Reliable low-molecular-weight heparin reversal in a child undergoing emergency surgery: a case report.

Low-molecular-weight heparin neutralization using protamine alone can be unreliable, especially in cases of immediate reversal for emergency surgery. Here, we describe a unique case of a 17-month-old girl with a history of glioneuronal tumor and corresponding hydrocephalus status post debulking and ventriculoperitoneal shunt placement, who was placed on enoxaparin after the development of a sagittal sinus thrombosis. Patient presented for emergency craniectomy and evacuation of subdural bleed after a fall while on therapeutic dose of enoxaparin. Protamine and fresh frozen plasma were used in the patient's perioperative course providing a reliable reversal of enoxaparin.

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay.

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 µg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

Homogeneous low-molecular-weight heparins with reversible anticoagulant activity.

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Postinjury hyperfibrinogenemia compromises efficacy of heparin-based venous thromboembolism prophylaxis.

BACKGROUND: Venous thromboembolism (VTE) prophylaxis remains debated following trauma, and recommendations have not been established. Although hyperfibrinogenemia is a marker of proinflammatory states, it also contributes to thrombus formation. Postinjury hyperfibrinogenemia is common, but the effect of hyperfibrinogenemia on VTE prophylaxis has not been fully elucidated. Therefore, we hypothesized that heparin is less effective for VTE prophylaxis following severe injury due to hyperfibrinogenemia. METHODS: In vitro studies evaluated thromboelastography (TEG) parameters in 10 healthy volunteers after the addition of fibrinogen concentrate and heparin. Data from a recent randomized controlled trial, conducted at an academic level I trauma center surgical intensive care unit, were reviewed. Critically injured patients were randomized to standard VTE prophylaxis (5,000 U low-molecular-weight heparin daily) or TEG-guided prophylaxis (up to 10,000 U low-molecular-weight heparin daily) and were followed up for 5 days. Analysis was performed to evaluate the relationship between fibrinogen levels, measures of anticoagulation, and TEG parameters. RESULTS: In vitro studies revealed increased fibrinogen reversed the effects of heparin as measured by TEG. Fifty patients were enrolled in the clinical study with 25 in each arm. Thromboelastography parameters, fibrinogen, platelet count, and anti-Xa levels did not differ between groups despite treatment provided. Fibrinogen levels increased over the 5-day study period (597 ± 24.0 to 689.3 ± 25.0), as well as clot strength (9.8 ± 0.4 to 14.5 ± 0.6), which had a significant correlation coefficient (P < 0.01). Moreover, there was a moderate inverse correlation between fibrinogen level and the effect of heparin (RF), which was significant on study days 1 and 3, implicating hyperfibrinogenemia in heparin resistance. CONCLUSIONS: Hypercoagulability and heparin resistance are common following trauma. The preclinical and clinical relationships between fibrinogen levels and hypercoagulability implicate hyperfibrinogenemia as a potential factor in heparin resistance.

Neutralizing the anticoagulant activity of ultra-low-molecular-weight heparins using N-acetylglucosamine 6-sulfatase.

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight: unfractionated (UF, average molecular weight 13 000), low molecular weight (average molecular weight 5000) and ultra-low-molecular-weight heparin (ULMWH, average molecular weight 2000). An overdose of heparin may lead to very dangerous bleeding in patients. Protamine sulfate may be administered as an antidote to reverse heparin's anticoagulant effect. However, there is no effective antidote for ULMWH. In the current study, we examine the use of human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells, as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra(®) (fondaparinux), effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to developing human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation.

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity.

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

Protamine neutralisation of low molecular weight heparins and their oligosaccharide components.

Protamine sulphate is an effective inhibitor of heparin and is used clinically to neutralise both low molecular weight heparins (LMWH) and unfractionated heparin (UFH). However, protamine sulphate does not fully counter the anti-Xa effect of LMWH, even in excess (>40 µg to 1 IU/ml). To investigate the molecular basis for this observation, the residual potencies in the presence and absence of plasma as well as the molecular weight profiles of commercial LMWH neutralised with increasing amounts of protamine were measured. Materials over 5000 Da are preferentially neutralised by protamine. To further investigate this molecular weight dependence, monodisperse oligosaccharides were prepared from three commercial LMWHs. The specific anti-Xa activity for the fractions increased with molecular weight, and was found to vary between the three preparations for oligosaccharides of the same molecular weight. Our results indicate that protamine sulphate neutralisation is largely dependent on molecular weight, leading to the implication that LMWHs containing a larger proportion of small oligosaccharides will not be as effectively neutralised. Protamine sulphate neutralisation of any given LMWH is also affected by the specific anticoagulant activities of its low molecular weight components, which varies between LMWH products, presumably with the method of manufacture.

In vivo neutralization of unfractionated heparin and low-molecular-weight heparin by a novel salicylamide derivative.

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are widely used anticoagulants for surgical and interventional use. Currently, the anticoagulant and bleeding effects of heparin are neutralized by protamine sulfate. There are several problems associated with the use of protamine sulfate, including allergic reactions, cardiovascular effects, heparin rebound, and incomplete neutralization of LMWHs. The objective of this investigation is to characterize the effectiveness of a novel salicylamide-derived heparin antagonist, PMX 60056, in neutralizing the antithrombotic and bleeding effects of UFH and LMWHs. Animals were first anticoagulated using an intravenous injection of UFH or LMWH followed by protamine sulfate or PMX 60056. Established animal models of hemorrhage (rat-tail transection) and thrombosis (jugular vein clamping) were then performed. Blood samples were collected for ex vivo analysis using activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa assays. We demonstrate that PMX 60056 neutralized the antithrombotic, anticoagulant, and bleeding effects of heparins as effectively as protamine sulfate and may be slightly more efficacious against LMWHs. These results suggest that PMX 60056 may provide an improved approach for the neutralization of UFH and LMWHs.

CB-01-05-MMX, a novel oral controlled-release low molecular weight heparin for the potential treatment of ulcerative colitis.

CB-01-05-MMX (LMW Heparin MMX), being developed by Cosmo Pharmaceuticals SpA, is a novel oral parnaparin sodium formulation for the potential treatment of ulcerative colitis. At the time of publication, clinical trial data were limited and the mechanism of action had not been elucidated. However, in one phase I and one phase IIb trial in patients with left-sided ulcerative colitis, CB-01-05-MMX had an acceptable safety profile and was not associated with bleeding complications, which is a known side effect of unfractionated heparin and low molecular weight heparin compounds previously evaluated for the treatment of ulcerative colitis. Evaluable efficacy data from the phase IIb trial revealed significant improvements in the clinical activity index values in the CB-01-05-MMX group, but this was not accompanied by mucosal healing. More research, including placebo-controlled trials in patients with left-sided ulcerative colitis and trials in patients with pancolitis, would determine the role of CB-01-05-MMX in the current spectrum of available medications for this condition.

Detection and neutralisation of heparin by a fluorescent ruthenium compound.

Heparin and low-molecular-weight heparin (LMWH) are commonly monitored by determination of activated clotting times or chromogenic assays. Despite their wide use, these assays determine the biological activity and not the concentration of the anticoagulants. They may be inaccurate in some circumstances such as certain disease states. In addition, there is a significant interest in alternative tests for the point-of-care detection of heparin and LMWH. Their binding to small molecules for the detection in biological matrices is poorly explored. We describe here a new optical molecular probe for the detection of LMWH in serum samples. The polycationic ruthenium compound 1 is applicable to the quantification of heparin by monitoring 630 nm fluorescence. In addition, compound 1 is a rare example of a non-polymeric low molecular weight compound which neutralises the anticoagulant activity of heparin and LMWH in plasma samples. Limitation of the method is its low sensitivity currently being improved by structural modification of compound 1.

Mechanism of low-molecular-weight heparin reversal by platelet factor 4.

Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. Within a low range of LMWH concentration (<0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.

[Effect of chronic intramuscular administration of low molecular weight heparin-collagen complex on hemostatic indices and development of alloxan-induced diabetes].

Repeated intramuscular administration of low molecular weight heparin-collagen complex proved to increase fibrinolytic activity and to decrease platelet aggregation in the blood of rats (11 months) with depressed anticoagulant system. Administration of diabetogenic alloxan dose induced no diabetes mellitus in such animals.

Beneficial effect of low-molecular-weight heparin against lipopolysaccharide-induced disseminated intravascular coagulation in rats is abolished by coadministration of tranexamic acid.

OBJECTIVE: We examined the role of coagulation and fibrinolysis in lipopolysaccharide (LPS) induced disseminated intravascular coagulation (DIC) in rats, studying their contribution to fibrin deposition and organ failure in rats with LPS-induced DIC by concurrent administration of low molecular weight heparin (LMWH) with or without tranexamic acid (TA). METHODS: DIC was induced in male Wistar rats by a 4-h infusion of LPS (30 mg/kg) via the tail vein (LPS group). In the LPS+LMWH group LMWH (200 u/kg) was administered to rats from 30 min before the infusion of LPS for 4.5 h. In the LPS+LMWH+TA group LMWH (200 microg/kg) and TA (50 mg/kg) were administered to rats from 30 min before the infusion of LPS for 4.5 h. RESULTS: In the LPS+LMWH group lower plasma levels of TAT, D dimer, creatinine, and alanine aminotransferase were observed, along with less glomerular fibrin deposition and improved survival over rats administered LPS alone. However, these effects of LMWH were completely eliminated and damage beyond that observed in rats administered LPS alone resulted from combined administration of TA (LPS+LMWH+TA group), except that TAT and D dimer levels remained lower than in the group administered LPS alone. CONCLUSIONS: Suppression of fibrinolysis by TA (despite coadministration of LMWH) resulted in increased organ damage in this study, suggesting that depressed fibrinolysis plays a large role in organ failure resulting from LPS-induced DIC, even though hemostatic activation is moderately suppressed by LMWH.

Neutralization of DHG, a new depolymerized holothurian glycosaminoglycan, by protamine sulfate and platelet factor 4.

The neutralization of depolymerized holothurian glycosaminoglycan (DHG), unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH) by protamine sulfate (PS) or platelet factor 4 (PF4) was studied. In in vitro studies, the prolongation of thrombin clotting time (TCT) by these glycosaminoglycans was completely neutralized by PS, whereas activated partial thromboplastin time (APTT) was relatively resistant to neutralization. In rats, prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT ex vivo. Heparin-cofactor-II-dependent antithrombin activity of DHG or UFH was neutralized by PF4 at a high concentration to the same extent; however, prolongation of the APTT by DHG was more resistant to neutralization by PF4 at a physiological plasma level than that by UFH. In conclusion, since the prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT, and since PF4 neutralized the antithrombin activity of DHG, these proteins may be useful as antidotes for DHG to prevent bleeding in case of an overdose.

[Low molecular weight heparins. Implications in anesthesia and resuscitation]. / Heparinas de bajo peso molecular. Implicaciones en anestesia y reanimación.

Low molecular weight heparins are a group of drugs that have only recently been introduced in clinical practice. The are widely used for prophylaxis in thromboembolic disease and are being employed increasingly to treat established venous thrombosis. One way in which these drugs are often used is for prophylaxis in the perioperative period for patients at high risk of developing venous thromboembolism, and the anesthesiologist must therefore be familiar with the main aspects of this application. We review pharmacological characteristics of these drugs as well as the literature on low molecular weight heparins, stressing points of main interest to the anesthesiologist and intensive care recovery unit specialist, namely adverse effects (mainly bleeding) and the implications that use of low molecular weight heparin will have on choice of anesthetic (in particular the dilemma of whether to use local/regional anesthesia).