Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

scholarly journals Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.M Engelen ◽  
C Van Laer ◽  
M Jacquemin ◽  
C Vandenbriele ◽  
K Peerlinck ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Heart Valves ◽  
Thrombus Formation ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Mechanical Heart Valves ◽  
Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3271-3271
Author(s):  
Teresa M Brophy ◽  
Jasimuddin Ahamed ◽  
Barry S. Coller
Keyword(s):  
Transforming Growth Factor ◽  
Coagulation Factor ◽  
Transforming Growth Factor Β1 ◽  
Affinity Column ◽  
Factor V ◽  
Disulfide Exchange ◽  
Control Peptide ◽  
Tgf Β1

Abstract Abstract 3271 Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation. Disclosures: Coller: Centocor/Accumetrics/Rockefeller University:.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

scholarly journals Cardiac Myosin Acts Is a Potent Procoagulant in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3632-3632
Author(s):  
Jevgenia Zilberman-Rudenko ◽  
Hiroshi Deguchi ◽  
Mohammed Hayat ◽  
Meenal Shukla ◽  
Jennifer Nagrampa Orje ◽  
...  
Keyword(s):  
Blood Loss ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Xa ◽  
Mouse Plasma ◽  
Gla Domain ◽  
Coated Surfaces ◽  
Prothrombinase Activity

Thrombin generation and fibrin formation can cause occlusive thrombosis and myocardial infarction is caused by occlusive thrombi. Exposure and release of cardiac myosin (CM) are linked to myocardial infarction, but CM has not been accorded any thrombotic functional significance. Skeletal muscle myosin (SkM), which is structurally similar to CM, was previously shown to exert procoagulant activities (Deguchi H et al, Blood. 2016;128:1870), leading us to undertake new studies of the in vitro and in vivo procoagulant activities of CM. First, the setting of hemophilia A with its remarkable bleeding risk was used to evaluate the procoagulant properties of CM. In studies of human hemophilia plasma and of murine acquired hemophilia A plasma, CM was added to these plasmas and tissue factor (TF)-induced thrombin generation assays were performed. Plasmas included human hemophilia A plasma and C57BL/6J mouse plasma with anti-FVIII antibody (GMA-8015; 5 microgram/mL final). CM showed strong procoagulant effects in human hemophilia A plasma, which is naturally deficient in factor VIII (<1% FVIII). The addition of only CM (12.5-200 nM) greatly increased thrombin generation in a manner comparable to addition of only recombinant FVIII. In the wild-type C57BL/6J mouse plasma, anti-FVIII antibody greatly reduced TF-induced thrombin generation, as reported. When CM (12.5-200 nM) was added to mouse plasma containing anti-FVIII antibodies, TF-induced thrombin generation was concentration-dependently restored. To study the in vivo hemostatic ability of SkM, an acquired hemophilia A mouse model was employed. Intravenous injection of anti-FVIII antibody (GMA-8015; 0.25 mg/kg) or control vehicle was given retro-orbitally to wild type C57BL/6J mice at 2 hours prior to tail cutting. The distal portion of the tail was surgically removed at 1.5 mm tail diameter to induce moderate bleeding. Tails were immersed in 50 mL of saline at 37 degrees. Total blood loss was measured as the blood volume collected during 20 min normalized for mouse weight (microL/g). Mice given only anti-FVIII antibody had more blood loss (median = 6.7 microL/g) compared to control mice (median < 2 microL/g) (Figure). In this mouse model receiving anti-FVIII antibody, CM (5.4 mg/kg) injected at 15 min prior to tail cutting significantly reduced the median blood loss from 6.7 to 2.0 and 3.2 microL/g, respectively (p < 0.001 for each myosin) (Figure). Thus, these studies provide in vivo proof of concept that both CM and SkM can reduce bleeding and are procoagulant in vivo. Second, studies of the effects of CM on thrombogenesis ex vivo using fresh human flowing blood showed that perfusion of blood over CM-coated surfaces at 300 s-1 shear rate caused extensive fibrin deposition. Addition of CM to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of CM's thrombogenicity. Further studies showed that CM enhanced thrombin generation in platelet rich plasma and platelet poor plasma, indicating that CM promotes thrombin generation in plasma primarily independently of platelets. To address the mechanistic insights for CM's procoagulant activity, purified coagulation factors were employed. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, CM greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for CM's prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. Binding studies showed that CM directly binds factor Xa. In summary, here we show that CM is procoagulant due to its ability to bind factor Xa and strongly promote thrombin generation. In summary, CM acts as procoagulant by its ability to bind factor Xa and strongly promote thrombin generation both in vivo an in vitro. These provocative findings raise many questions about whether and how the protective pro-hemostatic properties or the pathogenic prothrombotic properties of CM contribute to pathophysiology in the coronary circulation. This discovery raises many questions about CM and coronary pathophysiology, and future CM research may enable novel translations of new knowledge regarding CM's procoagulant activities for coronary health and disease. Figure Disclosures Mosnier: The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

scholarly journals Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

2020 ◽  
Vol 26 ◽  
pp. 107602962096082
Author(s):  
Dalia Qneibi ◽  
Eduardo Ramacciotti ◽  
Ariane Scarlatelli Macedo ◽  
Roberto Augusto Caffaro ◽  
Leandro Barile Agati ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
In Vivo Studies ◽  
In Vitro Tests ◽  
Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

scholarly journals Characterization of an inducible endothelial cell prothrombin activator

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2989-2994 ◽  
Author(s):  
L Liu ◽  
GM Rodgers
Keyword(s):  
Thrombin Generation ◽  
Interleukin 1 ◽  
Factor Xa ◽  
Apparent Molecular Weight ◽  
Factor V ◽  
Factor X ◽  
Exogenous Factor ◽  
Km Value ◽  
Prothrombin Activation

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

Absolute hypoxic exercise training enhances in vitro thrombin generation by increasing procoagulant platelet-derived microparticles under high shear stress in sedentary men

2013 ◽  
Vol 124 (10) ◽  
pp. 639-649 ◽  
Author(s):  
Yu-Wen Chen ◽  
Yi-Ching Chen ◽  
Jong-Shyan Wang
Keyword(s):  
Shear Stress ◽  
Exercise Training ◽  
Thrombin Generation ◽  
Coagulation Factor ◽  
Strenuous Exercise ◽  
Factor V ◽  
High Shear ◽  
High Shear Stress ◽  
Vigorous Exercise

HS (high shear) stress associated with artery stenosis facilitates TG (thrombin generation) by increasing the release of procoagulant PDMPs (platelet-derived microparticles). Physical exercise and hypoxia may paradoxically modulate vascular thrombotic risks. The aim of the present study was to investigate how exercise training with/without hypoxia affected TG mediated by PDMPs under physio-pathological shear flows. A total of 75 sedentary males were randomly divided into five groups (n=15 in each group): 21% O2 [NC (normoxic control)] or 15% O2 [HC (hypoxic control)] at rest or were trained at 50% of peak work rate under 21% O2 [NT (normoxic training)] or 15% O2 [HAT (hypoxic-absolute training)], or 50% of HR (heart rate) reserve under 15% O2 [HRT (hypoxic-relative training)] for 30 min/day, 5 days/week for 4 weeks. The PDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, strenuous exercise markedly increased the PDMP count (14.8%) and TG rate (19.5%) in PDMP-rich plasma at 100 dynes/cm2 of shear stress (P<0.05). After the interventions, both NT and HRT significantly attenuated the enhancement of HS-induced PDMPs (4.7 and 4.9%) and TG rate (3.8 and 3.0%) (P<0.05) by severe exercise. Conversely, HAT notably promoted the PDMP count (37.3%) and TG rate (38.9%) induced by HS (P<0.05), concurrent with increasing plasma TF (tissue factor) and coagulation factor V levels at rest or following exercise. We conclude that both HRT and NT depress similarly HS-mediated TG during exercise, but HAT accelerates the prothrombotic response to vigorous exercise. These findings provide new insights into how exercise training under a hypoxic condition influences the risk of thrombosis associated with stenotic arteries.

scholarly journals Prothrombin Membrane Binding and Gla-Dependent Function Are Not Required for Effective Hemostasis In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 124-124
Author(s):  
Lindsey A. Greene ◽  
Nabil K. Thalji ◽  
Harlan Bradford ◽  
Sriram Krishnaswamy ◽  
Rodney M. Camire
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Membrane Binding ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Full Length ◽  
Vessel Occlusion ◽  
Gla Domain

Abstract Prothrombin, like other vitamin K-dependent coagulation factors, undergoes γ-carboxylation of its Gla domain, a posttranslational modification critical for membrane binding. In patients on anticoagulant treatment with warfarin, the INR has historically been correlated with the degree of des-gamma-carboxy-prothrombin (DCP or PIVKA-II). PIVKA-II can be measured readily and used as a marker for vitamin K deficiency or warfarin therapy and is thought to be useful in detecting subclinical disease. Long-standing dogma suggests prothrombin γ-carboxylation is necessary for prothrombin membrane binding facilitating engagement with prothrombinase leading to rapid thrombin generation and effective hemostasis. However, recent studies indicate that despite an inability to bind membranes, uncarboxylated (desGla) full-length prothrombin demonstrated an unexpected modest decrease in the rate of thrombin generation (J Biol Chem 2013, 288:27789-800). Thus, it is possible loss of prothrombin γ-carboxylation, and thereby membrane binding, is far less significant for prothrombin activation than previously appreciated. Instead warfarin's effect on other coagulation factors (FX, FIX, and FVII) may be the primary causative determinant impairing hemostasis in these anticoagulated patients. To test these ideas, we first analyzed thrombin generation using recombinant full-length fully carboxylated and desGla prothrombin in vitro. Human prothrombin deficient plasma (Factor II activity <4%) was reconstituted to normal levels (100 μg/mL) with desGla or carboxylated prothrombin. DesGla prothrombin generated approximately half the amount of thrombin observed in carboxylated prothrombin plasma and normal human plasma controls. We next analyzed full-length desGla prothrombin's in vivo hemostatic function. A prothrombin anti-sense oligonucleotide (ASO) was administered to hemostatically normal mice to knock down endogenous murine prothrombin expression (<0.1-1μg/mL, 0.1-1%) and confirmed by ELISA analysis. Hemostasis was analyzed by the ferric chloride (FeCl3) carotid artery injury model. In mice treated with an ASO control, vessel occlusion occurred at approximately 8 minutes while mice treated with the prothrombin ASO did not clot during the 30-minute post injury observation period. In additional experiments two minutes following injury, prothrombin ASO treated mice were administered either carboxylated or desGla recombinant prothrombin to restore plasma concentrations to the normal range (100 μg/mL). Remarkably, administration of either desGla or carboxylated prothrombin restored vessel occlusion to ASO control findings, with minimal variability observed between desGla and carboxylated prothrombin treated mice (Figure 1). Warfarin treatment results in impaired prothrombin γ-carboxylation. However, if prothrombin γ-carboxylation, is, in fact, not necessary for prothrombin activation, fully carboxylated Factor Xa (FXa) should reverse the effects of warfarin by efficiently activating the un/under-carboxylated prothrombin thereby bypassing the other warfarin-affected factors. To study this, we used a "zymogen-like" factor Xa (FXaI16L) molecule previously developed by our group (Nat. Biotech 2011, 29:1028-33) that has a greater half-life than the wild-type protein. In thrombin generation assays, addition of 1nM FXaI16L to plasma from patients anticoagulated with warfarin, irrespective on INR (2.8, 4,4 7.1), resulted in thrombin generation comparable to that of normal human plasma. Importantly, similar results were obtained in vivo in warfarin-anticoagulated mice (INR 2-3). Administration of 3 mg/kg FXaI16L to 8 out of 8 warfarin mice corrected the time to carotid artery occlusion in the FeCl3 injury model. In two separate in vitro and in vivo model systems, we demonstrated that prothrombin membrane binding is not absolutely required for thrombin generation. Thrombin is unique among the coagulation serine proteases in that it does not have a Gla domain once fully processed by prothrombinase; thus, the absence of a Gla domain in the protease (thrombin) may explain the lack of a requirement for membrane binding by the zymogen (prothrombin) precursor. Our findings may also have clinical relevance, since they suggest that FXa (or a variant) could be used as a novel warfarin bypass strategy to rapidly achieve hemostasis in the setting of warfarin anticoagulation. Figure 1. Figure 1. Disclosures Greene: Baxter: Research Funding. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding.

Differential Effects of TAK-442, a Factor Xa Inhibitor, and Ximelagatran, a Thrombin Inhibitor, on Bleeding: Possible Role of Differences in Effects on Factor V-Mediated Feedback on Blood Coagulation Cascade

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5460-5460
Author(s):  
Noriko Konishi ◽  
Katsuhiko Hiroe ◽  
Yasuhiro Imaeda ◽  
Takuya Fujimoto ◽  
Keiji Kubo ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Rat Model ◽  
Positive Feedback ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Factor V ◽  
Differential Effects

Abstract Thrombin generation serves to amplify the coagulation cascade via positive feedback activation of factor V (FV) and factor VIII. We hypothesized that factor Xa (FXa) inhibitors, unlike thrombin inhibitors, would not block the feedback activation of the coagulation cascade but would have a favorable anticoagulating profile—sufficient to prevent thrombus formation, yet not interfere with hemostatic plug formation. TAK-442 is a newly synthesized, selective FXa inhibitor that strongly inhibits FXa (with a Ki value of 1.8 nM), and displays more than 440x selectivity toward FXa than other serine proteases. In the present study, we compared the effects of TAK-442 versus ximelagatran on FV-mediated positive feedback in vitro, and on their antithrombotic and hemorrhagic effects in a rat model of venous thrombosis. In vitro, TAK-442 gradually inhibited thrombin generation and prolonged prothrombin time (PT) in a dose-dependent manner, while melagatran, an active form of ximelagatran, exhibited a steeper effect at higher doses tested. The PT prolonging potency was increased in FV–deficient human plasma, with CT2 values (the concentration that causes 2 times prolongation of clotting times) of 120 nM for TAK-442 and 32 nM for melagatran, compared with 500 nM and 360 nM for TAK-442 and melagatran, respectively in normal plasma. In the rat model of venous thrombosis, TAK-442 (10 mg/kg, po) prevented thrombus formation by 55% and prolonged PT by 1.3 times of control values; a similar effect was observed in ximelagatran-treated (3 mg/kg, po) animals, with 59% inhibition of thrombus formation and 1.2 times prolongation of PT. TAK-442 at 100 mg/kg, prolonged PT by 2.1 times, with no significant change in bleeding time (BT); in contrast, increasing the dose of ximelagatran to 10 mg/kg, po prolonged PT by 3.9 times and significantly (P&lt;0.025) increased BT. Our data suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

The Effect of the Synthetic Pentasaccharide SR 90107/ORG 31540 on Thrombin Generation Ex Vivo Is Uniquely due to ATIII-Mediated Neutralization of Factor Xa

1995 ◽  
Vol 74 (06) ◽  
pp. 1474-1477 ◽  
Author(s):  
J C Lormeau ◽  
J P Herault
Keyword(s):  
Thrombin Generation ◽  
Graphical Representation ◽  
Ex Vivo ◽  
Subcutaneous Administration ◽  
Factor Xa ◽  
Significant Rise ◽  
Time Courses ◽  
The One

SummaryThe inhibition of thrombin generation (TG) was studied in plasma from human volunteers after single subcutaneous administrations of 4000, 8000 or 12,000 anti-Xa units (i.e., 6, 12 or 18 mg) of the synthetic pentasaccharide (SR 90107/ORG 31540) (SP).SP impaired TG in plasma for up to 18 h after injection, and the time-courses of TG and factor Xa inhibitions were similar.In untreated plasma supplemented in vitro with SP to obtain the same anti-Xa activity as in ex vivo samples, equivalent TG inhibitions were observed thus showing that no transformed SP molecules were involved in the TG inhibition ex vivo.Functional as well as immunological assay of TFPI indicated that subcutaneous injection of 12,000 anti-Xa units of SP did not induce any TFPI release, whereas under the same conditions, 13,000 IU of Fraxiparine® produced a significant rise of TFPI in plasma.The plotting of TG inhibition versus SP concentration could be fitted with a good correlation (r = 0,94) to the graphical representation linking [ATIII-SP] to [SP].These results demonstrate that following subcutaneous administration to man, SP inhibits TG ex vivo and likely in vivo exclusively through the same selective ATIII-mediated inhibition of factor Xa as the one elicited in vitro.

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