scholarly journals Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2460-2460
Author(s):  
Amey Barakat ◽  
Reema Jasuja ◽  
John E. Murphy ◽  
Debra D Pittman
Keyword(s):  
Blood Loss ◽  
Mouse Model ◽  
Tissue Factor ◽  
Low Dose ◽  
Hemophilia A ◽  
Factor Xa ◽  
Prothrombin Complex Concentrates ◽  
Activated Prothrombin Complex Concentrates ◽  
Dose Dependent ◽  
Tail Clip

Abstract BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Tissue Factor Pathway Inhibitor Causes Unresponsiveness to Activated Prothrombin Complex Concentrates for Hemophilia A Patients with Inhibitors.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3663-3663
Author(s):  
Kenichi Ogiwara ◽  
Keiji Nogami ◽  
Ichiro Tanaka ◽  
Katsumi Nishiya ◽  
Nobuyuki Tsujii ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Poor Response ◽  
Free Form ◽  
Prothrombin Complex Concentrates ◽  
Tissue Factor Pathway ◽  
Bypassing Agents ◽  
Activated Prothrombin Complex Concentrates

Abstract Abstract 3663 Bypassing agents such as activated prothrombin complex concentrates (APCC, FEIBA®) and recombinant activated factor VII (rFVIIa, NovoSeven®) are effective for most hemophiliac patients with inhibitors. While, some patients exhibit unresponsiveness to the treatment with APCC and/or rFVIIa, but their mechanisms remain unknown. We had a severe hemophilia A patient with inhibitor whose bleeding worsened despite of consecutive infusion of APCC. Switching from APCC to rFVIIa was very effective for his bleeding symptoms, and one-week cessation of bypassing agents had restored good response for APCC. Comprehensive coagulation assay such as thromboelastometry and thrombin generation test (TGT) provided us clear evidence of unresponsiveness to APCC. In particular, tissue factor (TF)-triggered TGT showed two significant features, the prolonged lag time and reduced peak thrombin level even after APCC infusion. Although FII, FVII(a), FIX, FX(a) and protein C contained in APCC were elevated in his plasmas after APCC infusion, increased amounts of these factors did not affect the parameters of TGT described above in vitro. We focused on a natural anticoagulant, tissue factor pathway inhibitor (TFPI), since the prolonged lag time in TF-triggered TGT might result from the impairment of FVII/TF-induced initial reaction of blood coagulation. In vitro experiment on the addition of TFPI to FVIII-deficient plasma with APCC showed similar inhibitory pattern in TGT. TFPI antigen levels (total and free forms) in his plasma actually increased above normal range after APCC infusion, whilst these levels unchanged after rFVIIa infusion and returned to the normal range after one-week cessation, speculating that TFPI might contributes to unresponsiveness to APCC. To confirm this, plasmas from several hemophiliac patients with APCC and/or rFVIIa infusion, including 4 patients with poor response pattern in TGT, were prepared. Among 12 pairs of plasmas (a pair; pre and post bypassing agents), each of 4 pairs were for APCC-poor response (APCC-PR), APCC-good response (APCC-GR), and rFVIIa-good response (FVIIa-GR). Free form TFPI antigen levels (normal; 15–35 ng/ml) increased after infusion in APCC-PR (pre/post; 38±4/51±3 ng/ml, p<0.05) and APCC-GR (28±4/37±4 ng/ml, p<0.05), but not increased in FVIIa-GR (23±3/21±3 ng/ml, p>0.05). Post-infusion levels in APCC-PR were significantly higher than those in APCC-GR (p<0.05). By adding anti-TFPI antibody, plasmas in APCC-PR showed marked increase of peak thrombin levels than those in APCC-GR in TGT, supporting that APCC-PR possessed more TFPI activity. Unexpectedly, ELISAs revealed that total TFPI were contained in APCC at 24±4 ng/unit (corresponded to 25≂f50% of physiological concentration), and 34% of them were free form, speculating that APCC infusion with ≂f90 units/kg appeared to increase free TFPI by ≂f15 ng/ml in plasma. Taken together, our results supported that TFPI contained in APCC attenuated the potentials of thrombin generation in hemophilia A patients with inhibitors, and some patients exhibited APCC-resistance due to TFPI accumulated by the consecutive use of APCC. Disclosures: Ogiwara: Baxter Hemophilia Scientific Research and Education Fund in Japan 2009: Research Funding. Nogami:Bayer Hemophilia Award Program 2009: Research Funding.

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

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

Blood ◽  
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.

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.

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 (&lt;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 &lt; 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 &lt; 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.

Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

1981 ◽  
Author(s):  
E Lechler ◽  
B Eggeling ◽  
D Meyer-Börnecke ◽  
H Stoy
Keyword(s):  
Hemophilia A ◽  
Gel Filtration ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Minor Degree ◽  
Activated Prothrombin Complex Concentrates ◽  
A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽  
2011 ◽  
Vol 117 (20) ◽  
pp. 5514-5522 ◽  
Author(s):  
Emily K. Waters ◽  
Ryan M. Genga ◽  
Michael C. Schwartz ◽  
Jennifer A. Nelson ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Long Term Dose-Dependent Correction of Hemophilia A Dogs Using AAV-8 and AAV-9-Mediated FVIII Gene Transfer.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 999-999
Author(s):  
Denise E. Sabatino ◽  
Amy M. Lange ◽  
Melinda Mucci ◽  
Rita Sarkar ◽  
Aaron M. Dillow ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Dose Group ◽  
Low Dose ◽  
Hemophilia A ◽  
Clotting Factor ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Aav Vector ◽  
Dose Dependent

Abstract Hemophilia A (HA) is an X-linked bleeding disorder characterized by deficiency in clotting factor VIII (FVIII). Current treatment for hemophilia is protein replacement therapy while a gene-based therapy would provide continuous expression of even low levels of FVIII protein (&gt;1% of normal) that is likely to improve the disease phenotype. It is challenging to utilize an AAV-mediated gene transfer approach for the FVIII cDNA (4.4kb) since the AAV vector can only efficiently accommodate a &lt;5.3kb transgene cassette. The FVIII protein is composed of 2 chains -the heavy chain (HC) and the light chain (LC). FVIII undergoes proteolytic cleavage and processing of the 2 individual chains that form the active FVIII protein. In other studies in HA dogs (n=8), no dose-response and AAV serotype-dependent FVIII expression has been documented, which illustrates the difficulties in using a FVIII single-chain approach. We have utilized a 2-chain approach in which the 2.4kb LC cDNA is packaged in one AAV vector while the 2.5kb HC is packaged into a second AAV vector. Each construct contains a 695bp thyroxine-binding globulin gene promoter/enhancer fused to a 175bp intron along with a 263bp SV40 poly A signal. For this approach the LC and HC vectors packaged into either AAV8 or AAV9 were administered to HA dogs via the hepatic artery. Two male HA dogs received HC and LC in AAV8 and 2 male dogs received HC and LC in AAV9 at doses of 6x1012gc/vector/kg (low dose) or 1.25x1013gc/v/kg (high dose). At 150 days after vector infusion, the high dose group expressed FVIII at levels of 4.8% (AAV8) and 3% (AAV9) as detected by a functional assay (Coatest assay). FVIII remained stable for 797 days (AAV8) and &gt;200 days (AAV9) (the longest time points to date) without any evidence of antibody formation to the transgene. In the low dose group at 150 days, FVIII levels were 1.5% (AAV8) and 0.5% (AAV9) cFVIII activity and were maintained in a follow up period of &gt;150 days (AAV8) and &gt;700 days (AAV9) without formation of antibodies to FVIII. Thus, no major differences between AAV8 and AAV9 vectors were observed. The transgene product is also functional based on shortening of whole blood clotting time (baseline values &gt;50 min), in a dose-dependent manner, 10–15 min and 16–20 min for the high and low dose cohorts, respectively. Interestingly, high dose injection of AAV8 to 2 female HA dogs (1.25x1013 and 3x1013gc/v/kg) results in FVIII levels of 1–2%, which is consistent with data obtained in mice on the poor performance of AAV in mediating gene transfer to liver in female animals. Liver function tests and other blood chemistries were transiently elevated after the surgical procedure and were in normal limits within 4 days. Importantly, all dogs did not develop antibodies to FVIII. These findings suggest that FVIII chains efficiently assemble in vivo without increasing the protein immunogenicity. The 4 male dogs have remained asymptomatic with no spontaneous bleeds, whereas &gt;20 bleeding episodes were expected for this group since untreated dogs require 5.5 plasma infusions/year. These data demonstrate for the first time, dose-dependent sustained expression of functional cFVIII in HA dogs by AAV8 and AAV9 vectors without formation of antibodies to cFVIII.

A Native Whole Blood Thrombin Generation Assay Allows Discrimination of Whole Blood Samples with FVIII Levels below 1%

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4215-4215
Author(s):  
Christina K Baumgartner ◽  
Jonathan C Roberts ◽  
Paula M Jacobi ◽  
Sandra L Haberichter ◽  
Qizhen Shi ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Blood Samples ◽  
Time Points ◽  
Fviii Activity ◽  
Post Infusion ◽  
Whole Blood Samples ◽  
Dose Dependent

Abstract Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%. Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (>6 half-lives, calculated FVIII levels <1%) significantly differed from healthy control samples in all thrombin generation parameters. Compared with healthy controls (6.9 ± 0.6 min; mean ± SEM) the hemophilic patient had a lag time (LT) of 24.8 ± 3.4 min. Peak time in healthy controls and the patient was 10.1 ± 1 min and 35 ± 5 min, peak thrombin was 528 ± 78 nM and 124 ± 20 nM, endogenous thrombin potential (ETP) was 1949 ± 117 nM and 1201 ± 50 nM, and thrombin generation rate was 196 ± 58 nM/min and 21 ± 6 nM/min, respectively. While previous studies on thrombin generation in plasma samples mainly reported on differences in peak thrombin and ETP, spiking of hemophilic blood with increasing concentration of recombinant FVIII in vitro revealed that the LT was FVIII dose dependent in our assay. When hemophilic blood was reconstituted with FVIII to a 2%, 5% and 50% level, the LT was 22.5 ± 1.6 min, 16.1 ± 1.7 min and 8.8 ± 0.6 min, respectively. All other thrombin generation parameters were FVIII dose dependent as well. A FVIII dependent LT was also apparent in vivo, when we monitored the patient after FVIII infusions. LT was 6.4 ± 0.2 min at 15 min, 8.5 ± 0.4 min at 24 hrs, and 13.8 ± 0.5 min at 48 hrs post FVIII treatment. We identified the lack of tissue factor as being key to a FVIII dose dependent LT. At all post infusion time points the LT was approximately 5 min when tissue factor was added to the assay. To our surprise, looking at specific time points equal to or greater than 72 hrs post infusion enabled us to discriminate these samples based on the LT (72 hrs: LT= 13.0 ± 0.6 min, 84 hrs: LT= 19.5 ± 0.8 min, 96 hrs: 36.0 ± 4.4 min). The ETP, commonly used as a variable parameter in previous thrombin generation reports, however, was not different among 72, 84 and 96 hrs post FVIII infusion samples. Strikingly, FVIII activity determined by chromogenic and one stage clotting assay was below detection limit (1% FVIII:C) in samples obtained 72 hrs post infusion or later. Thus, the patient in our study displayed considerable thrombin generation determined by the nWB-TGA at post FVIII infusion time points when FVIII levels were considered undetectable with currently available technology. Our data suggest that the different LT observed in 72, 84 and 94 hrs post infusion samples is possibly related to differences in residual FVIII levels below 1%. In conclusion, the nWB-TGA provides a useful tool to monitor efficacy of FVIII replacement therapy and might assist in tailoring individual FVIII treatment regimens. This close to physiological whole blood assay allows distinguishing blood samples with FVIII levels below 1% in vivo, and might help to explain the heterogeneity in bleeding phenotypes observed in severe hemophilia A patients. This assay may also be useful in assessing therapeutic benefit of “long acting” FVIII or FIX products. Disclosures No relevant conflicts of interest to declare.

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