Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation

2011 ◽  
Vol 22 (5) ◽  
pp. 369-373 ◽  
Author(s):  
Sonia Ben-Hadj-Khalifa ◽  
Nathalie Hézard ◽  
Wassim Y. Almawi ◽  
Marie G. Remy ◽  
Bernadette Florent ◽  
...  
1993 ◽  
Vol 69 (03) ◽  
pp. 227-230 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
H Merk ◽  
T H Müller ◽  
M R Buchanan ◽  
W G Eisert

SummaryWe compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely deendothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p <0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins. We conclude that, i) mild vessel wall injury (selective de-endothelialization) in veins results in a thrombogenic vessel wall; ii) the thrombogenecity of which is not inhibited by prophylactic doses of heparin; but iii) is inhibited by annexin V, which binds to injured vessel wall surface, and inhibits thrombin generation independently of antithrombin III.


Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4197-4205 ◽  
Author(s):  
J.M. Herbert ◽  
J.P. Hérault ◽  
A. Bernat ◽  
R.G.M. van Amsterdam ◽  
J.C. Lormeau ◽  
...  

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.


1993 ◽  
Vol 70 (03) ◽  
pp. 423-426 ◽  
Author(s):  
Rika ohishi ◽  
Naoko watanabe ◽  
Masaharu Aritomi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  

SummaryThrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4171-4171
Author(s):  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Evangelos Litinas ◽  
Brigitte Kaiser ◽  
Jawed Fareed

Abstract Abstract 4171 AVE5026 (Sanofi-Aventis, Paris, France) represents an anti-Factor (F) Xa enriched ultra low molecular weight heparin (ULMWH) (Mw=2.4 Kda; anti-FXa activity ∼160 U/mg). In comparison to Enoxaparin it has a lower anti-FIIa activity (∼2 U/mg). The oligosaccharide composition of AVE5026 also differs from Enoxaparin and other LMWHs. Besides the molecular and compositional differences, the biologic half-life of AVE5026 (18-20 hours) is significantly longer than Enoxaparin (4-6 hours). In order to compare the other pharmacodynamic differences between AVE5026, Enoxaparin and unfractionated heparin (UFH), a primate model (macaca mulatto) was used since its tissue factor pathway inhibitor (TFPI) profile is comparable to the human response. Individual groups of primates (n=6) were administered with 1 mg/kg SC of either AVE5026, Enoxaparin or UFH. Heptest and APTT measurements were determined on whole blood (WB) and plasma was analyzed for APTT, Heptest, thrombin time (TT), anti-FXa and anti-FIIa effects at varying periods up to 28 hours. TFPI antigen was measured using the assay from Stago (Parsipanny, NJ). Functional TFPI measurements were determined using the kit from American Diagnostica (Stamford, CT). In contrast to UFH, in the WB assays, neither the AVE5026 nor the Enoxaparin produced a strong effect on the APTT and TT, however both demonstrated a strong effect on the heptest assay. AVE5026 produced a much stronger effect with a longer half-life (T½=11 hrs) in comparison to Enoxaparin (T½=6 hrs). In the plasma based systems only UFH produced a measurable effect on the APTT and TT. However, in the heptest and anti-FXa assays, both AVE5026 and Enoxaparin produced a stronger effect, which was much longer with AVE5026 (2-3 fold increase). The plasma time course of TFPI antigen release was longer with AVE5026 in comparison to Enoxaparin and UFH. The ratios of immunologic to functional TFPI levels were also higher in the primates administered with AVE 5026. In the thrombin generation test, AVE5026 produced a sustained effect which lasted longer than Enoxaparin (T½ =16.8 hrs vs. 9.2 hrs.). These results show that AVE5026 produces stronger anti-FXa effects in primates which are associated with a higher circulating level of TFPI and more pronounced suppression of thrombin generation compared to Enoxaparin and UFH. Disclosures: Hoppensteadt: Sanofi-Aventis: Research Funding.


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

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.


2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Yang Chen ◽  
Tao Sun ◽  
Junzhen Wu ◽  
Bill Kalionis ◽  
Changcheng Zhang ◽  
...  

The aim of the study was to investigate the effect of icariin (ICA) on cardiac aging through its effects on the SIRT6 enzyme and on the NF-κB pathway. Investigating the effect of ICA on the enzymatic activity of histone deacetylase SIRT6 revealed a concentration of 10−8 mol/L ICA had a maximum activating effect on histone deacetylase SIRT6 enzymatic activity. Western analysis showed that ICA upregulated SIRT6 protein expression and downregulated NF-κB (p65) protein expression in animal tissues and cell models. ICA upregulated the expression of SIRT6 and had an inhibitory effect on NF-κB inflammatory signaling pathways as shown by decreasing mRNA levels of the NF-κB downstream target genes TNF-α, ICAM-1, IL-2, and IL-6. Those effects were mediated directly or indirectly by SIRT6. We provided evidence that inflammaging may involve a novel link between the effects of ICA on SIRT6 (a regulator of aging) and NF-κB (a regulator of inflammation).


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