Dose response relationships of anticoagulant activities after subcutaneous administration of two low molecular weight heparins in healthy individuals

This study was performed to estimate appropriate dosages of two low molecular weight heparins (LMWH) for clinical trials on subcutaneous perioperative thrombosis prophylaxis. Anticoagulatory activities and platelet function were investigated after single doses of two LMWH and of unfractionated sodium heparin (UFH) in 24 healthy individuals. Twelve subjects received subcutaneous injections of 1000, 1500, and 2500 i.u. (aPTT) of LMHW 1, and the other 12 received LMWH 2 at same dosages. The following parameters were determined before 30 min, 1 h, 90 min, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after either LMWH or 5000 i.u. (aPTT) UFH: aPTT, thrombin time, anti-Xa activity (S 2222, Coatest heparin), and anti-IIa activity (Chromozym TH). Bleeding time, platelet count, and adrenalin-, collagen-, and ADP-induced platelet aggregation were assessed before and 3 h after administration. After application of 1500 i.u. LMWH 1 and LMWH 2, the anti-Xa and anti-IIa levels were already significantly higher than after 5000 i.u. UFH. 2500 i.u. LMWH 1 and LMWH 2 evoked significantly greater prolongations of aPTT and thrombin time values than did 5000 i.u. UFH. This was not the case after 1000 and 1500 i.u. LMWH. The half-lives of anticoagulatory effects after LMWH were markedly longer than after UFH. Platelet function was not altered by any of the heparins tested. Our results indicate that LMWH cause anticoagulatory effects in vivo that cannot be predicted by in vitro studies and that the appropriate single dosages of LMWH in subcutaneous perioperative thrombosis prophylaxis have to be estimated by dosage determinations in healthy subjects.     

Antithrombotic efficacy in a rat model of the low molecular weight heparin, reviparin sodium, administered by the oral route

Previous studies in rats show that unfractionated heparin and the low molecular weight heparin logiparin have a dose-dependent antithrombotic effect and are found in endothelium and plasma when administered orally. Objectives of the present study were to determine if similar evidence of absorption could be observed with oral reviparin sodium. Thrombosis incidence was determined 4 h after application of 10% formalin in methanol to the exposed jugular vein. A dose-dependent antithrombotic effect was observed when 0.01 to 7.5 mg/kg (20 rats/group) was administered by stomach tube immediately following thrombus initiation. Thrombotic incidence was also significantly reduced when 0.025 mg/kg was given 4 and 2 h prior to, immediately after, and 2 and 3 h following thrombus initiation. Reviparin was recovered from endothelium and plasma in trace amounts at all doses. At 0.025 mg/kg, peak aortic endothelial reviparin concentrations were found at 1 and 2 h and peak plasma anti-Xa activity was detected at 2 h. Trace amounts of plasma TFPI were found only at 8 h after administration. Dose-dependent antithrombotic activity and recovery from endothelium and plasma support the hypothesis that orally administered reviparin sodium is absorbed.     

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

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

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.     

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.     

Study of anticoagulant mechanism of low molecular weight heparin

The binding ability of low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) to factor Xa (F.Xa), thrombin and ATIII was investigated using FR-860- and UF-heparin-Sepharoses. FR-860 could not bind directly to F.Xa. FR-860 bound to thrombin and ATIII with stronger affinity to ATIII than to thrombin. On the other hand, UF-heparin bound to F.Xa, thrombin and ATIII with the strongest affinity to AT III followed by thrombin and F.Xa. AT III mediated the binding between F.Xa and FR-860 and accelerated the reaction between F.Xa and UF-heparin. On the other hand, ATIII did not affect the binding between thrombin and FR-860 or UF-heparin. Diisopropyl fluorophosphate-treated thrombin inhibited the binding between ATIII and FR-860, but not that between ATIII and UF-heparin. These results suggest that the anti-F.Xa activity of FR-860 is mediated by AT III. Furthermore, the difference of antithrombin activity between FR-860 and UF-heparin depends on the capability to form ternary complex of FR-860 or UF-heparin, ATIII and thrombin.     

Higher specificity of the activity of low molecular weight fucoidan for thrombin-induced platelet aggregation

Algal fucoidans possess a wide variety of biological activities, including anticoagulation and antithrombosis, making them potential candidates for clinical use. We assessed the antiaggregant, anticoagulant and antithrombotic activities and the underlying mechanism of the low- and high-molecular weight fucoidans from the seaweed Laminaria japonica of Qingdao, China (F-Q and HMWF-Q). In the platelets of rats and humans, HMWF-Q demonstrated a pro-aggregation response, whereas F-Q (like the commercially purchased fucoidan (F-S) and heparin), showed an inhibitory effect on thrombin-induced aggregation with an IC₅₀ of 8 µg/mL, approximately five times lower than those of F-S and heparin. In the activated partial thromboplastin time test, F-Q (40 μg/mL) demonstrated less potent effect than F-S (40 μg/mL) and heparin (7 μg/mL); 162 ± 2.4 s vs. 250 ± 13.2 s and > 300 s, p < 0.01, respectively. It was also less effective than F-S on inhibiting thrombin catalyzed fibrinogen cleavage (IC₅₀ 10 μg/mL vs. 2.8 μg/mL) in vitro and rat thrombosis in vivo at 3 mg/kg (i.v.). The inhibitory effects of F-Q and heparin on thrombin activity were strikingly enhanced by either antithrombin (AT) or heparin cofactor II (HCII). A direct interaction of F-S with thrombin, and F-Q or heparin with AT was demonstrated in both fluorescence quenching and PAGE analysis. Additionally, a pro-aggregation effect and an enhancement of thrombin activity were also observed with F-S, but not with F-Q or heparin, treatment. These results indicate that F-Q inhibits thrombin via activation of AT and HCII, whereas F-S mainly interacts directly with thrombin. Importantly, F-Q shows a higher specificity for hypoaggregation and a weaker effect for anticoagulant profiles than heparin and F-S. Therefore, F-Q could be a promising candidate for the treatment of thrombosis-related cardiovascular diseases.     

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.     

Heparin and fibrinolysis--comparison of subcutaneous administration of unfractionated and low molecular weight heparin

The effects on the fibrinolytic system after a single s.c. bolus injection (at 9 a.m.) of either 5000 IU conventional heparin or 5000 anti-Xa U of a fractionated low molecular weight heparin (Fragmin, KabiVitrum, Sweden) were investigated in 9 healthy volunteers. The effects were compared to those of an injection of normal saline in 6 volunteers. Samples for biochemical analyses were taken regularily during 6 hours after drug or placebo administration. In the coagulation system the following parameters were measured: Activated partial thromboplastin time (APTT), anti-Xa activity, thrombin time and fibrinogen. The fibrinolytic system was monitored by analysing: plasminogen, alpha 2-antiplasmin, fibrin(ogen) degradation products (FDP), euglobulin clot lysis time (ECLT), tissue plasminogen activator (t-PA) activity, t-PA antigen and plasminogen activator inhibitor (PAI) activity. Injection of the 2 drugs was followed by elevations in APTT and anti-Xa activity, and were more pronounced for Fragmin than heparin. The fibrinolytic system exhibited a diurnal variation with decreasing PAI activity and increasing t-PA activity during the day. Volunteers receiving normal saline (placebo) showed a similar pattern. The results were unrelated to heparin. It is concluded from this study that neither heparin nor Fragmin had any significant effect on the fibrinolytic parameters when measured after a single s.c. bolus injection since the observed variations were within the diurnal range.     

Antithrombotic and anticomplementary properties of a very low molecular mass dermatan sulfate

INTRODUCTION: Dermatan sulfate, a sulfated glycosaminoglycan, acts as an anticoagulant by accelerating the inhibition of thrombin by heparin cofactor II. MATERIALS AND METHODS: A low molecular mass dermatan sulfate was obtained by peroxy-radical depolymerization from a parent dermatan sulfate. Chemical characterization of this low molecular mass dermatan sulfate shows a material of approximately 5 kDa that conserves sulfated sequences (2-O-sulfation of the iduronic acid units and/or 4 or 6 positions of galactosamina N acetyl) essential for dermatan sulfate-heparin cofactor II interaction with more sulphated proportion (27.7+/-1.9 microg% vs 11.5+/-0.8 microg%, P<0.05 n=6, low molecular mass dermatan sulfate vs dermatan sulfate). RESULTS: After a single intravenous administration of low molecular dermatan sulfate in rats, fibrinolytic activity increased simultaneously with thrombin clotting time prolongation. Low molecular dermatan sulfate showed an inhibitory effect on classical complement activation pathway reaching a maximum during the first hour. Furthermore, low molecular dermatan sulfate was as effective as dermatan sulfate to prevent thrombus formation and to diminish thrombus weight in a rat venous thrombosis model. CONCLUSIONS: The results indicate that peroxy-radical depolymerization of dermatan sulfate produced a low molecular dermatan sulfate with profibrinolytic, thrombolytic, antithrombotic and anticomplement properties. We conclude that low molecular dermatan sulfate may be an effective adjunct in the management of thrombotic events.     

Pregnancy associated with lupus anticoagulant and heparin induced thrombocytopenia: management with a low molecular weight heparinoid.

The management of pregnant patients with coagulopathies and heparin induced thrombocytopenia is difficult and poorly defined. We report the case of a patient who was treated with a low molecular weight heparinoid. The treatment was complicated by the delayed occurrence of thrombocytopenia and a thrombotic event. This is the first report of thrombocytopenia caused by heparinoid. It is possible that this complication could have been avoided by a shorter duration of treatment with heparinoid and the use of Vitamin K antagonists during the second trimester of pregnancy.     

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.     

On the conversion of high molecular weight urokinase to the low molecular weight form by plasmin

The conversion of high molecular weight urokinase (HMW-UK) to low molecular weight urokinase (LMW-UK) by plasmin $\text{in vitro}$ has been studied. The two forms of urokinase were separated by SDS-polyacrylamide gradient gel electrophoresis and active enzyme was extracted from 5 mm gel segments into isotonic saline and analyzed by the fibrin plate method. Electrophoretic separation of the two forms was complete, as indicated by comparison with the migration of purified standards and by the absence of lytic activity in extracts of intervening gel segments. HMW-UK was incubated with plasminogen and fibrinogen for various time intervals, after which enzymatic activity was inhibited with aprotinin and the samples subjected to electrophoresis. Conversion from HMW-UK to LMW-UK was apparent in the first sample at 2.5 minutes and continued for the 10 minute duration of the experiments. Similar experiments starting with LMW-UK showed no change in molecular weight. Incubation of HMW-UK without plasminogen resulted in no conversion to LMW-UK, indicating that plasmin was the active agent and that the reaction was not autocatalytic.     

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.     

Antithrombotic effects of DX-9065a,a direct factor Xa inhibitor: a comparative study in humans versus low molecular weight heparin

BACKGROUND: Recent evidence suggests that TF may play a causal role in acute coronary syndromes, and may be an important therapeutic target. Several inhibitors of TF, coagulation factors VIIa and Xa are under investigation as novel antithrombotic approaches. We compared the antithrombotic effects of DX-9065a, a new FXa inhibitor, vs. enoxaparin. METHODS AND RESULTS: The protocol was an open-label crossover study. Subjects (n = 6) participated in 3 consecutive study-arms: a) enoxaparin + ASA (1 mg/Kg s. c + 162 mg/day x 3 days), b) three escalating doses of DX-9065a (1 mg bolus + 0.25 mg/h x 2 h, followed by an additional 1 mg bolus + 0.625 mg/h x 2 h and, a final 1 mg bolus + 1.25 mg/h x 2 h), and c) the same doses of DX-9065a in Arm 2 plus ASA pre-treatment. The antithrombotic effects were assessed using the Badimon perfusion chamber at each dose level. The administration of DX-9065a whether alone or combined with ASA significantly inhibited thrombus formation at high and low shear rate conditions while enoxaparin did not have a significant effect. Furthermore, these antithrombotic effects were obtained without significant prolongations of the standard coagulation parameters as those induced by enoxaparin. CONCLUSIONS: The direct inhibition of FXa by DX-9065a appears to be a safe and effective new approach for preventing the thrombotic complications of atherosclerotic disease. The clinical effectiveness of the direct FXa inhibitors should be further investigated.     

Antithrombotic efficacy of an oral low molecular weight heparin conjugated with deoxycholic asset on microsurgical anastomosis in rats

Introduction

Thrombogenic occlusion, at the anastomotic microvessels, contributed impaired blood flow to flap failure. The effect of an orally active low molecular weight heparin (LMWH) derivative conjugated with deoxycholic acid (DOCA) on the patency of anastomosis of the crushed rat artery was investigated, expecting its antithrombogenic effect.

Materials and Methods

60 Femoral arteries of 30 rats, divided into three groups of 20 each, were reanastomosed. LMWH-DOCA conjugate was orally administered prior to and after operation for 5 consecutive days. On the sixth day of operation, the patency of the anastomosed artery was evaluated.

Results

The patency of oral LMWH-DOCA (10 mg/kg) group was significantly enhanced from 15% to 45%, compared to non-treated control group. On the other hand, when the dosage of LMWH-DOCA was reduced to 1 mg/kg, its efficacy on anastomosis was not as efficacious in terms of patency. The intima of crushed artery was impaired and thrombus formation was examined in the control group. In the drug treated group, the patency was only compromised by a thin layer of thrombus that covered the inner layer of the vessel without causing any damage to the internal elastic lamina.

Conclusion

The medication of oral LMWH-DOCA conjugate has been vetted in microvascular anastomosis of the crushed artery. LMWH-DOCA was potentially useful for improving the patency in compromised vessels after microsurgery.