Thrombin Generation Is a Sensitive Measure of Low Molecular Weight Heparin Anticoagulant Activity.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1099-1099
Author(s):  
Alexander Gatt ◽  
Anne Riddell ◽  
Lesley Lanning ◽  
Saman Aghighi ◽  
Pamala Kanagasapathy ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Coagulation Cascade ◽  
Low Molecular Weight ◽  
Acute Thrombosis ◽  
Coagulation Proteins ◽  
Risk Patients ◽  
The Individual

Abstract Abstract 1099 Current guidelines recommend monitoring of low molecular weight heparins (LMWHs) using the anti-Xa assay in high risk patients like those with renal impairment, pregnancy or overweight and in children. However, it is well accepted that this test does not accurately predict the anticoagulant effect of these drugs1. This is understandable since the various LMWHs available affect the coagulation cascade in different ways relative to their different molecular structure. Notoriously, the larger LMWHs like Tinzaparin have a higher anti-IIa to anti-Xa ratio that is not detected by the anti-Xa assay. We hypothesized that thrombin generation (TG) is a better, more sensitive way how to monitor LMWH anticoagulant activity since it is a measure of the interplay of all coagulation proteins. Blood samples from patients with acute thrombosis, pregnancy and other conditions and having LMWH monitoring were analysed using a chromogenic anti-Xa assay and a TG assay as per Hemker et al2 together with a chromogenic anti-IIa and FVIII clotting assay (FVIII:C). A tissue factor trigger of 10pM Innovin was used for the TG experiments. Patient samples were divided into 2 groups: those on Tinzaparin (n=45) or Enoxaparin (n=39). There was no difference between the FVIII:C levels of the Tinzaparin and Enoxaparin groups (mean 250 vs 327IU/ml P>0.05). A higher lower mean anti-Xa and a higher mean anti-IIa level was achieved with Tinzaparin (0.48 vs 0.63 U/ml P= >0.05 for anti-Xa and 0.3 vs 0.15U/ml P=0.005 for anti-IIa respectively). The endogenous thrombin potential (ETP) was significantly lower with Tinzaparin than Enoxaparin despite a higher anti-Xa (705 vs 1216nM.min P=0.006). Linear regression analysing TG with anti-Xa of the two LMWHs shows that 1.0U/ml antiXa activity for Enoxaparin is as potent as 0.7U/ml Tinzaparin in suppressing TG to similar levels. Significant inter-individual variation in TG suppression was noted with both LMWHs. This study demonstrates that anti-Xa results achieved for different LMWHs do not have the same anticoagulant significance. Using TG, we achieved similar therapeutic anti-Xa levels as achieved from the individual LMWH clinical trials3. It is clear that TG is a better test to predict LMWH anticoagulant activity. This needs to be proven in clinical studies. 1. Baglin T et al British Journal of Haematol. 2006; 133(1): 19–34. 2. Hemker HC et al Pathophysiol Haemost Thromb, 33, 4-15. Boneu B and de Moorloose P. Semin Thromb Hemost 2001; 27(5): 519–522. Disclosures: No relevant conflicts of interest to declare.

In Vitro Characterization of a Homogeneous Low-Molecular Weight Heparin with Reversible Anticoagulant Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4263-4263
Author(s):  
Matthew F Whelihan ◽  
Yongmei Xu ◽  
Jian Liu ◽  
Nigel S. Key
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Thrombin Generation Assay ◽  
In Vitro Characterization ◽  
Fixed Concentration ◽  
Contact Pathway

Abstract Introduction. Due to their increased half-life over unfractionated heparin (UFH) and marked decrease in the incidence of heparin induced thrombocytopenia (HIT), low molecular weight heparins (LMWH) are the most widely prescribed heparin in the US. However owing to their incomplete reversibility with protamine, LMWHs (such as Enoxaparin) carry the risk of bleeding. The synthetic pentasaccharide, Fondaparinux, also lacks a specific antidote. We recently published (Xu et al. Nat. Chem. Biol. 2014) on a new class of synthetic LMWH that is not renal-excreted and offers the benefit of reversal by protamine. The new compound, dubbed “Super 12-mer”, is a 3,483 Da dodecasaccharide consisting of an antithrombin (AT) binding moiety with repeating units of IdoA2S-GlcNS6S (S is sulfate) and two 3-O-sulfate groups which afford the ability to bind protamine. We sought to characterize this new compound in a series of biochemical and global coagulation assays to better characterize its efficacy as a new reversible anticoagulant. Methods. Factor (F) Xa-AT inhibition assays were performed in both purified and plasma-based systems. The Super 12-mer was further tested in a purified prothrombinase system, as well as by tissue factor-initiated thrombin generation assays in contact pathway inhibited citrated plasma. Results. In vitro FXa inhibition studies indicated the IC50 to be 2-fold higher (49 ng/mL, 24 nM) than was previously reported. Nevertheless, the Super 12-mer anti FXa activity was approximately 2-fold greater than Enoxaparin at identical concentrations. However, the anti FXa activities of the Super 12-mer and Enoxaparin in plasma-based systems were roughly equivalent. Prothrombinase experiments indicated that both the Super 12-mer and Enoxaparin were equivalent in their ability to inhibit FXa in complex with FVa. When the two heparinoids were compared in a plasma-based thrombin generation assay (TGA), their effects on thrombin generation were nearly identical with a 50% reduction in peak thrombin generation occurring at approximately 325 nM heparinoid. When protamine is titrated against a fixed concentration of Super 12-mer (625 nM), the Super 12-mer displays a complete reconstitution of thrombin generation. Conclusions. In plasma and purified systems, the Super 12-mer displayed virtually identical efficacy in FXa inhibition compared to Enoxaparin. In buffered systems, the Super 12-mer was approximately 2-fold more active than Enoxaparin against FXa suggesting the Super 12-mer may have other binding partners in plasma. Interestingly, FXa inhibition in prothrombinase was essentially identical between the two heparinoids. Unlike Enoxaparin however, the Super 12-mer displayed near complete reversibility with protamine in TGAs. A significant lag in thrombin generation was observed when protamine was added, consistent with a previous report (Ni Ainle et al. Blood 2009) that protamine itself can act as an anticoagulant by interfering with FV activation. These data show that the Super 12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the fact that this compound can be safely used in renal-impaired patients, the Super 12-mer is a promising new heparanoid anticoagulant with a potentially enhanced safety profile. Disclosures No relevant conflicts of interest to declare.

Direct inhibitors of coagulation proteins – the end of the heparin and low-molecular-weight heparin era for anticoagulant therapy?

2009 ◽  
Vol 102 (11) ◽  
pp. 892-899 ◽  
Author(s):  
Elisabeth Perzborn ◽  
Stefan Heitmeier ◽  
Georges von Degenfeld ◽  
Elke Dittrich-Wengenroth ◽  
Anja Buchmüller ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Vitamin K Antagonists ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Thrombin Inhibitor ◽  
Low Molecular Weight ◽  
Direct Factor ◽  
New Anticoagulants ◽  
Coagulation Proteins ◽  
The Eu

SummaryHeparins, either unfractionated or low-molecular-weight (UFH and LMWHs), and vitamin K antagonists (VKAs) are currently the anticoagulants of choice for the prevention of post-operative venous thromboembolism (VTE) and for the treatment of acute venous and arterial thromboembolism. While VKAs are widely used in the US, LMWHs are the standard of care in the EU. Although efficacious, these agents are associated with a number of drawbacks, such as the risk of heparin-induced thrombocytopenia, the need for frequent coagulation monitoring in the case of UFH and VKAs, and the parenteral mode of administration in the case of heparins, which can lead to problems associated with patient compliance. There is a need for new anticoagulants that overcome these limitations. Direct, small-molecule inhibitors of coagulation proteins targeting a single enzyme in the coagulation cascade – particularly thrombin or Factor Xa – have been developed in recent years. Two agents, the direct thrombin inhibitor dabigatran and the direct Factor Xa inhibitor rivaroxaban, have recently been approved in the EU and several other countries for the prevention of VTE after total hip or knee replacement surgery. Here we will review data that suggest that the antithrombin-independent mechanism of action of these agents, particularly that of direct Factor Xa inhibitors, leads to increased efficacy with similar safety profiles compared with the antithrombin-dependent heparins. Although the end of the heparins era is not to be expected, the new anticoagulants presented in this review potentially represent the future of anticoagulation.

Generic and Branded Low Molecular Weight Heparins Can Be Differentiated Based on Biologic and Pharmacologic Assays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1885-1885
Author(s):  
Walter P. Jeske ◽  
Paul Ackerman ◽  
Amanda Drenth ◽  
Jeanine M. Walenga ◽  
Mamdouh Bakhos
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Complex Nature ◽  
Low Molecular Weight ◽  
Functional Assay ◽  
Low Molecular Weight Heparins ◽  
Biologic Drugs ◽  
Branded Drug

Abstract Several generic versions of the low molecular weight heparins enoxaparin and dalteparin have become available in different parts of the world and several products are currently under review by the FDA. At this time, there are no regulatory guidelines for defining equivalence of complex biologic drugs. Because of their complexity and hybrid nature (biologic and chemical), the available generic versions may not be acceptable by using currently applicable generic criteria. Due to their complex nature, the pharmacologic effect of these may not be similar to the branded product. Therefore, additional methods are needed to equate these drugs with the branded products. In this study, multiple lots of Lovenox or generic enoxaparin such as Lupenox (Lupin Pharma, India), Dripanina (Ariston, Brazil), Dilutol (Lazar, Argentina), Clenox (Pharmayect, Columbia), and Cutenox (Gland Pharma, India) and generic versions of dalteparin such as Daltehep (Gland Pharma, India) were supplemented to human plasma in the absence or presence of protamine sulfate (PS). Anticoagulant activity was measured using the aPTT, Heptest, PiCT assays. Antithrombin and anti-factor Xa activity were assessed by amidolytic assays. Effects on thrombin generation and the activation of thrombin activatable fibrinolytic inhibitor (TAFI) were determined by functional assay. At prophylactic levels of LMWH, no differences in anticoagulant or antiprotease activities were observed between generic and branded LMWHs. At therapeutic concentrations, significantly higher anticoagulant and antiprotease activities were observed with Lovenox. Assay-dependent variations in PS neutralization of antiprotease and anticoagulant activities were observed at higher concentrations of the generic versions of enoxaparin and dalteparin. Significant differences were noted in the effects of these agents on thrombin generation and the activation of TAFI. Current generic LMWHs possess some differences from branded LMWHs in known biologic properties of heparin. Variations in PS neutralization raise the question as to whether the interaction with other plasmatic proteins also differs from that of the branded drug. These results emphasize a need to consider multiple functional parameters when defining bioequivalence of complex biologic drugs and underscore the importance of further pharmacologic studies involving animal models and human clinical trials. Such approaches can be incorporated in the recommendations to develop guidelines for the approval of the generic versions of low molecular weight heparins.

Suppression of Markers of Thrombin Generation and Inflammation in Patients Receiving Low Molecular Weight Heparin Compared to Unfractionated Heparin for TEE-Guided Cardioversion of Atrial Fibrillation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3621-3621
Author(s):  
Debra Hoppensteadt ◽  
Jawed Fareed ◽  
Allan Klein ◽  
Susan Jasper ◽  
Carolyn Apperson-Hansen ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Anticoagulant Effect ◽  
Low Molecular Weight ◽  
Blood Samples ◽  
Markers Of Inflammation

Abstract Introduction: Atrial fibrillation (AF) represents a cardiovascular syndrome which can lead to embolic stroke. Several AF clinical trials have shown a decrease risk of stroke with anticoagulation using warfarin. Currently, there are two approaches for anticoagulation in AF patients. For outpatients, warfarin is used with an INR goal 2.0–3.0. For inpatients requiring cardioversion, unfractionated heparin (UFH, IV) is used as a bridge to therapeutic warfarin. More recently, anticoagulation with a low molecular weight heparin, enoxaparin, has been reported to give a more predictable anticoagulant response than UFH in TEEguided cardioversion. The present study compares the markers of inflammation and thrombin generation in patients included in the ACUTE II study. Methods: This was a randomized multicenter trial of 155 patients from 17 clinical sites, the anticoagulant activity of LMWH (enoxaparin, 1 mg/kg sc bid, Sanofi-Aventis, n=76) was compared to that of UFH (APTT 1.5 – 2.5 x control, n=79). Blood samples were drawn at enrollment (baseline), day 2 (peri cardioversion) and day 4 in both groups. Day 2 and day 4 samples were taken 3–4 hours after the injection of LMWH in patients in the LMWH group. Blood samples were evaluated for inflammation: C-reactive protein (CRP), CD 40 ligand (CD 40L), monocyte chemotactic protein 1 (MCP-1) and anticoagulant effect: thrombin antithrombin complex (TAT) and prothrombin fragment (F 1.2). Results: The APTT and anti-Xa levels indicated therapeutic anticoagulation was achieved (previously reported). In addition, both TAT and F1.2 levels were increased at baseline and were significantly decreased with LMWH (p<0.01) in comparison to UFH. The CRP, CD40L, MCP-1 were decreased after treatment in both groups of patients. These results suggest that AF patients treated with LMWH demonstrate a stronger anticoagulant effect as measured by a significant reduction in the markers of thrombin generation. Furthermore, anticoagulation with either UFH of LMWH results in a decrease in inflammation which was not different between groups. Conclusion: In the ACUTE II trial, anticoagulation with LMWH in AF patients may be a better alternate than UFH during TEE-guided cardioversion due to a a stronger anticoagulant.

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4324-4324
Author(s):  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Jeanine M. Walenga ◽  
Josephine Cunanan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Confidence Interval ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Serotonin Release ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Safety And Efficacy ◽  
Response Data

Abstract Abstract 4324 Introduction: Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods: Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results: A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions: These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures: Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

1993 ◽  
Vol 70 (04) ◽  
pp. 625-630 ◽  
Author(s):  
Edward Young ◽  
Benilde Cosmi ◽  
Jeffrey Weitz ◽  
Jack Hirsh
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Plasma Proteins ◽  
Low Molecular Weight Heparin ◽  
Specific Binding ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

Characterisation of Persistent Anti-Xa Activity Following Administration of the Low Molecular Weight Heparin Enoxaparin Sodium (Clexane)

1994 ◽  
Vol 72 (02) ◽  
pp. 275-280 ◽  
Author(s):  
David Brieger ◽  
Joan Dawes
Keyword(s):  
Molecular Weight ◽  
Enoxaparin Sodium ◽  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Active Material ◽  
Biologically Active ◽  
Size Exclusion ◽  
Low Molecular Weight ◽  
Exclusion Chromatography ◽  
Liver And Kidney

SummaryIt is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected 125I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

Preparation of low molecular weight Sargassum fusiforme polysaccharide and its anticoagulant activity

2018 ◽  
Vol 36 (3) ◽  
pp. 882-891 ◽  
Author(s):  
Yuhao Sun ◽  
Xiaolin Chen ◽  
Song Liu ◽  
Huahua Yu ◽  
Rongfeng Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Sargassum Fusiforme
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