USP Standardized Mixtures of Bovine, Ovine and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single Sourced Heparins and May be Interchangeable,

Abstract Introduction: Unfractionated heparin (UFH) is the first line anticoagulant for the management of medical indications. UFH complexes with antithrombin to produce strong inhibition of thrombin and factor Xa. The UFHs are standardized using USP compliant amidolytic anti-Xa and IIa methods in defined conditions. Clinically used UFH is solely sourced from porcine mucosal tissue. Because of the shortage of porcine tissue and the African Swine Fever, the supply chain of this anticoagulant is compromised. Thus, there is a need for resourcing of this anticoagulant. Bovine and ovine mucosal sources represent alternate material for production of UFH. Previous studies have shown that bovine and ovine UFH exhibit anticoagulant effects which can be standardized by using the USP method. Additionally, the standardized heparins from various sources can be blended and their potency can be adjusted to exhibit comparable effects as the single sourced UFH. The purpose of this study is to evaluate the pharmacologic profile of the blended heparin and compare these activities to that of the single sourced porcine, ovine and bovine heparins. Methods: Two groups of heparins were evaluated in this study, porcine, ovine, bovine, and the blended heparin in gravimetric measurements (ug/ml) and these same four in potency adjusted measurements (U/ml). The pharmacologic profiles of the heparins in this study were investigated via global anticoagulant assays and anti-protease assays performed in plasma. Clot based assays such as the activated partial thromboplastin time (aPTT) and thrombin time (TT) were used to study the anticoagulant effects of the single source and blended heparins. The amidolytic anti-Xa and IIa assays were used to assess the inhibitory effects of these heparins on these proteases. USP compliant anti-Xa and IIa assays were used to determine potencies of the various heparins. Protamine sulfate (PS) neutralization studies were performed to evaluate the reversal of anticoagulant effects in each of the heparins. Results: The aPTT assay showed that at final concentrations of 5 ug/ml and 2.5 ug/ml porcine heparin significantly (p < .01) prolonged the aPTT compared to ovine, bovine, and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable aPTT values at all concentrations (U/ml). The TT assay showed that porcine and ovine heparins prolonged the TT at 1.25 ug/ml compared to bovine and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable TT values at all concentrations (U/ml). The anti-Xa assay showed that at all final concentrations between 10 ug/ml and 0.625 ug/ml porcine, ovine, and blended heparins produced significantly (p <.001) stronger Xa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-Xa inhibition at all concentrations (U/ml). The anti-IIa assay showed that at final concentrations 2.5 ug/ml, 1.25 ug/ml, and 0.625 ug/ml porcine and ovine heparins produced significantly (p < .05) stronger IIa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-IIa inhibition at all concentrations (U/ml). The USP compliant anti-Xa assay with gravimetric heparins showed potencies of 201, 201, 150, and 184 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-Xa assay with potency adjusted heparins showed comparable potencies for all four heparins. The USP compliant anti-IIa assay with gravimetric heparins showed potencies of 204, 196, 127, and 167 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-IIa assay with potency adjusted heparins showed comparable potencies for all four heparins. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for all of the potency adjusted heparins in the aPTT, TT, anti-Xa, and anti-IIa assays. Conclusion: These studies support the hypothesis that a blended heparin product from bovine, ovine, and porcine tissue, when standardized in USP unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species heparins. These findings suggest that there is a potential for development of blended heparin to stabilize supply chain of this important anticoagulant and warrant clinical validation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Management of Large Intentional Overdose of Enoxaparin with Protamine Sulfate

Blood ◽

10.1182/blood.v118.21.4330.4330 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4330-4330

Author(s):

Samantha N Gomez ◽

Jack B. Alperin ◽

Wendy L Russo ◽

Barbara J. Bryant

Keyword(s):

Plasma Levels ◽

Clinical Symptoms ◽

Conflicts Of Interest ◽

Peak Plasma ◽

Factor Xa ◽

Protamine Sulfate ◽

Management Decision ◽

Self Administration ◽

Laboratory Assessment ◽

Emergency Situations

Abstract Abstract 4330 Introduction: Enoxaparin, a low molecular weight heparin (LMWH), is used for prophylaxis and treatment of venous and arterial thromboembolism. Due to its safety profile, there has been little literature regarding management and treatment of overdoses. Two reports of enoxaparin overdose have been described in the literature; a neonate received a 10-fold overdose and a 64-year old man received an extra daily dose. Both cases were treated with an approximate 1 to 1 ratio of protamine sulfate to LMWH (30 mg and 20 mg, respectively) and showed incomplete reversal of anticoagulation. Plasma levels of LMWH can be monitored by measuring anti-factor Xa levels; however, studies correlating large overdoses have not been performed. Peak plasma levels of LMWH are found 3–4 hours after administration, and the half-life of the drug is 4.5–7 hours. Anti-factor Xa levels, however, are not readily available at most institutions. We report a challenging case of a large overdose of LMWH, successfully treated despite the lack of appropriate laboratory monitoring tests. Case Report: A 50-year old Caucasian man with a past medical history significant for bipolar disorder, schizophrenia, hypertension, and pulmonary embolism secondary to a hypercoagulable state caused by tetrahydrofolate reductase deficiency was found unresponsive and transported to the emergency department. Upon admission, he reported self-administration of 20 subcutaneous injections of 100 mg of enoxaparin (2000 mg) and 8 tablets of quetiapine (400 mg/tablet) in an attempt to commit suicide. He was somnolent but arousable with bruising noted around multiple injection sites on his abdomen. The remainder of his physical exam was unremarkable, and he exhibited no overt signs of bleeding. Initial laboratory assessment, approximately 2 hours post-suicide attempt, revealed an elevated aPTT of >150 seconds (reference range 23–38 seconds). Anti- factor Xa levels were not available at the time of treatment. An initial dose of protamine sulfate, 250 mg mixed in 500 ml of 0.9% sodium chloride, was infused over 3–4 hours. Repeat aPTT was 61 seconds. A second infusion of protamine sulfate was administered 2 hours later, and the aPTT decreased further to 49 seconds. Rebound phenomenon was noted 3–8.5 hours later as the aPTT increased (70 to 89 seconds), however, no additional protamine sulfate was given. At 24 hours, the aPTT was 46 seconds. The patient did not exhibit any signs of bleeding during treatment, and tolerated the infusions of protamine sulfate without any adverse effects. Subsequent testing for anti-factor Xa revealed an initial level of 4.72 IU/mL upon admit (therapeutic range 0.50 – 1.10 IU/mL), decreasing to 4.52 and 3.82 IU/mL after the infusions of protamine sulfate. The anti-factor Xa level at 24 hours was 1.39 IU/mL. Conclusion: The reversal of an enoxaparin overdose is challenging and frequently difficult to manage. Reversal with protamine sulfate is often incomplete, and infusion of the recommended dosage of 1 mg for each 1 mg of enoxaparin can be risky in large dosages. Ideally, Factor Xa levels, when available, can assist in management decision, however in emergency situations as described here, the aPTT and clinical symptoms must be used to guide treatment. Disclosures: No relevant conflicts of interest to declare.

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Idarucizumab, a Specific Antidote for Dabigatran, Cross-Reacts with Melagatran and May Also Interact with Other Benzamidine-Containing Compounds

Blood ◽

10.1182/blood.v128.22.3836.3836 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3836-3836

Author(s):

Jawed Fareed ◽

Larissa Reikensmeyer ◽

Amanda Walborn ◽

Debra Hoppensteadt ◽

Jeanine M. Walenga ◽

...

Keyword(s):

Whole Blood ◽

Conflicts Of Interest ◽

Dabigatran Etexilate ◽

Factor Xa ◽

Cross Reactivity ◽

Thrombin Time ◽

Fab Fragment ◽

Strong Concentration ◽

Procoagulant Effect ◽

Us Fda

Abstract Introduction: Dabigatran etexilate is a pro-drug which is used to prevent embolic stroke in patients with atrial fibrillation. This oral anticoagulant is also approved for other indications in Europe. As with all anticoagulants, there is a potential for serious hemorrhage with dabigatran usage which may require antidotes to control bleeding. Idarucizumab is an anti-dabigatran Fab fragment (Boehringer-Ingelheim) that binds to the benzamidine group on dabigatran and inhibits its anti-thrombin activity. Idarucizumab has recently been approved by the US FDA for the control of bleeding associated with dabigatran. Materials and Methods: Such antithrombin agents as argatroban, melagatran, hirudin, and bivalirudin, human antithrombin, thrombomodulin, heparin cofactor II, and heparin-AT complex were commercially obtained. Anti-factor Xa agents (rivaroxaban, apixaban and DX-9065a were also obtained from various sources To test the specificity of the inhibitory effects of idarucizumab, each of these agents were supplemented to whole blood and citrated plasma at concentrations ranging from 0.1 to 100 µg/mL. Idarucizumab was added to each mixture at a concentration of 1 mg/mL and anticoagulant activities were assessed using PT, aPTT, thrombin time and chromogenic anti-IIa/Xa and flurometric thrombin generation assays. Results: Idarucizumab itself did not produce any anticoagulant effects on whole blood or plasma clotting profile. However it showed a slight procoagulant effect in the whole blood and plasma based assays. It produced a strong concentration dependant inhibition of both dabigatran and melagatran. The antibody showed strong specificity for the inhibition of dabigatran amd melagatran and did not affect the anticoagulant and other effects of the other synthetic and natural thrombin and FXa inhibitors. The prolongation of the PT, APTT and thrombin time by melagatran was completely inhibited by idarucizumab. Idarucizumab more effectively inhibited the prolongation of thrombin time by dabigatran than the prolongation induced by melagatran. Discussion: The cross-reactivity of idarucizumab with melagatran may result from the presence of a common benzamidine pharmacophore which is present in both of these anticoagulant agents. Since the benzamidine pharmacophore is present in a number of serine protease inhibitors as well as drugs such as pentamidine, propamidine and dibromopropamidine. These observations suggest that simultaneous administration of idarucizumab may compromise the pharmacodynamics profile of benzamidine derived drugs such as the anti-malarials, anti-psychotic, anti-fungal and other compounds. Thus there is a need for a systemic screening of idarucizumab for its potential interactions with drugs containing benzamidine based therapeutic agents. Disclosures No relevant conflicts of interest to declare.

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Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Blood ◽

10.1182/blood-2019-127072 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1158-1158

Author(s):

Fakiha Siddiqui ◽

Alfonso J Tafur ◽

Debra Hoppensteadt ◽

Jeanine Walenga ◽

Walter Jeske ◽

...

Keyword(s):

Research Funding ◽

Thrombin Generation ◽

Surrogate Marker ◽

Factor Xa ◽

Bleeding Complications ◽

Dependent Manner ◽

Thrombin Time ◽

Inhibitory Effects ◽

Biologic Effects ◽

Inhibition Of Thrombin

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

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Lyso-Sulfatide Binds Factor Xa and Potently Inhibits Thrombin Generation.

Blood ◽

10.1182/blood.v116.21.1130.1130 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1130-1130

Author(s):

Subramanian Yegneswaran ◽

Yajnavalka Banerjee ◽

Jose A. Fernandez ◽

Hiroshi Deguchi ◽

John H. Griffin

Keyword(s):

Free Amino ◽

Conflicts Of Interest ◽

Factor Xa ◽

Coagulation System ◽

Amino Group ◽

Thrombin Time ◽

Binding Studies ◽

Binding Interaction ◽

Dependent Inhibition ◽

Gla Domain

Abstract Abstract 1130 Although phospholipids are well-recognized for their effects on coagulation reactions, little is generally known about the effects of sphingolipids on clotting pathways. Negatively-charged sulfatides can potently initiate the intrinsic pathway of coagulation system by binding and autoactivating factor (f) XII. Sphingosine potently inhibits the ability of factor Xa (fXa) to generate thrombin (fIIa) in the prothrombinase complex (II-ase) (fXa/fVa/phospholipids) by interacting directly with fXa's Gla domain. Here we report that lyso-sulfatide (lyso-SF) (sulfogalactosyl sphingosine), a lipid of minor abundance in plasma that is primarily in HDL particles, exhibits potent anticoagulant activity. Lyso-SF dose-dependently prolonged clotting in fXa-1-stage but not thrombin-time clotting assays. Lyso-SF inhibited II-ase activity by > 90 % in purified reaction mixtures (fXa/fVa/II) in the presence of 6 or 30 μM phospholipids (PL). However, lyso-SF did not inhibit fIIa generation by fXa/fVa in the absence of PL, suggesting the absolute requirement of PL for lyso-SF-dependent inhibition of fIIa generation. Lyso-SF inhibited fIIa generation by fXa/PL in the absence of fVa. Additionally, lyso-SF inhibited fIIa generation by Gla-domainless (gd)-fXa in the presence but not in the absence of fVa and PL. Lyso-SF-dependent inhibition of fIIa generation was also observed for fXa/fVa/PL when gd-II was used as the substrate instead of II. However, no inhibition by lyso-SF was observed when using gd-fXa/PL and gd-II/PL in the presence or absence of fVa. Lyso-SF had no effect on fXa or fIIa amidolytic activity. These data plus other studies suggested that ≥ two components of the II-ase complex needed to be PL-bound for potent inhibition of fIIa generation by lyso-SF. PL surfaces bind and assemble each the II-ase protein components; however, PL's and lyso-SF may also alter the conformations of fXa, fVa and II. To gain mechanistic insights for lyso-SF inhibition of II-ase activity, Surface Plasmon Resonance (SPR) and fluorescence spectroscopy were used to define molecular interactions. Remarkably, SPR binding studies showed that lyso-SF binds to immobilized fXa (KD = 83 μM) and gd-fXa (KD = 36 μM). Controls using SPR showed no binding of lyso-SF to immobilized fVIIa or fIXa whereas SPR confirmed the ability of fXa, fVIIa and fIXa to bind PL's. Fluorescence binding assays confirmed SPR data showing that lyso-SF bound to and altered the dansyl fluorescence of dansyl-GluGlyArg-labeled fXa (DEGR-fXa) both in the presence (KD = 50 μM) and absence (KD = 75 μM) of PL and that this binding required calcium ions. Thus, lyso-SF binds fXa outside the Gla domain. Fluorescence monitoring of fVa binding to DEGR-fXa in the presence of PL showed that lyso-SF inhibited this binding interaction. To characterize structure-activity relationships for lyso-SF inhibition of II-ase, different analogs of lyso-SF were tested for their ability to inhibit fIIa generation by gd-fXa/fVa/PL. Psychosine (galactosyl sphingosine), glucosyl sphingosine and lyso-sphingomyelin each inhibited fIIa generation showing that the sulfate ester moiety and the sugar group in lyso-SF were not essential for the anticoagulant effects of lyso-SF. However, acetylation of the free amino group in lyso-SF ablated its inhibition of fIIa generation showing that the free amino group on carbon 2 is essential for the inhibitory activity of lyso-SF. In conclusion, these findings show that lyso-SF and several of its analogs are potent anticoagulant lipids and that the mechanism for inhibition of fXa by lyso-SF may involve its binding to fXa at sites outside fXa's Gla domain. This suggests that certain sphingolipids may exert allosteric downregulation of fXa activity without inhibiting the enzyme's active site or the binding of the Gla domain to PL surfaces. Disclosures: No relevant conflicts of interest to declare.

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Management of Major Bleeding Caused By Rivaroxaban and the Use of Desmopressin

Blood ◽

10.1182/blood.v124.21.5099.5099 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5099-5099

Author(s):

Ahmad Jajeh

Keyword(s):

Major Bleeding ◽

Large Scale ◽

Conflicts Of Interest ◽

Factor Xa ◽

Fresh Frozen Plasma ◽

Thrombin Time ◽

Gi Bleeding ◽

Complex Product ◽

Packed Red Blood Cells ◽

Treatment And Prevention

Abstract Rivaroxaban is a new anticoagulant that is substituted for Coumadin on a large scale in the treatment and prevention of Deep Vein Thrombosis DVT and Pulmonary Embolism PE. It is an oral agent that inhibits Factor Xa. The most attractive attribute of this new anticogulant is the lack of monitoring PT/INR. However, out of many cases put on Rivaroxaban a few reports of major and threatening bleed that could be fatal. Particularly, the the GI bleeding. Unfortunately, no set standard antidote or management is available when such catastrophic bleeds happen. This abstract present our experience with three major bleeding cases that presented with massive GI bleeding. Two are associated with peptic ulcer upon Upper GI endoscopy. Two males and one female age 60, 71 (males) and 71 (female). The first two patients were treated with Prothrombin complex product. The female patient presented with sever anemia of 4 grams of Hb with hematemesis and bright red blood per rectum. The Prothrombin complex product was not readly available . She was given multipe doses of Fresh Frozen Plasma FFP and multiple units of packed red blood cells. She was also given a product Profilnine which contains Factor II, IX and VII. Patient's coagulation profile of PTT, PT and Thrombin time were corrected. However, she continue to have bright blood per NG suction. Upon receiving D-DAVP Desmopressin 0.3 micrograms per Kg she stopped bleeding and EGD was done later with sclerosing treatment of gastric ulcer and ligation. Patient was given later a small dose of Prothrombine complex when was available since the last dose of Rivaroxaban was given less than 13 hours from her presentation to the hospital. All of the mentioned patients had prolongation of PT/INR/PTT at presentation. Thrombin time was monitored in all of them. All patients had survived the magor GI bleeding. D-DAVP were given to all of them. In conclusion D-DAVP Desmopressin should be considered as an adjuvant drug in patient presentong with major GI bleeding secondary to Rivaroxaban. Disclosures No relevant conflicts of interest to declare.

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The Venous Antithrombotic Profile of Naroparcil in the Rabbit

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648977 ◽

1994 ◽

Vol 72 (06) ◽

pp. 874-879 ◽

Cited By ~ 11

Author(s):

Jean Millet ◽

Jocelyne Theveniaux ◽

Neil L Brown

Keyword(s):

Oral Administration ◽

Bleeding Time ◽

Dermatan Sulfate ◽

Factor Xa ◽

Bleeding Risk ◽

Thrombin Time ◽

Heparin Cofactor Ii ◽

Antithrombotic Activity ◽

Thrombin Inhibition ◽

Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

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Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649704 ◽

1993 ◽

Vol 70 (06) ◽

pp. 0942-0945 ◽

Cited By ~ 18

Author(s):

Job Harenberg ◽

Marietta Siegele ◽

Carl-Erik Dempfle ◽

Gerd Stehle ◽

Dieter L Heene

Keyword(s):

Tissue Factor ◽

Binding Sites ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Factor X ◽

Rebound Phenomenon ◽

Tissue Factor Pathway ◽

Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

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The Measurement of Heparin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649125 ◽

1973 ◽

Vol 30 (03) ◽

pp. 471-479 ◽

Cited By ~ 31

Author(s):

K. W. E Denson ◽

John Bonnar

Keyword(s):

Degradation Products ◽

Factor Xa ◽

Assay Method ◽

Thrombin Time ◽

Fibrinogen Degradation Products ◽

Low Levels

SummaryA method for the measurement of heparin utilising the potentiating effect of heparin on the action of anti-factor Xa is described. The effect on the assay of platelet contamination of plasma, the presence of fibrinogen degradation products and low levels of anti-factor Xa have been studied. The assay method has been compared with the calcium thrombin time method and a group of obstetrical patients have been studied using both methods.

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Chrono-Pharmacological Study of Once Daily Curative Dose of a Low Molecular Weight Heparin (200IU antiXa/kg of Dalteparin) in Ten Healthy Volunteers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649794 ◽

1995 ◽

Vol 74 (02) ◽

pp. 660-666 ◽

Cited By ~ 15

Author(s):

P Mismetti ◽

J Reynaud ◽

B Tardy-Poncet ◽

S Laporte-Simitsidis ◽

M Scully ◽

...

Keyword(s):

Molecular Weight ◽

Healthy Volunteers ◽

Low Molecular Weight Heparin ◽

Pharmacological Study ◽

Area Under The Curve ◽

Factor Xa ◽

Similar Efficacy ◽

Low Molecular Weight ◽

Thrombin Time ◽

Once Daily

SummaryLow molecular weight heparin (LMWH) is currently prescribed for the treatment of deep vein thrombosis at the dose of 100 IU antiXa/kg twice daily or at a dose of 175 IU antiXa/kg once daily with a similar efficacy. We decided to study the chrono-pharmacology of curative dose of LMWH once daily administrated according to the one previously described with unfractionated heparin (UFH).Ten healthy volunteers participated in an open three-period crossover study according to three 24 h cycles, separated by a wash-out interval lasting 7 days: one control cycle without injection, two cycles with subcutaneous injection of 200 IU antiXa/kg of Dalteparin (Fragmin®) at 8 a.m. or at 8 p.m. Parameters of heparin activity were analysed as maximal values and area under the curve.Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and tissue factor pathway inhibitor (TFPI) were higher after 8 p.m. injection than after 8 a.m. injection (p <0.05) while no chrono-pharmacological variation of anti factor Xa (AXa) activity was observed. Thus the biological anticoagulant effect of 200 IU antiXa/kg of Dalteparin seems to be higher after an evening injection than after a morning injection.A chrono-therapeutic approach with LMWH, as prescribed once daily, deserves further investigation since our results suggest that a preferential injection time may optimise the clinical efficacy of these LMWH.

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Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657178 ◽

1982 ◽

Vol 47 (03) ◽

pp. 244-248 ◽

Cited By ~ 47

Author(s):

D P Thomas ◽

Rosemary E Merton ◽

T W Barrowcliffe ◽

L Thunberg ◽

U Lindahl

Keyword(s):

Antithrombin Iii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Blood Levels ◽

Thrombin Time ◽

High Affinity ◽

Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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