Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro - modulatory action of glycosaminoglycans

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC₅₀ 255 ng/ml) was more pronounced than that of factor Xa (IC₅₀ 684 ng/ ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 μg/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3–40 μg/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40–50 % was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.     

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.     

Heparin enhances thrombin-stimulated prostaglandin I2 production by cultured endothelial cells

Endothelial cells from human umbilical vein were incubated in vitro with thrombin in the absence or presence of unfractionated heparin (UFH), low molecular weight heparin (LMWH) or dermatan sulfate (DS) to clarify the effect of these glycosaminoglycans on thrombin-stimulated prostaglandin I2 (PGI2) production. Although DS did not affect the thrombin stimulation, UFH and LMWH each enhanced it about 1.5-fold. Pretreatment of cells with either UFH or LMWH also enhanced the thrombin stimulation. In addition, UFH and LMWH each enhanced PGI2 production stimulated by calcium ionophore A23187, but did not affect that stimulated by arachidonic acid. From these results, it was suggested that UFH and LMWH might cause a physical change of cell membrane, which would caused an easier liberation of arachidonic acid by phospholipases.     

Bemiparin and fluid flow modulate the expression, activity and release of tissue factor pathway inhibitor in human endothelial cells in vitro.

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.     

Heparin-induced thrombocytopenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. MATERIALS AND METHODS: By photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. RESULTS AND CONCLUSIONS: The main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and--for a given degree of platelet activation (PF4 availability)--as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.     

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.     

Plasma levels of total and free tissue factor pathway inhibitor (TFPI) as individual pharmacological parameters of various heparins

The release of circulating tissue factor pathway inhibitor (TFPI) into plasma by heparins is thought to contribute to their overall antithrombotic activity. In the presented study in healthy volunteers, we measured the heparin-induced increase of circulating total and free TFPI antigen and the aXa- and aIIa activity after subcutaneous (s.c.) injection of 9000 aXa-U of four different heparins: unfractionated heparin (UFH) (13.0 kDa), a medium molecular weight (MW) heparin with a narrow MW range (HF) (10.5 kDa), certoparin (6.0 kDa) and enoxaparin (4.5 kDa). Based on the administration of equi-active aXa doses, certoparin induced the highest increase in total TFPI determined as AUC (p <0.01). The lowest effect was observed for UFH (p <0.0001). However, the AUC of released free TFPI significantly increased in the order: enoxaparin < UFH < certoparin < HF, showing MW dependency with the exception of UFH. Comparing the effects of equi-gravimetric heparin doses, the MW dependency becomes even more pronounced. The mismatch of UFH may be due to its poor bioavailability, which becomes obvious from its low ex vivo aXa activity. In contrast to the TFPI releasing potency, the ex vivo aXa activity continuously decreased with increasing MW. Although the ex vivo aIIa activity of the heparins increased in the same order like the release of free TFPI, there was no clear correlation. This is attributed to the fact that the aIIa activity of heparin is not only dependent on the MW, but, in contrast to its TFPI releasing effect, also on the percentage of material with high affinity to AT. In conclusion, besides the aXa- and aIIa activity, the TFPI releasing effect of heparins is an additional parameter of their individual pharmacological profile.     

高糖及高糖条件培养液对血管内皮细胞的影响

目的探讨高糖对血管内皮细胞分泌高迁移率族蛋白B1(HMGB1)的影响,以及高糖条件培养液对血管内皮细胞氧化应激水平的影响。方法 1.采用不同糖浓度的培养基(5.6mmol/L,20mmol/L,33mmol/L)体外培养人脐静脉血管内皮细胞株(Ea.hy926),台盼兰排斥法检测48h以内细胞的存活率,使用Western blot检测培养液上清中HMGB1的表达水平。2.分别用33mmol/L高糖培养基、混合液(33mmol/L组上清液与33mmol/l高糖培养基混合),刺激血管内皮细胞,采用分光光度比色法检测血管内皮细胞内丙二醛(MDA)和超氧化物岐化酶(SOD)的水平。结果 1.与正常对照组相比,高糖组培养液上清中HMGB1表达均明显增加(P<0.05)。台盼蓝排斥法检测不同糖浓度48h内对细胞生存率影响较小。2.与单纯高糖培养基刺激组相比,混合液刺激组的血管内皮细胞内MDA、SOD水平有显著差异(P<0.05)。结论高糖可诱导体外血管内皮细胞表达分泌HMGB1。高糖环境下,机体可能通过增加分泌HMGB1,加强氧化应激反应而促进脑血管病的发生。     

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.     

Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation

INTRODUCTION: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic-hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). MATERIALS AND METHODS: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. RESULTS: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. CONCLUSIONS: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP).     

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.     

Endothelial capillary tube formation and cell proliferation induced by tumor cells are affected by low molecular weight heparins and unfractionated heparin

BACKGROUND: Clinical studies suggest a survival advantage in cancer patients receiving low molecular weight heparin (LMWH). A suggested mechanism for this beneficial effect may reside in the antiangiogenic activity of heparins. OBJECTIVES: In this study we investigated whether two different LMWHs, i.e. enoxaparin and dalteparin, and unfractionated heparin (UFH), affect the angiogenic potential of human microvascular endothelial cells (HMEC-1) promoted by tumor cells. METHODS: HMEC-1 cells were incubated with tumor cell conditioned media (TCM) derived from human breast cancer and leukemic cells (i.e. MCF-7, MDA.MB.231, and NB4 cell lines) or recombinant cytokines (i.e. VEGF, FGF-2, TNF-alpha) +/-heparins. Capillary-like tube formation in Matrigel and cell proliferation were evaluated. RESULTS: All three TCM induced a significant (p<0.05) increase in total length of tubes formed by HMEC-1 in Matrigel. These increases were significantly counteracted (62 to 100% mean inhibition) by enoxaparin and dalteparin, but were significantly less affected by UFH. Similarly, the tube formation induced by standard VEGF, FGF-2, or TNF-alpha was 100% inhibited by enoxaparin, and 70-90% by dalteparin, whereas minor or no inhibition was observed with UFH. VEGF was the most active cytokine in TCM of both breast cancer and leukemic cells. EC proliferation was significantly increased by standard angiogenic factors, and slightly affected by breast cancer TCM (p=ns). The addition of heparins significantly counteracted the proliferative stimuli. CONCLUSIONS: These results support a major role for LMWH compared to UFH in inhibiting the proangiogenic effect exerted by tumor cells or purified angiogenic factors on microvascular endothelium.     

Interaction of thrombin with heparin cofactor II and antithrombin III on prostacyclin production by cultured endothelial cells

To investigate the physiologic function of heparin cofactor II (HCII), endothelial cells from human umbilical vein were incubated in vitro for 20 min with 0.5 NIH U/ml thrombin in the presence of HCII or antithrombin III (ATIII), and prostacyclin production determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Although ATIII at 20 mInh.U/ml slightly but significantly inhibited thrombin-induced prostacyclin production, neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) at 1 U/ml accelerated the inhibitory effect of ATIII. HCII at 10 and 20 mInh.U/ml did not decrease thrombin stimulation of prostacyclin production in the presence or absence of UFH or LMWH. However, HCII caused a marked decrease in the thrombin-stimulated prostacyclin prostacyclin production in the presence of 2 mg/ml dermatan sulfate (DS). The significant inhibition by HCII occurred when the DS concentrations were 0.2 microgram/ml and higher. From these results we suggest that HCII may prevent a prostacyclin-induced inhibition of platelet aggregation for hemostasis when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of DS.     

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.     

Hemostatic markers with bolus versus prolonged heparin after carotid artery stenting

BACKGROUND: The evolving technique of carotid stenting (CS) requires optimal antithrombotic strategies to reduce periinterventional thromboembolic risk. In animal models of balloon injury, tissue factor (TF) was shown to be the major procoagulant of the atherosclerotic plaque mediating prolonged procoagulant activity. METHODS: We analyzed TF and TF-dependent hemostatic markers before and 2, 6 and 24 h after CS with two antithrombotic drug regimens. Group A (n=20) received prolonged unfractionated heparin (UFH) for 18-20 h starting at intervention next to aspirin and thienopyridine. In group B (n=16), single bolus UFH was administered next to combined antiplatelet therapy. Natural anticoagulants were determined at baseline. RESULTS: Patients with symptomatic and asymptomatic cerebrovascular disease did not differ in plasma TF levels. Furthermore, no statistically significant difference for TF, TFPI/Xa-complex and prothrombin fragment F1.2 was observed between bolus and prolonged heparin treatment. No significant change was found in time course for these parameters. Two patients (5.5%; one in each treatment group) suffered periinterventional minor stroke associated with increased levels of F1.2 and TFPI/Xa-complex. Both were resistant to activated protein C (APC ratio<1.9) due to heterozygous factor V Leiden mutation. CONCLUSIONS: No significant activation of the TF pathway was seen with both antithrombotic regimens suggesting that single bolus UFH combined with antiplatelet therapy is generally sufficient to control TF-dependent procoagulant activity after CS. However, patients with resistance to activated protein C may be at increased periinterventional stroke risk.     

Concentration-dependent roles for heparin in modifying lipopolysaccharide-induced activation of mononuclear cells in whole blood

In addition to their anticoagulant activity, unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) have important immunomodulatory properties. However, different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. It was the objective of this study to determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide-binding protein (LBP) in mediating this effect. Whole blood was pre-treated with UFH or LMWH (0.1-200 IU/ml), prior to stimulation with LPS (10 ng/ml). After six hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1beta, IL-6, IL-8, and TNF-alpha) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of LBP (50-100 nM). Under serum-free conditions, heparin demonstrated dose-dependent anti-inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1beta, IL-6, IL-8, and TNF-alpha) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1beta and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10-200 IU/ml). Our data demonstrate that in the absence of LBP, the direct effect of heparin on LPS-stimulated monocytes is anti-inflammatory. However in whole blood, the immunomodulatory effects of heparin are significantly more complex, with either pro- or anti-inflammatory effects dependent upon heparin concentration.     

A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro

The influence of unfractionated (Heparin–Natrium) and low-molecular heparin (Fragmin®) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.     

Fixed-dose, body weight-independent subcutaneous LMW heparin versus adjusted dose unfractionated intravenous heparin in the initial treatment of proximal venous thrombosis. EASTERN Investigators

BACKGROUND: Body weight-adjusted subcutaneous low-molecular-weight heparin (LMWH) has been proven to be at least as effective and safe as dose-adjusted intravenous unfractionated heparin (UFH) for the treatment of patients with venous thromboembolism. However, body weight-adjusted dosage of low-molecular-weight heparin may be cumbersome and could lead possibly to incorrect dosing. Therefore a fixed LMWH dose, independent of body-weight, might rationalize initial treatment for venous thromboembolism. METHODS: Patients with proven proximal deep-vein thrombosis were randomly assigned to fixed dose subcutaneous LMWH Certoparin (8,000 anti-factor Xa U b.i.d.; 265 patients) or to adjusted dose i.v. UFH (273 patients) for 12 days. Vitamin K antagonists were started between day 3 and 7 and continued for up to 6 months. The primary outcome measure was a 30 percent or greater improvement in the Marder Score, as revealed by repeated venography on day 12 (end of the initial treatment). The secondary composite outcome measure included death, recurrent venous thromboembolism and major bleeding and was assessed at day 12 and after 6 months by a blinded adjunction committee. RESULTS: The Marder score improved by 30% or more in 30.3% and 25.0% of patients assigned to LMWH (198 paired venograms) and UFH (192 paired venograms), respectively (2p = 0.26). At the end of the initial treatment, the composite outcome was observed in 4 of the 265 patients (1.5%) randomized to LMWH, as compared with 14 of the 273 patients (5.1%) randomized to UFH (2p = 0.03). At 6 months these figures were 6.8% and 12.8%, respectively (risk reduction 0.53, confidence interval 0.31-0.90, 2p = 0.02). CONCLUSION: Fixed dose subcutaneous LMWH certoparin is at least as efficacious as UFH in resolving proximal vein thrombosis.     

Heparin induces mobilization of osteoprotegerin into the circulation

Heparin treatment may induce osteoporosis by an unknown mechanism. Osteoprotegerin (OPG), a glycoprotein with a heparin-binding site, is a decoy receptor for RANKL which is responsible for osteoclast development. The objective was to investigate the effect of unfractionated heparin (UFH) and lowmolecular-weight heparin (LMWH; dalteparin) on plasma levels of OPG. Twenty-two male students were allocated to the following treatment regimens; A) one bolus of 5,000 IU UFH iv followed by infusion of 450 IU/kg/24 h for 72 hours (n = 7), B) sc administration of LMWH (200 IU/kg) once daily for 72 hours (n = 8), C) sc administration of 100 IU/kg LMWH once (n = 8), D) sc administration of 250 IU/kg UFH once (n = 7), E) control infusion of saline for 12 hours (n = 7). UFH boluses of 5,000 IU were given 4 and 24 hours after cessation of regimens A and B. Bolus injection of UFH iv caused a prompt increase in plasma OPG from 0.68 ng/ml (SD = 0.09) to 1.13 ng/ml (SD = 0.30) (p = 0.003) which declined during the continuous UFH infusion and reached baseline values after 8 hours (regime A). Similar increases in plasma OPG was obtained by repeated UFH boluses after cessation of treatment. Subcutaneous administration of LMWH (200 IU/kg) caused a modest, but significant (p = 0.002) increase in plasma OPG similar to the mobilization by 250 IU/kg UFH sc, but the LMWH treatment caused a three-fold higher anti-Xa activity (p < 0.001). We conclude that UFH causes a more pronounced vascular mobilization of OPG than LMWH, indicating that UFH has a higher affinity for OPG than LMWH.