Successful reconstruction of an ankle defect with free tissue transfer in a hemophilia A patient with repetitive hemoarthrosis:A case report

BACKGROUND Hemophilia, an uncommon yet consequential hereditary bleeding disorder, manifests as two clinically indistinguishable forms that hinder the normal functioning of the coagulation cascade. This impairment renders individuals more susceptible to excessive bleeding during significant surgical interventions. Moreover, individuals with severe hemophilia frequently encounter recurring hemarthrosis, resulting in progressive joint destruction and, subsequently, the need for hip and knee replacement surgeries.CASE SUMMARY The patient was a 53-year-old man with hemophilia A as the underlying disease and had self-injected factor Ⅷ twice weekly for several decades. He had undergone ankle fusion surgery for recurrent hemarthrosis at the Department of Orthopedic Surgery 1 mo prior and was referred to our department because of skin necrosis after a hematoma at the surgical site. An anterolateral thigh perforator free flap was created after three cycles of factor Ⅷ administration in addition to the concomitant administration of tranexamic acid(TXA)(Transamin 250 mg cap, 1 cap tid, q8h). After the operation, from postoperative days(PODs) 1-5, the factor Ⅷ dose and interval were maintained, and q12h administration was tapered to q24h administration after POD 6. Because the patient’s flap was stable 12 d after the operation, factor Ⅷ administration was tapered to twice a week. At 6 mo follow-up, the patient recovered well without any complications.CONCLUSION To the best of our knowledge, there are very few reports of successful free flaps in patients with hemophilia, and none have been reported in patients with hemophilia A. Moreover, there are several reports on the efficacy of TXA in free flaps in general patients;however, there are no case reports of combining factor Ⅷ and TXA in patients with hemophilia. Therefore, we report this case to contribute to future academic research.

Characterization of Bleeding and Laboratory Phenotype in Hemophilia A Carriers: A Cross-Sectional Study in Benin

Background: In Africa, hemophilia is underdiagnosed and carriers have long been considered free from bleeding symptoms. However, recent research has begun to reveal hemostatic abnormalities and bleeding manifestations in carriers of hemophilia A, particularly due to excessive inactivation of normal X chromosomes. Objective: To describe the bleeding symptoms and hemostatic abnormalities in carriers of hemophilia A (HA) in Benin. Methods: This study was conducted as a prospective cross-sectional investigation between April 2021 to March 2022. The study population consisted of identified through pedigrees of persons with hemophilia A being treated in various hospitals in Benin. Data were collected through interviews conducted by trained physician and each carrier underwent a biological workup. Results: A total of 71 hemophilia A carriers were included and 38 of whom were obligatory carriers. Thirty-one carriers (43.7%) reported abnormal bleeding symptoms. Menorrhagia has (71%) being the most important manifestation, followed by bleeding during or after childbirth (45.2%). Among the 71 carriers, 45 were of reproductive age. Of whom 22 (48.8%) had a Higham score exceeding 100. Activated partial thromboplastin time was prolonged in 7 carriers (9.9%). The mean activity factor VIII:C (FVIII:C) levels were 68.8 ± 34.9 IU/dL. The average FVIII:C level in obligatory carriers was 56.9% and among potential carriers, the average FVIII:C level was higher at 80.4%. However twelve female carriers (16.9%) had FVIII:C levels < 40%. The FVIII:C/FvWAg ratio was below 0.7 in 73.2% of female drivers. Obligatory carriers (p = 0.00003) and FVIII;C/FvWAg ratio = 0.003) were statistically associated with abnormal bleeding symptoms, while blood group O (p = 0.0002) and FVIII/FvWAg ratio = 0.0016) were associated with a higher risk of menorrhagia. Conclusion: In Benin, carriers of haemophilia A present bleeding symptoms and haemostatic abnormalities. Further studies on a larger number of carriers are needed to better characterize and manage these patients.

Acquired hemophilia A in solid cancer: Two case reports and review of the literature

Acquired hemophilia A （AHA） is a rare, hemorrhagic autoimmune disease, whose pathogenesis involves reduced coagulation factor Ⅷ （FⅧ） activity related to the appearance of inhibitors against FⅧ. Common etiological factors include autoimmune diseases, ma-lignancy, and pregnancy. We report two cases of AHA in solid cancer. The first case is a 63-year-old man who developed peritoneal and intestinal bleeding after gastrectomy for gastric cancer. He was diagnosed with AHA, and was treated with prednisone, followed by cyclophosphamide. In the second case, a 68-year-old man developed a subcutaneous hemorrhage. He was diagnosed with AHA in hepatocellular carcinoma on CT imaging, and treated with rituximab alone. Hemostasis was achieved for both patients without bypassing agents as the amount of inhibitors was reduced and eradicated. However, both patients died within 1 yeardue to cancer progression. Successful treatment for AHA in solid cancer can be diffcult because treatment of the underlying malignancy is also required.

A Retrospective Analysis of Intracranial Hemorrhage in Children with Hemophilia A

To investigate the incidence,risk factors,clinical manifestations and prognosis of intracranial hemorrhage (ICH)in children with hemophilia A in a center of China, we conducted a retrospective analysis of 126 children with hemophilia A at our hospital in recent 4 years.Thirty-six children with hemophilia A (including 19 severe cases,and 17 moderate cases complicated with joint diseases)received low dose factor Ⅷ (FⅧ) prophylaxis,and none of them had ICH.However,13 cases of hemophilia A not given prophylaxis were complicated with ICH (12 severe cases,and 1 moderate case)and demonstrated an incidence of 10.3%(13/126)in all patients,and 28.6%(12/42)in severe cases.Of the 13 cases,9 severe ICH cases had a definite history of head injury,accounting for 69.2%.Headache was common in children >3 years,but somnolence,irritability, gaze or convulsions in children <3 years.The most common findings of cranial CT scan included intracranial hematoma (9/13),and less commonly observed were subependymal hemorrhage and intraventricular hemorrhage.After administration of FⅧ,all patients survived.Hematoma of 6 cases was observed during CT reexamination after 1-3 months. During the follow-up period,only one case had slight activity limitation on one side of the limb,but steadily recovered.Besides the decreased concentration of FⅧ,trauma is the most common risk factor of ICH in children with hemophilia A.The active treatment can improve the prognosis of ICH in children with hemophilia A.

Thromboelastography Use in the Perioperative Transfusion Management of a Patient with Hemophilia A Undergoing Liver Transplantation

The thrombelastogram is a method used to monitor clotting dynamics. Thrombelastography (TE) has been used to guide therapy of coagulation disorders mostly in cardiac surgery but also in liver surgery. TE is a useful tool for perioperative management of patients at risk for coagulopathy. There are several reports describing the use of the thrombelastogram in patients undergoing orthotopic liver transplantation (OLT), but only few cases include patients with both liver disease and inherited bleeding disorders. We describe the use of TE in a patient with hemophilia A and advanced cirrhosis undergoing OLT.

Hepatitis C treatment with triple therapy in a patient with hemophilia A

We report a case of successful treatment of chronic hepatitis C infection with telaprevir-based triple therapy in a patient with hemophilia A complicated by factor Ⅷ inhibitor. A twenty-two years old male with hereditary hemophilia A and high-titer factor Ⅷ inhibitor was taking maintenance doses of recombinant factor Ⅷ. He visited our clinic for treatment of his chronic hepatitis C with the newly instituted protease inhibitor based therapy. He was diagnosed with hepatitis C genotype 1a at one year of age. He was initiated on telaprevir, ribavirin and peg-interferon for treatment of hepatitis C and qualified for response-guided therapy. He completed treatment at 24 wk with minimal adverse effects. Notably, after 4 wk of hepatitis C treatment, his factor Ⅷ inhibitor screen was negative and the dose for recombinant factor Ⅷ decreased by half of the initial dosing before he was treated for hepatitis C. We suspect that suppressing hepatitis C may help decrease factor Ⅷ inhibitor level and the need for recombinant factor Ⅷ.

Practical Guidance for Non-Specialist Physical Therapists Managing People with Hemophilia and Musculoskeletal Complications

People with hemophilia (PWH) have impaired blood coagulation and hemostasis. Bleeding into joints and muscles is the main manifestation of the disorder, causing severe joint damage and muscle complications. Lifelong, regular physical therapy is therefore important for PWH, in order to support recovery from bleeds and reduce recurrences. Ideally, these individuals should be in the care of a hemophilia treatment center (HTC), where a physical therapist (PT) should be a regular part of the comprehensive team. However, not all PWH have convenient access to an HTC, and they may be referred to local non-specialist PTs, who may have only limited experience with the condition, for day-to-day physical therapy treatment. This article, written by four experienced hemophilia PTs, introduces key aspects of the pathology of the hemophilic joint in adult PWH with musculoskeletal complications or established joint disease (or those with inhibitors) that are relevant to PTs. The characteristic differences between the joints of PWH and those of people with similar pathologies are clarified. Practical advice on how the physical therapy consultation can be adjusted for PWH is given, and the importance of communicating with the HTC emphasized. The aim of this narrative review is to raise awareness of hemophilia and joint disease for non-specialist PTs, and serve as helpful background information for PTs who encounter PWH with musculoskeletal complications or established joint disease in their clinics.

Dental Treatment with 30% Trichloroacetic Acid in a Patient with Moderate Hemophilia A

We present the case of a 51-year-old moderate hemophiliac with gingival bleeding due to papillary inflammation and adherence in the molar area 47. After dental diagnosis a calculus was removed and 30% trichloroacetic acid was placed by a pressurized applicator for five seconds;the procedure was repeated until the bleeding stopped, then tranexamic acid was placed by a gauze. The patient chewed the gauze for 30 minutes and was discharged after repeating the same procedure for 30 minutes more. We observed how local treatment with trichloroacetic acid combined to tranexamic acid placed on the hemorrhagic site was able to stop the bleeding.

Clinical Analysis and Mental Health Survey of Hemophilia Carriers:a Cross-sectional Study

Objective:Hemophilia carriers(HCs),who are heterozygous for mutations in the clotting factor VIII/clotting factor IX gene(F8 or F9),may have a wide range of clotting factor levels,from very low,similar to afflicted males,to the upper limit of normal,and may experience mental health issues.The purpose of this study was to provide genetic information on mothers of hemophilia patients and to understand the clotting factor activity and phenotype of HCs.Additionally,we aimed to investigate the mental health status of HCs in China.Methods:A total of 127 hemophilia mothers,including 93 hemophilia A(HA)mothers and 34 hemophilia B(HB)mothers,were enrolled in this study.Long distance PCR,multiplex PCR,and Sanger sequencing were used to analyze mutations in F8 or F9.Coagulation factor activity was detected by a one-stage clotting assay.The Symptom Checklist 90(SCL-90,China/Mandarin version)was given to HCs at the same time to assess their mental health.Results:A total of 90.6%of hemophilia mothers were diagnosed genetically as carriers,with inversion in intron 22 and missense mutations being the most common mutation types in HA and HB carriers,respectively.The median clotting factor level in carriers was 0.74 IU/mL(ranging from 0.09 to 1.74 IU/mL)compared with 1.49 IU/mL(ranging from 0.93 to 1.89 IU/mL)in noncarriers,of which 14.3%of HCs had clotting factor levels of 0.40 IU/mL or below.A total of 53.8%(7/13)of HA carriers with low clotting factor levels(less than 0.50 IU/mL)had a history of bleeding,while none of the HB carriers displayed a bleeding phenotype.The total mean score and the global severity index of the SCL-90 for surveyed HCs were 171.00(±60.37)and 1.78(±0.59),respectively.A total of 67.7%of the respondents had psychological symptoms,with obsessive-compulsive disorder being the most prevalent and severe.The pooled estimates of all nine factors were significantly higher than those in the general population(P<0.05).Conclusions:The detection rate of gene mutations in hemophilia mothers was 90.6%,with a median clotting factor level of 0.74 IU/mL,and 14.3%of HCs had a clotting factor level of 0.40 IU/mL or below.A history of bleeding was present in 41.2%of HCs with low clotting factor levels(less than 0.50 IU/mL).Additionally,given the fragile mental health status of HCs in China,it is critical to develop efficient strategies to improve psychological well-being.

Low-dose immune tolerance induction for severe hemophilia A inhibitor patients:Immunosuppressants are generally not necessary for inhibitor-titer below 200 BU/mL

Importance:It remained unclear that the efficacy comparison between low-dose immune tolerance induction(LD-ITI)incorporating immunosuppressants(IS)when severe hemophilia A(SHA)patients had inhibitor-titer≥200 Bethesda Units(BU)/mL(LD-ITI-IS^(200) regimen)and LD-ITI combining with IS when SHA patients had inhibitor-titer≥40 BU/mL(LD-ITI-IS^(40) regimen).Objective:To compare the efficacy of the LD-ITI-IS^(200) regimen with that of the LD-ITI-IS^(40) regimen for SHA patients with high-titer inhibitors.Methods:A prospective cohort study on patients receiving LD-ITI-IS^(200) compared to those receiving LD-ITI-IS^(40) from January 2021 to December 2023.Both received LD-ITI[FVIII 50 IU/kg every other day].IS(rituximab+prednisone)was added when peak inhibitor tier≥200 BU/mL in the LD-ITI-IS^(200) regimen and≥40 BU/mL in the LD-ITI-IS^(40) regimen.Success is defined as a negative inhibitor plus FVIII recovery≥66%of the expected.Results:We enrolled 30 patients on LD-ITI-IS^(200) and 64 patients on LD-ITI-IS^(40),with similar baseline clinical characteristics.A lower IS-use rate was discovered in the LD-ITI-IS^(200) regimen compared to the LD-ITI-IS^(40) regimen(30.0%vs.62.5%).The two regimens(LD-ITI-IS^(200) vs.LD-ITI-IS^(40))had similar success rate(70.0%vs.79.7%),median time to success(9.4 vs.10.6 months),and annualized bleeding rate during ITI(3.7 vs.2.8).The cost to success was lower for LD-ITI-IS^(200) than for LD-ITI-IS^(40)(2107 vs.3256 US Dollar/kg).Among patients with peak inhibitor-titer 40-199 BU/mL,10 non-IS-using(on LD-ITI-IS^(200) regimen)and 28 IS-using(on LD-ITI-IS^(40) regimen)had similar success rates(70.0%vs.78.6%)and time to success(9.0 vs.8.8 months).Interpretation:In LD-ITI,IS are not necessary for inhibitor titer<200 BU/mL.

Extra Dural Hematoma of the Dorso-Lumbar Region in a Hemophiliac: A Rare Entity

Introduction and objective: Hemophilia is a genetic bleeding disorder inherited as a recessive train linked to the male gender. Bleeding into the central nervous system in patients with hemophilia is an extremely dangerous condition that can be directly life-threatening, if left untreated. Extradural hematoma of the dorso-lumbar region is rare but potentially deadly disease in children. This condition can result in severe neurological deficits. We aim to discuss the clinical, radiological and progressive clinical aspects of this illness. Case report: We report the case of a 5-year-old child with severe hemophilia A treated for extradural hematoma of the dorso-lumbar region resulting from trauma. A spinal magnetic resonance imaging revealed an extradural hematoma. The patient was successfully treated with intensive replacement therapy and did not require surgical intervention. Conclusion: Extradural hematoma is a rare complication of hemophilia, that needs to be looked for in children who have bleeding disorders. For the best neurological outcome, early diagnosis is crucial.

Short-term outcome of total knee replacement in a patient with hemophilia:A case report and review of literature

BACKGROUND Hemophilia A is a rare inherited bleeding disorder caused by mutations in the factor Ⅷ gene. This clotting factor plays an intrinsic role in the blood coagulation pathway. Patients with hemophilia may develop orthopedic manifestations such as hemarthrosis, but multiple malunion of fractures over the knee is rare and difficult to treat.CASE SUMMARY We report a patient with hemophilia A who developed severe knee osteoarthritis along with fracture malunion and nonunion. Total knee replacement was performed using a custom-made modular hinged knee prosthesis(cemented) equipped with extended distal and proximal stems. At 3 years’ follow-up, the patient exhibited excellent clinical function and remained satisfied with the surgical outcome. Surgical intervention was accompanied by rigorous coagulation factor replacement.CONCLUSION This case highlights various unique scenarios specific to individuals with hemophilia and fracture deformity.

Carrier detection and prenatal diagnosis of hemophilia A

Objective To establish an effective laboratory examination system for carrier detection and prenatal diagnosis of haemophilia A (HA) with a variety of molecular biological methods which are simple, rapid and easy to use. Methods Detection of inversion involving intron 22 in the FVIII gene was completed by long distance polymerase chain reaction ( PCR) and linkage analysis was performed by using several genetic polymorphisms including an intragenic Bcl I RFLP, 2 STRs and an extragenic St14 VNTR. Results Intron 22 inversion was observed in 10 out of the 21 (47.6%) pedigrees examined. Prenatal diagnosis was completed in 3 pedigrees. A further combination of the four intragenic and extragenic polymophic loci gave an informative rate of 94.7%. Conclusions Female relatives in HA families with inversion can be detected with direct diagnostic procedure. The application of long distance PCR makes the detection much more simple and rapid. For families without inversions, it is easier and more cost-effective to undertake linkage analysis of genetic polymorphism based on PCR.

Pharmacokinetic Studies of Factor VIII in Chinese Boys with Severe Hemophilia A： A Single-Center Study

Background： Although much attention has been paid to the pharmacokinetics （PKs） of different factor VIII （FVIlI） concentrates in persons with hemophilia A （HA）, limited information is available in young boys with severe HA. In this study, we aimed to assess the PK parameters of FVIII products in boys with severe HA in China. Methods： A total of 36 boys （plasma-derived [pd]-FVIII, n= 15; recombinant [r] FVIII, n = 21） were enrolled between January 2015 and May 2016 in Beijing Children＇s Hospital. PK characteristics of FVIII products were studied according to a reduced 4-sampling time point design （1 h, 9 h, 24 h, and 48 h postinfusion）. Results： The mean FVIII half-life （t1/2） was 10.99 ± 3.45 h （range 5.52-20.02 h）, the mean in vivo recovery （IVR） was 2.01 ± 0.42 IU/dl per IU/kg （range 1.24-3.02 IU/dl per IU/kg） and mean clearance （CL） of FVIII is 4.34 ± 1.58 ml·kg^-1·h ^-1 （range 2.29-7.90 ml·kg^-1·h·^-1）. We also analyzed the influence of several parameters that potentially modulate FVIII PK. The age was closely associated with FVIII half-life （R^2 = 0.32, P 〈 0.01 ）. The t1/2 of FVIII increased by 0.59 h per year. Besides age, yon Willebrand factor antigen （VWF：Ag） also was associated with FVIII half-life （R^2 = 0.52, P 〈 0.01）. Patients with blood Group O had a shorter FVIII halt-life than patients with non-O blood group （9.40 ± 0.68 h vs. 12.3 ± 0.79 h, t = 2.70, P = 0.01）. The FVIII IVR correlated with age （R^2 =0.21, P 〈 0.01） and VWF：Ag level （R^2 = 0.28, P 〈 0.01 ）. CL rates were taster in young patients and in those with low-VWF：Ag levels. CL rates of FVIII are higher in blood Group O versus non-blood Group O persons （5.02 ± 0.38 vs. 4.00 ± 0.32 ml·kg^-1·h^-1 , t = 2.53, P = 0.02）. Conclusions： Chinese boys with severe HA have similar PK values to other ethnic groups and large differences in FVIII PK between individual patients. Age, blood group, and VWF：Ag levels are important determining factors for FVlll CL.

The Mesenchymal Stem Cells Derived from Transgenic Mice Carrying Human Coagulation Factor Ⅷ Can Correct Phenotype in Hemophilia A Mice

Hemophilia A （HA） is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII （FVIII） deficiency. Previous studies showed that introduction of mesenchymal stem cells （MSCs） modified by FVIll-expressing retrovims may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-de/eted human FVIll （hFVHIBD） vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVlllBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak （77 ng/ mL） at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT （activated partial thromboplastin time） value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice （45.5 s vs. 91.3 s）. Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

Original article ：Identification of seven novel mutations in the factor Ⅷ gene in 18 unrelated Chinese patients with hemophilia A

Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. Methods Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTERS database. A clotting method was used to assay the FⅧ activity level and the Bethesda assay was used to detect the FⅧ inhibitor. Results A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation （4382-3 AC deletion） was associated with inhibitor development. Conclusion These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FⅧ inhibitor.

Value of Contrast-enhanced Ultrasound in Evaluating Synovitis and Predicting Recurrent Joint Bleeding of Hemophilia

Objective Contrast-enhanced ultrasound(CEUS)is advantageous for evaluating microcirculation,and has been applied to assess arthritis in previous studies.However,CEUS examinations have not been studied for hemophilia arthritis.Hemophilia arthritis is different from other arthritis,because it is induced by spontaneous joint bleeding.Hence,CEUS may have special value in evaluating hemophilia arthritis.The present study assessed the value of CEUS in evaluating synovial hypertrophy and predicting recurrent joint bleeding in severe hemophilia A patients.Methods From August 2016 to January 2017,81 severe hemophilia A patients,who were referred to our hospital for ultrasound joint assessment with conventional ultrasound,were enrolled.Among these 81 patients,46 patients consented for CEUS examinations on the same day.Results Compared to color Doppler flow imaging(CDFI),four more joints presented with a blood flow signal under CEUS mode.In addition,the synovial hypertrophy measured by CEUS was thicker than that measured by conventional ultrasound.The ultrasound scores(including the total grey-scale ultrasound score,joint effusion/hemarthrosis,synovial hypertrophy,CDFI semi-quantitative score,and CEUS semi-quantitative score)were significantly higher in the joint bleeding group than in the no joint bleeding group(P<0.05).Furthermore,these ultrasound scores were positively correlated with the joint bleeding frequency,and had the highest correlation with the CEUS score(r=0.620,P<0.05).Conclusion CEUS can more accurately assess the degree of synovial hypertrophy and vascularization,and diagnose synovitis,when compared to conventional ultrasound.In addition,CEUS appears to be essential for evaluating the possibility of recurrent joint bleeding,and providing more reliable evidence for individualized treatment.

Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FⅧcomposed of the human heavy chain and the rat light chain

Conventional therapies for hemophilia A(HA)are prophylactic or on-demand intravenous FⅧinfusions.However,they are expensive and inconvenient to perform.Thus,better strategies for HA treatment must be developed.In this study,a recombinant FⅧcDNA encoding a human/rat hybrid FⅧwith an enhanced procoagulant potential for adeno-associated virus(AAV)-delivered gene therapy was developed.Plasmids containing human FⅧheavy chain(hHC),human light chain(hLC),and rat light chain(rLC)were transfected into cells and hydrodynamically injected into HA mice.Purified AAV viruses were intravenously injected into HA mice at two doses.Results showed that the hHC+rLC protein had a higher activity than the hHC+hLC protein at comparable expression levels.The specific activity of hHC+rLC was about 4-to 8-fold higher than that of their counterparts.Hydrodynamic injection experiments obtained consistent results.Notably,the HA mice undergoing the AAV-delivered hHC+rLC treatment exhibited a visibly higher activity than those treated with hHC+hLC,and the therapeutic effects lasted for up to 40 weeks.In conclusion,the application of the hybrid FⅧ(hHC+rLC)via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice.These data might be of help to the development of optimized FⅧexpression cassette for HA gene therapy.