ABSTRACT
Protamine sulphate is an effective inhibitor of heparin and is used clinically to neutralise both low molecular weight heparins (LMWH) and unfractionated heparin (UFH). However, protamine sulphate does not fully counter the anti-Xa effect of LMWH, even in excess (>40 µg to 1 IU/ml). To investigate the molecular basis for this observation, the residual potencies in the presence and absence of plasma as well as the molecular weight profiles of commercial LMWH neutralised with increasing amounts of protamine were measured. Materials over 5000 Da are preferentially neutralised by protamine. To further investigate this molecular weight dependence, monodisperse oligosaccharides were prepared from three commercial LMWHs. The specific anti-Xa activity for the fractions increased with molecular weight, and was found to vary between the three preparations for oligosaccharides of the same molecular weight. Our results indicate that protamine sulphate neutralisation is largely dependent on molecular weight, leading to the implication that LMWHs containing a larger proportion of small oligosaccharides will not be as effectively neutralised. Protamine sulphate neutralisation of any given LMWH is also affected by the specific anticoagulant activities of its low molecular weight components, which varies between LMWH products, presumably with the method of manufacture.
Subject(s)
Anticoagulants/antagonists & inhibitors , Anticoagulants/chemistry , Heparin Antagonists/pharmacology , Heparin, Low-Molecular-Weight/antagonists & inhibitors , Heparin, Low-Molecular-Weight/chemistry , Protamines/pharmacology , Anticoagulants/metabolism , Dalteparin/antagonists & inhibitors , Dalteparin/chemistry , Dalteparin/metabolism , Factor Xa/metabolism , Factor Xa Inhibitors , Heparin, Low-Molecular-Weight/metabolism , Humans , Molecular Weight , Oligosaccharides/antagonists & inhibitors , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Partial Thromboplastin Time , Prothrombin/antagonists & inhibitors , Prothrombin/metabolism , TinzaparinABSTRACT
In a double-blind, randomized, cross-over study the neutralizing action of protamine towards unfractionated heparin (UFH, 150 U/kg i.v.) and a low molecular weight heparin (LMWH, Fragmin, 100 anti-Xa U/kg i.v.) was investigated in 15 healthy subjects in vitro by measuring activated partial thromboplastin time (APTT), thrombin time (TT) and anti factor Xa activity (anti-Xa) in venous blood and in vivo by determination of prothrombin fragment 1.2 (f1.2) and thrombin-antithrombin III complexes (TAT) in venous blood and in shed blood. UFH and LMWH caused a prolongation of APTT and TT, an increase in anti-Xa and significantly inhibited f1.2 and TAT formation in shed blood, whereas only a minimal effect on TAT and f1.2 formation in venous blood was noted. Administration of 1 mg protamine/100 U UFH resulted in a near complete reversal of APTT, TT and anti-Xa, whereas lower doses (0.25 and 0.5 mg) were less effective. The effects of UFH on f1.2 and TAT generation in shed blood were partially (60-70%) neutralized only by the high dose (1.0 mg). Application of 1 mg protamine/100 anti-Xa U LMWH caused a near complete reversal of both APTT and TT but had only a weak effect on anti-Xa. In shed blood, the effect of LMWH on TAT and f1.2 formation was reversed by protamine only by 14% and 23% respectively. Our data do not support the concept that to reduce the incidence of protamine's potential clinical side effects, the administration of a lower dose of protamine than 1 mg protamine/100 U UFH is justified.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Blood Coagulation/drug effects , Dalteparin/antagonists & inhibitors , Heparin/pharmacology , Protamines/pharmacology , Adult , Antithrombin III/analysis , Cross-Over Studies , Double-Blind Method , Factor Xa Inhibitors , Humans , Male , Partial Thromboplastin Time , Peptide Fragments/analysis , Peptide Hydrolases/analysis , Prothrombin/analysis , Thrombin TimeABSTRACT
Fragmin and heparin were studied in pigs during 120 min of cardiopulmonary bypass (CPB) and up to 240 min postoperatively, with respect to clotting, bleeding and the effects of protamine. Thirty-three pigs received bolus injections of 300 IU/kg with or without additional dosage during CPB and with or without subsequent protamine sulphate. Doses of Fragmin 60% higher were necessary to prevent clotting. These had 100% higher anti-FXa levels but about 50% shorter activated coagulation time (ACT) compared with heparin. Anti-FXa increased with cumulative doses of heparin and Fragmin but ACT and activated partial thromboplastin time (aPTT) did not, indicating a larger loss of thrombin inhibition compared with anti-FXa in both drugs during CPB. Thrombin inhibition was crucial for prevention of clotting. Protamine efficiently normalized ACT in the Fragmin group but left a residual 20% anti-FXa, which did not increase the bleeding tendency. Fragmin could adequately be monitored with ACT and would be a safe alternative to heparin in CPB.
