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.     

Differential effects of heparin and low molecular weight heparin on osteoblastogenesis and adipogenesis in vitro

We have previously demonstrated that heparin produces cancellous bone loss in rats due in part to a decrease in the number of osteoblasts lining the trabecular bone surface. In the present study, we use a stromal-derived cell culture system together with measurements of alkaline phosphatase (ALP) activity, to compare the effects of heparin and the low molecular weight heparin (LMWH), Fragmin, on osteoblast differentiation in vitro. In addition, we examined the possibility that both heparin and LMWH can induce adipogenesis in our stromal cell culture system. Both heparin and LMWH were found to produce a statistically significant (P < 0.01) and concentration-dependent decrease in the number of osteoblasts while increasing the number of adipocytes. When the effects of gravimetrically equivalent amounts of heparin and LMWH were compared, heparin had a 4-fold greater effect than LMWH. In contrast to heparin, N-desulfated heparin was found to have minimal effects on both osteoblast and adipocyte differentiation indicating that the heparin effect is not only chain-length dependent but also charge-dependent. The observation that LMWH has less of an effect on bone formation than heparin is compatible with the results of clinical trials indicating that LMWH produces less bone loss after long-term administration.     

Pharmacokinetic of a very low molecular weight heparin in chronic renal failure.

The pharmacokinetic characteristics of a low molecular weight heparin (LMWH) (Cy 222; mean mw: 2500 daltons) are studied in 24 patients with 3 degrees of chronic renal failure (CRF) stage I (creatinine clearance between 50 and 30 ml/mn), stage 2 (creatine clearance between 30 and 10 ml/mn), stage 3 (creatinine clearance below 10 ml/mn). Patients with CRF have significantly higher values of anti Xa activity at 3 hours (p less than 0.05), 5 hours (p less than 0.05), and at 8 hours (p less than 0.03) after injection than controls, CMAX values, VDSS and AUC do not differ, whereas patients with the highest stage of CRF are characterised by the most important t1/2 a (p less than 0.001) and the smallest total body clearance (p less than 0.01). Consequences of these disturbances of pharmacokinetic characteristics have to be evaluated before adequate posology of heparin fragments could be determined in patients with CRF.     

Neutralisation of heparan sulphate and low molecular weight heparin by protamine

The neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.     

Platelets of patients with peripheral arterial disease are hypersensitive to heparin

We sought to verify earlier reports of increased platelet reactivity in patients with peripheral arterial disease (PAD) during perioperative heparin administration, and to test the hypothesis of platelet hypersensitivity to heparin in these patients. Before and after incubation of platelet rich plasma with unfractionated (UH), low molecular weight heparin (LMWH), and a low molecular weight heparinoid, real-time quantitative assessment of platelet function was performed by stagnation point flow adhesio-aggregometry (SPAA) in 21 patients with PAD and 14 healthy volunteers. With SPAA the occurrence of spontaneous aggregation is pathological. In the 15 patients requiring operation, platelet function and count were measured at regular intervals. To detect heparin dependent antibodies, the heparin induced platelet activation assay (HIPA) was performed preoperatively and after 10 days of heparin therapy. Mean baseline platelet adhesion in patients was double that observed in controls (p < 0.001). Spontaneous aggregation was seen in 9 (43%) patients and no controls (p < 0.001). In controls heparinoid reduced, whereas UH and LMWH slightly increased adhesion. Spontaneous aggregation was observed once with UH. Platelets from patients showed significantly enhanced adhesiveness and aggregability (p < 0.05) with UH and LMWH when compared to controls. Effects with the heparinoid were less pronounced and non-significant. In patients requiring operation, postoperative increases in platelet function and reductions in count were significant (p < 0.001). Ten (67%) experienced a fall in platelet count of > 50%. Preoperatively the HIPA assay showed no evidence of antibodies, whereas after heparin administration antibodies were verified in 4 (32%) patients and could not be ruled out in 6 (40%). Three developed postoperative thrombosis, in one case fatal. A hypersensitive in vitro and in vivo platelet response to heparin was verified in patients with PAD and a large number developed the immunological type of heparin-associated thrombocytopenia. Our findings suggest that a thrombin antagonist which does not interact with platelets may give the best perioperative protection in these patients.     

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.     

Effect of standard heparin and a low molecular weight heparin on thrombolytic and fibrinolytic activity of single-chain urokinase plasminogen activator in vitro

The effect of unfractioned heparin (UH) and low molecular weight heparin (LMWH) (Kabi 2165 - Fragmin) on in vitro scu-PA thrombolytic and fibrinogenolytic activity was investigated. Thrombolytic activity was evaluated by following lysis of radiolabeled plasma clot immersed in plasma in presence of scu-PA alone or with either form of heparin. A 200 IU/ml scu-PA concentration produced clot lysis within 7 hr. UH or LMWH led to a slightly faster clot lysis which was statistically significant only at the 2nd and 3rd hour. No significant difference could be evidenced between UH and LMWH effect. During clot lysis, plasmin, generated within the clot led to a gradual transformation of scu-PA to tcu-PA, specially after a 4-hr incubation. Appearance of tcu-PA activity in the plasma surrounding the clot was significantly inhibited by either form of heparin. This finding contrasts with results observed in purified systems and suggests the presence of heparin-dependent plasma factor(s) inhibiting tcu-PA formation or its activity. Possible candidates might be anti-thrombin III and PAI-3. No fibrinogen breakdown was observed when plasma was incubated for 7 hr at 37 degrees C in presence of scu-PA alone (200 IU/ml) or with either form of heparin. However, in presence of a plasma clot, an important fibrinogen breakdown was observed during clot lysis reflecting the action of plasmin and/or tcu-PA generated within the clot, in the surrounding plasma. Fibrinogenolysis was less pronounced in the presence of both heparin preparations possibly as a consequence of the reduction in the tcu-PA level. These results underline the importance of plasma factors in the interaction of heparin with plasminogen activators such as scu-PA.     

The effects of post-heparin plasma lipases on anti-Xa clotting activity

The effects of hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL) on the anti-Xa clotting activity of plasma were studied. LPL had no effect, but HTGL enhanced anti-Xa activity. This enhancement was shown to be due to a time-dependent action of HTGL on lipoproteins. These results could explain the increases in anti-Xa clotting activity previously observed after injection of heparin analogues, SSHA and SP54, which are potent releasers of lipase enzymes.     

A human pharmacological study comparing conventional heparin and a low molecular weight heparin fragment

The pharmacokinetics of a heparin fragment of low molecular weight (LMWH) of 4000-5000 D and unfractioned standard heparin (UFH) have been studied after i.v. injections of different doses and infusions in 8 humans. The heparin activity was significantly higher and the effect on APTT lower after LMWH fragment as compared to UFH in the same doses. The half-life of heparin activity was about 1 hr for UFH and about 2 hr for LMWH. LMWH was found to be eliminated according to first order kinetics and there were no signs of dose dependency.     

Inhibition of low molecular weight heparin by protamine chloride in vivo

To determine the antagonization of anticoagulant and lipolytic effects of a low molecular weight [LMW] heparin preparation protamine chloride was given intravenously after i.v. injection of LMW or normal heparin. The effects of normal heparin on factor Xa, thrombin, aPTT, lipoprotein [LPL] and hepatic triglyceride lipase [HTGL] activities were neutralized immediately by i.v. protamine. The inhibition of thrombin and aPTT by LMW heparin were also abolished, whereas the effects on LPL and HTGL were counteracted to 80% and on factor Xa only to 40% by i.v. protamine chloride. No rebound of the anticoagulant or lipolytic effect was detected. It is assumed that haemorrhagic complication during therapy can be antagonized by protamine chloride. The incomplete inhibitory effect of protamine chloride on LPL, HTGL and factor Xa activities of LMW heparin indicate that protamine chloride requires more than 14 saccharide units in the heparin molecule for interaction.     

Effect of tissue factor pathway inhibitor (TFPI) in the HEPTEST assay and in an amidolytic anti factor Xa assay for LMW heparin.

Both the HEPTEST and amidolytic anti factor Xa assays are currently being used for heparin activity detection in plasma from patients receiving standard heparin or low molecular weight heparin (LMWH). In this study we have investigated the influence of recombinant and endogenous Tissue Factor Pathway Inhibitor (TFPI) on these assays. The HEPTEST determinations were performed on an ACL 300 R Clottimer using the APTT program which resulted in a longer incubation time with factor Xa than recommended by the manufacturer. rTFPI added to plasma prolonged the HEPTEST clotting time markedly, but had only a little effect in the amidolytic assay. Antibodies against TFPI (anti-TFPI) abolished these effects. The effect of adding rTFPI and Logiparin was additive. When anti-TFPI IgG was added to samples of normal plasma, a statistically significant shortening of the HEPTEST clotting time was seen. When anti-TFPI was added to plasma samples from volunteers who had received Logiparin by subcutaneous or intravenous injection, then the HEPTEST clotting time was shortened considerably. For some samples the clotting time was halved. These experiments show that the HEPTEST clotting time is prolonged not only by heparin-antithrombin III, but also by TFPI released by heparin injection.     

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.     

Heparin-induced thrombocytopenia platelet aggregation studies in the presence of heparin fractions or semi-synthetic analogues of various molecular weights and anticoagulant activities

One case of heparin-induced thrombocytopenia is reported. Aggregation was observed in the platelet-rich plasma of this patient in the presence of two commercial standard heparin preparations (from a final concentration of 0.025 IU/ml upwards), of two semi-synthetic heparin analogues (0.1 APTT U/ml) and of three low-molecular weight heparin (LMWH) fractions (0.1 anti-Xa U/ml) but not in the presence of five other LMWH fractions. The patient's isolated platelets no longer aggregated in the The patient's isolated platelets no longer aggregated in the presence of heparin but the phenomenon recurred after addition of the patient's platelet poor plasma (PPP). Furthermore, addition of patient's PPP to control platelets led to heparin-induced aggregation. The phenomenon was associated with thromboxane generation and could be blocked by in vitro addition of aspirin, PGI2, and PGD2 whereas the lag phase was dose-dependently prolonged by adenosine. It is concluded that platelet aggregation may be induced in some patients by standard heparin and by certain LMWH fractions or semi-synthetic analogues, independently of their molecular weight and anticoagulant activity.     

Comparison of the effects of low molecular weight heparin and unfractionated heparin on activation of human platelets in vitro

An unfractionated heparin (UFH) and a depolymerised derivative of low molecular weight heparin (LMWH) have been compared for their ability to activate platelets suspended in citrated plasma (PRP) or after washing and suspension in hepes buffered tyrode containing fibrinogen. Neither heparin alone induced aggregation of washed platelets, but UFH and to a much lesser extent LMWH, induced aggregation of platelets in PRP. Both heparins caused significant enhancement of a low concentration of ADP-induced activation of PRP and, again, the effect of LMWH was somewhat less than that of UFH. UFH produced a marked potentiation of ADP-induced activation of washed platelets and LMWH was about a third as potent. In addition, UFH induced a potentiation of PAF-induced aggregation and dense-granule release in PRP, a property not shared by LMWH. In PRP, UFH was three times more potent at inhibiting thrombin-induced aggregation and dense-granule release, as might be expected from their specific activities in the KCCT and thrombin time assay. However, with washed platelets, both heparins were equivalent at inhibiting thrombin-induced aggregation, dense-granule release and elevation of cytosolic free calcium ([Ca++]i) as monitored by quin 2 fluorescence. UFH and LMWH alone did not induce a change in [Ca++]i, nor had they any effect on ADP- or PAF-induced elevation of [Ca++]i.     

Heparin and a low molecular weight fraction enhances thrombolysis and by this pathway exercises a protective effect against thrombosis

In human volunteers unfractionated heparin and a low molecular weight fraction of heparin (LMWH) caused an increase in plasma plasminogen activator (PA) which peaked at 3 hours after subcutaneous injection. Using a perfused isolated rabbit ear model the enhancement of PA activity was confirmed and was related to the anti-Xa activity of both products infused. Using a modified rabbit Wessler model for thrombus formation it was found that, when using doses of heparin and LMWH sufficient to give a 100% antithrombotic effect, antifibrinolytic drugs (eg. ε-ACA and aprotinin), negated this protective effect. It is concluded that the effect of heparin and LMWH on haemostasis is mediated in part through the enhancement which these drugs have on fibrinolysis, the latter being arguably a major defence against fibrin formation during thrombosis.     

Fibrinolytic and anticoagulant activity after a single subcutaneous administration of a low dose of heparin or a low molecular weight heparin-dihydroergotamine combination

The anticoagulant and potential profibrinolytic effect of a combination of low molecular weight heparin with dihydroergotamine (LMWH-DHE) and of unfractionated heparin was studied in eight healthy volunteers. Each volunteer received a subcutaneous injection of either LMWH-DHE (1,500 U anti-Xa of LMWH + 0.5 mg DHE), unfractionated heparin (5,000 IU) or of placebo (saline) between 7 and 8 h in the morning on three different occasions. Anti-Xa activity, and fibrinolytic activity measured by the euglobulin clot lysis time (ECLT) and by the fibrin plate assay were determined before and at different times after administration of the three substances. Anti-Xa activity in plasma reached a maximum four hours after injection of both LMWH-DHE and unfractionated heparin. LMWH-DHE showed a better bioavailability when compared to unfractionated heparin; the anti-Xa activity peak was two and a half fold higher after LMWH-DHE despite injection of a three fold lower dose of anti-Xa units. The half-life of anti-Xa activity was approximately 4 hours for LMWH-DHE but only 90 min for unfractionated heparin. The fibrinolytic activity measured by ECLT as well as by fibrin plate assay, showed a significant increase during the day reaching a peak 8-12 h after injection regardless of the product administered (including the placebo). The profile of the diurnal fibrinolytic activity curve was identical for all three substances. The increase in fibrinolytic activity, observed after administration of LMWH-DHE or unfractionated heparin, was therefore not due to these drugs but reflected the circadian physiological fluctuation of fibrinolysis.     

Localization of heparin and low-molecular-weight heparin in the rat kidney

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are cleared, at least in part, by the kidneys through a poorly understood process.This study was undertaken to explore the mechanism of renal clearance of these drugs. Rats were given fluorescein-5-isothiocyanate (FITC)-labeled UFH or LMWH intravenously. At intervals after injection, rats were euthanized and the kidneys were harvested and subjected to immunohistochemical analysis and fluorescence microscopy. Both UFH and LMWH were localized to renal tubular cells and no immunoperoxidase staining or fluorescence was detected in glomeruli. Autoradiography demonstrated similar intracellular distribution of radio-labeled UFH suggesting that this phenomenon is independent of the method used to label heparin. Fluorescence in the tubules increased as a function of time after UFH injection, but reached a plateau after LMWH injection suggesting that the rate of renal tubular uptake depends on the molecular size of the heparin. When administered prior to FITC-labeled UFH or LMWH, probenecid, a renal organic anion inhibitor, decreased the renal tubular uptake of the heparins, whereas cimetidine, a renal organic cation inhibitor, had no effect.These findings suggest that renal excretion of UFH and LMWH primarily reflects tubular uptake via an organic anion transport mechanism.     

Release of lipoprotein lipase and hepatic triglyceride lipase in rats by heparin and other sulphated polysaccharides

The ability of parenteral heparin to release the capillary-bound enzymes lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) into the circulation is shared by several other sulphated polysaccharides. The lipase-releasing activity in rats of two unfractionated heparins has been compared with that of characterised preparations of other glycosaminoglycans and of two partially synthetic polysaccharides. The results suggest that whilst both molecular weight and degree of sulphation are important in determining the potency, there is also some specific structural element associated with the heparin class. The clearance of lipases was also investigated. The half-lives of LPL (29 mins) and of HTGL (36 mins) did not appear to be affected by the nature of the releasing agent.     

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.     

Effects of low molecular weight heparin and unfragmented heparin on induction of osteoporosis in rats

A comparison between the effect of low molecular weight heparin (LMWH) and unfragmented heparin (UH) on induction of osteoporosis was made in 60 rats treated with either UH (2 IU/g bw), LMWH in 2 doses (2 XaI U/g or 0.4 XaI U/g) or placebo (saline) for 34 days. Studied variables were: bone mineral mass in femora; fragility of humera; zinc and calcium levels in serum and bone ash and albumin in plasma. A significant reduction in bone mineral mass was found in all heparin-treated rats. There was no difference between UH and LMWH in this respect. The effect was dose-dependent in LMWH-treated animals. The zinc contents in bone ash were decreased in all heparin-treated rats as compared with controls. No recognizable pattern was seen in alterations of zinc or calcium in serum. The fragility of the humera, tested as breaking strength did not differ between treatment groups and controls. In conclusion, if dosed according to similar factor Xa inhibitory activities, LMWH induces osteoporosis to the same extent as UH and in a dose-dependent manner. The zinc content in bone ash was decreased after heparin treatment, irrespective of type of heparin given.