Modulation of antithrombin-protease interactions by semisynthetic low-molecular-weight heparins with different sulfation patterns

Heparin, a natural glycosaminoglycan (GAG), is widely used for the treatment of thrombotic diseases. Most of its side effects are related to its ability to bind to different proteins, thus interfering with its target biological activity. To gain insight into structure-activity relationships, we investigated the interaction of a homogeneous series of sulfated polysaccharides, derived from controlled desulfation of a supersulfated low-molecular-weight heparin (LMWH) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation process of the serpin AT against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the polysaccharide sulfation degree was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the LMWH derivatives in activating AT toward the proteases was generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated heparin showed a serpin activation ability higher than expected from the affinity studies. Examination of the sulfation pattern in the light of the above results suggests a key role of the substitution of the iduronic acid residue in the heparin-mediated serpin binding and activation processes. Indeed, sulfation at position 2 of the uronic acid is beneficial, whereas 2,3-disubstitution generates unfavorable contacts between the GAG and AT. Glucosamine sulfation at position 6 appears to grant increased catalytic efficiency. These results indicate that chemical modification of the heparin sulfation pattern can be used to modulate binding specificity and activity toward its biological targets.     

Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit

Our purpose was to determine the relative contribution of the antifactor Xa and antithrombin activities of heparin to its antithrombotic potency. The antithrombotic activities of unfractionated heparin (UH), two low molecular weight heparins (LMWH, CY 216 and CY 222) with increasing anti-factor Xa/antithrombin ratio and a synthetic pentasaccharide (PS) with high affinity to antithrombin III and no antithrombin activity were evaluated. In the Wessler-thromboplastin model, the most potent antithrombotic agent, on a weight basis, was UH followed by CY 216, CY 222 and the PS which was 40 times less potent than UH. On an antithrombin unit basis, the antithrombotic potencies of UH, CY 216 and of CY 222 were equivalent. Thus, in this model, the antithrombotic effect results from the catalytic action of UH or LMWH on thrombin inhibition. In the Wessler-serum model, on a weight basis, the antithrombotic effectiveness of UH was unchanged, those of CY 216 and CY 222 were doubled, and that of the PS was increased 10 times. On an anti-factor Xa unit basis, CY 216 was as effective as UH, and PS as effective as CY 222. On an antithrombin unit basis, CY 216 and CY 222 were equivalent and more potent than UH. Thus, in this model, the antifactor Xa activity of heparin becomes important for its antithrombotic property. After a single subcutaneous injection of 1000 antifactor Xa U/kg, the antithrombotic effects of UH were maintained for more than 14 h in the two models. After injection of the same dose of CY 216 significant antithrombotic effects were observed only for 9 h, in the Wessler-thromboplastin model but for 18 h in the Wessler-serum model. At that time, no detectable antithrombin activity was measurable in the plasma while 0.11 units of antifactor Xa activity/ml was detected. Thus, the relative contribution of the anti-factor Xa and antithrombin activities to the antithrombotic effect of a LMWH differs according to the nature of the thrombogenic stimulus.     

Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The mechanism and molecular target for intrinsic tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced V(max(app))), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa-phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC(50) 35-fold lower than soluble LWMH. Analysis of intrinsic tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity and the potency of LMWH for intrinsic tenase. DHG also accelerated decay of the intact intrinsic tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interactions.     

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.     

Low molecular weight heparin restores antithrombin III activity from hyperglycemia induced alterations

Alteration of antithrombin III (ATIII) activity, glycemia level dependent, exists in diabetes mellitus. In this study the ability of a low molecular weight heparin (LMWH) (Fluxum, Alfa-Wassermann S.p.A., Bologna, Italy), as well as unfractioned héparin, to preserve ATIII activity from glucose-induced alterations, both in vitro and in vivo, is reported. The subcutaneous and intravenous LMWH and heparin administration increases basal depressed ATIII activity in diabetic patients. Heparin shows an equivalent effect on both anti-IIa and anti-Xa activity of ATIII, while LMWH is more effective in preserving the anti-Xa activity. Similarity, heparin preserves ATIII activity from hyperglycemia-induced alterations, during hyperglycemic clamp, and LMWH infusion is able to preserve a significant amount of anti-Xa activity from glucose-induced alterations. Since diabetic patients show a high incidence of thrombotic accidents, LMWH appears to be a promising innovation for the prevention of diabetic thrombophylia.     

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.     

Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism

The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the β-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.     

Effects of a heparin-binding protein on blood coagulation and platelet function.

The objective of this study was to characterize the heparin-binding properties of a protein secreted by mouse myeloma cells. The characterization was performed using clinical assays, such as heparin activity assays and heparin-induced thrombocytopenia (HIT) platelet activation assays. The tests were performed in the presence of heparin, low-molecular-weight heparins (LMWH), or heparinoids and either heparin-binding protein (HBP) or saline to determine whether the HBP affects the activity of heparins. The characterization of the HBP using heparin activity assays showed that the HBP shortened the prolonged clotting times of the activated partial thromboplastin time (aPTT) and thrombin clotting time induced by high concentrations of unfractionated heparin. The chromogenic assays for antithrombin (AT), thrombin inhibition, and factor Xa inhibition demonstrated that this effect is related to heparin concentrations below 0.5 IU/ml. The Heptest assay did not detect these differences. The HBP did not modify the anticoagulant effect of any LMWH or low- or high-sulfated glycosaminoglycans in the aPTT assay. Activation of donor platelets in the presence of unfractionated heparin, platelet factor 4 (PF4), and HIT-serum was not counteracted by the HBP in any of the assays. The characterization of the HBP using a PF4-enzyme-linked immunosorbent assay (ELISA) confirmed the lack of structural identity with PF4. However, the optical density data indicated that the protein structure may be similar to PF4 by binding to a PF4 antibody. These data suggest that the HBP isolated from mouse myeloma cells has a low affinity to heparin and interacts with the secondary binding site to AT and also perhaps to PF4.     

Effects of sulfation on antithrombin-thrombin/factor Xa interactions in semisynthetic low molecular weight heparins.

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.     

Physiological function of tissue factor pathway inhibitor and interaction with heparins

Tissue factor pathway inhibitor (TFPI) is now recognized as a major physiological anticoagulant. Its main role is to modulate factor VIIa/tissue factor catalytic activity. Another important role is to potentiate the effect of heparins. TFPI is released from the vascular endothelium after injection of either unfractionated heparin (UFH) or low-molecular-weight heparins (LMWHs), which may then provide high concentrations of TFPI at sites of tissue damage and ongoing thrombosis. In dilute prothrombin-time-based assays, released TFPI contributes approximately one-third to the anticoagulant effect of heparin, the remaining being accounted for by antithrombin. Released TFPI, but not plasma TFPI, contains the basic carboxy-terminal tail which is important for the anticoagulant effect. UFH and LMWH exert differential effects on intravascular TFPI. UFH, but not LMWH, given in therapeutic doses, is associated with a progressive depletion of TFPI, which is associated with a strong rebound activation of coagulation after cessation of treatment. Such depletion may explain the apparent superior efficacy of LMWH observed in clinical trials.     

Beneficial effect of JTV-803, a new synthetic inhibitor of activated factor X, against both lipopolysaccharide-induced and tissue factor-induced disseminated intravascular coagulation in rat models.

We examined whether JTV-803, a specific activated factor X inhibitor independent of antithrombin III (ATIII), is effective against disseminated intravascular coagulation (DIC) in rat models induced by tissue factor (TF) or lipopolysaccharides (LPS). In male Wistar rats, DIC was induced by a 4 h infusion of thromboplastin (3.75 U/kg) or LPS (50 mg/kg). The rats were given JTV-803 (0.3 or 3 mg/kg, bolus intravenously) (JTV-803 groups) or low molecular weight heparin (LMWH groups) (200 U/kg, bolus intravenously) prior to an injection of TF or LPS. The results showed that JTV-803 was dose-dependently effective against DIC in both TF-induced and LPS-induced rat models. This anti-DIC effect of JTV-803 at higher doses was almost equivalent to that of LMWH in both types of DIC. Plasma ATIII activity was more prominent in the group treated with JTV-803 than in that treated with LMWH. None of rats died in the TF-induced DIC model with or without drug administration. On the contrary, seven of 22 rats died (mortality rate, 31.8%) in the LPS-induced DIC model without drug administration. Although the mortality rate of rats induced with LPS and treated with LMWH was quite high (6/16, 37.5%), none of the LPS-induced rats treated with JTV-803 died. These findings suggested that JTV-803 can treat both TF-induced and LPS-induced DIC models, and that this drug has greater potential in preserving ATIII and in improving the prognosis of DIC.     

Modulation of the enzymatic activity of alpha-thrombin by polyanions: consequences on intrinsic activation of factor V and factor VIII.

The polyanions heparin and dermatan sulfate catalyze alpha-thrombin inhibition and can delay the onset of factor VIII and factor V necessary for intrinsic prothrombin activation to begin in plasma. These polyanions bind alpha-thrombin at its anion-binding exosite(s), structural domain(s) occupancy of which may alter the properties of the fibrin(ogen) recognition exosite of alpha-thrombin. We compared how such four polyanions influenced factor VIII and factor V activation during intrinsic coagulation. A pentasaccharide with high affinity for antithrombin III and the C-terminal dodecapeptide fragment of hirudin (hirugen) which occupy the anion-binding and fibrin(ogen) recognition exosites of alpha-thrombin, respectively, could not significantly inhibit factor VIII and factor V activation. In contrast, heparin and a bis-lactobionic acid, both of which catalyzed alpha-thrombin inhibition, could effectively inhibit factor VIII and factor V activation. These results suggest that occupancy of fibrin(ogen) or anion-binding exosites by itself does not provide a necessary and sufficient condition for catalysis of thrombin inhibition or the inhibition thrombin-mediated amplification reactions.     

Low Molecular Weight Heparin and Coronary Intervention

For more than 25 years, basic experiments using cell culture and animal experimental techniques have predicted superior thrombin inhibition with use of low molecular weight heparin (LMWH) rather than unfractionated heparin (UFH). This should have application in the cardiac laboratory, where thrombin control is essential to securing a safe and optimal therapeutic angioplasty for patients with atherosclerotic heart disease. Despite much interest, few meaningful clinical studies have been completed and there are limited clinical data to establish the magnitude of benefit actually conferred through use of LMWH rather than UFH among most patients undergoing elective or urgent cardiac catheterization procedures. At this point, the available data suggest minimal clinical advantage is to be gained through the use of LMWH over UFH. Also, obtaining this modest advantage may require an extended period of drug therapy before and after intervention. Any benefit conferred is likely restricted to the period shortly after coronary intervention. On the other hand, available data suggest that serious bleeding should not be problematic despite difficulties measuring that antithrombin effect among patients receiving LMWH, even when it is combined with potent platelet inhibitor therapy. Although cell culture and animal experiments suggest that heparin compounds should be effective in limiting intimal hyperplasia (and therefore clinical restenosis) after angioplasty, randomized clinical trials have failed thus far to show any restenosis benefit for either UFH or LMWH. Ongoing studies will clarify the use of combining LMWH with potent antiplatelet agents as combined medical adjuncts to coronary intervention.     

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.     

Role of accessory components in the activation of vitamin K-dependent coagulation factors

Kinetic studies of prothrombin activation and intrinsic factor X activation carried out in the absence and presence of phospholipids and the protein cofactors Va or VIIIa have provided insight in the mechanism by which the accessory components enhance coagulation factor activation. In intrinsic factor X and prothrombin activation, phospholipids cause a drastic drop of Km for the substrates factor X and prothrombin, whereas the protein cofactors factor Va and factor VIIIa increase Vmax of the prothrombin- and factor X-activating reactions. The mode of action of factor Va in prothrombin activation is however somewhat more complex. Besides its stimulatory effect on the catalytic activity of factor Xa, Factor Va also plays an important role in the assembly of the prothrombin-activating complex at phospholipid surfaces especially when the latter have a low affinity for vitamin-K-dependent coagulation factors. This effect is likely accomplished by promoting the binding of both prothrombin and factor Xa to the procoagulant surface.     

Comparison of the anticoagulant effect of a direct thrombin inhibitor and a low molecular weight heparin in an acquired antithrombin deficiency in children with acute lymphoblastic leukaemia treated with L-asparaginase: an in vitro study

Thrombosis occurs in 37% of children with acute lymphoblastic leukaemia (ALL) and is related to an L-asparaginase-induced acquired antithrombin (AT) deficiency. The incidence dictates the need for anticoagulant prophylaxis. Direct thrombin inhibitors (DTI) are independent of AT for effect and may thus have advantages in this population. The objective of this study was to determine the interaction of an AT deficiency with the anticoagulant effects of a DTI and a low molecular weight heparin (LMWH). Plasma samples from children with ALL were pooled (mean AT 0.53 U/ml). LMWH 0.3 and 0.7 U/ml or melagatran 0.3 and 0.5 micromol/l were added to the pools, then divided and AT was added back to one aliquot. In additional experiments, AT was added to AT immuno-depleted plasma. Endogenous thrombin generation capacity (ETGC) was assessed by the continuous method. In plasma with LMWH, there was a 66-88% decrease in ETGC in AT-normalised samples compared with neat. Conversely, no significant difference in ETGC with or without AT added for melagatran was seen. Experiments with AT-depleted plasma showed no effect of AT level on anticoagulant activity of DTI, but a significant relationship for LMWH. By contrast to LMWH, DTI provides a consistent anticoagulant response independent of AT levels in children with AT deficiency.     

Non-ST elevation acute coronary syndrome: changes of parameters of hemostasis during and after treatment with unfractionated heparin and enoxaparin

AIM: To compare changes of parameters of hemostasis in patients with non-ST elevation acute coronary syndrome (NSTEACS) during and after treatment with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) enoxaparin. MATERIAL: Control groups of 2 randomized studies of effects of thienopyridines in NSTEACS with similar inclusion criteria. METHODS: In patients (n=18) of study one UFH was infused intravenously for 54.2+/-22.3 h, in patients of study two subcutaneous enoxaparin 1 mg/kg b.i.d. was used for 76.2+/-28.3 h. Levels of D-dimer (DD), thrombin-antithrombin complex (TAT), prothrombin fragment 1+2 (F1+2), von Willebrand factor (vWF), fibrinogen, tissue plasminogen activator (TPA) and activity of its inhibitor (PAI), soon after start of treatment with heparins and in 1, 3, 7 and 14 days. RESULTS AND CONCLUSION: Short term lowering of DD and TAT levels occurred during UFH infusion apparently reflecting primary antithrombin action of UFH. During LMWH use DD level remained unchanged however its lowering was observed after cessation of LMWH (on days 7 and 14). The use of LMWH was not associated with increases of thrombinemia (TAT), thrombin generation (F1+2), and fibrinogen which were registered after end of UFH infusion (days 3, 7 and 14). Neither UFH nor LMWH prevented acute phase vWF elevation. The use of both heparins was associated with changes that could be interpreted as profibrinolytic. In LMWH treated patients these changes (elevations of TPA level) became manifested early (on day 3) and were short lived while in UFH treated patients they appeared later (on day 7) and were prolonged (as lowered PAI activity on day 14).     

Determination of the levels of unfractionated and low-molecular-weight heparins in plasma: their effect on thrombin-mediated feedback reactions in vivo. Preliminary results after subcutaneous injection.

We define a standard independent unit (SIU) of heparin as that amount that, in plasma containing 1 mumol of ATIII, raises the (pseudo-)first-order breakdown constant of factor Xa by 1 min-1. These units measure all material with a high affinity for ATIII (HAM); only material above the critical chain length of 17 monosaccharide units (above critical chain length material; ACLM) catalyzes the inactivation of thrombin. An SIU of ACLM is therefore analogously defined as the amount that, in plasma containing 1 mumol of ATIII, will raise the (pseudo-)first-order breakdown constant of thrombin by 1 min-1. Of any given heparin preparation one can determine the specific HAM and ACLM activities in terms of SIU/mg. On the basis of the factor Xa and thrombin breakdown constants found in a plasma sample one can then determine the levels of HAM and ACLM. Preliminary experiments were carried out in plasma samples obtained after subcutaneous injection of unfractionated heparin (UFH) and of two types of low-molecular-weight heparin (LMWH). About three times more of UFH activity than of LMWH activity has to be injected to obtain the same levels of ACLM in the plasma. Only with the LMWHs significant amounts of BCLM are found, which rises higher and persists longer than the ACLM. We determined the course of thrombin generation in platelet-rich plasma (PRP) and in platelet-poor plasma (PPP), as well as in the PPP factor Xa generation curve and the course of prothrombin conversion. The observed inhibitions correlated much better with the levels of ACLM than with those of below critical chain length material. The difference between UFH and LMWHs can therefore not be explained in terms of antithrombin and anti-factor-Xa activity. The essential difference between UFH and LMWH appears in the feedback effect of thrombin in PRP, where thrombin generation is both inhibited and retarded by LMWH, while it is only retarded but hardly inhibited by UFH.     

Effects of low molecular weight heparin,alone or combined with antithrombin III,on mortality,fibrin deposits and hemostatic parameters in endotoxin-induced disseminated intravascular coagulation in rabbits

The effect of low molecular weight heparin (LMWH) with or without antithrombin III (AT III) has been studied in a rabbit model of disseminated intravascular coagulation (DIC) induced by continuous infusion of 100 μg/kg/hr of Escherichia coli endotoxin for 6 hr. LMWH (5 and 10 IU/kg/hr/6 hr), alone or in combination with AT III (20 U/kg/hr/6 hr), or saline were administered simultaneously with endotoxin. Hemostatic markers at 0, 2, and 6 hr as well as kidney fibrin deposits and the mortality rate at 24 hr were determined. Rabbits receiving only endotoxin showed an impairment in hemostasis, as well as high kidney fibrin deposits and a high mortality rate. LMWH alone did not exert any effect. The simultaneous infusion of LMWH and AT III exerted a beneficial effect on the hemostatic markers and reduced the kidney fibrin deposits as well as the mortality rate in a LMWH dose-dependent manner. Fibrinogen and protein C consumption were significantly higher and renal fibrin deposits more intense in the rabbits that had died in the first 24 hr. There was also a significant positive correlation between kidney fibrin deposits and platelets, fibrinogen, and protein C consumption, taking the whole rabbit population. It is concluded that the simultaneous infusion of LMWH and AT III is useful in this DIC model and would make it possible to reduce significantly the AT III doses used when AT III is given alone. Am. J. Hematol. 60:6–11, 1999. © 1999 Wiley-Liss, Inc.