AND LOW MOLECULAR WEIGHT HEPARINS (LMWH) EFFECTS ON BLEEDING TIME AND ANTI-Xa ACTIVITY IN HEALTHY VOLUNTEERS. EMPogliani, P.Bucciarelli. I.Baragetti. E.Bragani. lstituto di Scienze Biomediche, Universita di Milano, Div. Ematologia, Ospedale S.Gerardo.Monza Bleeding time and pharmacokinetlc parameters of Lh4WH heparins CY 216 SO75 Anti-Xa IU, Enoxaparin 2OOCland 4OW Anti-Xa IU and calcium heparin (Calciparina 5ooo IU t.i.d.) administered by subcutaneous route were Investigated In 12 healthy volunteers in a cross over study according to uncompleted balanced blocks design. The pharmacological effects were evaluated by measuring bleeding tiome (BT), chromogenic antlfactor Xa activii (S-2222, S-2732), activated partial thromboplastin time (APlT) and antithrombin Ill (AT Ill) after single and repeated administration (5 days). Calcium heparln did not prolong BT in a statlstically significant way after the first admlnlstratlon. while Lh4Wheparins CY 216 3075 AXa IU and enoxaparine 4OLXIAXa IU, significantly prolonged BT (p < 0.01) at Tmax After repeated doses, all heparins showed no statistical significant action on BT prolungation either at the basal or Tmax values in comparison with the same evaluation time at the fifth day of treatment. In fact, the increasing in ET, measured as the difference between Tmax and basal value, was not statistically significant different after repeated administrations, both for Lh4W heparins and calcium heparin. These results support the notion that heparins do not create an accumulation after repeated administrations of prophylactic dosages. No significant correlation has been found between the delta of BT and anti-Xa activity, APTT and anti-Xa/APTT ratio between the three different dosages of LMWH heparins expressed by IU. This observation confirms that LMW heparins are drugs with different biological and physical characteristics between each other.