Metabolism of liposome-encapsulated heparin

In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or 14C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of 14C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.     

A low molecular weight heparin compared with unfractionated heparin

A 6000 daltons low molecular weight heparin (LMWH) was compared with unfractionated mucosal heparin in vitro and in vivo. Despite unimpressive specifications by clotting assays in vitro, the LMWH gave high and sustained activity in vivo by anti-Factor Xa assays, following subcutaneous injection. However, activity measured by APTT and calcium thrombin time assays was at least as high as occurred following unfractionated heparin. On the basis of clotting assays, there seems no reason to expect a lower incidence of haemorrhagic side-effects following the clinical use of this LMWH. The study also strikingly demonstrates the inadequacy of in vitro clotting assays for assessing the in vivo behaviour of LMWH.     

The disappearance of a low molecular weight heparin fraction (CY 216) differs from standard heparin in rabbits

In previous studies, we have reported that standard heparin (SH) was cleared by two mechanisms, a saturable mechanism which predominated at low doses (<100 anti-factor Xa U/kg) and a non-saturable mechanism which predominated at higher doses, when the first mechanism became saturated. In this study, we examined the importance of these two mechanisms in the disappearance of a low molecular weight heparin fraction (LMWH) (CY 216), by comparing the pharmacokinetics and the pharmacodyna-mics of a wide range of doses of SH and CY 216 (1.5 to 500 anti-factor Xa U/kg) Pharmacokinetics was measured as the disappearance of ¹²⁵I-radiolabelled SH or CY 216. Pharmacodynamics was measured as the disappearance of the anti-factor Xa activity of SH and CY 216. We found that the saturable mechanism contributed little to the disappearance of CY 216 and that it was cleared predominantly by the non-saturable mechanism at all doses tested. Thus, at low doses (<100 anti-factor Xa U/kg), SH was cleared more rapidly than CY 216, whereas at higher doses, CY 216 was cleared more rapidly than SH. We conclude that the mechanism of disappearance of LMWH's differ significantly from those of SH, and that this difference may explain the apparent prolonged anticoagulant activity of LMWH's within the therapeutic range doses.     

Studies on a highly active anticoagulant fraction of high molecular weight isolated from porcine sodium heparin

We have studied heparin fractionation using gel filtration and ion-exchange chromatographic methods. The starting material was commercial grade porcine mucosal sodium heparin (PSH). The fractionation was monitored employing synthetic substrates for assaying both antithrombin (with H-D-Phe-Pip-Arg-pNA ; S-2238) and anti-FXa (with Bz-Ileu-Glu-Gly-Arg-pNA ; S-2222) activities. The resulting fractions were evaluated in different amidolytic and coagulation methods used to determine heparin potency by comparison with PSH. By gel filtration of PSH on Ultrogel AcA 54, both strong anti-FXa and antithrombin activities were associated with the fractions eluted in the high molecular weight range (MW 20 × 10³). These fractions also had potent anticoagulant action when assayed by conventional clotting methods. PSH was also subjected to fractionation by an ion-exchange technique (DEAE-Sephacel) with increasing salt molarity. The patterns for antithrombin and anti-FXa activities were again closely related, if not identical. Four fractions were usually distinguished, with respectively negligible, intermediate, high and very high activities when compared to PSH. The very highly active fraction (HAF), approximately 15% by weight, was eluted at high salt molarity (> 0.8 M NaCl). On a weight basis its anticoagulant activity was 2–3 times that of PSH as determined by amidolytic as well as clotting methods. Intravenous injection of HAF to rabbits and dogs (1.0 and 2.5 mg/kg) produced a much stronger anticoagulant response than PSH, also showing an effect which persisted for a longer duration.     

Effects of heparin and a semi-synthetic heparin analogue on platelet aggregation, lipoprotein lipase and other laboratory tests in surgical patients

Platelet aggregation, lipoprotein lipase activity, coagulation parameters and routine blood chemistry were measured in a randomised study of 21 surgical patients before, immediately after and 3 months after operation. Sodium heparin 5000 IU was given subcutaneously to 11 patients every 12 hours for 7 days, the first injection 2 hours preoperatively; 10 patients received a semi-synthetic heparin analogue (SSHA 75 mg) in the same manner. The groups were sex and age matched. No conclusive changes were found in platelet aggregation. The increase in lipoprotein lipase activity in SSHA patients 2 hours after injection was significantly greater than in heparin patients. Neither of the two drugs induced significant changes in coagulation parameters or routine blood chemistry. The results indicate a difference in the effect on lipoprotein lipase release between heparin and SSHA at the used dosage schedules.     

Thrombin inactivation on surfaces with covalently bonded heparin

About 8000 Daltons porcine mucosa heparin fragments were covalently bonded by end-point attachment to polyethylene. The interaction between the immobilized heparin, added thrombin, and antithrombin III [AT] was investigated.

The heparin surface was adsorbed with either albumin, AT dissolved in albumin or Tyrode, or platelet free plasma. Irrespective of the pre-treatment procedure, exposure of the surface to thrombin resulted in the same substantial decrease of thrombin in solution and the same degree of surface-confined thrombin activity. It was concluded that the heparin surface has a large capacity to bind thrombin and that the thrombin inhibitory capacity of high affinity heparin fragments is limited.

On exposure of the thrombin-loaded surfaces to defibrinogenated plasma or AT, the surface-confined thrombin was inhibited within 30 seconds. Successive dilutions of plasma or AT decreased the inhibition rate but not the inhibition capacity. It is concluded that inhibition of thrombin adsorbed on the heparin surface occurs as follows: Added AT adheres to high affinity heparin fragments on the surface whereupon adsorbed thrombin migrates in the hydrophilic heparin coating towards the reaction site of AT and becomes inhibited. The inactivated thrombin-AT complex leaves then the surface, thus enabling the process to be repeated.     

Laboratory monitoring of thromboprophylaxis with low molecular weight and standard heparin

This study was made to evaluate assays for monitoring of low dose heparin thromboprophylaxis and to evaluate its efficacy in reduction of hypercoagulation. Patients with medical diseases scheduled for routine thromboprophylaxis were subcutaneously treated with either 5.000 anti XaU low molecular weight (LMW) heparin once daily (n=20) or 5.000 IU standard (ST) heparin 3 times daily (n= 19). On days 1,2,3, before, 1 and 4 hours after heparin injection APTT, TCT, anti Xa, Heptest, thrombin-antithrombin complexes (TAT), and D-Dimer levels were measured. In the LMW heparin group, median values of APTT and TCT slightly increased after heparin and the ranges of pre- and postinjection values showed extensive overlap. However, values of anti Xa and Heptest markedly increased, showing complete separation of ranges. In the ST heparin group neither APTT, TCT, anti Xa, nor Heptest were significantly different comparing pre- and postheparin values. Half of the patients in both groups had subclinical hypercoagulation at baseline (TAT>5ng/ml, D-Dimer>200ng/ml). On day 3 of prophylaxis this percentage was not significantly decreased. Moreover, several patients in both groups increased in TAT and D-Dimer. In the LMWheparin group, negative correlations between body weight and 4 h postinjection heparin levels were found (anti Xa R=−0.50, Heptest R=−0.31) and between 1 h postinjection heparin and TAT and D-Dimer levels 3 h later (TAT-anti Xa R=−0.58, TAT-Heptest R=−0.64, D-Dimer-anti Xa R=−0.32, D-Dimer-Heptest R=−0.33). These results show that low dose LMW but not ST heparin therapy can be monitored by the anti Xa test or the Heptest.     

Inactivation of heparin cofactor II by polymorphonuclear leukocytes

Inactivation of purified human heparin cofactor II by polymorphonuclear leukocytes was investigated. A proteolytic mechanism of inactivation was demonstrated by SDS-polyacrylamide gel electrophoresis. Inactivation and related proteolysis did not occur in the presence of unstimulated leukocytes and were prevented by various protease inhibitors. Heparin cofactor II was inactivated more rapidly than antithrombin III. However, heparin (110 ug/ml) strongly accelerated the rate of antithrombin III inactivation and slightly protected heparin cofactor II, thus reversing the order of inactivation. Dermatan sulfate had no effect on this process.     

Signs of thrombin generation in pediatric cardiac catheterization with unfractionated heparin bolus or subcutaneous low molecular weight heparin for antithrombotic cover

Introduction: Thrombosis is one of the most frequent adverse events after cardiac catheterization, which can be reduced by anticoagulation with unfractionated heparin (UFH) in both children and adults. Low molecular weight heparin (LMWH) might possibly offer advantages. Laboratory signs of thrombin generation during pediatric cardiac catheterization, with unfractionated heparin (UFH) bolus or subcutaneous LMWH for thrombosis prophylaxis, were determined in a first step to investigate the potential of LMWH for antithrombotic cover. Materials and methods: Signs of thrombin generation (D-dimer and F₁₊₂), anti-Xa activity and activated clotting time (ACT) were measured in 65 patients with congenital heart disease. A total of 40 patients were treated with a UFH bolus of 100 IU/kg bodyweight and, in 25 children, enoxaparin was subcutaneously administered at a dosage of 1/1.6 mg/kg bodyweight. Results: The dose to plasma activity of enoxaparin was more consistent than in the UFH group. Only a slight elevation of F₁₊₂ was found in some patients, which was a little higher in the enoxaparin group, but no difference of incidence of increased F₁₊₂ generation was detected between the two groups. D-dimer was elevated in three children after UFH bolus application, but no such effect was observed in any child after LMWH administration. Conclusions: Application of LMWH was equally efficacious during pediatric cardiac catheterization than UFH bolus administration, as determined by plasma levels and markers of clotting activation. In contrast to UFH bolus, no further monitoring was necessary after the application of LMWH during cardiac catheterization due to a consistent dose to plasma activity.     

Evidence for a saturable mechanism of disappearance of standard heparin in rabbits

This work demonstrates that after bolus intravenous injection standard heparin (SH) disappearance results from the combination of a saturable and a non saturable mechanism. Pharmacokinetics and pharmacodynamics of SH were studied by measuring the disappearance of increa-sing doses (5 – 500 anti-factor Xa U/kg) of ¹²⁵I-heparin and of its biological effects. CPM curves allowed to determine the half lives of heparin according to the dose injected. The half lives were clearly dose dependent and reached a plateau over 100 anti-factor Xa U/kg. The complex curve which describes the amount of heparin cleared per time unit after any given dose has been resolved into its two components reflecting a saturable and a non saturable mechanism of disappearance. For the doses <100 anti-factor Xa U/kg the saturable mechanism was preeminent and the anti-factor Xa activity disappearance followed an exponential pattern ; for the doses >100 anti-factor Xa U/kg the contribution of the non saturable mechanism becames more important and the anti-factor Xa activity disappearance followed a concave-convex pattern. Further experiments showed that the heparin half life shortened as the circulating anti-factor Xa activity decreased ; this phenomenon may explain the concave-convex pattern of the curve of the anticoagulant effect observed after injection of large doses of SH.     

Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute to the action of this drug in blood coagulation.     

Study on the mechanism of action of heparin and related substances on the fibrinolytic system: Relationsship between plasminogen activators and heparin

It is now generally well accepted that heparin and related substances increase the fibrinolytic activity . The stimulation of the amidolytic, plasminogenolytic and fibrinogenolytic activity of tissue plasminogen activator and urinary plasminogen activator through heparin was investigated . A concentration-dependent stimulation of the plasminogenolytic and fibrinogenolytic activity of both urinary and tissue-type plasminogen activators was observed in the presence of heparin. No heparin dependence was observed in the amidolytic assay. Heparin stimulates the plasminogenolytic activity of tissue plasminogen activator in the same manner as fibrin. Both activators form complexes with heparin; the heparin-binding-site seems to be identical or related with the fibrin-binding-site of tissue plasminogen activator. The physiological role of these interactions is discussed.     

The effect of heparin fragments of different molecular weights on experimental thrombosis and haemostasis

The effect of heparin fragments of different molecular weights has been compared with that of conventional sodium heparin on experimental thrombosis in vivo and ex vivo and experimental haemostasis in vivo. In the first part of the study fragments of different molecular weights were given (4,900, 6,500, 9,500 and 22,200 dalton). All preparations including the control gave a significant prolongation of the haemostatic plug formation time in the rabbit mesenteric microcirculation, and all except the fragment with the lowest molecular weight reduced the frequency of jugular vein thrombosis (induced by a combination of endothelial denudation and stasis). There was a correlation between the XaI activity of the different heparin fragments and frequency of thrombosis. Using an ex vivo method (modification of Chandler's model) a dose dependent lag phase until start of thrombus formation was found. In the second part of the study a dose response investigation was made comparing different doses of a fragment (6,500 dalton) with conventional heparin in the same XaI doses (10, 30 and 60 units/kg). Sodium heparin in the highest dose prolonged the haemostatic plug formation time whereas none of the fragment doses did. The lowest dose both of the fragment and conventional heparin did not reduce the frequency of thrombosis, whereas the two higher doses did. Thus it may be possible to obtain preventive effect on thrombus formation with a heparin fragment.