Plasma lipolytic activity after subcutaneous administration of heparin and a low molecular weight heparin fragment

The effect of heparin and a low molecular weight heparin fragment (LMWH, mean molecular weight 4000-6000) on plasma anticoagulation and lipolysis was studied in eight healthy men. The activities of antifactor Xa (antiFXa), lipoprotein lipase (LPL), hepatic lipase (HL) and plasma levels of free fatty acids (FFA) were analysed after the injection of 5000 antiFXa units of heparin or LMWH subcutaneously. In comparison with heparin, the administration of LMWH resulted in a significantly higher antiFXa activity (p less than 0.001) but a lower release of LPL and HL (p less than 0.001), which did not increase plasma FFA. It is concluded that subcutaneous injection of LMWH in men elicits an adequate anticoagulant effect measured as antiFXa activity but has a negligible effect on plasma lipolytic activity.     

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.     

A comparison of the antithrombotic and haemorrhagic effects of a low molecular weight heparin (LHN-1) and conventional heparin

The antithrombotic effects after intravenous administration of a low molecular weight heparin (LHN-1) and conventional heparin were compared in a rabbit model of experimental thrombosis, where thrombus formation was induced by a combination of endothelial damage and stasis. Both compounds were able to prevent thrombosis completely. However, LHN-1 was significantly less potent than conventional heparin, the ratio between doses with the same antithrombotic effect being 2.4:1 on a weight basis. Bleeding times after administration of LHN-1 and conventional heparin were determined by tail transsection in anaesthetized rats and by template bleeding in the ear of conscious pigs. Given intravenously at a dose ratio of 2.4:1 (w/w), LHN-1 affected APTT less than conventional heparin, whereas the effects on haemostasis were not significantly different. In conclusion, it was found that after intravenous administration LHN-1 prevented experimental thrombosis as effectively as conventional heparin. However, the correlation between antithrombotic and haemorrhagic effects of LHN-1 was the same as that of conventional heparin. The corresponding relation in man remains to be determined.     

Neutralization of a low molecular weight heparin (LHN-1) and conventional heparin by protamine sulfate in rats

The neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized. In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.     

The relative antithrombotic effectiveness of heparin, a low molecular weight heparin, and a pentasaccharide fragment in an animal model

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes' stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes' stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.     

The binding of unfractionated heparin and low molecular weight heparin to thrombin-activated human endothelial cells

The binding of unfractionated heparin to endothelium is thought to be responsible for the rapid and saturable phase of unfractionated heparin clearance. Thrombin can induce endothelial cells to express and/or secrete a number of heparin binding proteins that have the potential to increase the binding of unfractionated heparin and to a lesser extent the binding of low molecular weight heparin. To explore this possibility, we examined the binding of unfractionated heparin and low molecular weight heparin to thrombin-activated endothelial cells. Cultured human umbilical vein endothelial cells were used to determine the binding of 125I-labeled unfractionated heparin and low molecular weight heparin to untreated and to thrombin-activated cells. After thrombin treatment, we obtained a time-dependent increase in the binding of radio-labeled unfractionated heparin. In contrast, there was much less binding of low molecular weight heparin, and a time-dependent increase was not apparent. After 30, 45, and 60 minutes of thrombin treatment, the binding of unfractionated heparin was significantly higher than that of low molecular weight heparin. The increase in binding of unfractionated heparin to thrombin-activated cells also was demonstrated using fluorescently labeled unfractionated heparin followed by fluorescence microscopy. The average fluorescence intensity of thrombin-treated cells increased by 44% when compared with resting cells. The present results indicate that thrombin can increase the binding of unfractionated heparin to human umbilical vein endothelial cells. Thus, an activated endothelium may contribute to the variability of the anticoagulant response to unfractionated heparin. In contrast, the binding of low molecular weight heparin is much less affected, which may account for its better bioavailability and longer half-life.     

A new low molecular weight heparin fragment (PK 10169): in vitro and in vivo studies

The depolymerized heparin fragment PK 10169 was compared with conventional mucosal sodium heparin. The inhibition of factors Xa and IXa by heparin and by PK 10169 was similar on a weight base whilst the inhibition of thrombin by PK 10169 was at least 5 times weaker than by heparin. Subcutaneous injection of PK 10169 was not followed by prolongation of the thrombin time. The APTT was considerably less prolonged after PK than after heparin. Platelet reaction was increased by heparin but was not influenced by PK 10169. In vitro the euglobulin lysis time (ELT) was shortened after addition of heparin to plasma but not after addition of PK 10169. After injection, however, there was an equal shortening of the ELT by both substances. Advantages of PK 10169 over heparin are therefore a weaker anticoagulant effect and the missing influence on platelet functions.     

Pharmacokinetics of heparin and low molecular weight heparin fragment (Fragmin) in rabbits with impaired renal or metabolic clearance

The kinetics and tissue distribution of 3H-heparin and a 3H-labelled low molecular weight heparin fragment were compared in normal rabbits as well as in rabbits with blocked renal function or reticuloendothelial system (RES). Radioactivity in plasma, urine, liver and kidneys, as well as anti-FXa activity in plasma were determined. The plasma elimination of heparin was, when compared to normal controls, prolonged both in rabbits with renal dysfunction as well as in rabbits with blocked RES, while renal dysfunction was the only parameter that significantly prolonged the plasma half-life of Fragmin. Studies on tissue distribution in normal rabbits revealed that about 60 per cent of the radioactive heparin dose accumulated in the liver and kidney three hours after the injection, whereas the corresponding value was less than 10 per cent for the Fragmin-derived radioactivity. The recovery of radioactivity in urine within three hours was 5 and 35 per cent of the dose, respectively, for 3H-heparin and 3H-Fragmin. It is concluded from the present study that the rapid plasma elimination of heparin in the rabbit (t1/2 = 17 minutes) is mainly due to a high tissue distribution (liver and kidney) while the plasma elimination of Fragmin (t1/2 = 28 minutes) is mainly caused by renal excretion.     

Anticoagulant and lipolytic effects of a low molecular weight heparin fraction

The lipolytic and anticoagulant actions of a 4000 dalton low molecular weight (LMW) heparin were compared with unfractionated mucosal heparin after intravenous and various subcutaneous doses in man. I.v. injection of 100 USP units/kg body weight lipoprotein lipase (LPL) activity, and inhibition of factor Xa decreased with a half life twice as long after LMW heparin compared to normal heparin (p < 0.05). There were no differences in half lives for HTGL activity, thrombin inhibition and on aPTT. The area under the activity time curve (AUC) of LPL and factor Xa was double with LMW heparin (p < 0.05). S.c. administration showed that the AUC of LMW heparin on the factor Xa inhibition was 10 times larger compared to normal heparin. LPL activity was released comparable to normal heparin. The effects on HTGL were three times larger compared to normal heparin. There were no differences in half lives. The data show that in contrast to normal heparin LMW heparin is rapidly and completely absorbed from the subcutaneous depots. The pharmacodynamic data of LPL activity and factor Xa inhibition suggest similar release mechanisms.     

Prevention of deep vein thrombosis after major knee surgery--a randomized, double-blind trial comparing a low molecular weight heparin fragment (enoxaparin) to placebo.

Consecutive patients undergoing knee arthroplasty or tibial osteotomy at four participating hospitals received either enoxaparin, 30 mg subcutaneously every 12 h (n = 66) or an identical-appearing placebo (n = 65). All study medications started the morning after the operation and were continued up to a maximum of 14 days. Patients underwent surveillance with 125I-fibrinogen leg scanning and impedance plethysmography. Bilateral contrast venography was performed routinely at Day 14 or at time of discharge, if sooner. Deep vein thrombosis was detected by venography in 35 of 54 patients (65%) in the placebo group and in 8 of 41 patients in the enoxaparin group (19%), a risk reduction of 71%, P less than 0.0001. For the entire study group, deep vein thrombosis was detected by either venography of non-invasive tests in 37 of 64 patients (58%) in the placebo group and in 11 of 65 patients (17%) in the enoxaparin group, a risk reduction of 71%, P less than 0.0001. Proximal vein thrombosis was found in 19% of the placebo patients and in none of the enoxaparin patients, a risk reduction of 100%, P less than 0.001. Bleeding complications occurred in 5 of 65 patients (8%) in the placebo group and in 4 of 66 patients (6%) in the enoxaparin group, P = 0.71. There were no differences in the amount of blood loss, minimum hemoglobin levels and number of units of packed red cells given between the two treatment groups. We conclude that a fixed dose regimen of enoxaparin, started post-operatively, is an effective and safe regimen for reducing the frequency of deep vein thrombosis after major knee surgery.     

In vivo effects of a low molecular weight heparin fragment on platelet aggregation and platelet dependent hemostasis in dogs

Plasma defibrinogenated dogs were used to study the influence of conventional heparin and a low molecular weight heparin fragment (Fragmin, mean MW 5,000 d) on platelet dependent hemostasis. The heparins were given intravenously in gravimetrically equal doses. The bleeding from standardized skin flap wounds and platelet aggregation (ADP and thrombin) was studied. In comparison, higher doses of the fragment than of heparin were required to increase the bleeding. ADP-induced aggregation in defibrinogenated platelet rich plasma (after addition of normal dog plasma) was potentiated by both heparins. After injection of heparin or the fragment, ADP induced platelet aggregation without prior addition of normal plasma to the test-tube. In conclusion the heparin fragment affected bleeding to a less extent than conventional heparin. One explanation might be a weaker inhibition of thrombin-induced platelet aggregation.     

An international standard for low molecular weight heparin

An international collaborative study has been carried out with the aim of establishing an international standard for low molecular weight (LMW) heparin. Three preparations of LMW heparin were assayed against the International Standard for unfractionated heparin (UFH) by 25 laboratories in 13 countries, using nine different assay methods. The results confirmed previous findings of non-parallel assays, wide interlaboratory variability and differences between methods when LMW heparins are assayed against a UFH standard. Use of one of the LMW heparins as a standard for the other two gave parallel assays and much closer agreement between laboratories. The preparation in ampoules coded 85/600 was selected as likely to give the best agreement with the largest number of LMW heparins; potencies were assigned by taking the mean of all the anti-Xa assays, and the mean of the thrombin and APTT assays, to represent the two major groups of activities. Preparation 85/600 has been established by WHO as the 1st International Standard for LMW heparin, with potencies of 1,680 iu/ampoule by anti-Xa assays and 665 iu/ampoule by thrombin inhibition and APTT assays.     

Heparin-associated thrombocytopenia treatment with low molecular weight heparin

Eight patients with heparin associated thrombocytopenia (HAT) were treated by a low molecular weight heparin derivative (LMW). Biological and clinical improvement occurred in all patients. This efficiency confirms the antithrombotic activity of LMW and allows its use in patients with HAT.     

Prevention of postoperative venous thrombosis: a randomized trial comparing unfractionated heparin with low molecular weight heparin in patients undergoing total hip replacement

A double blind randomized trial comparing subcutaneous enoxaparin (40 mg once daily) with standard unfractionated calcium heparin administered at a dose of 5,000 units every 8 hours in patients undergoing elective hip replacement has been performed. Treatment regimens began 12 hours preoperatively with enoxaparin, 2 hours preoperatively with standard unfractionated calcium heparin, and were continued for 15 days or until discharge. Venography was performed in all patients. Two hundred thirty-seven patients were included in the study: 113 received unfractionated heparin and 124 received enoxaparin. The incidence of proximal deep vein thrombosis was reduced from 18.5% in the unfractionated heparin group to 7.5% in the enoxaparin group (p = 0.014), and the incidence of total deep vein thrombosis was similarly reduced from 25% to 12.5% (p = 0.03). There were two major bleeding episodes and one minor bleed in the enoxaparin group compared to two minor bleeds in the unfractionated heparin group. Patients who received enoxaparin required fewer red blood cell transfusions and had a significantly higher hemoglobin on postoperative days 3 and 4. Thus prophylaxis with enoxaparin, 40 mg once daily, is simple, safe and more effective than standard low dose unfractionated heparin in preventing deep vein thrombosis in patients undergoing elective hip replacement.     

A pilot study on systemic thrombolysis followed by low molecular weight heparin in ischemic stroke

Low molecular weight heparin (LMWH) administered immediately after intravenous thrombolysis (IT) may reduce the risk of arterial re-occlusion. Its benefit, however, may not outweigh the risk of intracranial hemorrhage (ICH). We sought preliminary data regarding safety of this combined therapy in an open-label, non-randomized study. The patients received either a standard anticoagulation (AC) starting 24 h after IT (the standard AC group) or AC with 2850 IU of nadroparin, given every 12 h immediately after IT (the early AC group). Sixty patients received IT treatment: 25 in the standard AC group [mean age 66, median National Institutes of Health Stroke Scale (NIHSS) 13, 64% men] and 35 in the early AC group (mean age 68, median NIHSS 13, 69% men). Symptomatic ICH occurred in one patient (4%) in the standard AC group and three patients (8.6%) in the early AC group [odds ratio (OR) 1.8; 95%CI 0.2–12.8]. At 3 months, nine patients in the standard AC group (36%) and 16 patients in the early AC group (45.7%) achieved a modified Rankin scale 0 or 1 (OR 1.2; 95%CI 0.5–3.2). Our study suggests that treatment with LMWH could be associated with higher odds of ICH, although it may not necessarily lead to a worse outcome. This justifies larger clinical trials.     

Dose finding study of a low molecular weight heparin, Innohep, in haemodialysis.

A pilot investigation was performed with Innohep, a low molecular weight (LMWH) preparation (peak maximum molecular mass 3,000-6,000), to determine possible dose regimens for patients undergoing regular maintenance haemodialysis for chronic renal failure. Results from this study suggested that suppression of macroscopic clot formation and fibrinopeptide A (FPA), a marker of fibrin formation, could be achieved following bolus injections rather than bolus injections and an infusion. On the basis of these preliminary findings, a randomised crossover study was performed in eight patients undergoing regular maintenance haemodialysis for 5-7 h to determine the effective antithrombotic dose of this LMWH. Single i.v. bolus doses of 1,250 AFXa u, 2,500 AFXa u and 5,000 AFXa u (n = 7-8) were compared to an UFH regime of 5,000 iu + 1,500 iu/h. Excessive clot formation in the dialyser bubble trap, necessitating additional UFH to enable completion of a prolonged (up to 7 h) dialysis, was observed in all patients on the 1,250 AFXa u dose (mean duration of dialysis prior to UFH, 3 h) but in a single patient only receiving the other LMWH doses. A dose-related response in the AFXa activity, measured by chromogenic substrate (CS) assay was seen in the three LMWH groups, with levels declining significantly (p less than 0.05) from 1-7 h. This contrasted with the constant levels maintained during dialysis with UFH. FPA levels were significantly elevated after 2 h following the 1,250 AFXa u bolus and after 4 h following the 2,500 AFXa u bolus. There was no significant difference in FPA levels between the 5,000 AFXa u bolus and UFH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of standard heparin and a low molecular weight heparin (Kabi 2165) on fibrinolysis

Previous and recent reports have suggested a fibrinolysis-enhancing property of standard heparin and low molecular weight heparins, but these observations have never been confirmed in a study fulfilling appropriate methodological criteria. The aim of this study was to evaluate the effect of standard heparin and a low molecular weight heparin (Kabi 2165) on fibrinolysis in a randomized cross-over double blind placebo controlled study. Six healthy volunteers received intravenously a bolus dose of the following treatments: placebo; standard heparin, 5,000 I.U.; Kabi 2165, 5,000 anti-Xa U; Kabi 2165, 10,000 anti-Xa U. Before the injection and at established times thereafter, blood samples were collected for the following assays in plasma: t-PA activity, PA inhibitor activity, fibrin plate lysis area (FPLA), plasminogen, alpha 2-antiplasmin, fibrinogen and anti-Xa activity. Placebo and Kabi 2165, 5,000 anti-Xa U, had no effect on t-PA plasma level. Standard heparin and Kabi 2165, 10,000 anti-Xa U, produced a statistically significant increase in t-PA level at 1 hour after the infusion. This increase lasted for at least 1 hour after the infusion. No effect of any treatment on PA inhibitor, plasminogen, FLPA, alpha 2-antiplasmin and fibrinogen was observed. We conclude that an intravenous bolus dose of both standard heparin, 5,000 I.U. and Kabi 2165, 10,000 anti-Xa U produces a delayed and sustained increase in plasma t-PA.