In vitro effect on Heptest of low molecular weight heparin fractions and preparations with various anti-IIa and anti-Xa activities

The Heptest heparin assay has recently been introduced, and evaluated for the laboratory monitoring of patients receiving low molecular weight heparins (LMWH). The aim of the present study was to elucidate the relative role on the Heptest assay of the anti-factors Xa and IIa activities present in the three types of compounds that possess: 1. exclusively anti-Xa activity (LF1: LMWH fractions with MW ranging from 1,200 to 4,200 D.); 2. both anti-Xa and anti-IIa activities (LF2 with MW from 4,800 to 12,000 D.); 3. exclusive anti-IIa activity (Hirudin and Dermatan Sulfate). All compounds studied demonstrated dose-dependent activities in both amidolytic and clotting assays. The LF2 in contrast to the LF1, additionally enhanced the clotting times of Heptest. This enhancement was shown to be due to the anti-Factor IIa activity of the agents. Heptest does not exclusively reflect Anti-Xa activity since it is influenced by agents containing exclusive anti-IIa activity like Hirudin and Dermatan Sulfate. At low concentrations of LF2, Heptest measures predominantly the anti-factor Xa activity while at higher concentrations it is influenced by the combined activity of anti-factor Xa and anti-factor IIa. However, Heptest sensitivity to anti-factor IIa is significantly lower than for anti-Xa activity.     

Discordance between the anti-Xa activity and the antithrombotic activity of an ultra-low molecular weight heparin fraction

The relationship between the in vivo antithrombotic effect of heparin and ex vivo anti-Xa activity has been investigated using an animal thrombosis model. Three low molecular weight heparins were compared with the standard heparin from which they were fractionated. All four heparins showed a dose-dependent antithrombotic effect enabling the relative antithrombotic and anti-Xa activities to be compared over a dosage range. A correlation between ex vivo anti-Xa heparin levels and antithrombotic effect was demonstrated for the standard (MW 16,000), intermediate (MW 7,600) and low (MW 4,600) molecular weight heparins but not for the ultra-low molecular weight (MW 3,000) fraction. The lack of relationship between anti-Xa activity and inhibition of thrombosis for the very low molecular weight fraction indicates that a very high anti-Xa activity (measured in vitro or ex vivo) is not always predictive of in vivo antithrombotic efficacy. These findings suggest that other properties of low molecular weight heparins contribute to their antithrombotic effectiveness.     

通心络与低分子肝素联合治疗急性脑梗死临床分析

目的 探讨通心络与低分子肝素联合治疗急性脑梗死的疗效.方法 将256例急性脑梗死病人随机分为2组,对照组常规治疗;治疗组在常规治疗的基础上加用通心络胶囊4粒/次,tid,连用4周为一个疗程.低分子肝素钙5000U,皮下注射,q12h,连用7d.结果 治疗组总有效率87.5%,优于对照组的74.2%,2组比较差异有统计学意义(P<0.05).结论 通心络与低分子肝素合用治疗急性脑梗死有显著疗效.     

Does protamine chloride neutralize low molecular weight heparin sufficiently?

The heparin neutralizing properties of protamine chloride on conventional heparin (porcine mucosa) and on low molecular weight heparin (Kabi 2165) were studied in vitro. Protamine chloride neutralized 99% of the delaying effect of conventional heparin on the activated partial thromboplastin time, whereas only 70% of the effect of low molecular weight heparin was neutralized. The neutralizing effect of protamine chloride on the inhibition of factor Xa (clot test) was 95% for conventional heparin and 55% for low molecular weight heparin, whereas the effect of both heparin preparations on the thrombin inhibition could be completely neutralized. We conclude that conventional heparin is neutralized more effectively in vitro by protamine chloride than is the low molecular weight heparin. The findings do not exclude that protamine chloride is able to suppress in vivo bleedings caused by low molecular weight heparin.     

Measurement of low molecular weight heparin ex vivo activities in clinical laboratories using various anti-Xa assays: interlaboratory variability and requirement for an agreed low molecular weight heparin standard

The only sensitive and convenient assay to assess the biological activity of low molecular weight heparins (LMWHs) is based on the potentiation of activated factor Xa inhibition. Several procedures for measuring the socalled anti Xa activity have been proposed. In this collaborative study including eight laboratories, we have used four different assays (three amidolytic and one clotting based methods) for measuring the anti Xa activity of ex vivo samples obtained after injecting three different LMWHs. The dispersion of the results obtained by calibration against standard heparin could be reduced by using any of the three LMWHs for calibration. A coefficient of variation less than 0.20 between values obtained in different laboratories using a variety of methods seems acceptable. However it is necessary to refer to a common international standard for expressing the results in units and to define, for each of the three products, the therapeutic range.     

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.     

低分子肝素钙治疗急性脑梗死的疗效观察

目的观察低分子肝素钙（LMWHCa）治疗急性脑梗死的疗效和安全性。方法选用48例急性脑梗死病人,其中24例用常规治疗为对照组,治疗组24例除常规治疗外,加用LMWHCa4100抗Xa国际单位腹部皮下注射,bid,连续10d为1疗程。治疗前后分别作神经功能评分、血液流变学观察和PT、AFTT、TT、Fg。结果治疗组神经功能恢复、血液流变学改善均明显优于对照组（P〈0．05）。治疗组用药后Fg降低,PT延长,与对照组比较差异显著（P〈0．01或P〈0．05）,两组治疗前后的TT、APTI变化不明显;治疗组有2例出现皮下淤斑。结论脑梗死急性期给予低分子肝素钙治疗安全和有效。     

In vitro and in vivo studies of the anti-Xa activity of heparin

Several heparin preparations have been assayed using the Denson and Bonnar anti-Xa assay. The heat defibrination step involved in this assay has been shown to introduce discrepancies into the results, due mainly to co-precipitation of the heparin with fibrinogen. The amount of heparin lost is dependant on the molecular weight. The anti-Xa activity of ex vivo samples from subjects given heparin was much more resistant to loss during defibrination than in vitro samples, resulting in artificially high potency estimates. A modified assay has been proposed, omitting the defibrination step. The results provide further evidence that the anti-Xa activity observed after subcutaneous injection of heparin differs from that measured when the drug is added to plasma in vitro.     

Influence of heparin;of different heparin fractions and of a low molecular weight heparin-like substance on the mechanism of fibrinolysis

The influence of different heparin fractions and of a synthetic polysulfated polysaccharide (SP54) on the fibrinolytic mechanism was examined. In vitro,a significant shortening of the euglobulin lysis time (ELT) was found after addition of standard mucosa heparin, of high MW heparin and of SP 54 respectively. Low MW heparin fractions had no influence on the ELT. Simultaneously with the shortening of the ELT an activation of factor XII and of kallikrein was observed. A similar effect was found in groups of volunteers after i.v. or s.c. injections and even after oral administration of SP 54. The effect of venous occlusion on the ELT and on the activation of factor XII was considerably increased when heparin or SP 54 was injected 2 h prior to the test. When a comparable concentration of the test substances was added to plasma samples before and after venous occlusion,the effect on the ELT was much less pronounced than after injection whilst the effect on factor XII was comparable in both tests. From these results the conclusion was drawn that activation of fibrinolysis by polysulfated polysaccharides is achieved by an endogenous pathway as well as by an increased availability of the vascular activator. The magnitude of the activation of fibrinolysis partly depends on the MW of the substance but apparently also on the degree of sulfatation since a low MW substance with a high number of sulfate bonds such as SP 54 was considerably more active than the low MW fraction of standard heparin.     

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.     

Study of low molecular weight heparin effect on the relation between anticoagulant activity and antithrombin III affinity.

Low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) were fractionated by rabbit antithrombin III (AT III)-Sepharose, and the effects of each affinity fraction on the coagulation and fibrinolytic activities were investigated. FR-860 was fractionated to no-affinity, low-affinity (LA) and high-affinity (HA) fractions, and UF-heparin to LA and HA fractions. The HA fractions showed higher activities regarding the prolongation of activated partial thromboplastin time, anti-factor Xa activity and antithrombin activity compared with those of LA. The HA and LA fractions exhibited the enhancement of heparin cofactor II (HC II) activity and fibrinolytic activity in a dose-dependent manner. These results suggest that the antithrombotic activity of FR-860 is exerted through AT III and other mechanism such as HC II-mediated system.     

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.     

In vivo release of anti-Xa clotting activity by a heparin analogue

The parenteral injection of a semi-synthetic heparin analogue (SSHA) releases anti-Xa clotting activity, lipoprotein lipase activity and PF₄ antigen. The increased anti-Xa activity is not neutralized by PF₄ or protamine sulphate. A second injection of the drug after 90 minutes, or an increase in dose, does not increase the level of induced anti-Xa clotting activity. Possible mechanisms of action include the release by SSHA of endogenous glycosaminoglycans with anti-Xa activity, and interference by released lipoprotein lipase of a modulator of anti-Xa activity. It is concluded that a drug with weak anticoagulant activity in vitro may nevertheless have significant antithrombotic potential.