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.     

Comparative effects of heparin and PK 10169, a low molecular weight fraction, in a canine model of arterial thrombosis

The comparative properties of heparin and PK 10169, a low molecular weight fraction, were studied using an antithrombotic test in anaesthetized dogs. The antithrombotic properties of the two compounds were evaluated by measuring inhibition of thrombus formation following transluminar stimulation of coronary artery with anodal current and by measuring anticoagulant properties, anti Xa and anti IIa activities. The results show that PK 10169 displayed significant antithrombotic activities above 0.625 mg/kg and was equipotent at 2.5 mg/kg s.c. with heparin 10 mg/kg s.c. No correlation could be observed between antithrombotic/anti Xa ratio of both compounds. Moreover it was shown that, unlike heparin, PK 10169 s.c. was devoid of obvious anticoagulant properties and induced a negligible anti IIa activity contrasting with a high anti Xa level. A similar dissociation between anti Xa and anti IIa activities was observed following i.v. administration of 2.5 mg/kg of PK 10169 but not with heparin. This low molecular weight heparin fraction might thus be regarded as a potential arterial antithrombotic agent devoid of appreciable anticoagulant effect.     

Could non steroidal anti-inflammatory drugs be used to potentiate L.M.W.H activity in thrombosis? (Part I).

Heparin fractions are antithrombotic drugs prescribed for preventive treatment but their efficacy must be optimized to permit curative use without side effects. The present study was performed on 144 rats receiving a low molecular weight heparin (L.M.W.H), Fraxiparine, and a non steroidal anti-inflammatory drug (Phenylbutazone), which were injected simultaneously or separately. Neither Phenylbutazone nor L.M.W.H at their lowest dose (1 mg/kg) reduced thrombus size. However, administered together, they produced a significant limitation of thrombus growth. Variation in anti Xa activity limitation was only observed with the highest dose of Fraxiparine alone or in combination with Phenylbutazone (1 mg/kg) corresponding to its antithrombotic effect.     

A comparison between low molecular weight heparin (KABI 2165) and standard heparin in the intravenous treatment of deep venous thrombosis

In order to study whether a low molecular weight heparin (LMWH) of mw 4000 D is effective in the treatment of deep venous thrombosis (DVT), patients with DVT verified by phlebography were randomized to treatment by continuous intravenous infusion of either unfractionated heparin (UFH) or LMWH. The initial dose was 240 U (anti F Xa)/kg/12 h. This study (study I) was stopped because of major bleeding in 2 newly operated patients in the LMWH group after 27 patients had been treated. The heparin activity measured as F Xa inhibition assayed in retrospect, was found to be much higher in the LMWH group (mean 1.6-2.0 anti F Xa U/ml) than in the UFH group (mean 0.5-0.8 anti F Xa U/ml). A second study was therefore initiated in which the DVT patients were randomly given UFH (240 U/kg/12 h) or LMWH only 120 U (anti F Xa)/kg/12 h, as initial doses (study II). In this study 27 patients could be evaluated, the mean heparin activity still being higher in the LMWH group (0.9-1.2 anti F Xa U/ml) than in the UFH group (0.5-0.7 anti F Xa U/ml). A second phlebographic investigation showed progression of thrombus size in 3 (11%) of the UFH patients of studies I and II (n = 29) and improvement in 14 (48%). There was no progression in any LMWH patient, 6 (50%) had improved in study I and 10 (77%) in study II. The mean decrease of thrombus size score (according to Marder) during treatment did not differ between the 3 groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The influence of a new low molecular weight sulphated polysaccharide from mussel broth (Org 30016) on haemostasis

The effect on haemostasis of a new low molecular weight sulphated polysaccharide (4300 dalton) from mussel broth (Org 30016) was investigated. In an APT time test system it had an anticoagulant effect corresponding to 30-40% of that of heparin. It had no antithrombin effect. It exerted its effect only in test systems containing phospholipids. It did not prolong haemostatic plug formation time in the rabbit mesenteric microcirculation as did heparin. Formation of ex vivo Chandler thrombi was significantly inhibited. The practical interest of the substance comes from the combination of an antithrombotic effect and little influence on primary haemostasis.     

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.     

The effect of low molecular weight heparin on experimental thrombosis and haemostasis--the influence of production method

Three low molecular weight heparins prepared by enzymatic depolymerization, chemical degradation, and fractionation, respectively were studied in experimental thrombosis and haemostasis models in vivo and in biological assays in vitro. The three low molecular weight heparins, which had comparable molecular weight distributions, showed very similar activities both in vitro and in vivo. All three showed dose dependent thromboprophylactic effect. The antithrombotic effects of the low molecular weight heparins and conventional heparin administered in the same dose (30 XaI u/kg b.w.) did not differ. Neither LMW heparin nor conventional heparin (60 or 90 XaI u/kg b.w.) showed significant effects on the haemostatic plug formation time in the rabbit mesenteric microcirculation.

These experiments confirm that low molecular weight heparins are potential antithrombotic drugs, which by intravenous administration have effects similar to those of standard heparin. The method of preparation seems to be of no or minor importance, at least if the molecular weight distributions of the products are similar.     

Influence on the clotting mechanism of sodium pentosan polysulfate (SP54) in comparison to commercial beef lung sodium heparin

Heparin consists of a series of fractions of different molecular weight. Whilst low MW fractions exert a strong anti Xa-activity, high MW fractions mainly enhance thrombin inhibition. A semi-synthetic polysulfated polysaccharide of a MW of 2,000 (Polyanion SP 54) also enhanced the anti Xa-activity. This effect was dose dependent and was comparable to the action of heparin when the substances were tested in a purified system. In plasmatic environment,the anti Xa-activity was lower than the anti Xa-effect of heparin when bovine factor Xa was added to the test system but was less evident when activation of factor Xa was carried out in the test plasma. The antithrombin effect of SP 54 was negligible and was about 4 per cent of the antithrombin effect of heparin. After subcutaneous injection of SP 54,its anti Xa-effect was superior to the effect of a comparable dose of heparin. As a consequence of the low antithrombin potentiating effect,the results of coagulation tests with exception of the aPTT were not influenced by SP 54.     

Anti Xa monitoring during treatment with low molecular weight heparin or danaparoid: inter-assay variability.

If laboratory monitoring of low molecular weight heparin (LMWH) therapy is required the test of choice is the anti Xa activity assay. The relationship between anti Xa results obtained using different techniques is unknown. The aim of the present study was to compare anti Xa results obtained with eight different commercially available anti Xa activity assays (five chromogenic and three clotting based assays) in samples from patients receiving either therapeutic or prophylactic LMWH (enoxaparin or dalteparin) or danaparoid. We have demonstrated that highly significant differences exist between results obtained using different techniques. The mean anti Xa activity in patients receiving treatment or prophylaxis with enoxaparin ranged from 0.28 to 0.64 iu/ml. A similar relationship was present in samples from patients treated with dalteparin, mean anti Xa results ranging from 0.43 to 0.69 iu/ml. The Heptest clotting assay as used here in combination with the Automated Coagulation Laboratory instrument, was associated with lower results than other clotting or chromogenic techniques. In patients receiving danaparoid for heparin induced thrombocytopaenia (HIT) mean results with three clotting based assays were 0.30 to 0.36 u/ml, compared to mean results of 0.47 to 0.65 u/ml for chromogenic assays. Our data clearly indicate that the selection of anti Xa assay method could influence patient management since the dose required to achieve the therapeutic range would differ according to the assay employed. This is particularly important since the frequency of haemorrhagic side effects has been shown by others to be dose dependent, irrespective of the concomitant anti Xa activity results. In danaparoid therapy the clotting assays studied here should not be employed for monitoring without a modified target range, unless it can be demonstrated that the higher doses required to achieve the therapeutic range are safe.     

Heparin-induced platelet aggregation: Dose/response relationships for a low molecular weight heparin derivative (pk 10169) and its subfractions

Addition of heparin or heparin derivatives to citrate anticoagulated platelet-rich plasma caused platelet aggregation in a dose-dependent manner. Utilizing heparin, a low molecular weight heparin derivative (PK 10169) and its various subfractions, we determined dose/response relationships for platelet aggregation and found that the ability of these agents to cause platelet aggregation was dependent upon the molecular weight of the individual subfraction used. In comparison to unmodified porcine mucosal heparin, the lower molecular weight derivative (PK 10169) yielded a dose/response curve that was shifted down and to the right, and indicated that this agent was less potent in causing platelet aggregation. In addition, as the molecular weight of PK 10169 subfractions decreased, their dose/response curves were progressively shifted down and to the right. The lowest molecular weight subfraction was essentially without platelet aggregating activity. We also measured the anti IIa and anti Xa activities of these agents and concluded that these activities did not appear to correlate with platelet aggregating activity. Platelet aggregation studies with PK 10169 subfractions of high and low affinity for antithrombin III (AT III) indicated that the platelet aggregating activity of these compounds may not be related to their affinity for AT III, but results were not definitive.     

A new low molecular weight heparin derivative. In vitro and in vivo studies

We present here the in vitro and in vivo evaluation of a new heparin low molecular weight (LMW) derivative that can be prepared on an industrial scale. The in vitro anti activated factor Xa (anti Xa) activity was 67 per cent that of standard heparin while anti-thrombin and anticoagulant activities were respectively limited to 16 and 10 per cent. The same discrepancy between anti Xa and anticoagulant activity was found after intravenous (IV) or subcutaneous (SC) injection to ten healthy volunteers. From the kinetic of the anti Xa activity disappearance it can be assumed that the mechanism of elimination of LMW heparin is not strongly different from that of traditional heparin.     

Low molecular weight heparin (KABI 2165) as thromboprophylaxis in elective visceral surgery. A randomized, double-blind study versus unfractionated heparin

In two randomized double-blind studies perioperative bleeding complications and thromboembolic events were assessed in 189 patients (pts) undergoing elective visceral surgery after subcutaneous administration of a low molecular weight (LMW) heparin fragment (KABI fragment 2165) or unfractionated (UF) heparin. The first study comparing 1 X 7'500 anti-factor Xa IU LMW heparin daily with 2 X 5'000 IU UF heparin was interrupted because of excessive bleeding complications (LMW heparin: 11/23 pts, UF heparin: 2/20 pts, p less than 0.01). In the second study (146 pts) the dose of LMW heparin was reduced to 1 X 2'500 anti-factor Xa IU. Bleeding complications (LMW heparin: 14.9%, UF heparin: 15.3%) and thromboembolic events (LMW heparin: 2.86%, UF heparin: 2.94%) were equal among the two groups. 2'500 anti-factor Xa IU/day of this LMW heparin fragment, corresponding to 15 mg/day, is the lowest dose of a LMW heparin used in a randomized clinical trial and was found to be a safe and efficient regimen in perioperative thrombosis prophylaxis. An advantage of LMW heparin over UF heparin is its once daily administration.     

A double-blind randomized placebo controlled trial of thromboprophylaxis in major elective general surgery using once daily injections of a low molecular weight heparin fragment (Fragmin)

The safety and efficacy of the low molecular weight heparin fragment (Fragmin) administered as a single daily injection of 2,500 anti Xa units has been evaluated in 183 patients undergoing major elective general surgery. The study was double-blinded and placebo controlled. The active agent, or placebo, was given subcutaneously with the preoperative medication and continued postoperatively for 5-9 days. Ninety five patients received Fragmin and 88 were randomized to receive the placebo. The clinical characteristics of the two treatment groups were similar. Fragmin significantly reduced the incidence of deep venous thrombosis, as detected by a positive 125I fibrinogen leg scan, relative to the placebo treated patients (4/95, 4.2% v. 14/88, 15.9%; p = 0.008). The thrombotic events occurred predominantly (73%) amongst patients with malignancy. Haemorrhagic endpoints necessitating discontinuation of the trial treatment were 4% in each group. No severe adverse reactions or drug related deaths occurred. These results indicate that 2,500 anti Xa units of Fragmin given only once daily is effective thromboprophylaxis for patients undergoing major elective abdominal surgery.     

Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate.

In general, the in vitro anti Xa activity of low molecular weight heparins is neutralized to a lesser degree than the anti Xa activity of unfractionated heparin. To determine whether these differences occur in vivo, a rabbit stasis thrombosis model and a rat laser-induced thrombosis model were utilized. In the laser model, a similar degree of neutralization of the antithrombotic activity of heparin and Fraxiparin was obtained. However, in the stasis thrombosis model, significant antithrombotic activity of Fraxiparin remained after equigravimetric protamine administration. Ex vivo APTT, thrombin time, Heptest, amidolytic anti Xa and anti IIa assays were performed. A coefficient (r = .806) was obtained for the correlation of Heptest activity to antithrombotic effect in the stasis thrombosis model, while the coefficients obtained for the other tests ranged from .152-.570. However, after neutralization by protamine, the thrombin time exhibited the highest correlation coefficient (r = .685) between ex vivo activity and residual antithrombotic effect. Since Fraxiparin retains antithrombotic activity after protamine administration, clinical benefit may be observed for this low molecular weight heparin as compared to unfractionated heparin after neutralization.     

Effect on primary haemostasis of prophylactic regimens of low molecular weight heparin, unfractionated heparin, dextran and their combinations. An animal experimental study.

The aim of the study was to determine whether an impairment of the haemostasis could be observed experimentally when thromboprophylactic substances, which act differently on the haemostatic mechanism, were given single or in combination in prophylactic doses. In 36 rabbits we measured the primary haemostatic plug formation time (PHT), rebleedings and total haemostatic plug formation time (THT) after transection of venules and arterioles using an intravital microscope. We combined unfractionated heparin (UH) and low molecular weight heparin (LMWH) in low dose with either dextran 70 or polygeline (placebo volume expander) in a randomized double-dummy set up. In the placebo group (NaCl and polygeline) the median PHT was 55 and 101 seconds for arterioles and venules respectively, which are well-comparable to earlier results from our group. Most prolonged PHT and THT for arterioles were seen for dextran+NaCl, actually less prolongation was seen for UH+dextran. We did not observe any differences, except for a prolongation of THT for venules in rabbits given dextran+NaCl (p less than 0.05). Thus, in thromboprophylactic doses used, there does not seem to be an impaired or additive effect between heparins and dextran 70 in primary haemostasis in rabbits.     

Effects of sulfated polysaccharides on inhibition of thrombus formation initiated by different stimuli

To test the possibility that different doses of heparin or other sulfated polysaccharides are required to inhibit thrombosis initiated by different stimuli, we compared the effects of heparin (HEP), pentosan polysulfate (SP54) and dermatan sulfate (DS) on the inhibition of thrombus formation induced by either I) tissue thromboplastin; II) thrombin; or III) factor Xa. Inhibition of thrombus formation induced by the stimuli was measured in a rabbit jugular vein hypercoagulation/stasis model. First, we determined the minimum dose of each sulfated polysaccharide which inhibited tissue thromboplastin-induced thrombus formation by approximately 75%, and then compared the relative effectiveness of this dose to prevent thrombus formation initiated with the other two stimuli. HEP and SP54 were less effective when thrombin was the thrombogenic stimulus, while DS was more effective. HEP was the most effective agent when factor Xa was the stimulus. We conclude that the antithrombotic effectiveness of a given dose of a sulfated polysaccharide may vary depending on the stimulus which initiates thrombus formation.     

A comparison of the anti thrombotic and haemorrhagic effects of low molecular weight heparin fractions: The influence of the method of preparation

Bleeding is an important complication of heparin therapy. A number of low molecular weight heparin fractions produce less bleeding than standard heparin for an equivalent antithrombotic effect in experimental animals. Low molecular weight heparin fractions and fragments are produced by a number of different procedures but their relative effects on haemostasis and thrombosis have not been evaluated. We have compared the antithrombotic and haemorrhagic effects of two low molecular weight heparin fragments and of a heparinoid with porcine mucosa heparin and related these in vivo findings to the results of ex vivo tests of blood coagulation and in vitro tests of platelet function. Haemorrhage was assessed using a rabbit ear bleeding model. The antithrombotic effects were assessed by measuring inhibition of a tissue thromboplastin-induced jugular vein thrombus and by inhibition of fibrin and platelet accumulation in an arterial-venous shunt. The ex vivo anticoagulant effects were     

Purification, characterization and in vivo studies of salmon heparin

Heparin was purified from gills and intestines from farmed Atlantic salmon (Salmo salar).Heparin activity was determined after size exclusion chromatography in the molecular weight range from above 8,000 to near 1,500. A specific activity of 110.1 antifactor Xa units/mg was measured in the less than 3,500 molecular weight fraction while 136.8 antifactor Xa units/mg was detected in a 8,000-3,500 molecular weight fraction.The presence of high affinity salmon heparin was demonstrated by using chromatography on antithrombin-Sepharose. Heparin with molecular weights lower than 3,500 was found both in high and low affinity fractions. NMR-analysis detected N- and O-sulfated oligosaccharides essential for heparin activity.The amount of salmon heparin with molecular weight lower than 8,000 varied from 12% to almost 100%. The factors determining this variation is not known, but appears to reside in the fish at the time of slaughter.The in vivo effect of salmon heparin was tested in rabbits using dalteparin as control. Salmon heparin activity was recovered in plasma samples expressed as antifactor Xa activity after intravenous administration. Based on a small number of samples and animals, the results indicate that in vivo half-life time of salmon heparin was higher than that of dalteparin.     

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.