scholarly journals The Prognostic Advantage of Calreticulin Mutations in Myelofibrosis Might be Confined to Type 1 or “type 1-like” Calreticulin Variants

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3166-3166
Author(s):  
Ayalew Tefferi ◽  
Terra L Lasho ◽  
Alexander Tischer ◽  
Emnet A Wassie ◽  
Christy Finke ◽  
...  
Keyword(s):  
Leukocyte Count ◽  
Alpha Helix ◽  
Wild Type ◽  
C Terminus ◽  
Helix Propensity ◽  
The Difference ◽  
Calr Mutations ◽  
Propensity Scale

Abstract Background : Approximately 25% of patients with primary myelofibrosis (PMF) harbor calreticulin (CALR) mutations, which have been associated with longer survival (Klampf et al. NEJM 2013). More than 80% of CALR mutated patients harbor one of two mutation variants: type 1, a 52-bp deletion (p.L367fs*46) or type 2, a 5-bp TTGTC insertion (p.K385fs*47). Recent studies have suggested phenotypic and prognostic differences between these two variants (Tefferi et al. Blood 2014, Leukemia 2014 and AJH 2014). Furthermore, data are emerging that suggest functionally-relevant structural differences between type 1 and type 2 CALR variants, including a higher alpha-helix content of the mutant C-terminus in type 2, compared to type 1 (Eder-Azanza et al. Leukemia 2014). Objectives : We used statistical models to calculate helix propensity for thirty-one unique amino acid sequences that were altered by CALR mutations and used the results to subclassify non-type 1/2 CALR mutations into “type 1-like” and “type 2-like” variants. Subsequently, we examined the prognostic relevance of these subgroups. Methods : Calculation of helix propensity, which is the percentage of residues that are predicted to be involved in the formation of an alpha-helix, was performed using AGADIR, which is a statistical approximation algorithm (Munoz et al. Biopolymers 1997). The helix tendency calculations were performed using conditions of pH 7.0, 5 and 25 °C, an ionic strength of 0.1 M and no N- or C-terminal protection. Results : 532 PMF patients were screened for JAK2, CALR and MPL mutations; the respective mutational frequencies were 58%, 24.6% and 7.3%. Among the 131 CALR-mutated cases, 98 (74.8%) harbored type 1, 15 (11.5%) type 2 and 18 (13.7%) other variants. Based on predicted helix propensity scale, the “other” CALR mutations were subclassified as type 1-like (n=12) or type 2-like (n=6) and respectively grouped with type 1 and type 2 variants, for purposes of phenotypic and prognostic comparisons. The AGADIR-derived predicted helix propensity scale was 29.69 for wild-type CALR and 8.6 or 34.17 for type 1 and type 2 mutant CALR, respectively; accordingly, CALR variants with values that are close to or above the value for wild-type CALR were classified as “type 2-like” (range 26.47-36.12) and those with values close to or below the value for type 1 as “type 1-like” (range 2.11-17.3). Comparison of “type 1/type 1-like” (n=110) and “type 2/type 2-like” (n=21) CALR mutations showed the latter to be associated with higher DIPSS-plus score (p=0.01), EZH2 mutations (p<0.01), leukocyte count >25 x 10(9)/L (p<0.01), higher circulating blast percentage (p=0.02) and palpable spleen size >10 cm (p<0.01). Comparison of “type 1/type 1-like” CALR and JAK2 mutations (n=309) showed the former to be associated with younger age, higher platelet count, lower transfusion need, higher hemoglobin level, lower leukocyte count and lower DIPSS-plus score (p<0.01 for all comparisons). None of these associations was evident during comparison of “type 2/type 2-like” CALR with JAK2 mutations. Survival was similar between patients with type 1 and “type 1-like” (p=0.8) and between type 2 and “type 2-like” (p=0.63) CALR mutations. In contrast, survival was significantly shorter in patients with type 2 (HR 2.4, 95% CI 1.2-4.8) and “type 2-like” (HR 3.2, 95% CI 1.0-10.6), when compared to those with type 1 CALR mutations. Survival was also significantly shorter with “type 2/type 2-like” vs “type 1/type 1-like” CALR mutations (p=0.003; HR 2.5, 95% CI 1.4-4.5) and the difference remained significant when analysis was adjusted for age (p=0.047), ASXL1 (p=0.003) or EZH2 (p=0.001) mutations. Similarly, compared to JAK2-mutated cases (n=309), survival was longer in patients with “type 1/type 1-like” (HR 0.4, 95% CI 0.3-0.5) but not in those with “type 2/type 2-like” (HR 0.9, 95% CI 0.5-1.6) CALR mutations; the difference in survival between JAK2 and “type 1/type 1-like” CALR mutated cases remained significant (P<0.01) when analysis was adjusted for age, ASXL1 or EZH2 mutations or DIPSS-plus score. Conclusions : CALR mutations in PMF might be subclassified into type 1-like and type 2-like variants, based on predicted helical propensity of their mutant C-terminus. The favorable impact of CALR mutations in PMF might be restricted to type 1 or “type 1-like” variants. Disclosures No relevant conflicts of interest to declare.

scholarly journals Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4319-4319 ◽  
Author(s):  
Wei Wang ◽  
Tiansu Wang ◽  
Andriana G. Kotini ◽  
Camelia Iancu-Rubin ◽  
Ronald Hoffman ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Mutant Type ◽  
C Terminus ◽  
Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Polymorphisms of the Plasminogen Activator Inhibitor- 1 Gene in Type 1 and Type 2 Diabetes, and in Patients with Diabetic Retinopathy

1994 ◽  
Vol 71 (06) ◽  
pp. 731-736 ◽  
Author(s):  
M W Mansfield ◽  
M H Stickland ◽  
A M Carter ◽  
P J Grant
Keyword(s):  
Diabetic Retinopathy ◽  
Plasminogen Activator Inhibitor ◽  
Allelic Frequency ◽  
Repeat Sequence ◽  
Dinucleotide Repeat ◽  
Plasminogen Activator Inhibitor 1 ◽  
The Difference ◽  
Pai 1

SummaryTo identify whether genotype contributes to the difference in PAI-1 levels in type 1 and type 2 diabetic subjects and whether genotype relates to the development of retinopathy, a Hind III restriction fragment length polymorphism and two dinucleotide repeat polymorphisms were studied. In 519 Caucasian diabetic subjects (192 type 1, 327 type 2) and 123 Caucasian control subjects there were no differences in the frequency of the Hind III restriction alleles (type 1 vs type 2 vs control: allele 1 0.397 vs 0.420 vs 0.448; allele 2 0.603 vs 0.580 vs 0.552) nor in the allelic frequency at either dinucleotide repeat sequence. In 86 subjects with no retinopathy at 15 years or more from diagnosis of diabetes and 190 subjects with diabetic retinopathy there was no difference in the frequency of Hind III restriction alleles (retinopathy present vs retinopathy absent: allele 1 0.400 vs 0.467; allele 2 0.600 vs 0.533) nor in the allelic frequencies at either dinucleotide repeat sequence. The results indicate that there is no or minimal influence of the PAI-1 gene on either PAI-1 levels or the development of diabetic retinopathy in patients with diabetes mellitus.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

CLINICAL TRIALS IN INFANTS OF ORALLY ADMINISTERED ATTENUATED POLIOMYELITIS VIRUSES

PEDIATRICS ◽  
1959 ◽  
Vol 23 (6) ◽  
pp. 1041-1062
Author(s):  
Stanley Alan Plotkin ◽  
Hilary Koprowski ◽  
Joseph Stokes
Keyword(s):  
Clinical Trials ◽  
Fecal Excretion ◽  
Jackson Type ◽  
Poliomyelitis Virus ◽  
Minor Illness ◽  
The Difference ◽  
Antibody Levels ◽  
Type 3

Forty-six infants, ranging from less than 1 day to 6 months of age, were given more than 100 feedings of living, attenuated poliomyelitis viruses without the occurrence of major or minor illness. The strains used were CHAT (type 1), Wistar (type 1), Jackson (type 2), P-712 (type 2) and Fox (type 3). All strains except the Jackson strain were found to be antigenic on oral administration. Response to vaccination was demonstrated in these infants by the presence after vaccination of antibody levels significantly in excess of those attributable to transplacentally acquired antibodies, and by the detection of fecal excretion of poliomyelitis virus. Infants less than 2 months old were more difficult to immunize than older infants. The evidence suggests that biologic immaturity rather than transplacental antibodies caused the difference. When the three types of poliomyelitis virus were fed at 3-week intervals, responses occurred to all types. No interference between types was observed when they were fed in all possible sequences. Three infants given a second feeding of homotypic, attenuated poliomyelitis virus 3 to 5 months after a successful vaccination showed resistance to intestinal reinfection.

Anatomic patterning in the expression of vestibulosympathetic reflexes

2000 ◽  
Vol 279 (1) ◽  
pp. R109-R117 ◽  
Author(s):  
I. A. Kerman ◽  
B. J. Yates ◽  
R. M. McAllen
Keyword(s):  
Relative Proportion ◽  
Vestibular Stimulation ◽  
Nerve Fibers ◽  
Spike Shape ◽  
The Face ◽  
Nerve Fascicle ◽  
The Difference ◽  
Vestibulosympathetic Reflexes

To investigate the possibility that expression of vestibulosympathetic reflexes (VSR) is related to a nerve's anatomic location rather than its target organ, we compared VSR recorded from the same type of postganglionic fiber [muscle vasoconstrictor (MVC)] located at three different rostrocaudal levels: hindlimb, forelimb, and face. Experiments were performed on chloralose-anesthetized cats, and vestibular afferents were stimulated electrically. Single MVC unit activity was extracted by spike shape analysis of few-fiber recordings, and unit discrimination was confirmed by autocorrelation. Poststimulus time histogram analysis revealed that about half of the neurons were initially inhibited by vestibular stimulation (type 1 response), whereas the other MVC fibers were initially strongly excited (type 2 response). MVC units with types 1 and 2 responses were present in the same nerve fascicle. Barosensitivity was equivalent in the two groups, but fibers showing type 1 responses fired significantly faster than those giving type 2 responses (0.29 ± 0.04 vs. 0.20 ± 0.02 Hz). Nerve fibers with type 1 responses were most common in the hindlimb (21 of 29 units) and least common in the face (2 of 11 units), the difference in relative proportion being significant ( P < 0.05, χ2 test). These results support the hypothesis that VSR are anatomically patterned.

scholarly journals Protective Effects of Konjac and Inulin Extracts on Type 1 and Type 2 Diabetes

2019 ◽  
Vol 2019 ◽  
pp. 1-12
Author(s):  
Tianle Gao ◽  
Yue Jiao ◽  
Yang Liu ◽  
Tao Li ◽  
Zhiguo Wang ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Blood Glucose ◽  
Dose Group ◽  
Vehicle Control ◽  
Continuous Treatment ◽  
High Risk Population ◽  
Wild Type ◽  
Group A

Objective. The present study was designed to determine whether konjac and inulin extracts or their combination, konjac-inulin (KI) composition, as diet supplementary, can exert beneficial effects against type 1 diabetes and type 2 diabetes using animal models. Methods. A total of 60 diabetic (type 1) rats induced by streptozotocin (STZ) were randomly assigned to five groups: vehicle control (STZ group), KI combination at low dose group (KI-L group), KI combination at medium dose group (KI-M group), KI combination at high dose group (KI-H group), konjac extract group (konjac group), and inulin extract group (inulin group). A sham group (without STZ) was also included. Levels of blood glucose were monitored at each week. After continuous treatment of each diet for 24 days, a glucose tolerance test was performed. After 28 days of treatment, plasma biochemical indicators including glycated serum proteins, total cholesterol, and triglycerides were measured and immunohistochemistry staining of the rat pancreas was performed, to study the insulin expressions. Type 2 diabetes was developed in db/db mice. A total of 28 db/db mice were divided into 4 groups: vehicle control (db/db group), KI composition group (KI group), konjac extract group (konjac group), and inulin extract group (inulin group). A wild-type control group (wild-type group) for db/db mice was also included. Levels of blood glucose, body weight, and blood triglycerides were monitored at each week. Results. Daily use of the KI composition significantly decreased levels of blood glucose and blood triglycerides, as well as improved the insulin production in islets or reduced development of obesity in STZ-induced diabetic rats or in db/db mice. Such effects from KI composition were better than single ingredient of konjac or inulin extract. Conclusion. The results of this study suggest that daily use of KI composition has a protective role on type 1 and 2 diabetes and provided experimental basis for further development of KI composition as a food supplement for diabetic or diabetic high-risk population.

scholarly journals Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

2012 ◽  
Vol 80 (4) ◽  
pp. 1593-1605 ◽  
Author(s):  
Mary Ann Pohl ◽  
Sabine Kienesberger ◽  
Martin J. Blaser
Keyword(s):  
Helicobacter Pylori ◽  
Wild Type ◽  
Sequence Analyses ◽  
Upstream Gene ◽  
Content Type ◽  
Lewis Antigen ◽  
Mutant Strains ◽  
H Pylori

ABSTRACTLewis (Le) antigens are fucosylated oligosaccharides present in theHelicobacter pylorilipopolysaccharide. Expression of these antigens is believed to be important forH. pyloricolonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galTis essential for production of type 1 (Leaand Leb) antigens. The upstream genejhp0562, which is present in many but not allH. pyloristrains, is homologous to β-(1,3)galTbut is of unknown function. BecauseH. pyloridemonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galTnull mutant, but neither native nor recombinantjhp0562can. Mutagenesis ofjhp0562revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galTexpression in all wild-type (WT) and mutant strains tested, whereasjhp0562was not expressed injhp0562null mutants, as expected. Sincejhp0562unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whethergalT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed thatgalTis essential for Lebproduction. In total, these results demonstrate thatgalTandjhp0562have functions that cross the expected Le synthesis pathways and thatjhp0562provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

scholarly journals Resistance to Acetaminophen Interference in a Novel Continuous Glucose Monitoring System

2018 ◽  
Vol 12 (2) ◽  
pp. 393-396 ◽  
Author(s):  
Peter Calhoun ◽  
Terri Kang Johnson ◽  
Jonathan Hughes ◽  
David Price ◽  
Andrew K. Balo
Keyword(s):  
Type 2 Diabetes ◽  
Real Time ◽  
Monitoring System ◽  
Continuous Glucose Monitoring ◽  
Interference Effect ◽  
Glucose Monitoring ◽  
Post Dose ◽  
The Difference

Acetaminophen (APAP) can cause erroneously high readings in real-time continuous glucose monitoring (rtCGM) systems. APAP-associated bias in an investigational rtCGM system (G6) was evaluated by taking the difference in glucose measurements between rtCGM and YSI from 1 hour before to 6 hours after a 1-g oral APAP dose in 66 subjects with type 1 or type 2 diabetes. The interference effect was defined as the average post-dose (30-90 minutes) bias minus the average baseline bias for each subject. The clinically meaningful interference effect was defined as 10 mg/dL. The G6 system’s overall mean (±SD) interference effect was 3.1 ± 4.8 mg/dL (one-sided upper 95% CI = 4.1 mg/dL), significantly lower than 10 mg/dL. The G6 system’s resistance to APAP interference should provide reassurance to those using the drug.

Effects of Anti-FVIII Inhibitors On Factor VIIa/Tissue Factor-Catalyzed Activation and Inactivation of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3169-3169
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Normal Individuals ◽  
The Difference ◽  
Functional Inactivation

Abstract Abstract 3169 Poster Board III-110 Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.

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