Subcutaneous treatment of deep venous thrombosis with low molecular weight heparin. A dose finding study with LMWH-Novo

Treatment of deep venous thrombosis with low molecular weight heparin (LMWH-Novo, Logiparin) was carried out with two different doses of Logiparin, 75 XaI U/kg b.w. twice daily and 150 XaI U/kg b.w. once daily subcutaneously for 5 days. Simultaneously warfarin was given from the first day of heparin treatment. Mean age of the twenty patients was 65 years and one third was females. No serious side effects, hematomas, pulmonary emboli or signs of recurrent thrombosis occurred during treatment with either dose regime. Venografic assessment with Marder scoring one week after initiation of Logiparin treatment showed a slight not significant improvement apparent in 40 % of the patients. The activities of F-IIaI and F-XaI in the blood plasma were found to increase after injection of Logiparin. These two parameters seem to be the most suitable for monitoring the effect during treatment. For future studies on the therapeutic effect of Logiparin in deep venous thrombosis a single dose of 150 to 200 F-XaI activity per 24 hours seems to be most suitable.     

Influence of thromboembolism prophylaxis by low molecular weight heparin CY 216 (fraxiparine®) on several parameters of haemostasis in patients under dialysis and receiving either unfractionated heparin or CY 216

The hemorragic risk of an association of the low molecular weight (LMWH), Fraxiparine® injected intravenously at the dose of 7.500 AXaICU or of unfractionated heparin (UFH) injected intravenously at the usual dose used during hemodialysis (3.750 ± 1.280 IU + 1.000 IU after 2 hours of dialysis) to the subcutaneous administration once daily of a thromboembolism preventive dose of Fraxiparine (7.500 AXaICU) was evaluated on the modification of the following hemostasis parameters : thrombin time, activated partial thromboplastin time (APTT), anti Xa activity, in 13 uremic patients on hemodialysis. The association of intravenous and subcutaneous Fraxiparine prevented efficiently the clotting of the extracorporeal circulation without inducing a detectable antithrombinic activity. In contrast, the association of I.V. UFH to subcutaneous Fraxiparine induced a significant increase of the thrombin time and of the APTT, so explained by the activity of UFH. It is concluded that subcutaneous Fraxiparine at the thromboembolism preventive dose can be associated as well to I.V. Fraxiparine as to UFH without increasing the potential hemorrhagic risk. Nevertheless the association of SC and IV Fraxiparine 7 500 AXaIC u seems preferable to the association of SC Fraxiparine with UFH.     

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

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.     

Clinical evaluation of low-molecular-weight heparin (FR-860) on disseminated intravascular coagulation (DIC) - a multicenter co-operative double-blind trial in comparison with heparin -

This study was evaluated the effectiveness, safety and utility of FR-860 and to compare those with heparin in patients with Disseminated intravascular coagulation (DIC). A diagnosis of DIC was made based on the criteria proposed by the Research Committee on DIC in the Ministry of Health and Welfare of Japan. FR-860 (FR group,75 anti-factor Xa international units/kg/day) and Heparin (HP group, 240 units/kg/day) were administered for 5 days by continuous intravenous infusion. The total number of enrolled patients was 126 cases, and after excluding 1 case a total of 125 cases. Moderate or higher improvement of bleeding symptoms was 33.3% in the FR group and 18.5% in the HP group. On the organic symptoms, FR group showed a significantly higher improvement rate than the HP group, 20.5% and 8.2% respectively. On the overall efficacy of cases with pretreatment plasma AT III levels of less than 21 mg/dl or less than 70%, FR group showed a significantly higher improvement rate than the HP group. The safety rate of FR-860 (93.4%) was a significantly higher than that of the HP group (79.7%). Our report demonstrates that FR-860, as a therapeutic agent for the treatment of patients with DIC, is significantly higher safety and clinical utility as compared with heparin.     

Studies on a highly active anticoagulant fraction of high molecular weight isolated from porcine sodium heparin

We have studied heparin fractionation using gel filtration and ion-exchange chromatographic methods. The starting material was commercial grade porcine mucosal sodium heparin (PSH). The fractionation was monitored employing synthetic substrates for assaying both antithrombin (with H-D-Phe-Pip-Arg-pNA ; S-2238) and anti-FXa (with Bz-Ileu-Glu-Gly-Arg-pNA ; S-2222) activities. The resulting fractions were evaluated in different amidolytic and coagulation methods used to determine heparin potency by comparison with PSH. By gel filtration of PSH on Ultrogel AcA 54, both strong anti-FXa and antithrombin activities were associated with the fractions eluted in the high molecular weight range (MW 20 × 10³). These fractions also had potent anticoagulant action when assayed by conventional clotting methods. PSH was also subjected to fractionation by an ion-exchange technique (DEAE-Sephacel) with increasing salt molarity. The patterns for antithrombin and anti-FXa activities were again closely related, if not identical. Four fractions were usually distinguished, with respectively negligible, intermediate, high and very high activities when compared to PSH. The very highly active fraction (HAF), approximately 15% by weight, was eluted at high salt molarity (> 0.8 M NaCl). On a weight basis its anticoagulant activity was 2–3 times that of PSH as determined by amidolytic as well as clotting methods. Intravenous injection of HAF to rabbits and dogs (1.0 and 2.5 mg/kg) produced a much stronger anticoagulant response than PSH, also showing an effect which persisted for a longer duration.     

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.     

Effect of a synthetic thrombin-inhibitor MD805 on the reaction between thrombin and plasma antithrombin-III

MD805, a synthetic thrombin-inhibitor, effectively retarded the time-dependent inactivation of thrombin which was generated endogeneously or added exogeneously in human plasma. The kinetical study of the time-dependent inactivation indicated that the type of inhibition was competitive and the obtained Ki of MD805 for thrombin was 3 × 10⁻⁸M. MD805 also inhibited the formation of thrombin-ATIII complex. These results indicated that the active site of thrombin was involved in the reaction between thrombin and ATIII, and that MD805 competed with ATIII for thrombin in exactly the same manner as it competed with fibrinogen or synthetic peptide substrates. As a result, MD805 would serve as a protective agent for ATIII from being consumed, in addition to its potent thrombin-inhibitory activity without the aid of ATIII. By contrast, heparin accelerated the time-dependent inactivation rate of thrombin and the formation of thrombin-ATIII complex, which indicates that heparin accelerates the consumption of ATIII.     

Human pharmacological studies of a defined low molecular weight heparin fraction (Fragminr) evidence for a simultaneous inhibition of factor Xa and IIa (thrombin)

The effect of a low molecular weight heparin (FragminR) (20–80 U/kg) was studied in 10 healthy male volunteers after intravenous administration. Clotting ab-abnormalities are extensive shortly after injection, lasting for 1 – 2 hours depending on heparin dosage. Dilute thrombin time, showing a close correlation to anti-F-Xa activity, is as sensitive as anti-F-Xa. The parallel time course of dilute thrombin time and anti-F-Xa activity clearly demonstrates that LMWH has an effect on F-IIa lasting as long as that of F-Xa. After subcutaneous administration clotting abnormalities are only slight with 40–60 U/kg, in contrast to the intravenous study. The platelet system remains unchanged.     

Pharmacokinetics of a low molecular weight heparin, Logiparin, after intravenous and subcutaneous administration to healthy volunteers

In six healthy volunteers we have estimated the pharmacokinetic parameters of the anti factor Xa (AXa) and anti factor IIa (AIIa) activities of a LMW heparin, Logiparin. For the AXa the following parameters were estimated in a 1-compartment model (mean and 95% confidence limits in brackets): elimination half life 82 minutes (60–127 min), absorption half life (s.c.inj.) 200 minutes (137–368 min), bioavailability 90% (24–156 %), and apparent volume of distribution 3.9 1 (3.1–5.2 1). The plasma activity was linearly correlated to the dose given and to the body weight of the volunteer. For the AIIa the parameters estimated in a 1-compartment model were: elimination half life 71 minutes (52–115 min), absorption half life 257 minutes (133–3442 min), bioavailability 67% (44–90 %), and apparent volume of distribution 10.1 1 (7.2–16.7 1). The plasma activity was dependent on dose and body weight but it also seemed to be influenced by individual factors. This study shows that the absorption rate is the rate limiting factor and the explanation for the long lasting effect of this LMW heparin after subcutaneous injection. The slow absorption rate and the high bioavailability are probably the major advantages of LMW heparins compared to conventional heparin.     

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.     

At high heparin concentrations, protamine concentrations which reverse heparin anticoagulant effects are insufficient to reverse heparin anti-platelet effects

Combined effects of heparin and protamine on plasma clot structure and platelet function were studied. Anticoagulant effects were monitored as changes in aPTT. Clot structure was defined in terms of fibrin fiber mass/length ratio (μ) and clot elastic modulus (EM). Platelet function was studied utilizing platelet aggregation and platelet force development (PFD) measurements. Heparin (1 U/ml) prolonged the aPTT from 30 to >300 seconds, reduced PFD from 5,100 to 0 dynes, decreased μ (in batroxobin-induced gels) from 1.36 to 1.08 × 10¹³ daltons/cm and decreased clot EM from 9,600 to 2000 dynes/cm². Varying amounts of protamine reversed these effects: 16 μg/ml normalized the aPTT, 20 μg/ml normalized PFD, 32 μg/ml corrected μ, and 20 μg/ml returned EM to baseline. At high heparin concentrations (4 U/ml), protamine concentrations which corrected anticoagulant effects were inadequate to reverse antiplatelet effects. A protamine concentration of 40 μg/ml normalized the aPTT and μ, but 140 μg/ml of protamine was required to reverse heparin suppression of force development and clot elastic modulus. Excess protamine inhibited clotting and platelet function. In plasma containing 1 u heparin/ml, 140 μg protamine/ml reduced PFD by 83%, prolonged the aPTT by 63%, and reduced clot EM by 75%. In heparin free plasma, >75 μg protamine/ml prolonged the aPTT. Thus, platelet function and clot structure are sensitive to protamine during heparin neutralization, and anti-platelet effects of heparin may persist when the aPTT is completely corrected. Excess protamine inhibits platelet function and compromises clot structure.     

Pravastatin potentiates the anticoagulant effects of low molecular weight heparin

Introduction: Statins have been shown in randomized trials to reduce coronary events independent of baseline LDL-C level. In the case of pravastatin sodium (PS), there is conflicting evidence as to what is the actual mechanism of its non-lipid lowering beneficial effects. Because pravastatin has been found to prolong the clotting time in vitro, we conducted a study to determine if pravastatin plus low molecular weight heparin (LMWH) would result in a synergistic effect on the in vitro clotting time, thus supporting the hypothesis that pravastatin exerts antithrombotic effects through reduction of fibrin formation. Materials and methods: Aliquots of PS were combined with dalteparin, a LMWH, in 500 μl of human whole blood. The clotting time in seconds was analyzed on a Sonoclot® Coagulation Analyzer, a miniviscometer that is sensitive to early fibrin generation. Results: PS and LMWH, each resulted in a significant prolongation of the clotting time compared with control. The combination of PS and LMWH resulted in a significantly prolonged clotting time compared with either given alone. All values were significantly different from each other (p<0.05). Our results showed that the combination of PS and a LMWH prolongs the clotting time to a significantly greater degree when compared to either administered alone. Conclusions: The synergistic effect of PS and LMWH on prolongation of the clotting time suggests that PS exerts its effect by inhibition of the coagulation cascade and fibrin formation.     

Anticoagulant effects of two types of low molecular weight heparin administered subcutaneously

Two types of LMW heparin were prepared by gel filtration of standard heparin (LMW fraction) and by degradation of heparin by nitrous acid (LMW fragment), respectively. The effects on factor Xa inhibition (XaI), APTT, platelet aggregation and AT III level of these preparations were studied after subcutaneous administration to humans and compared with those of standard heparin. At a dose of 5000 IU (XaI) the LMW fraction and LMW fragment induced peak plasma XaI activity of 0.32 IU/ml and 0.41 IU/ml respectively, compared to 0.07 IU/ml for heparin. Still 11.5 h after administration both LMW preparations gave higher activities than heparin ever induced. Following administration of 10,000 IU (XaI) of the LMW fragment the plasma peak XaI activity was 0.81 IU/ml. This prolonged the APTT from 36 sec to 46 sec only. The half-lives of the XaI activity in plasma were between 3 and 4 hours. No effect on platelet aggregation or AT-III level was demonstrated.     

Study on the mechanism of action of heparin and related substances on the fibrinolytic system: Relationsship between plasminogen activators and heparin

It is now generally well accepted that heparin and related substances increase the fibrinolytic activity . The stimulation of the amidolytic, plasminogenolytic and fibrinogenolytic activity of tissue plasminogen activator and urinary plasminogen activator through heparin was investigated . A concentration-dependent stimulation of the plasminogenolytic and fibrinogenolytic activity of both urinary and tissue-type plasminogen activators was observed in the presence of heparin. No heparin dependence was observed in the amidolytic assay. Heparin stimulates the plasminogenolytic activity of tissue plasminogen activator in the same manner as fibrin. Both activators form complexes with heparin; the heparin-binding-site seems to be identical or related with the fibrin-binding-site of tissue plasminogen activator. The physiological role of these interactions is discussed.     

Prevention of experimental venous thrombosis in rabbits with low molecular weight heparin, dextran and their combinations, administered before or during induction of venous endothelial trauma

An in vivo experimental venous thrombosis model based on endothelial damage and flow reduction was used to investigate the effect of low molecular weight heparin (LMWH) alone and in combination with dextran and the effect of surgical and endothelial trauma on thrombus formation, formation of occlusive thrombi and thrombus weights. Five groups with 15 rabbits in each were studied. Two groups received dalteparin (50 anti-Xa IU/kg i.v.) before surgical trauma or after, during the endothelial trauma and two groups received dalteparin (50 anti-Xa IU/kg i.v.) with dextran 70 (1 g/kg i.v.) before surgical trauma or after, during the endothelial trauma. Compared to a control group (saline) all treatment regimes reduced significantly the frequency of thrombosis and occlusive thrombi as well as thrombus weights. No significant difference was observed between the identical treatment groups when the substances were introduced before respective after surgical trauma. It is concluded from the present study that thromboprophylaxis with LMWH in this particular in vivo model, given before or after surgical trauma is equally effective. Dextran has a certain augmenting thromboprophylactic effect when added to LMWH in this model.     

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.     

Thrombin inactivation on surfaces with covalently bonded heparin

About 8000 Daltons porcine mucosa heparin fragments were covalently bonded by end-point attachment to polyethylene. The interaction between the immobilized heparin, added thrombin, and antithrombin III [AT] was investigated.

The heparin surface was adsorbed with either albumin, AT dissolved in albumin or Tyrode, or platelet free plasma. Irrespective of the pre-treatment procedure, exposure of the surface to thrombin resulted in the same substantial decrease of thrombin in solution and the same degree of surface-confined thrombin activity. It was concluded that the heparin surface has a large capacity to bind thrombin and that the thrombin inhibitory capacity of high affinity heparin fragments is limited.

On exposure of the thrombin-loaded surfaces to defibrinogenated plasma or AT, the surface-confined thrombin was inhibited within 30 seconds. Successive dilutions of plasma or AT decreased the inhibition rate but not the inhibition capacity. It is concluded that inhibition of thrombin adsorbed on the heparin surface occurs as follows: Added AT adheres to high affinity heparin fragments on the surface whereupon adsorbed thrombin migrates in the hydrophilic heparin coating towards the reaction site of AT and becomes inhibited. The inactivated thrombin-AT complex leaves then the surface, thus enabling the process to be repeated.