Diagnosis and Treatment of Acquired Hemophilia: One Single-Center Experience from PR. China

Objective: To deepen the understanding of the clinical manifestations of acquired hemophilia A for timely and correctly treatment. Methods: The clinical data of the acquired hemophilia A patients diagnosed in the hospital from Jan 2006 to Mar 2021 were retrospectively analyzed, and the relevant literature was reviewed. Results: 17 patients with acquired hemophilia A, male: female =10: 7, median age 61 years (19 to 78 years), were diagnosed and treated in the hospital with the median time from the onset to diagnosis 21 days (2 days to 6 months). Six patients had comorbidity, including hepatitis B carrying, chronic myelomonocytic leukemia, diabetes, hypertension and positive autoantibodies, pemphigoid and gastric cancer, respectively. Other 11 patients were healthy before the onset. All patients had large large ecchymosis of skin, and one case was combined with hematuria, and one case with retroperitoneal hematoma. All patients had APTT extension (45s-144.7s) and the prolonged APTT could not be corrected with normal mixed plasma with and without incubation at 37℃ for 2 hours. FVIII activity was 1% - 8.9% and inhibitor titer 2 - 128 Bu/ml. All patients with bleeding were with prothrombin complex/recombinant activated coagulation factor VII, some of them with pd-coagulation factor FVIII preparations. Inhibitors were removed with prednisone acetate (1 case) + chemotherapy (1 case), prednisone acetate / + CTX (11 cases) + chemotherapy (1 case), prednisone acetate/prednisolone + mabthera (2 cases) + CTX (1 case), respectively. The removal time of inhibitor was from 8 days to 4 years. During the treatment process, two patients developed lower extremity venous thrombosis, and one patient was complicated with lung infection. Conclusion: Patients with unexplained bleeding and prolonged APTT should be conducted normal mixed plasma correction test, coagulation factor activity and inhibitor titer examination. After correctly diagnosis, bypass agents /coagulation factor VIII preparations should be given timely for hemostasis, protocol based on glucocorticoid + CTX/mabthera to remove the inhibitor and symptomatic treatment for patients with primary comorbidity disease at the same time. Disclosures No relevant conflicts of interest to declare.

Subcutaneous Marzeptacog Alfa (Activated) Versus Intravenous rFVIIa for Treatment of Episodic Bleeding in FVIII Deficient Hemophilia a Rats

Background: FVIII deficient knock-out (F8 -/-) rats mimic the bleeding incidents seen in severe human hemophilia A (HA). Subcutaneous (SQ) marzeptacog alfa activated (MarzAA), a novel, engineered recombinant activated coagulation Factor VII (rFVIIa) has been shown to effectively treat episodic bleeding in a pilot experiment in HA rats. This study evaluated the effect of single SQ doses of MarzAA and a single intravenous (IV) dose of rFVIIa on episodic bleeding in F8 -/- rats. Moreover, it compared the effect of SQ MarzAA and IV rFVIIa directly. Methods: Animals were allocated to treatment with either SQ vehicle, SQ MarzAA (60, 180 or 385 µg/kg) or IV rFVIIa (NovoSeven ®) (580 µg/kg) immediately after the bleeding was diagnosed. Doses were based on allometric scaling from humans (Nair AB and Jacob S. J Basic Clin Pharm 2016; 7: 27-31). The primary endpoint of the study was clinical efficacy as rated by a well-defined 4-point scale (Excellent, Good, Fair or Poor), and the efficacy assessment was either treatment success (Excellent or Good) or treatment failure (Fair or Poor) at the 24-hour timepoint. All personnel handling or assessing animals were blinded to the treatment status of each animal except those dosing animals who knew the route of administration. Results: A total of 86 F8 -/- rats was enrolled in the study. Of these, 61 rats presented treatment eligible bleeds between 3 and 10 weeks of age. No statistically significant difference in bleeding severity score were found across groups on diagnosis. As assessed by the clinical outcome at 24 hours, all three SQ MarzAA dose groups exhibited a statistically significant effect on treatment response when compared to SQ vehicle treatment (Figure 1). Conversely, no statistically significant effect could be identified when the single IV rFVIIa 580 µg/kg dose group was compared to vehicle. The overall treatment success rates at 24 hours were: SQ vehicle: 8%, SQ MarzAA 60 µg/kg: 58%, SQ MarzAA 180 µg/kg: 67%, SQ MarzAA 385 µg/kg: 85%, and IV rFVIIa 580 µg/kg: 33%. When compared directly using allometric scaling of clinical doses, SQ MarzAA at 385 µg/kg exhibited a statistically superior effect compared to IV rFVIIa at 580 µg/kg (p=0.0154, Fischer's exact test). Conclusion: Single doses of SQ MarzAA were effective in treating episodic bleeding in HA rats and statistically distinguishable from vehicle at all dose levels tested. When clinically relevant doses were compared directly to rFVIIa, SQ MarzAA compared favorably to IV rFVIIa. Taken together, these data provide robust nonclinical evidence that a single dose of SQ MarzAA may be successful in treating episodic bleeding when administered after bleeding has started. Figure 1 Figure 1. Disclosures Knudsen: Catalyst Biosciences: Current Employment, Current holder of individual stocks in a privately-held company. Blouse: Catalyst Biosciences: Current Employment.

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

On ignoring instead of chasing after coagulation factor VII during warfarin management; an interrupted time series study

During warfarin management, prothrombin time (PT) based PT-INR variability is partly due to clinically inconsequential fluctuations of factor (F) VII. The new Fiix-PT and Fiix-normalized ratio (Fiix-NR), unlike PT-INR, is only affected by reduced FII and FX. Starting July 1st 2016 we replaced PT-INR monitoring of warfarin with Fiix-NR in our patients. Using interrupted time series methods, we retrospectively assessed if this affected thromboembolism (TE) and major bleeding (MB) incidence during 12 months prior to and 18 months after the replacement, months 13-18 being predefined as transitional months. The dynamic cohort comprised all our service´s 2,667 maintenance phase warfarin patients managed at any time during the 30 months. Using two-segmented regression, a breakpoint in total TE monthly incidence became evident six months after laboratory monitoring test replacement, followed by 56% reduction in TE incidence (from 2.82% to 1.23% per patient year, P=0.019 by ANOVA). Three-segmented regression found no significant TE incidence trend (slope +0.03) prior to test replacement but during months 13-18 and 19-30 the TE incidence gradually decreased (slope -0.12; R2=0.20;P=0.007). Based on segmented regressions, MB incidence (2.79% ppy) did not differ pre- or post-intervention. Incidence comparison during the 12 month Fiix- and PT-periods confirmed a statistically significant 55-62% reduction in TE. Fiix-monitoring reduced testing, dose adjustments and normalized ratio variability, and prolonged testing intervals and time in range. We conclude that ignoring FVII during Fiix-NR monitoring in real world practice stabilizes the anticoagulant effect of warfarin and associates with major reduction in thromboembolism without increasing bleeding.

Biochemical, molecular and clinical aspects of coagulation factor VII and its role in hemostasis and thrombosis

Activated factor VII (FVIIa), the first protease of clotting, expresses its physiological procoagulant potential only after complexing with tissue factor (TF) exposed to blood. Deep knowledge of the FVIIa-TF complex and F7 gene helps to understand the Janus-faced clinical findings associated to low or elevated FVII activity (FVIIc). Congenital FVII deficiency, the most frequent among the recessively inherited bleeding disorders, is caused by heterogeneous mutations in the F7 gene. Complete FVII deficiency causes perinatal lethality. A wide range of bleeding symptoms, from life-threatening intracranial hemorrhage to mild mucosal bleeding, is observed in patients with apparently modest differences in FVIIc levels. Though clinically relevant FVIIc threshold levels are still uncertain, effective management, including prophylaxis, has been devised, substantially improving the quality of life of patients. The exposure of TF in diseased arteries fostered investigation on the role of FVII in cardiovascular disease. FVIIc levels were found to be predictors of cardiovascular death and to be markedly associated to F7 gene variation. These genotype-phenotype relationships are among the most extensively investigated in humans. Genome-wide analyses extended association to numerous loci that, together with F7, explain >50% of FVII level plasma variance. However, the ability of F7 variation to predict thrombosis was not consistently evidenced in the numerous population studies. Main aims of this review are to highlight i) the biological and clinical information that distinguishes FVII deficiency from the other clotting disorders and ii) the impact exerted by genetically predicted FVII level variation on bleeding as well as on the thrombotic states.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4, and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.