Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins

Unfractionated heparin (UFH) has procoagulant activity in antithrombin/heparin cofactor II (HCII)‐depleted plasma. UFH prevents tissue factor pathway inhibitor alpha (TFPIα) from inhibiting the procoagulant enzyme complex, prothrombinase, providing a possible mechanism for its procoagulant activity. The procoagulant potential of UFH and various low molecular weight heparins (LMWHs) were characterized for TFPIα dependence, using thrombin generation assays performed with antithrombin/HCII‐depleted plasma. UFH, the LMWHs enoxaparin and dalteparin, and the low anticoagulant LMWH 2‐O, 3‐O desulphated heparin (ODSH) all promoted thrombin generation, but fondaparinux did not, and this activity was blocked by a TFPIα antibody. UFH, enoxaparin, and dalteparin were anticoagulant in reactions containing 1–2% normal plasma. In prothrombinase activity assays, UFH, enoxaparin, dalteparin and ODSH blocked prothrombinase inhibition by TFPIα, while again fondaparinux did not. In both the plasma and purified assays, LMWHs displayed greater procoagulant potential than UFH, even when normalized to saccharide concentration. These biochemical data reveal that UFH and LMWHs, but not fondaparinux, block prothrombinase inhibition by TFPIα, thereby producing their paradoxical procoagulant activity observed in the absence of antithrombin/HCII. The findings may help to understand the complex pathophysiology and treatment of patients that are simultaneously bleeding and clotting, such as those with disseminated intravascular coagulation.     

Conjectures and refutations on the mode of action of heparins. The limited importance of anti-factor xa activity as a pharmaceutical mechanism and a yardstick for therapy

Low-molecular-weight heparins (LMWHs), like unfractionated heparin (UFH), exert their action primarily by accelerating the interaction between antithrombin (AT) and thrombin. At the levels of aXa activity that are attained in human pharmacology, it does not cause significant (>15%) inhibition of the clotting system. The essential differences between LMWHs and UFH are: (a) LMWHs attain higher plasma concentrations after subcutaneous injection (high bioavailability), and (b) in contrast to LMWHs, UFH contains very large heparin molecules with a putative hemorrhagic action. The reputedly higher aXa activity of LMWH can be shown to be largely due to the absence of Ca(2+) using the current laboratory methods to estimate this activity. Via this artifact the apparently high aXa activity of LMWHs is correlated but not related to their favorable pharmacokinetic properties. Consequently dosage guidelines for the use of different LMWHs cannot be based upon their aXa activity. Until better laboratory methods are available, clinical results are the only reliable guideline to heparin dosage.     

The anticoagulant capacity of plasmatic unfractionated heparin decreases at 23 degrees C.

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are important clinical anticoagulants. As polynegative molecules they are potential triggers of the contact phase of coagulation. An incubation temperature lower than the physiological 37 degrees C favours intrinsic haemostasis activation by the polynegative molecule SiO2. The efficiency of UFH and LMWH after a plasmatic preincubation at 37 or at 23 degrees C is therefore studied. Samples (150 mul) of unfrozen pooled normal plasma supplemented with 0, 0.01, 0.1, or 1 IU/ml heparin or dalteparin in 5-ml polystyrole tubes were incubated for 10-70 min at 37 or at 23 degrees C. The extrinsic coagulation activity assay (EXCA) was then performed. Preincubation at 37 degrees C of 0.1 IU/ml plasmatic UFH does not result in any thrombin generation in EXCA-1, whereas preincubation at 23 degrees C results in a thrombin generation of about 0.1 IU/ml thrombin. Plasmatic UFH (0.01 IU/ml) at 23 degrees C acts nearly half as efficiently as 0.01 IU/ml plasmatic LMWH. Polynegatively charged niches particularly in the larger UFH molecule might trigger the contact system of haemostasis, especially at 23 degrees C. In contrast, the anticoagulant capacity of LMWH does not change significantly with temperature.     

The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma

The antifactor Xa activities of heparin fractions are widely used as an ex vivo index of their antithrombotic efficacy. Its clinical meaning, however, remains speculative. In the study reported, we measured the effects of standard heparin, a synthetic pentasaccharide heparin (antifactor Xa activity only), and a low molecular weight heparin (LMWH) on factor Xa, factor Va, and thrombin generation in thromboplastin-activated plasma. We clearly demonstrated that the antifactor Xa activity of heparin contributed little in its anticoagulant activity. The inhibition of factor Va generation, dependent on the heparin antithrombin activity only, is of prime importance to the inhibition of thrombin generation in plasma. The inhibition of thrombin generation by the LMWH was comparable with that of standard heparin on the basis of their respective antithrombin specific activities, but not on the basis of their antifactor Xa activities.     

Flow and the inhibition of prothrombinase by antithrombin III and heparin

Inhibition of prothrombinase by antithrombin III (ATIII) and heparin was investigated in a continuous-flow system. Phospholipid-coated capillaries, containing phospholipid-bound factor Xa and factor Va, were perfused with 1.0 mumol/L prothrombin and 0.5 nmol/L factor Va. At 25 degrees C and a flow rate of 32 microL/min (shear rate 28 seconds-1) the steady-state rates of prothrombin conversion depended linearly on the surface concentration of prothrombinase up to 2 fmol/cm2. The rate of thrombin generation was 952 +/- 43 (SE) mol/min/mol prothrombinase. When ATIII was included in the perfusate for 10 minutes, the free thrombin concentration at the outlet of the capillary was markedly reduced: a 50% neutralization was obtained at 0.7 mumol/L ATIII. However, the prothrombinase activity was not inhibited, as could be established after a subsequent perfusion with prothrombin and factor Va. At an ATIII concentration typical of normal plasma (2 mumol/L) a slight neutralization of prothrombinase was observed: 10% neutralization following a 10-minute perfusion. During a perfusion with ATIII in the absence of prothrombin, or in its presence with hirudin (2 mumol/L) also included in the perfusate, a more pronounced neutralization of prothrombinase was observed: 40% residual activity was obtained after a 10-minute perfusion. From this observation the suggestion comes forward that thrombin, continuously produced at the surface, consumes ATIII in the boundary layer. In this case the true ATIII concentration in the vicinity of surface-bound prothrombinase will be but a small fraction of the initial ATIII concentration in the bulk fluid. Unfractionated heparin and an ultra-low molecular weight heparin (pentasaccharide) did enhance the ATIII-dependent neutralization of prothrombinase, but to a much lesser extent than observed with small unilaminar phospholipid vesicles as the catalytic sites for prothrombinase assembly. The findings reported here support the notion that regulation of prothrombinase by heparin under in vivo conditions occurs at the stage of its formation, ie, through inhibition of free factor Xa and/or the generation of factor Va, rather than by direct inhibition of the prothrombinase activity.     

Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin.

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.     

Low‐Molecular‐Weight Heparins in Thrombosis and Cancer: Emerging Links

Heparin as well as low‐molecular‐weight heparins (LMWHs) have polypharmacological actions at various levels. Earlier studies focused on the plasma anti‐Xa and anti‐IIa pharmacodynamics (PD) for the different LMWHs. Other important PD parameters for heparin and LMWHs might explain the diverse clinical impacts of this class of agents in thrombosis and beyond: the release of the vascular tissue factor pathway inhibitor (TFPI), inhibition of key matrix‐degrading enzymes, and other mechanisms. There is much evidence for the key role of LMWHs in hypercoagulation in thrombosis and cancer, angiogenesis, and inflammatory disorders. Many cancer patients reportedly have a hypercoaguable state, with recurrent thrombosis due to the impact of cancer cells and chemotherapy or radiotherapy on the coagulation cascade. Studies have demonstrated that unfractionated heparin (UFH) or its low molecular weight fractions interfere with various processes involved in tumor growth and metastasis. Clinical trials have suggested a clinically relevant and improved efficacy of LMWHs, as compared to UFH, on the survival of cancer patients with deep vein thrombosis. Our laboratory has demonstrated a significant role for LMWHs and for LMWH‐releasable TFPI on the regulation of angiogenesis, tumor growth, and tumor metastasis; we have also seen potent inhibition of matrix‐degrading enzymes by LMWHs but not by TFPI. The antiangiogenesis effect of LMWHs or non‐anticoagulant LMWH derivatives was shown to be reversed by anti‐TFPI. Thus, modulation of tissue factor/Vila noncoagulant activities by LMWH‐releasable TFPI and the inhibitory effects on matrix‐degrading enzymes beside the anticoagulant efficacy have provided an expanded clinical utility for LMWHs in angiogenesis‐associated disorders, including human tumor growth and metastasis.     

A rationally designed heparin, M118, has anticoagulant activity similar to unfractionated heparin and different from Lovenox in a cell-based model of thrombin generation

Unfractionated heparin (UFH) enhances antithrombin (AT) inhibition of thrombin (IIa) and factor Xa (FXa). Low molecular weight heparins (LMWH) primarily enhance AT inhibition of FXa. M118 is a LMWH produced from UFH and retains its ability to promote both FXa and IIa inhibition. We tested the hypothesis that M118 has anticoagulant activities similar to UFH in an in vitro model of coagulation. Platelet IIa generation was assessed in a cell-based model that mimics aspects of coagulation in vivo. Inhibition of IIa generation as a function of concentration was steeper for UFH than Lovenox. The effect of M118 closely paralleled that of UFH. By contrast, M118 did not prolong the aPTT to as great a degree as UFH, though both prolonged the aPTT more than did Lovenox. Our data suggest that the ability to inhibit platelet surface IIa generation correlates with the therapeutic level of heparins and confirms similarities between the anticoagulant properties of M118 and UFH. Fred Spencer, MD served as a guest editor.     

Pharmacologic modulation of thrombin generation associated with human clots by human purified antithrombin alone or in the presence of low molecular weight heparin or unfractionated heparin.

Procoagulant activities associated with human clots may contribute to thrombus extension. We investigate the inhibition of clot-associated factor Xa and thrombin activities by purified human antithrombin either alone or as combination with a low molecular weight heparin (enoxaparin) as compared with unfractionated heparin (UFH). The standard clots were prepared by recalcification of frozen platelet-poor human plasma. Clot-associated thrombin was measured on the clot after clot incubation in recalcified buffer or recalcified prothrombin solution. The enzymatic reaction was measured using a specific substrate for thrombin (CBS 3447). The thrombin concentration was determined both on the clots and in the reaction mixtures. In parallel, prothrombin fragment 1.2 and thrombin-antithrombin complexes (TAT) were measured using enzyme-linked immunosorbent assay methods. We demonstrated that in the presence of purified human prothrombin and antithrombin (AT), a partial inhibition of clot associated thrombin activity correlated with an increase of TAT complexes. However, antithrombin was unable to inhibit thrombin generation induced by the clot-associated factor Xa. Enoxaparin (low molecular weight heparin) and UFH did not enhance clot-bound thrombin inhibition induced by AT. We conclude that clot-bound thrombin is accessible to human antithrombin alone. AT is also able to inhibit thrombin generated by factor Xa-associated clot. However, neither a low molecular weight heparin or UFH enhanced the effect of AT alone.     

Low molecular weight heparin in acute coronary syndromes

Traditionally, unfractionated heparin has played an important role in the treatment of acute coronary syndromes. Low molecular weight heparin (LMWH), is a promising new type of heparin, which is fractionated to include only heparin molecules of lower molecular weight. LMWHs are administered subcutaneously and do not require monitoring of the activated partial thromboplastin time, making them much easier to use. LMWHs are combined inhibitors of both thrombin and Factor Xa inhibitors. Several recent large trials in unstable angina and non-Q wave myocardial infarction have shown that LMWH is effective, and one agent has been shown to be superior to unfractionated heparin in reducing death, myocardial infarction, or recurrent angina. They also are very low cost (approximately $50 per day) and appear to be very cost effective in the treatment of unstable angina. Thus, LMWHs appear to be the new anticoagulant agent in acute coronary syndromes.     

Decreased prevalence of heparin-induced thrombocytopenia with low-molecular-weight heparin and related drugs

Heparin-induced thrombocytopenia (HIT) Type II represents a disease spectrum associated with a high risk of thrombosis leading to limb loss and death. The pathophysiology of HIT is based on the development of antibodies to the heparin-platelet factor 4 (PF4) complex. Unfractionated heparin (UFH) is heterogeneous in molecular chain length and degree of sulfation accounting in part, for, the heterogeneity of HIT antibodies. Because of its smaller size, low-molecular-weight heparin (LMWH) does not interact with PF4 and platelets as efficiently as does UFH. This translates into a lower risk of immune sensitization with LMWH than with UFH treatment. LMWH is less likely than UFH to cause antibody generation and thus patients do not develop clinical HIT at the same frequency with LMWH as with UFH treatment. The antibodies generated by LMWH treatment are more often immunoglobulin A (IgA) and IgM as opposed to IgG antibodies, which are associated with symptomatic clinical HIT generated by exposure to UFH. However, platelet activation/aggregation can occur from LMWHs in the presence of most pre-existing HIT antibodies that had been generated from UFH exposure, although the response is less than that caused by UFH plus HIT antibody. With the expanded use of LMWH, the frequency of clinical HIT may naturally decline, given that LMWHs are less likely to generate HIT antibody.     

Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross-over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti-factor Xa activity and anti-factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre-injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.     

Pharmacokinetic and pharmacodynamic characterization of a medium-molecular-weight heparin in comparison with UFH and LMWH

Despite the well-established medical use of heparins, the question arises whether the efficacy-safety ratio of the available heparins can still be improved. Therefore, a medium-molecular-weight heparin (MMWH), a new heparin with an average molecular weight of 10.5 kDa and a narrow molecular weight range (9.5 to 11.5 kDa) was developed. Its in vitro activities amount to 174.9 anti-factor Xa (aXa) U/mg and 170.0 antithrombin (aIIa) U/mg. In the presented randomized, double-blind, cross-over study in healthy volunteers, the pharmacokinetics and pharmacodynamics of MMWH are compared with those of an unfractionated heparin (UFH) and a low-molecular-weight heparin (LWMH; enoxaparin). After subcutaneous administration of 9000 aXa-U of either heparin in 16 volunteers, the prolongation of the activated partial thromboplastin time (aPTT), the aXa activity, and the aIIa activities were determined at 11 time points spread over 24 hours after injection. The ex vivo analysis revealed striking pharmacodynamic and pharmacokinetic differences between the three heparins. UFH had the lowest bioavailability regarding the aPTT, aXa, and aIIa activities. Enoxaparin exhibited only low aIIa activity but the highest aXa activity. Unlike UFH and enoxaparin, MMWH showed a high recovery of aIIa activity, which suggests that it combines the high potency to inhibit thrombin that characterizes UFH with the high bioavailability of the LMWHs. Consequently, substantially lower doses are needed to bring about effects comparable to those of UFH and LMWH.     

Low molecular weight heparins prevent thrombin-induced thrombo-embolism in mice despite low anti-thrombin activity. Evidence that the inhibition of feed-back activation of thrombin generation confers safety advantages over direct thrombin inhibition

BACKGROUND AND OBJECTIVES: Thrombin-induced thromboembolism in mice is a model in which the feed-back clotting activation produced by the injected enzyme greatly contributes to fibrin accumulation in lungs and to mortality. Using this model we have previously shown that activated human protein C (aPC), by interrupting endogenous clotting activation at a high level (factors Va and VIIIa), prevents mortality inducing only a minor hemostatic impairment. With the same model we have now compared the antithrombotic and prohemorrhagic effects of two low molecular weight heparins (LMWHs), reviparin and tinzaparin, which are expected to inhibit preferentially the positive feed-back triggered by thrombin (anti Xa activity), with those of unfractionated heparin (UFH) and PEG-hirudin, which inhibit mainly or exclusively thrombin activity (anti IIa activity). DESIGN AND METHODS: Pulmonary thromboembolism was induced in mice by i.v. injection of bovine thrombin (1,000U/kg). Drugs (from 0.12 to 1.2 mg/kg) were given as bolus injection 2 min prior to thrombin challenge and mortality was assessed within 15 min. The bleeding time was assessed by a tail tip transection model. Activated partial thromboplastin time (aPTT), thrombin clotting time (TcT), fibrinogen assay and anti Xa activity determination were performed in citrated plasma from saline- or drug-treated animals. RESULTS: All drugs protected mice from thrombin-induced mortality in a dose-dependent way. At comparable antithrombotic dosages, the anti IIa activity generated in plasma (assessed by TcT) was highest with UFH, intermediate with tinzaparin and very low with reviparin. Accordingly, the fibrinogen drop, which is caused mainly by the injected thrombin, was prevented by the heparins to an extent that was fairly well related to their anti IIa activity. aPTT and bleeding time, used as measures of hemorrhagic risk, were markedly more prolonged by UFH than by reviparin. Tinzaparin, instead, had an intermediate effect. Interestingly, PEG-hirudin, at equipotent antithrombotic dosages, caused a prolongation of bleeding time comparable to that observed with UFH. INTERPRETATIONS AND CONCLUSIONS: Our data show that, in our model, drugs acting at a high level of the blood clotting cascade, like LMWHs with a high anti Xa/anti IIa ratio, display a better antithrombotic/prohemorrhagic profile than drugs acting prevalently on thrombin.     

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)(n) tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [K(d)], approximately 50 nM), similar potencies in neutralizing anti-Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)(3)VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti-Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.     

Cellular effects of factor Xa on vascular smooth muscle cells--inhibition by heparins?

In addition to its key function as a clotting enzyme, factor Xa (FXa) also elicits cellular effects. In cultured human venous smooth muscle cells (SMCs), FXa induced a mitogenic response that was independent of thrombin and platelet-derived growth factor (PDGF). Unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) (enoxaparin) inhibited the mitogenic effects of FXa, thrombin and fetal calf serum (FCS), but did not reduce mitogenesis induced by PDGF. Similarly, both UFH and LMWH inhibited the activation of extracellular signal-regulated kinase (ERK-1/2) by FXa, thrombin and FCS, but not by PDGF. This indicates that heparins can influence cellular signaling in SMC via an antithrombin-II (AT-III)-independent mechanism. The inhibition of ERK-1/2 correlated with the inhibition of mitogenesis by the heparins. Thus, the inhibition of ERK-1/2 phosphorylation by heparins might predict an antimitogenai response in this system.     

Rebound thrombin generation after heparin therapy in unstable angina. A randomized comparison between unfractionated and low-molecular-weight heparin

OBJECTIVES: This study compared rebound coagulation in patients with acute coronary syndrome patients after discontinuation of unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). BACKGROUND: Up to a quarter of patients hospitalized for unstable angina experience recurrent ischemia after discontinuation of UFH or LMWH therapy, which may be the result of rebound coagulation activation and subsequent thrombosis. It is unknown whether UFH and LMWH differ in this respect. METHODS: We randomized 71 patients admitted with unstable angina to intravenous UFH or subcutaneous LMWH (dalteparin) and measured plasma markers of coagulation before, during, and after treatment. RESULTS: A complete series of measurements was obtained in 59 patients. Plasma prothrombin fragment 1+2 (F(1+2)) levels decreased in both groups during treatment. After loss of therapeutic plasma drug levels, F(1+2) increased (within 3 h) to a maximum level at 12 to 24 h that was higher than before or during treatment in both groups (p < 0.0001). In both groups, F(1+2) levels remained higher than pretreatment up to 24 h after discontinuation. Similarly, thrombin-antithrombin (TAT) levels exceeded treatment and pretreatment levels, at a slower rate after dalteparin than after UFH. However, after dalteparin a higher peak value of TAT was observed. CONCLUSIONS: Rebound coagulation activation occurs within hours after discontinuation of both UFH and dalteparin. With both drugs, thrombin generation is significantly greater after treatment than before or during treatment. A longer duration or weaning of treatment, or continuation with another anticoagulant treatment, may reduce rebound coagulation activation and ischemic events.     

How and when to monitor a patient treated with low molecular weight heparin.

Low molecular weight heparins (LMWHs) are as efficient as unfractionated heparin (UFH) for prevention and treatment of thromboembolism. There is no evidence that monitoring the dose improves the clinical efficacy. In contrast, any overdosage increases the risk of hemorrhage. Because renal function plays a significant role in the elimination of LMWH, curative treatment should be monitored with an anti-factor Xa assay in patients presenting renal insufficiency, in the elderly, and in patients presenting an increased hemorrhagic risk. It is advisable to sample the patient at peak activity (3 to 5 hours after the subcutaneous [sc] administration) and to target the mean anti-factor Xa activity that was found efficient and safe in the clinical trial. This target is different for each LMWH and each dose regimen.     

Intestinal absorption of low molecular weight heparin in animals and human subjects

INTRODUCTION: We had previously shown that the use of bile salts, which act as surfactants, facilitates the intestinal absorption of large molecules such as those of heparin and insulin. However, the bioavailability of unfractionated heparin (UFH) administered through the large intestine was low. The aim of the present study was to evaluate the absorption of low molecular weight heparin (LMWH) combined with bile salts through the gut mucosa in animals and human subjects. MATERIALS AND METHODS: LMWH (Fragmin, Kabi-Pharmacia, Stockholm) or UFH with or without sodium cholate (Sch) was administrated rectally in rats and healthy volunteers via a microenema. Absorption was estimated by the activated partial thromboplastin time (aPTT), the plasma anti-factor Xa activity and the plasma lipoprotein lipase (LPL) activation. RESULTS: In groups of 6 rats, LMWH at doses of 100--1,000 U with sodium cholate (10--20 mg/ml) was readily absorbed through the gut mucosa, as indicated by both, anti-factor Xa levels of up to 1 U/ml and a dose-dependent activation of LPL. The absorption was significantly superior to that of UFH with Sch or LMWH given without Sch (p < 0.001). The plasma anti-factor Xa levels in the 6 healthy volunteers who received a microenema containing 25,000 U of LMWH with 20 mg/ml of Sch were 0.38 U/ml at 15 min and 0.1 U/ml at 240 min. LPL activation and aPTT prolongation were also observed in these subjects. The plasma LMWH levels after rectal application were in the same range as those obtained after subcutaneous administration, however the elimination time (t 1/2) was shorter. There were no adverse reactions. CONCLUSIONS: Intestinal absorption of LMWH facilitated by Sch is both feasible and safe. A slow release formulation will be needed to prolong the plasma half-life.     

Heparin blunts endotoxin-induced coagulation activation

BACKGROUND: Lipopolysaccharide (LPS) is a major trigger of sepsis-induced disseminated intravascular coagulation (DIC) via the tissue factor (TF)/factor VIIa-dependent pathway of coagulation. Experimental endotoxemia has been used repeatedly to explore this complex pathophysiology, but little is known about the effects of clinically used anticoagulants in this setting. Therefore, we compared with placebo the effects of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on LPS-induced coagulation. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled trial, 30 healthy male volunteers received LPS 2 ng/kg IV followed by a bolus-primed continuous infusion of UFH, LMWH, or placebo. In the placebo group, activation of coagulation caused marked increases in plasma levels of prothrombin fragment F(1+2) (P<0.01) and polymerized soluble fibrin, termed thrombus precursor protein (TpP; P<0.01); TF-positive monocytes doubled in response to LPS, whereas levels of activated factor VII slightly decreased and levels of TF pathway inhibitor remained unchanged. UFH and LMWH markedly decreased activation of coagulation caused by LPS, as F(1+2) and TpP levels only slightly increased; TF expression on monocytes was also markedly reduced by UFH. TF pathway inhibitor values increased after either heparin infusion (P<0.01). Concomitantly, factor VIIa levels dropped by >50% at 50 minutes after initiation of either heparin infusion (P<0.01). CONCLUSIONS: This experimental model proved the anticoagulatory potency of UFH and LMWH in the initial phase of experimental LPS-induced coagulation. Successful inhibition of thrombin generation also translates into blunted activation of coagulation factors upstream and downstream of thrombin.