Efficacy of unfractionated heparin, low molecular weight heparin and both combined for releasing total and free tissue factor pathway inhibitor.

Unfractionated heparin (UFH) exerts its anticoagulant properties by increasing the inactivation of thrombin and activated factor X by antithrombin III. Apart from this main action release of tissue factor pathway inhibitor (TFPI) from endothelial cells could also be important for the antithrombotic activity of heparins. Four different heparin preparations were injected subcutaneously into 5 healthy volunteers 1 week apart: (1) UFH 2,500 IU fix dose (FixUFH), (2) 1 mg/kg body weight of low molecular weight heparin (LMWH), (3) the combined LMWH-adjusted dose plus UFH 2,500 IU fix dose (ComHep) and (4) UFH 2,500 IU/10 kg body weight (UFHvar). Plasma samples were drawn before and 1, 2, 4, 6, 12 and 24 h afterwards. FixUFH did not affect the concentration of total and free TFPI. Total TFPI increased in the 1st hour after LMWH injection from 74 to 124 ng/ml (p < 0.01), after ComHep from 82 to 144 ng/ml (p < 0.01), and after UFHvar from 91 to 113 ng/ml (p < 0.05). All observed elevations were significant at the peak value (+/- 2 h, p < 0.01 compared with baselines). The increase of free TFPI produced by UFHvar (74.5 and 70.5 ng/ml) was significantly higher than with LMWH (42.8 and 38.0 ng/ml) at 2 and 4 h (p < 0.001 and p < 0.01, respectively). UFHvar and ComHep but not LMWH produced a statistically significant increase of free TFPI compared with FixUFH at 2, 4 and 6 h (p < 0. 01). We concluded that at comparable therapeutic doses, subcutaneous UFHvar released more free TFPI than LMWH and ComHep. A synergism between LMWH and low dose of UFH was found in 4-, 6- and 12-hour blood samples.     

Unfractionated heparin and low-molecular-weight heparin for the initial treatment of acute venous thromboembolism

Anticoagulant drugs represent the therapy of choice for the initial treatment of venous thromboembolism. Unfractionated heparin (UFH) in adjusted doses and low-molecular-weight heparin (LMWH) in fixed doses are equally as effective and safe. Proper use of UFH requires considerable expertise, can cause inconvenience and has limitations. The use of LMWHs has multiple advantages over UFH including a more predictable dose-response and fixed administration dose. These properties make the treatment of suitable patients feasible in an outpatient setting. In two major clinical trials addressing the treatment of deep vein thrombosis, outpatient management with LMWH was associated with a substantial cost reduction compared with inpatient treatment using UFH. Recent studies have also shown that LMWHs are at least as effective and safe as UFH for the treatment of non-critical patients with pulmonary embolism. Whether or not home treatment of pulmonary embolism is feasible and safe remains to be demonstrated.     

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.     

Moderate alcohol intake in humans attenuates monocyte inflammatory responses: inhibition of nuclear regulatory factor kappa B and induction of interleukin 10

BACKGROUND: In contrast to the deleterious effects of chronic excessive alcohol consumption on the liver and cardiovascular system, modest alcohol intake, such as 1 to 2 drinks per day, has benefits on cardiovascular mortality. Little is known about the length of time or the amounts of alcohol consumed that may cause alterations in inflammatory cells such as monocytes that are crucial to atherosclerotic vascular disease. Here, we determine in vivo effects of acute alcohol consumption on inflammatory cytokine production and nuclear regulatory factor kappaB (NF-kappaB) binding in human monocytes. METHODS: Human blood monocytes were isolated by plastic adherence before and after acute alcohol consumption (2 ml vodka/kg body weight). Lipopolysaccharide (LPS)- and superantigen-induced tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1beta, and IL-10 production were then determined in monocytes by ELISA. Nuclear regulatory factor-kappaB activity of monocytes before and after alcohol consumption was estimated by electromobility shift assay and promoter-driven reporter activity. IkappaBalpha was determined by Western blotting in the cytoplasmic extracts. RESULTS: Eighteen hours after moderate alcohol consumption, we found a significant reduction in monocyte production of inflammatory mediators, TNF-alpha and IL-1beta, in response to LPS or staphylococcal enterotoxin B stimulation. Acute alcohol consumption inhibited LPS-induced DNA binding of the p65/p50 NF-kappaB in monocytes that regulates the expression of both the TNF-alpha and the IL-1beta genes. Consistent with this, acute alcohol treatment (25 mM) significantly reduced LPS-induced activation of an NF-kappaB-driven reporter gene suggesting inhibition of this proinflammatory signaling pathway. Further, LPS-induced IkappaBalpha degradation was not affected by acute alcohol consumption indicating an IkappaBalpha-independent mechanism, as observed earlier in the in vitro acute alcohol studies. In contrast, monocyte production of the anti-inflammatory cytokine, IL-10, was augmented by acute alcohol intake. CONCLUSIONS: Our findings suggest that acute alcohol consumption has dual anti-inflammatory effects that involve augmentation of IL-10 and attenuation of monocyte inflammatory responses involving inhibition of NF-kappaB. These mechanisms may contribute to the beneficial effects of moderate alcohol use on atherosclerosis.     

Orally active heparin and low-molecular-weight heparin

The heparins, (unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH)) given by subcutaneous or intravenous injection have been used extensively in the prevention and treatment of both venous and arterial thromboembolic disorders. The increasing use of the heparins, LMWHs in particular, in the out of hospital setting has stimulated interest in the development of orally absorbable antithrombotic agents that require little or no monitoring, and this includes the heparins. UFH or LMWH delivered orally has been shown to have an antithrombotic effect in animal thrombosis models although there is little change in plasma coagulation tests. The addition of a simple organic chemical N -(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) to UFH markedly enhances its absorption. A phase II study in patients undergoing total hip replacement indicated that SNAC heparin in two different doses was as effective and safe as UFH given subcutaneously. A phase III clinical trial comparing two doses of SNAC heparin given orally with LMWH by subcutaneous injection for the prevention of venous thromboembolism in patients undergoing total hip replacement is currently underway.     

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.     

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.     

Effect of ethanol on inflammatory responses. Implications for pancreatitis.

BACKGROUND/AIMS: Alcohol use alters inflammatory cell responses. While alcohol has direct effects on pancreatic acinar cells, activation of inflammatory cells is a major component of the pathology of alcoholic pancreatitis. METHODS: The effects of acute or chronic alcohol exposure were evaluated in human monocytes on the production of TNFalpha or IL-10 production, pro-inflammatory gene and nuclear factor-kappaB (NF-kappaB) activation. RESULTS: Moderate, acute alcohol consumption or equivalent doses of alcohol in vitro had anti-inflammatory effects on monocyte activation via inhibition of pro-inflammatory genes and NF-kappaB activation, inhibition of TNFalpha production and augmentation of the anti-inflammatory cytokine, IL-10. In contrast, acute alcohol treatment augmented NF-kappaB activation and TNFalpha production and inhibited IL-10 levels in the presence of complex stimulation with combined TLR2 and TLR4 ligands. Prolonged alcohol exposure also resulted in an increase in NF-kappaB and TNFalpha production in response to TLR4 stimulation with LPS. CONCLUSION: These results suggest that alcohol can either attenuate or promote inflammatory responses that are critical in pancreatitis. Our results support the hypothesis that both acute alcohol intake in the presence of complex stimuli (such as necrotic cells) and chronic alcohol exposure result in hyper-responsiveness of monocytes to inflammatory signals and may contribute to increased inflammation in pancreatitis.     

Effects of pre-analytical variables on the anti-activated factor X chromogenic assay when monitoring unfractionated heparin and low molecular weight heparin anticoagulation.

The purpose of this study was to determine whether the anti-activated factor X (anti-FXa) assay is less affected by pre-analytical variables in monitoring patients on unfractionated heparin (UFH) and low molecular weight heparin (LMWH) than the activated partial thromboplastin time (aPTT). Forty-six subjects receiving either enoxaparin (LMWH) or UFH were randomly selected. Each study subject had six vacutainer tubes (3.8% sodium citrate, 3.2% sodium citrate) drawn by an atraumatic venipuncture. One tube from each set had a blood to anticoagulant ratio of 9: 1. The other tube had an intentional "short-draw" of approximately 6: 1 blood to anticoagulant ratio. All specimens had an aPTT and a chromogenic anti-FXa assay performed on each specimen regardless of heparin type. The aPTT assay mean with the 3.8% sodium citrate tube short-draw tube was statistically different from the other aPTT assays (P = 0.06). However, all six of the mean anti-FXa assays for the UFH and LMWH heparin subjects were not statistically or clinically different (analysis of variance, P = 0.9878 for UFH and P = 0.9060 for LMWH). The intentional short-draw tube did not affect the anti-FXa assay regardless of the anticoagulant. The anti-FXa assay appears to be a better method for monitoring heparin subjects than the aPTT due to the lack of effect of pre-analytical variables.     

Contribution of tumour necrosis factor alpha and interleukin (IL) 1beta to IL6 production, NF-kappaB nuclear translocation, and class I MHC expression in muscle cells: in vitro regulation with specific cytokine inhibitors

OBJECTIVE: To evaluate the effect of tumour necrosis factor alpha (TNFalpha), interleukin (IL) 1beta, and their respective inhibitors the p75 TNFalpha soluble receptor (sTNFR) and the type II sIL1betaR (sIL1RII) on whole muscle and isolated myoblast activation. METHODS: Normal muscle samples were stimulated for 7 days with TNFalpha alone or in combination with IL1beta, and myoblasts from these samples for 48 hours. IL6 production was measured by ELISA. Nuclear translocation of NF-kappaB was analysed by immunofluorescent staining and class I MHC expression by FACS. RESULTS: TNFalpha and IL1beta induced IL6 production by normal muscle samples and myoblasts, the action of TNFalpha being more potent on muscle samples. Their soluble receptors (1 microg/ml) decreased this production. Suboptimal concentrations of TNFalpha and IL1beta induced NF-kappaB translocation. sTNFR markedly down regulated TNFalpha-induced translocation while sIL1RII was less potent on IL1beta-induced activation. NF-kappaB translocation induced by the combination of optimal concentrations of TNFalpha and IL1beta was completely inhibited by their soluble receptors. TNFalpha and to a lesser extent IL1beta induced class I MHC expression by myoblasts and this effect was completely inhibited by their respective soluble receptors. CONCLUSION: These results suggest that TNFalpha and IL1beta should be targeted for myositis treatment.     

Inhibitory activity of 1,8-cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes

BACKGROUND: The therapeutic value of secretolytic agents in COPD and asthma is still disputed. For this reason, in a preclinical study we aimed to test the potential anti-inflammatory efficacy of 1,8-cineol (eucalyptol) in inhibiting polyclonal stimulated cytokine production by human unselected lymphocytes and LPS-stimulated monocytes. METHODS: Cytokine production was determined following 20 h of incubation cells with 1,8-cineol simultaneously with the stimuli in culture supernatants by enzyme immunoassay. RESULTS: Therapeutic concentrations of 1,8-cineol (1.5 microg/ml=10(-5)M) inhibited significantly (n=13-19, p=0.0001) cytokine production in lymphocytes of TNF-alpha > IL-1beta> IL-4> IL-5 by 92, 84, 70, and 65%, respectively. Cytokine production in monocytes of TNF-alpha > IL-1beta> IL-6> IL-8 was also significantly (n=7-16, p<0.001) inhibited by 99, 84, 76, and 65%, respectively. In the presence of 1,8-cineol (0.15 microg/ml=10(-6)M) production of TNF-alpha>IL-1beta by monocytes and of IL-1beta> TNF-alpha by lymph-ocytes was significantly inhibited by 77, 61 and by 36, 16%, respectively. 1,8-cineol (10(-6)M) had a larger impact on TNF-alpha and IL-1beta-production in monocytes compared to lymphocytes (p<0.03) and similar effects (p>0.59) at therapeutically relevant concentrations of 1,8-Cineol (10(-5)M). CONCLUSION: These results characterize 1,8-cineol as strong inhibitor of TNF-alpha and IL-1beta and suggest smaller effects on chemotactic cytokines. This is increasing evidence for the role of 1,8-cineol to control airway mucus hypersecretion by cytokine inhibition, suggesting long-term treatment to reduce exacerbations in asthma, sinusitis and COPD.