scholarly journals Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation Via Direct Targeting of Factor IXa: Contribution of the Serpin-Independent Mechanism

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2242-2242
Author(s):  
Yang Buyue ◽  
John P. Sheehan
Keyword(s):  
Human Plasma ◽  
Mechanism Of Action ◽  
Thrombin Generation ◽  
Molecular Target ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Fluorogenic Substrate ◽  
Factor Ixa ◽  
Inhibition Of Thrombin

Abstract Abstract 2242 The relevance of factor IXa as a therapeutic target for heparin therapy remains incompletely defined. To evaluate the contribution of factor IXa inhibition, particularly serpin-independent inhibition of the intrinsic tenase complex (factor IXa-factor VIIIa), the ability of heparin preparations to inhibit tissue factor (TF)-triggered thrombin generation was examined in human plasma. Thrombin generation was determined by a fluorogenic substrate assay employing Technothrombin TGA evaluation software to determine lag time, time to thrombin peak, peak thrombin concentration, and velocity index (slope). The inhibitory potency (EC50) of each heparin was determined by plotting concentration versus relative velocity index (ratio of the velocity index in presence and absence of heparin). Inhibition of thrombin generation was initially compared under factor IX-dependent (limiting TF) and independent (excess TF) conditions, respectively, by addition of either 0.2 or 4 pM TF to pooled normal human plasma containing increasing concentrations of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, or unfractionated heparin (UFH). UFH and Fondaparinux demonstrated complete or near complete inhibition with identical EC50 values at both tissue factor concentrations, suggesting that inhibition of intrinsic tenase activity does not significantly contribute to their mechanism of action in plasma. In contrast, LMWH and ssLMWH demonstrated 2.9- and 5.1-fold lower EC50 values, respectively, in the presence of the limiting TF concentration. These results suggest that inhibition of intrinsic tenase activity contributes to the mechanism of action for LMWH in plasma. The effect of heparins on the time course of plasma thrombin generation by Western blotting under similar conditions correlated well with results of the fluorogenic substrate assay. At their EC50 values, LMWH, ssLMWH and Fondaparinux clearly reduced prothrombin/meizothrombin consumption in plasma, while UFH primarily accelerated formation of the thrombin-antithrombin complex. The contribution of antithrombin to inhibition of thrombin generation was determined by comparison of antithrombin- and mock-depleted human plasmas. As expected, antithrombin depletion markedly increased the EC50 values for UFH (62–fold) and Fondaparinux (42-fold) to levels that are ∼3–8 fold higher than their expected therapeutic ranges, confirming an antithrombin-dependent mechanism of action. In contrast, antithrombin depletion increased the EC50 values more modestly for LMWH (9.4-fold) and ssLMWH (2-fold), with the EC50 for LMWH (∼ 0.7 U/ml) remaining within the therapeutic range. In the absence of antithrombin, LMWH demonstrated partial inhibition of plasma thrombin generation with a plateau representing ∼8–10% of the starting activity, remarkably similar to the inhibition of intrinsic tenase activity with purified components. The molecular target for LMWH and ssLMWH was evaluated in dual factor IX/antithrombin-depleted plasma supplemented with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Based on both relative EC50 values for reduction in the velocity index and the maximal degree of inhibition, plasma supplemented with mutant factor IX demonstrated relative resistance to inhibition of thrombin generation by LMWH as follows: R233A > (K126A/R165A/K132A) > R170A > WT. Factor IX R233A demonstrated an 11.4-fold increase in the EC50 for LMWH relative to the wild type protease. The magnitude of differences between mutant factor IX proteins was reduced for ssLMWH relative to LMWH, but the rank order was similar. The effect of these mutations in human plasma recapitulates their effects on in vitro inhibition of intrinsic tenase activity, confirming the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of thrombin generation by LMWH and ssLMWH. The ability of therapeutic concentrations of LMWH to directly target factor IXa in antithrombin-depleted plasma suggests that this mechanism may contribute to antithrombotic effects. Furthermore, chemo-enzymatic synthesis of “non-anticoagulant” LMWH preparations acting via this exosite-mediated antithrombotic mechanism may offer advantages in high-risk populations, particularly patients with underlying malignancy. Disclosures: Buyue: Biogen Idec Hemophilia: Employment.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3081-3081
Author(s):  
Buyue Yang ◽  
John P. Sheehan
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Factor Ix ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Recombinant Factor Ix ◽  
Inhibition Of Thrombin

Abstract Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A> R170A> WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

scholarly journals Fucosylated chondroitin sulfate inhibits plasma thrombin generation via targeting of the factor IXa heparin-binding exosite

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3092-3100 ◽  
Author(s):  
Yang Buyue ◽  
John P. Sheehan
Keyword(s):  
Factor Viii ◽  
Chondroitin Sulfate ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Heparin Binding ◽  
Zymogen Activation ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Half Maximal Effective Concentration ◽  
Inhibition Of Thrombin

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC50) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mechanism. In the presence of excess TF, the EC50 for DHG was increased 13- to 27-fold, suggesting inhibition was dependent on intrinsic tenase (factor IXa-factor VIIIa) components. In factor VIII–deficient plasma supplemented with 700 pM factor VIII or VIIIa, and factor IX–deficient plasma supplemented with plasma-derived factor IX or 100 pM factor IXa, the EC50 for DHG was similar. Thus, cofactor and zymogen activation did not contribute to DHG inhibition of thrombin generation. Factor IX–deficient plasma supplemented with mutant factor IX(a) proteins demonstrated resistance to DHG inhibition of thrombin generation [factor IX(a) R233A > R170A > WT] that inversely correlated with protease-heparin affinity. These results replicate the effect of these mutations with purified intrinsic tenase components, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition by DHG. Glycosaminoglycan-mediated intrinsic tenase inhibition is a novel antithrombotic mechanism with physiologic and therapeutic applications.

scholarly journals The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3234-3241 ◽  
Author(s):  
Yang Buyue ◽  
Herbert C. Whinna ◽  
John P. Sheehan
Keyword(s):  
Saphenous Vein ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Factor Ix ◽  
Heparin Binding ◽  
Wild Type ◽  
Factor Ixa ◽  
Plasma Factor ◽  
The Times

Abstract The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the protease-factor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4- to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2- to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl3-induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P ≤ .003), and more than 45 minutes. These data support the role of the factor IXa heparin-binding exosite as a critical regulator of coagulation and novel antithrombotic target.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IXa in Activated Prothrombin Complex Concentrates Is the Dominant Contributor to Enhanced Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
State Level ◽  
Peak Level ◽  
Coagulation Factors ◽  
Major Effect ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa ◽  
Activated Prothrombin Complex Concentrates

Background. Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa and factor X has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to treating this bleeding in hemophilia A patients with inhibitors is to give an activated prothrombin complex concentrate (APCC; FEIBA) (disfavored in NHF MASAC #255). APCC contains a number of coaguation factors including prothrombin, factor X (FX), and factor IX (FIX). APCC also contains activated factor X (FXa) and factor IX (FIXa). Previous work has shown that when APCCs are added to hemophilia A plasma containing emicizumab there is a significant increase in thrombin generation [J Thromb Haemost 2018;16:1580-1591]. The goal of this work was to study thrombin generation in hemophilia A plasma with emicizumab and to examine the role of the zymogen and activated components of an APCC in the increased thrombin generation. Methods. In hemophilia A plasma, thrombin generation assays were done using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). The components of APCC were studied at concentrations that should mimic the levels seen in the plasma of a patient given a dose of 50 U/kg: prothrombin 1800 nM; FX 130 nM; FIX 90 nM; and FIXa 0.4 nM. Results. When initiated with low TF, hemophilia A plasma alone had essentially no thrombin generation. As expected, adding emicizumab enhanced thrombin generation. The addition of zymogen coagulation factors, prothrombin, FIX, and FX, separately or together gave a small increase in thrombin generation. However, addition of FIXa to emicizumab gave a large increase in peak thrombin. In hemophilia A plasma with emicizumab and FIXa, addition of prothrombin further increased thrombin generation and specifically increased the peak level of thrombin. Further addition of FX or FIX, separately or together, only minimally increased thrombin generation. Discussion. The strong contribution of factor IXa to the effects of APCCs on thrombin generation in hemophilia A plasma depends on the presence of emicizumab. In the absence of emicizumab, a study of the individual components of APCC showed that a combination of FXa and prothrombin at levels found in APCCs had the major effect on thrombin generation [Haemophilia. 2016;22:615-24]. That study found that FIXa did not increase thrombin generation. However, in the presence of emicizumab, despite the weak solution phase affinity of the bifunctional antibody for FIXa, small amounts of FIXa were the most significant contributor to thrombin generation. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

Differential inhibition of thrombin generation by vitamin K antagonists alone and associated with low-molecular-weight heparin

2009 ◽  
Vol 102 (07) ◽  
pp. 42-48 ◽  
Author(s):  
Grigoris T. Gerotziafas ◽  
Charlotte Dupont ◽  
Alex C. Spyropoulos ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Vitamin K ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Vitamin K Antagonists ◽  
Low Molecular Weight ◽  
Plasma Samples ◽  
Thrombin Generation Assay ◽  
International Normalised Ratio ◽  
Inhibition Of Thrombin

SummaryVitamin K antagonists (VKA) treatment starts with co-administration of low-molecular-weight heparin (LMWH). The anticoagulation induced by the two drugs is still not well determined. In the present study we used thrombin generation assay to evaluate the hypo-coagulation induced by treatment with VKA and by the combination of VKA with LMWH. Tissue factor triggered thrombin generation in platelet-poor plasma was assessed in samples from 15 healthy volunteers, 97 samples from patients treated with VKA and 41 samples from patients receiving enoxaparin and VKA. Patients were classified according to international normalised ratio (INR) level (<2, 2–3 and >3).In plasma samples from patients treated with VKA having INR 2–3 the inhibition of thrombin generation reached 50% compared to controls. In samples with INR>3 this inhibition was 80%. In samples from patients receiving both LMWH and VKA, thrombin generation was significantly decreased compared to the controls and VKA group. In samples with an INR 2–3 obtained from patients treated with LMWH and VKA, the inhibition of thrombin generation was similar to that observed in samples with an INR>3 obtained from VKA treated patients. Thrombin generation assay is sensitive to detect the global the anticoagulant effect produced by the association of LMWH and VKA. For equal INR dual anticoagulant treatment induces significantly more profound inhibition of thrombin generation compared to treatment with VKA alone. The clinical relevance of this observation merits to be studied in prospective studies in patients with defined indications of anticoagulant therapy.

Effect Of Combined Heparin- and Antithrombin-Binding Exosite Mutations On Human Factor IX(a) Coagulant Activity, Thrombin Generation, and Protease Half-Life In Human Plasma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2333-2333
Author(s):  
Pamela R. Westmark ◽  
Pansakorn Tanratana ◽  
John P. Sheehan
Keyword(s):  
Human Plasma ◽  
Half Life ◽  
Research Funding ◽  
Thrombin Generation ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulant Activity ◽  
Plasma Half Life

Abstract Introduction Hemophilia B is an X-linked genetic disorder characterized by defective factor IX activity. Recombinant factor IX (rFIX) is employed as protein replacement for the treatment and prophylaxis of bleeding episodes. Antithrombin is the primary plasma inhibitor of activated factor IX (FIXa), and inhibition is enhanced by heparin/heparan sulfate. We hypothesize that selective disruption of protease interactions with heparin and antithrombin via mutations in the respective heparin- and antithrombin-binding exosites may enhance rFIX(a) efficacy by prolonging protease half-life in vivo. Aim To assess the effect of mutations in the FIX(a) heparin- and antithrombin-binding exosites on traditional coagulant activity, thrombin generation, and protease half-life in human plasma. Methods Human FIX cDNA constructs with alanine substitutions (chymotrypsinogen numbering) in the heparin exosite (K126A, K132A, K126A/K132A), antithrombin exosite (R150A), or both (K126A/R150A, K132A/R150A, K126A/K132A/R150A) were expressed in HEK293 cell lines. Recombinant zymogens were purified from conditioned media, and a portion activated to protease with human factor XIa. Zymogen and protease forms were characterized in APTT-based clotting assays, and tissue factor (TF) and FIXa-initiated thrombin generation (TG) assays in pooled human FIX-deficient plasma, respectively. Comparisons were made with human plasma-derived factor IX (pFIX) and recombinant FIX wild type (WT). Protease half-life in pooled, citrated human plasma was determined using a novel assay that detects FIXa activity by TG response. Results Zymogen coagulant activities (% WT ± S.E) were: pFIX 105.2 ± 2.8, WT 100 ± 7.1, K132A/R150A 75.8 ± 3.4, K126A 63.3 ± 2.3, R150A 62.4 ± 4.0, K132A 30.9 ± 1.0, K126A/R150A 27.0 ± 2.1, K126A/K132A 20.6 ± 9.2, and K126A/K132A/R150A 7.3 ± 3.8. Similarly, protease coagulant activities were: WT 100 ± 6.1, pFIXa 98.4 ± 11.4, K132A 91.4 ± 1.6, K132A/R150A 84.9 ± 2.8, R150A 77.1 ± 5.8, K126A 39.5 ± 2.4, K126A/R150A 25.3 ± 2.8, K126A/K132A/R150A 10.9 ± 0.6, and K126A/K132A 9.3 ± 0.6. In contrast to their relative coagulant activities, FIX K126A (1.9-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased peak thrombin concentrations during TF-triggered TG; pFIX, FIX K132A and K126A/R150A were similar to WT; and FIX K126A/K132A/R150A (0.6-fold) and K126A/K132A (0.2-fold) demonstrated marked reductions in peak thrombin relative to WT. In the FIXa-initiated TG assay, FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased peak thrombin concentrations; pFIXa, FIXa K132A, R150A, and K126A (0.8-1.0 fold) were similar to WT; while FIXa K126A/K132A and K126A/K132A/R150A demonstrated markedly reduced (0.2-0.3 fold) and delayed peak thrombin concentrations. In pooled, citrated FIX-deficient plasma, FIXa WT (40.9 ± 1.4 min) and K126A/K132A (37.2 ± 0.7 min) demonstrated similar half-lives, while FIXa R150A, K126A/R150A, and K132A/R150A all had half-lives > 2 hr. Conclusions Single exosite mutations resulted in mild to moderate reductions in coagulant activity, while the double mutation in the heparin exosite (K126A/K132A) markedly reduced activity, likely due to a synergistic effect on cofactor binding. Traditional coagulant activity did not accurately represent the ability of the mutant proteins to support thrombin generation. Despite variable reductions in coagulant activity, FIX K126A, K132A, R150A, K126A/R150A and K132A/R150A supported levels of plasma thrombin generation that were equal to or greater than FIX WT. The plasma half-life of FIXa WT activity was remarkably lengthy, and while mutations in the heparin exosite had negligible effects, R150A in the antithrombin exosite substantially increased protease half-life, consistent with a primary role for antithrombin in the plasma inhibition of FIXa. Thus, single exosite mutations did not significantly disrupt the procoagulant function of human FIX(a), and combined exosite mutations (K126A/R150A and K132A/R150A) maintain or enhance plasma thrombin generation while disrupting exosite-mediated regulatory mechanisms. The combination of intact procoagulant function with disruption of antithrombin- and heparin-mediated regulation of FIX(a) will potentially enhance in vivo recovery, prolong plasma half-life, and enhance the efficacy of hemophilia B replacement therapy. Disclosures: Sheehan: Novo Nordisk Access to Insight Basic Research Grant: Research Funding; Bayer Hemophilia Awards Program: Research Funding; Diagnostica Stago: reagents, reagents Other.

Relieving Inhibition of Prothrombinase By TFPIα: a Procoagulant Activity of Unfractionated, Low Molecular Weight, and Nonanticoagulant Heparins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1478-1478
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Rodney M. Camire ◽  
Umesh R Desai ◽  
Alan E. Mast
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Research Funding ◽  
Thrombin Generation ◽  
Size Dependence ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Direct Inhibition ◽  
Acidic Region ◽  
Inhibition Of Thrombin

Abstract Introduction: Prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa), is inhibited by tissue factor pathway inhibitor (TFPI)α during the initiation of coagulation (Wood JP et al, PNAS 2013). Efficient inhibition of thrombin generation by prothrombinase requires an interaction between the TFPIα basic C-terminus and an acidic region of the FVa B-domain. This acidic region is present in FXa-activated FVa and FVa released from activated platelets, but is rapidly removed by thrombin. Thus, prothrombinase inhibition only occurs during the initiation phase of thrombin generation. As the exosite interaction is charge-dependent, large negatively charged molecules, including unfractionated heparin (UFH), block it, prevent prothrombinase inhibition, and promote thrombin generation. Studies using the negatively charged molecule polyphosphate have suggested a size requirement for blocking this TFPIα activity (Smith SA et al, Blood2010). A similar size-dependence may exist with heparins and could have clinical implications, as currently-used heparins range from long (unfractionated heparin; UFH) to medium (low molecular weight heparins; LMWHs) to short (the antithrombin-binding pentasaccharide fondaparinux). Studies were performed to assess the ability of the LMWHs enoxaparin and dalteparin, fondaparinux, and the nonanticoagulant heparin 2-O, 3-O desulfated heparin (ODSH) to block TFPIα and promote thrombin generation through this mechanism. Methods: TFPIα inhibition of thrombin generation by prothrombinase, assembled with a form of FVa containing the acidic region of the B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, fondaparinux, and ODSH. The effect of these compounds on the direct inhibition of FXa by TFPIα was measured using a FXa chromogenic substrate. The effect of these compounds on thrombin generation in plasma was measured by calibrated automated thrombography using human plasma immunodepleted of antithrombin III and heparin cofactor II (AT3/HCII-depleted plasma). Results: TFPIα inhibited prothrombinase activity (IC50 = 6.8 nM), and UFH blocked this inhibition (IC50 = 12.5 nM or 14.9 nM at 0.5 or 1 U/mL, respectively). Enoxaparin (0.8 U/mL; IC50 = 30.3 nM) and dalteparin (1 U/mL; IC50 = 29.7 nM) appeared to be more effective at reversing TFPIα inhibition. The reason for this apparent enhanced effect of LMWHs compared to UFH is not clear, as UFH and the LMWHs similarly enhanced the direct inhibition of FXa by TFPIα, and the differential activity was also observed when heparins were normalized to saccharide concentration. The same pattern was observed when measuring thrombin generation in AT3/HCII-depleted plasma, with LMWHs being more procoagulant than UFH. Consistent with TFPIα inhibition being charge-dependent, ODSH promoted thrombin generation similarly to LMWHs in both purified systems and AT3/HCII-depleted plasma. In contrast, clinical doses of fondaparinux had no effect in any assay. In a purified system, ~1000 times the clinical dose of fondaparinux was required to promote thrombin generation. Conclusion: There is a size-dependence for blocking TFPIα inhibition of prothrombinase using heparins, as the pentasaccharide has no effect. However, both LMWHs and UFH are sufficiently long to express this procoagulant activity at therapeutic doses. In addition, the nonanticoagulant heparin ODSH blocks prothrombinase inhibition by TFPIα. This procoagulant activity is likely most clinically relevant under conditions of antithrombin deficiency, which may result from sepsis, liver failure, or administration of L-asparaginase. Under any of these conditions, UFH, LMWHs, and ODSH may have unanticipated procoagulant activity mediated by blocking TFPIα. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast:Novo Nordisk: Research Funding.

Comparative Inhibition of Extrinsic and Intrinsic Thrombin Generation by Standard Heparin, a Low Molecular Weight Heparin and the Synthetic ATIII-Binding Pentasaccharide

1993 ◽  
Vol 69 (02) ◽  
pp. 152-156 ◽  
Author(s):  
Jean-Claude Lormeau ◽  
Jean-Pascal Herault
Keyword(s):  
Thrombin Generation ◽  
Marked Effect ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Vii ◽  
Low Molecular Weight ◽  
Inhibiting Effect ◽  
Selective Suppression ◽  
Factor Xa Inhibition

SummaryThe inhibiting effect of standard heparin, CY216 and the ATIII-binding synthetic pentasaccharide on extrinsic and intrinsic thrombin generation were quantified by evaluating the decrease of the total amount of active thrombin appearing in plasma after triggering coagulation.Heparin as well as CY 216 produced the same quantitative inhibition of extrinsic and intrinsic TGs whereas pentasaccharide inhibited more efficiently extrinsic TG.This pattern of inhibition was further confirmed on pure extrinsic or intrinsic coagulation respectively in factor IX- and factor VII-depleted plasmas.Furthermore, selective suppression of the anti-thrombin activity of CY 216 by limited amounts of PF4 affected the intrinsic TG inhibition more markedly than the extrinsic one.It was concluded that anticoagulant activity produced mainly through thrombin scavenging leads to similar quantitative impairment of extrinsic and intrinsic coagulation, while selective ATIII-mediated factor Xa inhibition results in a more marked effect against the extrinsic system.

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