Factor XIII Contributes to Clot Formation and Thrombin Generation

Abstract Background Factor XIII (FXIII) stabilizes fibrin clots, minimizing the amount of thrombin generation required to stop bleeding. It also has a long half-life (>7 days) and supratherapeutic levels are not associated with thrombosis making it an attractive therapeutic agent to augment factor replacement in hemophilia. FXIII is a transglutaminase that cross-links fibrin and localizes alpha 2-antiplasmin to fibrin inhibiting fibrinolysis. It also interacts with numerous other proteins, most of which have not been well studied and their physiologic significance is unknown. FXIII is classically considered to be activated by thrombin, which may limit its utility in low thrombin states such as hemophilia. However, activation also occurs physiologically through other mechanisms including calpain, neutrophil elastase and calcium influx. In addition, low levels of thrombin are generated by Factor Xa in the absence of the Factor VIII (FVIII)/Factor IX (FIX) complex that may contribute to both FXIII activation and clot formation. Thus, it is not clear if thrombin generated through the participation of FVIII and FIX is necessary for FXIII activation. Hypothesis We hypothesize that FXIII (recombinant FXIII-A2) may contribute to both clot formation and thrombin generation in hemophilia. Methods Animal models include Exon 16-deleted FVIII-deficient mice (FVIII-KO), wild-type mice (WT), and exon 16-deleted Factor VIII deficient/GP1bα-FVIII knock-in mice (PF). The PF mice are FVIII-deficient mice expressing human FVIII driven from the glycoprotein 1bα promoter resulting in approximately 3% circulating FVIII. Various combinations of factors were given via tail-vein injection. Citrated blood was collected by cardiac puncture 1.5 - 4 hours post-injection, depending on the factor type. Clotting was characterized using thromboelastography (TEG). Thrombin generation was measured on a fluorescence reader using a reagent comprised of a low concentration of phospholipid micelles containing tissue factor in HEPES buffer. Results TEG characterization shows differences in clotting times (R), speed of clot formation (K), and the kinetics of the formation of the clot (α angle) between the various groups without changes in overall clot stability (MA) or degree of fibrinolysis (LY30). R, K, and α angle are all measurements of clot formation, with prolongation of R and K and reduced α angle characteristic of hemophilia. PF mice have similar R, K, and α angle compared to FVIII-KO (p = 0.5, 0.68, and 0.89, respectively), and elongated R and K, and reduced α angle compared to WT (p = 4.0E-3, 2.0E-3, and 1.37E-6, respectively). Giving supratherapeutic FXIII to PF mice results in normalization of these values compared to WT, with a trend towards elongated K and α angle (p = 0.21, 0.08, and 0.13, respectively), and differences compared to FVIII-KO (p = 8.1E-3, 7.4E-3, and 2.1E-3, respectively). Administering FXIII to FVIII-KO mice did not alter R, K, and α angle compared to untreated FVIII-KO mice (p = 0.25, 0.37, and 0.67, respectively). PF mice have similar peak thrombin generation compared to FVIII-KO (p = 0.56) and reduced peak thrombin generation compared to WT (p = 6.69E-5). Giving supratherapeutic FXIII to the PF mice results in peak thrombin generation similar to that of WT (p = 0.97). In contrast, giving supratherapeutic FXIII to FVIII-KO mice did not alter peak thrombin generation levels compared to untreated FVIII-KO mice (p = 0.72). The administration of a cocktail containing both FVIII (2.5 U/kg) and FXIII resulted in a trend for improved peak thrombin generation when compared to an injection of FVIII alone (p = 0.12). Conclusions The function of FXIII has classically been considered to be secondary to its transglutaminase activity. With a direct impact on early clot formation and thrombin generation, these data suggest that FXIII has other roles beyond its known activities. The implication of these findings is that FXIII may be an effective hemostatic agent in mild and moderate hemophilia. Disclosures Taylor: Novo Nordisk: Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding.

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Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽

10.1182/blood.v128.22.3798.3798 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3798-3798

Author(s):

Lilley Leong ◽

Irina N. Chernysh ◽

Yifan Xu ◽

Cornell Mallari ◽

Billy Wong ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Neutralizing Antibodies ◽

Research Funding ◽

Thrombin Generation ◽

Hemophilia A ◽

Clot Formation ◽

Factor Vii ◽

Wild Type ◽

Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

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Evidence for a Factor IX-Independent Role for Factor XI in Thrombin Generation

Blood ◽

10.1182/blood.v118.21.2244.2244 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2244-2244

Author(s):

Anton Matafonov ◽

Qiufang Cheng ◽

Ingrid M. Verhamme ◽

Obi Umunakwe ◽

Erik I. Tucker ◽

...

Keyword(s):

Thrombin Generation ◽

Factor Ix ◽

Clot Formation ◽

Coagulation Cascade ◽

In Vitro Assays ◽

Dependent Manner ◽

Vessel Occlusion ◽

Fibrin Formation ◽

Equity Ownership ◽

Deficient Mice

Abstract Abstract 2244 In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV. We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not. In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX. Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures: Tucker: Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

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Pathogen Reduction Treatment of Whole Blood Inhibits Clot Lysis and Thrombin Generation Is Preserved to 14 Days by Storage At 4°C

Blood ◽

10.1182/blood.v118.21.2272.2272 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2272-2272

Author(s):

Alejandra G Mora ◽

Heather F Pidcoke ◽

Krystal K Valdez-Delgado ◽

Chriselda G Fedyk ◽

Heather L Reddy ◽

...

Keyword(s):

Factor Viii ◽

Ultraviolet Light ◽

Cold Storage ◽

Whole Blood ◽

Research Funding ◽

Thrombin Generation ◽

Post Treatment ◽

Clot Formation ◽

Clot Lysis ◽

Pathogen Reduction

Abstract Abstract 2272 Introduction: Fresh whole blood (WB) collected from a “walking blood bank” is used by the U.S. military to supplement component therapy when blood component supplies are exhausted. Currently, WB is used for emergency transfusion within 24 hours of collection, before results of pathogen testing are available. A pathogen reduction technology (PRT), which uses riboflavin and ultraviolet light to damage nucleic acids in pathogens, is being considered as a transfusion-transmitted disease (TTD) risk mitigation measure. The effect of this technology on the hemostatic properties of whole blood, particularly on clotting capacity and clot lysis, are poorly understood, and optimal storage conditions are not defined. We previously reported that activated partial thrombin time (aPTT) and prothrombin time (PT) are prolonged by treatment; however, these tests do not always correlate with clinical findings. Thromboelastography is a more robust measure of clot formation and stability over time; we previously found that maximum amplitude (MA), which represents clot strength, did not decrease with PRT treatment and was preserved by storage at 4°C. Here we explore the effects of PRT on other parameters important to clotting capacity and clot lysis, and present the effects of WB storage at 4°C compared to 22°C. Hypothesis: WB treated with PRT demonstrates similar hemostatic function to non-treated WB, and storage at 4°C reduces degradation of blood components essential to clotting capacity and clot lysis compared to 22°C. Methods: Under an IRB-approved protocol, 8 units per treatment group of WB were collected in CPD anticoagulant from healthy donors of normal hemostatic status according to standard blood donor guidelines. Pathogen reduction was performed using riboflavin and ultraviolet light (265–400nm phosphor; Mirasol® System, CaridianBCT) dosed at 80 J/mLRBC. Treatment groups included: control WB stored at 4° C (CON-04); control WB stored at 22° C (CON-22); PRT-treated WB stored at 4° C (PRT-04); and PRT-treated WB stored at 22° C (PRT-22). The hemostatic function of the blood was assessed at baseline, days 1–7, 10, 14, and 21. Factor VIII and fibrinogen were measured from assayed samples (BCS® XP system, Siemens). Thromboelastography (TEG®, Haemoscope Corp.) estimated total thrombin generation by calculating the first derivative of the TEG tracing, the Total Thrombus Generation variable (TTG). TEG was also used to measured lysis (LY30). Data were analyzed as repeated measures, followed by analysis of variance to assess interactions. Significance was set at p<0.05. Results: Treatment with PRT caused an initial drop in fibrinogen (baseline: 244 ± 77.5 mg/dL versus post-treatment: 185 ± 63.2 mg/dL, p≤0.04) and factor VIII (baseline: 96 ± 39% versus day post-treatment: 46 ± 23%, p≤0.001); however, levels stabilized thereafter (p≥0.987 and p≥0.871, respectively; see Fig. 1–2). Baseline fibrinogen levels were similar between groups p≥0.386). PRT-04 was the only group in which both fibrinogen and Factor VIII levels fell below clinical reference ranges (fibrinogen: p'0.039; factor VIII: p≤0.016). TTG was unaffected by PRT and was preserved through day 14 by storage at 4° C (p≥0.979, see Fig. 3), but only through day 10 when stored at 22°C (p≤0.290 at day 10). PRT treatment inhibited clot lysis (LY30) compared to storage at 22°C (p≤0.001), and variability was the lowest in the PRT-04 group (p≤0.001, see Fig. 4). Conclusions: Our data demonstrate that pathogen reduction inhibited clot lysis. Decreased clot formation could conceivably account for the smaller degree of lysis; however, we previously found that MA is unaffected, and now demonstrated that thrombin generation was preserved despite a treatment-related decrease in factor VIII levels. While fibrinogen levels were diminished in the PRT-04 group, they were preserved in the PRT-22 group, which also demonstrated the diminished lysis. Cold storage preserved WB clotting capacity compared to storage at room temperature. The clinical significance of these findings has yet to be established; a coagulopathic animal hemorrhage model could determine whether the effects of PRT-induced lysis inhibition and cold storage are beneficial. Disclosures: Mora: CaridianBCT: Research Funding. Pidcoke:CaridianBCT: Research Funding. Valdez-Delgado:CaridianBCT: Research Funding. Fedyk:CaridianBCT: Research Funding. Reddy:CaridianBCT: Employment, Research Funding. Goodrich:CaridianBCT: Employment, Research Funding. Cap:CaridianBCT: Research Funding.

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In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽

10.1182/blood-2020-139712 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Dougald Monroe ◽

Mirella Ezban ◽

Maureane Hoffman

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Plasma Levels ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor Ix ◽

Factor X ◽

Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) 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 safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma 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). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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Binding To Phospholipid Protects Factor VIII From Inhibition By Human Antibodies

10.1055/s-0038-1651982 ◽

1981 ◽

Author(s):

T W Barrowcliffe ◽

E Gray ◽

G Kemball-Cook

Keyword(s):

Factor Viii ◽

Thrombin Generation ◽

Total Phospholipid ◽

Fluid Phase ◽

Clinical Applications ◽

Factor Ix ◽

Two Stage ◽

Human Antibodies ◽

Thrombin Generation Assay ◽

Detectable Antigen

Previous studies with activated Factor IX concentrates have suggested that they may contain a form of Factor VIII clotting activity (VIII:C) which is partly protected from inactivation by antibodies. A possible mechanism for such protection is binding to phospholipid. The interaction between Factor VIII, phospholipid and human antibodies to Factor VIII was studied by a two-stage clotting assay, and by a fluid-phase immunoradiometric assay for Factor VIII clotting antigen (VIII C:Ag).In the two-stage thrombin generation assay, Factor VIII:C was rapidly destroyed by human antibodies, even in the presence of optimal phospholipid. However, preincubation of Factor VIII with phospholipid before addition of antibody protected the Factor VIII from inactivation, resulting in the production of much more thrombin.In assays of VIII C:Ag, pre-incubation of Factor VIII with phospholipid before addition of labelled antibody reduced the amount of detectable antigen. The reduction was greater with increasing phospholipid concentration, up to 60% of the original antigen being ‘lost’ at a total phospholipid concentration of around 250 μg/i.u.These results suggest that human antibodies to Factor VIII are directed largely at its phospholipid binding site. The protection of Factor VIII from inactivation by complexing with phospholipid could have important clinical applications in treatment of haemophiliacs with inhibitors.

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Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽

10.1182/blood.v95.4.1330.004k28_1330_1335 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1330-1335 ◽

Cited By ~ 98

Author(s):

Cornelis van 't Veer ◽

Neal J. Golden ◽

Kenneth G. Mann

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Plasma Concentrations ◽

Factor Xa ◽

Factor Vii ◽

Lag Phase ◽

Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

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Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

Blood ◽

10.1182/blood.v108.11.1751.1751 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1751-1751

Author(s):

Samit Ghosh ◽

Mirella Ezban ◽

Egon Persson ◽

Ulla Hedner ◽

Usha Pendurthi ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Surface ◽

Proteolytic Activity ◽

Thrombin Generation ◽

Factor Viia ◽

Factor Xa ◽

Factor Ix ◽

Factor X ◽

Wild Type

Abstract High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

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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 ◽

10.1182/blood.v122.21.2333.2333 ◽

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.

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

Blood ◽

10.1182/blood-2009-02-203604 ◽

2009 ◽

Vol 114 (2) ◽

pp. 452-458 ◽

Cited By ~ 96

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.

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Surveillance of Hemophilia in Indiana: A Preliminary Report

Blood ◽

10.1182/blood-2018-99-118849 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3516-3516

Author(s):

Amanda Okolo ◽

John M Soucie ◽

Scott D. Grosse ◽

Chris Roberson ◽

Isaac Janson ◽

...

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Research Funding ◽

Clinical Characteristics ◽

Claims Data ◽

Factor Ix ◽

Clotting Factor ◽

Advisory Committees ◽

Mortality And Morbidity ◽

Factor Ix Deficiency

Abstract Background and objectives: Hemophilia is a rare heritable bleeding disorder resulting from missing or deficient levels of factor VIII or factor IX. Hemophilia related complications result in high utilization of health care resources and include severe, debilitating chronic joint disease. In 1998, Soucie et al. published the results of a six-year surveillance study investigating the incidence and prevalence of hemophilia in six U.S. states. The study also described the relationship between mortality and morbidity and each patient's primary source of hematologic care (i.e. whether each patient had visited a federally designated hemophilia treatment center (HTC)). The Indiana Hemophilia Surveillance Project (IHSP) aims to identify all persons with hemophilia who resided in Indiana in 2011-2013, to calculate the prevalence and incidence of hemophilia in Indiana, and to determine the percentage of patients cared for at a federally recognized HTC. The IHSP further aims to compare morbidity and mortality data to the results of the Soucie et al. study. Methods: A hemophilia case in this study is defined as a male with physician-diagnosed hemophilia A or B and a measured baseline factor VIII or IX activity level less than 50%. A retrospective review of medical charts and other records was conducted to identify hemophilia cases during the surveillance years from 2011-2013. Case finding methods involved obtaining medical information from a variety of medical care resources including: HTCs in Indiana and surrounding states, hospitals, vital records, Indiana's birth defects registry, hospital and administrative claims data from the Regenstrief Institute, Medicaid claims data, clinical laboratories, specialty pharmacies, hematology/oncology clinics, and primary care physicians. Demographic and clinical data were collected on all identified cases. Data collected included: demographic information, clinical characteristics, joint health assessments, insurance status, clotting factor product utilization and cost, source of hemophilia care, hospitalizations and emergency room visits, and mortality information. Associations between clinical characteristics were assessed for statistical significance using chi-square and fisher's exact tests. Incidence was calculated by using the number of births from prevalent cases during the three surveillance years as the numerator and the number of live male births in Indiana for each year as the denominator. Results: 704 hemophilia cases were identified in Indiana in 2011-2013. Of those cases, 456 (64.8%) had factor VIII deficiency and 248 (35.2%) had factor IX deficiency. The median age of the population was 25 years. 453 cases (64.3%) were adult patients and 251 cases (35.7%) were pediatric patients under 18 years. Among those with known severity levels (n=685), 233 (33.1%) were severe, 185 (26.3%) were moderate, and 267 (37.9%) were mild. Overall, 81.7% of the hemophilia patients identified visited an HTC at least once during the three year study period, which was the minimum requirement for being considered a patient of an HTC. Age-adjusted prevalence was 21.8 cases per 100,000 males; 14.3 per 100,000 for factor VIII and 7.5 per 100,000 for factor IX. Mean incidence of hemophilia over the three year study period was 1:4059 live male births in Indiana. 24 cases (3.4%) died within the study period; 4.2% of the deaths occurred in HIV positive patients. Of those with a known cost of clotting factor (n=184), the average and median cost of factor over the three year period was $375,532 and $71,525 respectively. Conclusions: There was a significantly higher percentage of patients seen at an HTC (81.7%) as compared to the results of the Soucie et al. study (67%; p =<0.001). Indiana has a 62% higher prevalence and 24% higher incidence of hemophilia than in the Soucie et al. study. A high frequency of factor IX deficiency associated with a founder mutation in the Amish community contributes to the higher incidence of factor IX deficiency in Indiana, with a 64% higher percentage of factor IX deficiency among hemophilia cases. The higher prevalence likely reflects improved survival and increased utilization of HTCs in the past two decades. This report is the first follow-up study of the original Soucie et al. report using the same systematic approach. Further analysis on mortality and morbidity complications in this population will be completed and reported in future reports. Disclosures Shapiro: Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bioverativ, a Sanofi Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo Biosciences: Consultancy; Bio Products Laboratory: Consultancy; Prometic Life Sciences: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; BioMarin: Research Funding; Kedrion Biopharma: Consultancy, Research Funding; OPKO: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Healthcare: Other: International Network of Pediatric Hemophilia; Octapharma: Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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