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

Blood ◽  
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.

Towards the Care of Hemophilia a Patients Using Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Expressing Coagulation Factor (F) VIII

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2266-2266
Author(s):  
Randolph B Lyde ◽  
Li Zhai ◽  
Karen Vo ◽  
Danuta Jadwiga Jarocha ◽  
Spencer Sullivan ◽  
...  
Keyword(s):  
Whole Blood ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Hemophilia A ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Clot Formation ◽  
Patient Specific ◽  
Furin Cleavage ◽  
Furin Cleavage Site

Abstract We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. Disclosures Arruda: Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Whole Blood Thromboelastogram Assays Demonstrate Prolonged Factor VIIIa Potency for Recombinant Disulfide Bond-Stabilized Factor VIII Variants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2976-2976
Author(s):  
Klaus-Peter Radtke ◽  
Dean Chamberlain ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Disulfide Bond ◽  
Light Chain ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Disulfide Bridge ◽  
Clot Formation ◽  
Wild Type ◽  
Subunit Dissociation ◽  
A2 Domain

Abstract Following proteolytic activation of factor VIII (FVIII) by thrombin, the FVIIIa A2 domain, A3 domain and light chain (A3-C1-C2 domains) form a non-covalent hetero-trimer. Because spontaneous A2 subunit dissociation causes loss of FVIIIa activity, we previously made two mutants each with two new Cys to form a disulfide bond linking residues 662 (A2) and 1828 (A3) (FVIIIC662-C1828) or residues 664 (A2) and 1826 (A3) (FVIIIC664-C1826). Following thrombin activation, each FVIIIa mutant was stabile compared to wild type (wt) B-domain-deleted (BDD) FVIII. Previous SDS-PAGE data showed that the A2 domain was disulfide linked to the light chain. To show that this is true for undenatured FVIIIa, here we used surface plasmon resonance (SPR) to monitor A2 dissociation from thrombin-activated wild type and variant FVIII species that were bound to the sensor surface via a monoclonal antibody. Following passage of thrombin over sensor-bound FVIII, only wt FVIII showed a characteristic decrease of SPR reflecting A2 subunit dissociation and thrombin-treated FVIIIC662-C1828 and FVIIIC664-C1826 showed only minor decreases in SPR. Thus, SPR data directly demonstrate that engineered inter-domain disulfide bridges between the A2 and A3 domains prevent A2 domain dissociation from FVIIIa. In contrast to simple plasma coagulation assays of FVIIIa, rotational thromboelastogram (RoTEG) assays of whole blood provide multiple parameters reflecting clot formation, clot quality, and clot dissolution. RoTEG assays using fresh severe hemophilia A whole blood that was reconstituted with either wt FVIII, or FVIIIC662-C1828 or FVIIIC664-C1826 were performed to test the hypothesis that the disulfide-stabilized FVIIIa mutants would show improved potency for thrombin generation. After recalcification of hemophilia A blood with added FVIII, we measured the clotting time (CT), the rate of clot-formation, the clot-firmness time (CFT), defined as the time required to reach a specified clot firmness, and the clot firmness at 5 min (CF-A5), defined as the clot firmness at 5 min after the observed CT. Samples reconstituted with disulfide-bridge-stabilized FVIII mutants or wt-FVIII had comparable CTs at similar concentrations. However, in comparison to wild type BDD-FVIII, comparable rates of clot-formation, CFTs and CF-A5 were observed for up to 10-fold lower concentrations of each disulfide-bridge-stabilized FVIII mutant. The differences between wt and FVIII mutants were especially pronounced at very low FVIII concentrations whereas at FVIII concentrations >0.01 U/mL the differences were less apparent. Because clot formation occurs early relative to overall thrombin generation which is better reflected by CFT and CF-A5 values, we interpret these data to indicate that the disulfide-stabilized FVIIIa variants provide sustained thrombin generation in whole blood compared to wt FVIII and speculate that these FVIII variants may prove superior to wt FVIII for stabilizing a hemostatic plug by providing sustained thrombin generation capacity.

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

Blood ◽  
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.

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

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1330-1335 ◽  
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 &gt;100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (&lt;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.

Thromboelastography in Children with Coagulation Factor Deficiencies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2139-2139 ◽  
Author(s):  
Meera B. Chitlur ◽  
Indira Warrier ◽  
Madhvi Rajpurkar ◽  
Wendy Hollon ◽  
Lolita Llanto ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Response To Treatment ◽  
Factor Vii ◽  
P Value ◽  
Factor V ◽  
Mean Values ◽  
Significant Difference

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

Site-Specific PEGylation of Factor VIII (PEG-FVIII) Preserves Full Clotting Activity and Extends Therapeutic Efficacy in HemophiliaA Dogs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 511-511 ◽  
Author(s):  
Tongyao Liu ◽  
David Lillicrap ◽  
Xin Zhang ◽  
Andrea Labelle ◽  
Sandra Powell ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Half Life ◽  
Therapeutic Efficacy ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Volume Of Distribution ◽  
Pharmacokinetic Analysis ◽  
Site Specific ◽  
Intravenous Injections

Abstract To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

scholarly journals Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4662-4662
Author(s):  
Debnath Maji ◽  
Michael A Suster ◽  
Divyaswathi Citla Sridhar ◽  
Maria Alejandra Pereda ◽  
Janet Martin ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Point Of Care ◽  
Blood Samples ◽  
Healthy Group ◽  
Significant Difference ◽  
Bleeding Score ◽  
Bleeding History

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and > 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P < 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score >12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score <12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P < 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P < 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P < 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (<10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents & Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents & Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents & Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents & Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

RNA Aptamer Against FXa Synergizes with FXa Catalytic Site Inhibitors to Effectively and Reversibly Anticoagulate Blood in an Ex Vivo Oxygenator Circuit

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3823-3823
Author(s):  
Ruwan Gunaratne ◽  
James Frederiksen ◽  
Nabil K. Thalji ◽  
Michelle D Ho ◽  
Gowthami M Arepally ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Research Funding ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Catalytic Site ◽  
Clot Formation ◽  
Scanning Electron Micrographs ◽  
Rna Aptamer ◽  
Andexanet Alfa

Abstract Despite notable disadvantages, unfractionated heparin (UFH) remains the standard anticoagulant for clinical procedures requiring potent and reversible anticoagulation such as cardiopulmonary bypass (CPB). Limitations of UFH that contribute to patient morbidity in these settings include the fact that it permits thrombin generation and can cause the antibody-mediated syndrome Heparin-induced Thrombocytopenia (HIT), in addition to independent toxicities associated with its reversal agent, protamine. 11F7t is an anticoagulant RNA aptamer which inhibits FXa but unfortunately achieves less intense anticoagulation than UFH. The latter is also true for clinical FXa catalytic site inhibitors such as rivaroxaban, apixaban, or edoxoban. However, 11F7t does not inhibit FXa's catalytic activity but instead binds a FXa exosite to impede FVa binding and thus prothrombinase assembly. Owing to these different mechanisms, we previously reported that 11F7t can potently synergize with a FXa catalytic site inhibitor to prevent clot formation for >180 minutes (min) in whole blood thromboelastography (TEG) assays, thereby replicating the effect of UFH (5U/mL). Here we sought to determine whether combinations of 11F7t plus a FXa catalytic site inhibitor can also prevent clotting as effectively as UFH in flowing blood within an ex vivo CPB membrane oxygenator circuit. In addition, we investigated whether efficient and simultaneous reversal of both anticoagulants could also be achieved post-circulation using desGla-Xa-S195A, which is a Gla-domainless catalytically inactive recombinant FXa mutant analogous to Andexanet Alfa, a therapeutic currently in clinical trials as an antidote for several FXa inhibitors. We also quantified levels of thrombin generation during circulation based on Prothrombin fragment 1+2 (F1+2) measurement. Finally, we investigated whether purified IgG obtained from three HIT patients could induce platelet aggregation in the presence of 11F7t, as occurs with UFH. Human whole blood anticoagulated with either (A) UFH (5U/ml), or a combination of 11F7t (2µM) plus either (B) rivaroxaban (2µM), (C) apixaban (2µM), or (D) edoxaban (2µM) was circulated within a miniature oxygenator circuit at 33°C for 120 min at a 50 mL/min flow rate. While anticoagulation with either 11F7t or each of the FXa catalytic site inhibitors alone failed to maintain circuit blood fluidity, strategies (A) through (D) each prevented visible clot formation for 120 min and achieved therapeutic anticoagulation levels (>400 sec) as measured by the Activated Clotting Time (ACT). In addition, post-circulation scanning electron micrographs of the oxygenator membranes were similar for all four strategies and revealed minimal fibrinous and cellular debris. Successful normalization of the ACT was achieved upon administration of desGla-Xa-S195A (2µM) for strategies (B) through (D), similarly to that observed for UFH reversal by protamine. In addition, elevation of F1+2 levels post-circulation was significantly higher with UFH compared to each of the 11F7t plus FXa catalytic site inhibitor strategies (B-D). Finally, the HIT patient-derived purified IgG only induced platelet aggregation in the presence of UFH but not aptamer 11F7t (strategies B-D). We have shown that the anticoagulant synergy achieved by combining aptamer 11F7t with a FXa catalytic site inhibitor can prevent blood clotting within an ex vivo oxygenator circuit as effectively as UFH, and may be additionally advantageous in limiting thrombin generation. Moreover, administration of desGla-Xa-S195A or a similar inactive FXa decoy like Andexanet Alfa may enable simultaneous reversal of both anticoagulants. This dual anticoagulant strategy may provide a useful alternative to UFH in clinical settings like CPB that necessitate both potent and reversible anticoagulation, and may be especially valuable for patients with a prior history of HIT. Disclosures Arepally: Biokit: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Differential Dose Dependent Effects of Recombinant FVIIa and Enhanced Activity Analogue (NN1731) on Thrombin Generation, Platelet Function and Clot Structure in Blood from Normal Volunteers and Patients with Factor VIII Deficiency with or without Factor VIII Inhibitor Antibodies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4033-4033
Author(s):  
Donald F. Brophy ◽  
Erika J. Martin ◽  
Melinda Nolte ◽  
Jan G. Kuhn ◽  
Sashidhar N. Reddy ◽  
...  
Keyword(s):  
Factor Viii ◽  
Blood Sample ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viii Inhibitor ◽  
Severe Hemophilia ◽  
Clot Structure ◽  
Dose Dependent ◽  
Whole Blood Sample

Abstract Purpose: The onset of force production due to thrombin generation (TGT), the amount of force produced (PCF) and the elastic modulus (CEM) of clots have been shown to be dose dependent functions of increasing recombinant FVIIa (rFVIIa) concentrations in a variety of patient blood samples. We performed a dose dependent comparison of the effects of rFVIIa and NN1731, an enhanced activity analogue of rFVIIa, in blood from normal volunteers and from hemophilia patients with and without factor VIII inhibitors. Methods: Blood was obtained from six healthy controls, two patients with factor VIII deficiency, two patients with hemophilia and factor VIII inhibitors, and one patient with a spontaneous factor VIII inhibitor who were enrolled in an IRB approved study. The blood was spiked with increasing concentrations (0.32, 0.64, and 1.28 mcg/ml final) of either rFVIIa or NN1731. TGT, PCF and CEM were determined using the Hemodyne Hemostasis Analyzer. Results: rFVIIa (1.28 mcg/ml) shortened the TGT (from 7 to 4 mins), increased CEM (from 20 to 31 Kdynes/cm2) and increased PCF (from 5.6 to 6.6 Kdynes) in blood from healthy controls. The effects of NN1731 (1.28 mcg/ml) were similar for CEM and PCF parameters (31 Kdynes/cm2; and 6.8 Kdynes, respectively; however, the effects were slightly more pronounced for TGT (2 mins). Due to the prolongation of TGT, and the decrease in PCF and CEM in all patient samples, the effects of both rFVIIa and NN1731 were much more pronounced. In all cases, both agents shortened the TGT, and increased both the CEM and PCF. In all patients NN1731 was considerably more potent but the dose response to both agents showed significant patient dependent variability. An example of this variability is illustrated below in the kinetics of PCF development of two patients with hemophilia. At baseline both patients had profoundly prolonged TGT (&gt;20 mins) and no force development. Patient P01 showed minimal response to increasing doses of rFVIIa and good response to NN1731. Patient P02 showed a nice response to both agents. Conclusions: This study suggests that recombinant analogue NN1731 of rFVIIa may be more potent than rFVIIa in correcting the thrombin generation, platelet function and clot structure deficiencies noted in patients with hemophilia with or without factor VIII inhibitors. Additionally, this study showed significant patient dependent variability at baseline and in response to both rFVIIa and NN1731. Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P01) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P01) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P02) with Severe Hemophilia A Effects of NovoSeven and NN1731 in Whole Blood Sample of Patient (P02) with Severe Hemophilia A

Recombinant Porcine Factor VIII Corrects Thrombin Generation and Improves Clot Structure In Vitro in Plasma Containing Anti-Factor VIII Inhibitory Antibodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2282-2282 ◽  
Author(s):  
Claude Negrier ◽  
Shannon L. Meeks ◽  
Johannes Oldenburg ◽  
Uri Martinowitz ◽  
Jean-Claude Bordet ◽  
...  
Keyword(s):  
Factor Viii ◽  
Board Of Directors ◽  
Inhibitory Activity ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Cross Reactivity ◽  
Advisory Committees ◽  
Clot Structure

Abstract Abstract 2282 Introduction: Treatment of bleeding episodes in patients with hemophilia A who have developed inhibitory antibodies can be challenging. Using human factor VIII (FVIII) and, historically, porcine FVIII in patients with a low inhibitor titer are therapeutic options, and provide ease of monitoring. A B-domain deleted recombinant porcine FVIII (rpFVIII; OBI-1), which may possess low cross-reactivity to anti-human FVIII antibodies, is being investigated for the treatment of bleeding episodes in individuals with congenital hemophilia A and inhibitors, and in those with acquired hemophilia. The in vitro capacity of this molecule to correct hemostasis has been further characterized. Methods: This is an international, multicenter in vitro study. Individuals with hemophilia A and inhibitor antibodies were recruited during routine out-patient visits between January 2011 and March 2011. Written and signed informed consent was obtained prior to venepuncture. Blood was obtained from volunteers with congenital hemophilia A and inhibitors attending routine visits at participating hemophilia treatment centers. A single blood sample was obtained from consenting individuals under protocols approved by Institutional Review Boards/Ethical Committees. In vitro spiking experiments with OBI-1 were conducted using FVIII-deficient plasma with and without anti-FVIII inhibitory activity. Three control inhibitor plasmas were provided, composed of FVIII deficient plasma to which the anti-C1 monoclonal antibody (MAb) to human FVIII (Sanquin, Amsterdam, the Netherlands) was added at two concentrations to reach anti-human FVIII inhibitory activity of 4.9 Bethesda Units (BU)/mL and 32.8 BU/mL with anti-porcine anti-FVIII inhibitory activity of 2.7 BU/mL and 19.1 BU/mL, respectively; and FVIII deficient plasma to which “polyclonal” mixture of the anti-C1 MAb, along with an anti-A2 and 2 anti-C2 MAbs was added. Plasma from eight patients with hemophilia A and inhibitors was tested. Hemostatic correction by OBI-1 was assessed by thrombin generation measurement (Calibrated Analytical Thrombography assay, Synapse BV, Maastricht, The Netherlands) and clot structure using electron microscopy. Epitope mapping of the inhibitor patient plasma was undertaken at a central laboratory (Atlanta, Georgia, USA) using an Enzyme-Linked Immunosorbent Assay (ELISA) with human/porcine FVIII hybrids as the antigen. Results: The results showed a dose-dependent and anti-porcine titer dependent correction of thrombin generation parameters (peak and ETP) with OBI-1 at concentrations equivalent to 100 IU/dL, 200 IU/dL, and 400 IU/dL, which paralleled a correction of the clot structure (number and diameter of fibrin fibres). These results were only dependent on the anti-porcine titer. In samples with high titers of anti-porcine inhibitor (>10 BU), little or no restoration of the diminished thrombin generation was observed when various OBI-1 concentrations were added to the plasma. In the plasmas with high anti-human titers (≥10 BU/mL) the dominant epitope was C2 in 3 plasmas, A2 in 1 plasma, and indeterminate in 3 plasmas. The plasmas with no restoration of the thrombin generation with even the highest dose of OBI-1 all had antibody detected to more than one domain of FVIII or were not able to be mapped due to high porcine cross-reactivity. Conclusion: In vitro data obtained with spiking experiments using OBI-1 indicate that it has the potential to correct surrogate markers of hemostasis depending on the anti-porcine FVIII titer which may translate into in vivo effectiveness. Further investigation into the epitope specificity of responsive and non-responsive inhibitor plasmas correlation with effectiveness is warranted. Disclosures: Negrier: Inspiration Biopharmaceuticals: Honoraria, Research Funding. Meeks:Inspiration Biopharmaceuticals: Research Funding. Oldenburg:SOBI: Membership on an entity's Board of Directors or advisory committees; Catalyst: Membership on an entity's Board of Directors or advisory committees; Inspiration: Consultancy, Honoraria, Research Funding; LFB: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Honoraria; Octapharma: Consultancy, Honoraria, Research Funding. Bordet:Inspiration Biopharmaceuticals: Research Funding. Poetzsch:Inspiration Biopharmaceuticals: Research Funding. Al Dieri:Synapse BV: Employment. Dargaud:Inspiration Biopharmaceuticals: Research Funding. Hemker:Synapse BV: Employment. Eckmann:Sanquin Diagnostic Services: Employment. Gomperts:Inspiration Biopharmaceuticals: Employment. Lee:Inspiration Biopharmaceuticals: Employment.

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