Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

Haemophilia and cardiovascular disease in Japan: Low incidence rates from ADVANCE Japan baseline data.

INTRODUCTION: With the increasing life expectancy of people with haemophilia, the risk of cardiovascular disease (CVD) and thrombotic events has become a growing concern. Longitudinal studies on the incidence and risk factors of CVD in this population are limited, and optimal prevention and treatment strategies are yet to be established. AIM: This study aimed to present the baseline data of a prospective longitudinal study focusing on a subset of Japanese patients with haemophilia, specifically investigated the incidence, risk factors and treatment modalities for CVD and thrombotic diseases in people aged 40 years in Japan over 10 years through the ADVANCE Japan study. METHODS: The ADVANCE Japan study is a prospective multicentre cohort study involving 600 adult individuals with haemophilia A/B aged 40 years in Japan. The primary endpoint was the incidence of CVD, with secondary endpoints encompassing anticoagulant use, mortality rates, and comparison with the general population. RESULTS: Baseline data from the 600 participants revealed that thrombotic events occurred in 13 individuals (2.2%), mostly in those with haemophilia A. Atrial fibrillation was observed in 11 participants (1.8%). Hypertension and dyslipidaemia were identified as the prevalent risk factors. Various prophylactic treatments were employed, and no severe bleeding events were observed during the study period. CONCLUSION: This study provides vital baseline data for a 10-year prospective investigation of CVD and thrombotic disease risk in people with haemophilia. These findings will contribute to refining prevention and treatment approaches and improving patients' quality of life.

The usefulness of tranexamic acid for bleeding symptoms of chronic consumptive coagulopathy complicated by aortic disease: a single-institute, retrospective study of 14 patients.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail. METHODS: We evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival. RESULTS: Median age was 78.5 years (range, 66-89 years) and median observation period was 448 days (range, 0-2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4-11) and median platelet count was 64 × 109/L (range, 25-97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days. CONCLUSIONS: TXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.

The influence of hepatitis C virus eradication on hepatocarcinogenesis in patients with hemophilia.

INTRODUCTION AND OBJECTIVES: Hepatitis C virus (HCV) infections in patients with hemophilia lead to the development of hepatocellular carcinoma (HCC) at a relatively younger age than that in patients without hemophilia. Although recent progress in direct-acting-antivirals has facilitated a high rate of sustained virological response (SVR), the clinical influence of HCV eradication in hemophilia patients remains unclear. This study aimed to compare the clinical outcomes of SVR against HCV in patients with and without hemophilia. PATIENTS AND METHODS: The study enrolled 699 patients who achieved SVR after HCV antiviral treatment. Patients were divided into two groups: 78 patients with hemophilia (H group) and 621 patients without hemophilia (NH group). We evaluated patient characteristics, clinical outcomes, and the cumulative incidence of HCC after SVR. RESULTS: Compared with the NH group, patients in the H-group were significantly younger and had a lower hepatic fibrosis score. No difference was found in the incidence of liver-related disease or overall death between the two groups over a mean follow-up period of 7 years. Four patients in the H group and 36 patients in the NH group were diagnosed with HCC after SVR. Multivariate analysis showed that male sex, age, and cirrhosis were significant risk factors for HCC incidence. There was no significant difference in the cumulative incidence of HCC after propensity-score matching adjusting for the risk factors of HCC between the two groups. CONCLUSION: Hemophilia is not a significant risk factor for hepatocarcinogenesis after SVR against HCV.

Safety and efficacy of percutaneous radiofrequency ablation for hepatocellular carcinoma patients with haemophilia.

Haemophilia is an X-linked inherited bleeding disorder caused by coagulation factor deficiency. Hepatocellular carcinoma (HCC) is a major complication associated with the disease. No study thus far has investigated the safety and efficacy of percutaneous radiofrequency ablation (RFA) for HCC in patients with haemophilia. AIM: This study aimed to evaluate the safety and efficacy of RFA for HCC in haemophilia patients. METHODS: From July 2008 to June 2019, 217 patients with HCC underwent 300 RFA sessions. Of these, 18 sessions were performed in ten haemophilia patients (H group) and 282 in 207 non-haemophilia patients (NH group). The patients' characteristics, incidence of haemorrhagic complications and rates of local tumour recurrence were compared between the groups. RESULTS: A majority of the haemophilia patients received clotting factor concentrate replacement therapy before and after RFA treatment, with the aim of reaching a plasma clotting factor level of higher than 60%-80%. Twelve haemorrhagic complications were observed in the NH group (4.2%; 12/282). Major bleeding requiring control procedures was observed in two patients and minor bleeding with careful observation was noted in ten patients. No bleeding complications were observed in the H group (0/18). There were no significant differences in the 5-year local tumour recurrence rates after RFA treatment between the groups (35.0% in the H group and 32.1% in the NH group). CONCLUSION: RFA could be an effective and a safe method for HCC treatment in patients with haemophilia.

[Lupus anticoagulant-hypoprothrombinemia syndrome associated with follicular lymphoma].

A 56-year-old woman was referred to our hospital with symptoms of swelling, purpura, and pain in her limbs. Prior to referral, bleeding in her limbs had spontaneously appeared and disappeared several times. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged, and the factor II level was 17%. The plasma-mixing test indicated lupus anticoagulant (LA), which was confirmed using aPTT-LA and dilute Russell's viper venom time (dRVVT). Therefore, she was diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS). During screening for underlying disorders, chest computed tomography (CT) revealed a retrosternal mass. Biopsy was not performed because the administration of freshly frozen plasma failed to correct her coagulopathy. Prednisolone (PSL) treatment (1 mg/kg) was initiated, which normalized the coagulation tests. The retrosternal mass also disappeared. PSL was tapered without LAHPS recurrence; however, the follow-up CT revealed systemic lymphadenopathy. Follicular lymphoma was diagnosed using lymph-node biopsy. Considering the subsequent LAHPS recurrence, six cycles of bendamustine + rituximab were administered. Complete response with no LAHPS recurrence was observed at the time of drafting this report. LAHPS is rare and distinct from antiphospholipid syndrome because it can cause severe bleeding. Underlying disorders should be evaluated in cases of LAHPS.

Ischaemic events are rare, and the prevalence of hypertension is not high in Japanese adults with haemophilia: First multicentre study in Asia.

INTRODUCTION: With the increasing life expectancy of patients with haemophilia (PWH), the number of PWH with age-related comorbidities, such as ischaemic events, is increasing. AIM: We conducted this multicentre observational study to identify the risk factors for major ischaemic events in PWH. METHODS: This study was the first multicentre observational study, conducted with the participation of five haemophilia treatment centres in Japan, conducted in ≥30-year-old adult PWH. The latest data recorded in the medical charts between 1 January and 31 December 2016 were reviewed. Healthcare data collected from the National Health and Nutrition Survey were used as the control data. RESULTS: Data of a total of 711 patients were collected. Only two PWH (0.3%) had a history of ischaemic events. Age-adjusted analysis indicated that the prevalence of hypertension defined as a blood pressure of 140/90 mm Hg or over was similar in the PWH to that in the males of the general population. However, when hypertension was defined more strictly (≥130/85 mm Hg), the prevalence was significantly lower in PWH than in the general male population. The hypertension in PWH was associated with the age, BMI, CKD, HIV infection and inhibitors. In particular, the odds ratio for the presence of inhibitors was high (odds ratio = 7.529). CONCLUSION: Whether the present results can be attributed to Japanese ethnicity or to the presence of haemophilia per se remains uncertain. We propose to initiate a prospective study for further investigation.

Variability in combinations of APTT reagent and substrate plasma for a one-stage clotting assay to measure factor VIII products.

INTRODUCTION: An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma. AIM: A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP. METHODS: FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP. RESULTS: F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3. CONCLUSION: In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur.

Clinical course and prognosis of patients with hepatocellular carcinoma and haemophilia.

INTRODUCTION: Although patients with haemophilia are known to develop hepatocellular carcinoma (HCC) at a lower age than patients without, there are few reports on the clinical course and prognosis of HCC. AIM: We aimed to investigate the clinical course and prognosis of patients with HCC and haemophilia. METHODS: Twenty-two patients with haemophilia, who were initially diagnosed with HCC between 2003 and 2021, were included. Their clinical courses and prognoses were retrospectively analysed. The results were compared with those of the 24th Nationwide Follow-up Survey of Primary Liver Cancer. RESULTS: All 22 patients were male; of these, 20 patients had haemophilia A, and 2 had haemophilia B. The mean age of diagnosis was 63 years (range 45-78 years) which is lower than the mean of 72 years reported in the Nationwide Survey. The mean diameter of the largest tumour was 30 mm (range 11-70 mm), and 18 tumours (82%) were solitary at the initial diagnosis. Standard treatments for HCC were performed in all patients. Sixty-one transarterial chemoembolisation, 28 RFA, 10 hepatectomies, and 2 radiation treatments were performed, and molecular-targeted agents were administered to 5 patients during their clinical courses. No deaths were associated with complications of HCC treatments. The median survival time after initial treatment was 6.4 years (range 0.9-18.7 years) which did not differ much from the median survival time of 5.8 years in the Nationwide Survey. CONCLUSION: Standard treatment for HCC could improve the prognosis of patients with HCC and haemophilia.

VWF-Gly2752Ser, a novel non-cysteine substitution variant in the CK domain, exhibits severe secretory impairment by hampering C-terminal dimer formation.

BACKGROUND: Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C-terminal interchain-disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain-disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD-associated noncysteine substitution variants in the CK domain have not been reported. OBJECTIVE: To investigate the molecular mechanism of a novel non-cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. METHODS: Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS-agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. RESULTS: The patient was homozygous for VWF-Gly2752Ser. Plasma VWF enzyme-linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF-Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF-Gly2752Ser-transfected cell lines showed that rVWF-Gly2752Ser was severely impaired in its ER-to-Golgi trafficking. CONCLUSION: VWF-Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.

Development and validation of a novel qualitative test for plasma fibrinogen utilizing clot waveform analysis.

Plasma fibrinogen is commonly examined by Clauss fibrinogen assay, which cannot distinguish between quantitative and qualitative fibrinogen anomalies. However, our previously reported Clauss fibrinogen assay utilizing clot waveform analysis (Clauss-CWA) provides additional information that contributes to the classification of fibrinogen anomalies. In this study, we adopted the Clauss-CWA method for an autoanalyzer to automatically measure the antigenic estimate (eAg) of fibrinogen in addition to the functional amount (Ac), and to thus provide the Ac/eAg ratio as a qualitative indicator. Performance was validated by receiver operating characteristics (ROC) and precision recall (PR) curve analyses using a patient cohort, consisting of a training cohort (n = 519) and a validation cohort (n = 523), both of which contained cases of congenital (hypo)dysfibrinogenemia as qualitative defects. We obtained an optimal cutoff of 0.65 for Ac/eAg by ROC curve analysis of the training cohort, offering superior sensitivity (> 0.9661) and specificity (1.000). This cutoff was validated in the validation cohort, providing positive predictive value > 0.933 and negative predictive value > 0.998. PR curve analysis also showed that Clauss-CWA provided excellent performance for detecting qualitative fibrinogen anomalies. The Clauss-CWA method may represent a useful approach for detecting qualitative fibrinogen abnormalities in routine laboratory testing.

Protein S-Leu17Pro disrupts the hydrophobicity of its signal peptide causing a proteasome-dependent degradation.

INTRODUCTION: Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T > C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency. MATERIALS AND METHODS: The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry. RESULTS: In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol. CONCLUSION: PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B.

INTRODUCTION: Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. PATIENT AND METHODS: The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. RESULTS: A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. CONCLUSION: A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

Impact of variation in reagent combinations for one-stage clotting assay on assay discrepancy in nonsevere haemophilia A.

INTRODUCTION: Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C1st ) and CSA (FVIII:CChr ) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized. AIM: To clarify the variations in FVIII:C1st /FVIII:CChr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy. METHODS: Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C1st /FVIII:CChr ratio to define FVIII assay discrepancy for each reagent combination. RESULTS: Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C1st /FVIII:CChr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy. CONCLUSION: Our findings showed that the FVIII:C1st /FVIII:CChr ratio is dependent on the reagent combination used for OSA.

Bursitis, Bacteremia, and Disseminated Infection of Mycobacteroides (Mycobacterium) abscessus subsp. massiliense.

We herein report a 59-year-old woman with a 2-year history of chronic bursitis of the hand who took 50 mg/day prednisolone for several autoimmune diseases. Mycobacteroides abscessus subsp. massiliense was isolated from the abscess and blood culture. Combination therapy (imipenem/cilastatin, amikacin, and clarithromycin) was administered for a month. Two months later, M. massiliense was detected from a blood culture again, and disseminated lesions were found. Clarithromycin and sitafloxacin were administered following eight weeks of the same regimen. Six months after the diagnosis, M. massiliense was isolated from a blood culture, and she expired due to multiple organ failure.

Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice.

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusion bodies in granulocytes. However, whether these disorders cause any changes in erythroid cells has yet to be determined. This study analyzed the influence of Myh9 R702C, as one of the most commonly detected MYH9 disorders, on erythroid cells in a mouse model. Knock-in mice expressing Myh9 R702C mutation either systemically or specific to hematological cells (R702C and R702C vav1 mice, respectively) were used in this study. Both displayed lower hemoglobin and higher erythropoietin levels than wild-type (WT) mice, along with significant splenomegaly. Flow cytometric analysis revealed erythroblasts present at a higher rate than WT mice in the spleen. However, no obvious abnormalities were seen in erythroid differentiation from megakaryocyte/erythroid progenitor to erythrocyte. Cell culture assay by fetal liver and colony assay also showed normal progression of erythroid differentiation from erythroid burst-forming unit to red blood cell. In conclusion, R702C and R702C vav1 mice displayed erythroid abnormality with splenomegaly. However, erythroid differentiation showed no obvious abnormality. Further research is required to elucidate the underlying mechanisms.

Essential role of a carboxyl-terminal α-helix motif in the secretion of coagulation factor XI.

BACKGROUND: Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear. OBJECTIVE: To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient. PATIENT/METHODS: The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant. RESULTS: We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal-truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion. CONCLUSION: FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect resulting from aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.