Factor X Activity Research Articles

Phylogenetic analysis and pathogenesis study of a new deletion mutation causing inherited FⅩ deficiency

Objective: To analyze the F10 gene mutations in a Chinese pedigree affected with the deficiency of the hereditary coagulation factor X (FX), resulting from a new deletion mutation, and to study the associated molecular pathogenesis. Methods: Next generation sequencing (NGS) was performed to screen the genetic mutations in the proband which were then verified by Sanger sequencing. The FX activity (FX∶C) of probands and their family members was detected using the blood clotting method, and the mutation sites of the family members were analyzed using Sanger sequencing. The pathogenicity of the mutation site was predicted by using the online bioinformatics software, Mutation Taster. The SWISS-MODEL software was used for stimulating the three-dimensional models of the wild-type and mutant proteins for analyzing the influence of the mutation site on the structure and function of the proteins, and for analyzing the difference between the catalytic residues of the wild-type and the mutant proteins. The level of the F10 gene mRNA was quantitatively analyzed by qRT-PCR (quantitative reverse transcription polymerase chain reaction) method by constructing plasmids, transfecting human embryonic kidney 293T cells (HEK 293T), and analyzing the splicing of the mutated site by RT-PCR method. The levels of FⅩ∶Ag in cell lysates and cell culture media (both inside and outside the cells) were detected by the ELISA (enzyme linked immunosorbent assay) method. Results: A medium-grade factor X deficiency with a 36.42% FⅩ∶C ratio was detected in the proband by the coagulation method. NGS analysis demonstrated a heterozygous deletion mutation in exon 8:c.902_919del (p.Ala301_Glu306del) in the proband. Sanger sequencing analysis indicated that some members of the family (mother and grandfather) were also carriers of the corresponding deletion mutation. Online bioinformatics software predicted the pathogenic nature of the c.902_919del mutation, with a pathogenic score of 0.999. The 3D protein structure model analysis indicated that the c.902_919del mutation resulted in the disappearance of a segment of β-fold in the protein structure, thereby shortening the preceding segment of the β-fold and a subsequent loss of hydrogen bonds between adjacent amino acids with no significant difference in the side chain conformation of the key catalytic residues compared to the wild-type. mRNA splicing analysis indicated the absence of alternative splicing changes in the mutation, and qRT-PCR results indicated the absence of a statistically significant difference between the mRNA levels of F10 gene and wild-type mRNA in cells expressing c.902_919del mutant. The ELISA results indicated that there was no statistically significant difference in the FX∶Ag levels of the mutant cell culture medium and the lysate. Conclusions: In this pedigree, the heterozygous mutation in exon 8 of F10 gene (c.902_919del, p.Ala301_Glu306del) caused the hereditary factor Ⅹ deficiency.

Plasma-derived human factor X concentrate for the treatment of patients with hereditary factor X deficiency.

Hereditary factor X (FX) deficiency (HFXD) is an autosomal recessive rare bleeding disorder that leads to defects in the FX protein. Depending on the degree of deficiency, patients may be at risk of life-threatening bleeding episodes. Historical treatments for FX deficiency include prothrombin complex concentrates, which can increase the risk of thrombosis, and fresh frozen plasma, which can cause volume overload and transfusion reactions. Plasma-derived FX (pdFX), a single-factor, high-purity, high-potency human FX treatment, was approved in 2015 in the United States and in 2016 in Europe for on-demand treatment and prophylaxis of bleeding episodes and perioperative management of patients with HFXD. Five studies that examined the use of pdFX in patients with mild (plasma FX activity [FX:C] ≥5IU/dL), moderate (FX:C ≥1 and<5IU/dL), or severe (FX:C<1IU/dL) HFXD were reviewed: TEN01, TEN02 and TEN03 were prospective, open-label, multicentre, nonrandomised studies, and TEN05 and TEN06 were multicentre retrospective studies. When used as an on-demand treatment, pdFX was judged by investigators to be successful in treating 41/42 (97.6%), 2/3 (66.6%) and 79/79 (100%) bleeds in TEN01, TEN02 and TEN05, respectively. When used prophylactically, pdFX was judged 'excellent' for the prevention of bleeds in nine (100%) and eight (100%) patients in TEN02 and TEN05, respectively. Perioperative treatment and pharmacokinetics were also assessed. pdFX was safe and well tolerated. Together, these studies support the use of pdFX for on-demand treatment of bleeding, routine prophylaxis, and perioperative management of bleeding in patients with HFXD.

Features of disorders and methods of correction of the hemostasis system in women with antenatal fetal death

The improvement of the providing medical services quality to pregnant women remains an urgent issue in modern obstetrics. Women with perinatal losses deserve special attention, especially in the second half of pregnancy. Changes in the hemostasis system are an integral part of the development of pregnancy. The tendency to hypercoagulation has significant pathogenetic significance and can be the cause of a number of complications – miscarriage in the I trimester of pregnancy and preeclampsia, premature birth, antenatal fetal death (AFD) – in the II and III trimesters of pregnancy. Also, pregnancy is a background process for the activation of a number of diseases, in particular hereditary thrombophilia.The objective: to evaluate the changes in the hemostasis system in women with antenatal fetal death and the effectiveness of the use of low molecular weight heparins for the correction of disorders in the hemostasis system in this category of patients during childbirth and in the postpartum period.Materials and methods. 72 women were examined, including 42 pregnant women with AFD (main group) and 30 pregnant women with a physiological course of pregnancy, childbirth and the postpartum period (control group). The state of the hemostasis system was studied using a standard coagulogram. D-dimer was determined by immunoturbometric analysis.To assess the state of the hemostasis system, the following biochemical tests were used: procoagulant link – fibrinogen content and indicators: prothrombin index, activated partial thrombin time (APTT), thrombin time (TP), ancistrone time (AT), soluble fibrinogen-monomer complex (SFMC), factor X (FX); to evaluate the antithrombin system, the content of antithrombin-III (AT-III), protein C were determined; to characterize the state of the fibrinolytic system – the amount of plasminogen, α2-antiplasmin, fibrinogen degradation products (FDC).Thrombophilia markers and antiphospholipid antibodies were also determined. The preference was given to vaginal childbirth. During childbirth, mechanical compression of the lower limbs was applied using special compression stockings (compression level 2). In 12 hours after delivery thromboprophylaxis with low molecular weight heparins was started, the drug of choice being enoxaparin sodium. After 48 hours of the postpartum period, a comparative analysis of coagulogram indicators was performed to further determine the timing of thromboprophylaxis.Results. Analysis of family thrombotic anamnesis revealed risk factors in 12 (28.6%) patients of the main group. In close relatives of the first line, the presence of peripheral vein thrombosis was found – 5 (41.7%) cases, myocardial infarction under the age of 45 – 3 (25.0%) cases, pulmonary embolism – 1 (8.3%) case, transient ischemic attack of the brain – 3 (25.0%).In the control group only 2 (6.6%) patients had a family history of venous thromboembolism in first-line relatives. The analysis of the coagulation system shows significantly higher fibrinogen values (the main group – 5.3±0.2 g/l, the control group – 4.3±0.1 g/l; p&lt;0.05), functional FX activity (the main group – 149.3±3.1%, control group – 107.3±2.7%; p&lt;0.05), SFMC (main group – 15.9±1.2 μg/ml, control group – 7.8±0.9 μg/ml; p&lt;0.05) and D-dimer (main group – 4.4±0.25 μg/ml, control group – 0.7±0.2 μg/ml; p&lt; 0.05) in pregnant women of the main group. An increase in these indicators is a predictor of thrombus formation, activation of blood coagulation by the internal pathway with a decrease in the antithrombin reserve due to FX.During the correlation analysis, a strong direct relationship (r=0.8633) was established between the indicators of SFMC and FX in the blood serum of pregnant women of the main group, the combination of which determines the tendency to clot formation. Determination of markers for the most common types of thrombophilia shows the dominance of hereditary forms (prothrombin, Leiden mutation, MTHFR) in 43% of pregnant women of the main group.The implementation of the proposed treatment approach contributed to a significant decrease in the average concentration of fibrinogen 48 hours after delivery in postpartum women of the main group (main group: before delivery – 5.3±0.2 g/l, after 48 hours after delivery – 3.9±0.3 g/l; control group: 48 hours after delivery – 3.2±0.2 g/l; p&lt;0.05) in combination with a synergistic decrease in the average indicators of SFMC (main group: before delivery – 15.9±1.2 μg/ml, 48 hours after delivery – 6.2±0.2 μg/ml; control group: 48 hours after delivery – 5.4±0.3 μg/ml; p&lt;0.05) and FX (main group: before delivery – 149.3±3.1%, after 48 hours after delivery – 103.1±3.6%; control group: after 48 hours after delivery – 117.1±4.1%; p&lt;0.05).Conclusions. Increased coagulation function was confirmed in pregnant women with antenatal fetal death (AFD). Thanks to the proposed method of correcting disorders in the hemostasis system, a decrease in the frequency of postpartum thromboembolism in women with AFD is achieved, as well as an improvement in the main indicators of the hemostasis system. This prevents the emergence of a chronic form of the syndrome of disseminated intravascular blood coagulation which is developed in the cases of AFD.

The first reported case of factor X deficiency in a Filipino child – case study

Abstract Factor X (FX) deficiency is an extremely rare inherited bleeding disorder affecting one in 1,000,000 people. According to the most recent published census of the World Hemophilia Federation, to date there is no reported case of FX deficiency in the Philippines. Rare disorders like FX deficiency often go unrecognised or misdiagnosed. Here, we report the first case of FX deficiency in a Filipino child. A two-month-old male child with consanguinity was referred to our hospital due to bleeding episodes. On the third day of life, he had haematomas to the cervical area and upper extremities, and spontaneous bleeding of the umbilical cord was noted. Initial workup showed prolonged PT and aPTT. Factor deficiencies including FVIII and FIX were considered, however assays were normal. At six weeks of age, the child developed convulsions and deteriorating neurologic status. CT scan showed subarachnoid haemorrhage. The child was referred for further workup. Additional assay of clotting factors showed decreased FX activity at less than 1% and he was diagnosed with severe congenital FX deficiency. Following recurrent intracranial bleeding, the child has been observed to have permanent neurological deficit. This case highlights the importance of timely and accurate diagnosis to prevent life-threatening complications and the risk of permanent disability. Despite being an extremely rare disorder, the incidence of FX deficiency is estimated to be higher in populations where consanguineous marriages are common. Awareness of this rare condition must be emphasised. Families may benefit from screening through coagulation studies as well as genetic counselling, especially when planning future pregnancies. The rarity of this condition has not allowed for the establishment of evidence-based management guidelines, with treatment based on limited literature. Despite development of FX-specific clotting factor products, the high cost and limited availability impact their use in low-resource settings.

SUCCESSFUL MANAGEMENT OF SEVERE CONGENITAL FACTOR X DEFICIENCY DURING PREGNANCY AND LABOR WITH PCC IN TWO SISTERS

Factor X (FX) deficiency is an autosomal recessive disorder caused by quantitative or qualitative defects in the FX protein. FX deficiency has an estimated worldwide prevalence of one in 1000000. (1) Pregnancy in women with congenital FX deficiency has been associated with adverse fetal outcomes (abortion and preterm labor) (2,11). We report two cases of successful pregnancy with factor X deficiency. A 29-year-old woman with congenital factor X deficiency and prior abortion on prophylaxis PCC every 4 weeks. She was treated with PCC 25unit/kg twice weekly during the pregnancy course. At week 32 of pregnancy, she presented with labor pain. Lab showed PT 20.7 PTT 52.5 INR1.5 Fibrinogen 3.8 Hb13.8 platelet 195 WBCs 7.6 factor X 0.15. She was given PCC 25 units/kg until a level of 0.4 was achieved. She delivered a healthy, 1.9 kg baby by normal vaginal delivery. The estimated blood loss was 150 ml. She then received FX 15 units/kg for 3 days postpartum to maintain FX level >30% and INR <1.5. No episodes of abnormal bleeding were observed during pregnancy, labor or post-partum. A 36-years-old woman with congenital factor X deficiency and two prior abortions, on prophylaxis PCC every 4 weeks. She received prophylaxis PCC 25units/kg twice weekly during the course of this pregnancy. At week 38 of pregnancy, she delivered a healthy 3.2 kg baby by cesarean section (CS) after failing labor induction. Lab showed PT 23.7 PTT 50.4 INR1.7 Fibrinogen 2.3 CBC was normal.FX 0.13. She was given PCC 25 units/kg until a level of 0.4 was achieved. The estimated blood loss was 500 ml. She then received FX 15 units/kg for 7 days postpartum to maintain FX level >30% and INR <1.5. She was discharged on tranexamixc acid. No episodes of abnormal bleeding were observed during pregnancy CS or post-partum. Although FX activity increases during normal pregnancy, levels usually remain insufficient for hemostasis at delivery in women with severe FXD (4,5,6). FX replacement therapy with PCC or FX concentrate may be required to treat or prevent bleeding in FXD.Therefore, a therapeutic dose of PCC 20–30 iu/kg is expected to increase plasma FX activity by 0.4–0.6 iu/ml. Further infusions at 1- to 2-d intervals may be required if sustained treatment is necessary (3) . There are reports of FX replacement with PCC during pregnancy in women with previous adverse pregnancy outcomes (7,8) and FX replacement during labor with PCC or FFP, but with different regimens (9) . Our patients were treated with PCC prophylaxis during pregnancy and 25 units/kg during labor. No bleeding nor thrombosis was seen in both cases. The British guidelines recommend PCC 20–40 iu/kg during the third trimester for women with history of bleeding and with FX activity <03 iu/ml with the goal of achieving FX activity >04 iu/ml. They also recommend, to consider further PCC 10–20 iu/kg once daily to maintain FX activity >03 iu/ml for at least 3 days post-partum. (10) Prophylactic PCC resulted in excellent hemostasis in two of our patients, including one that delivered by C-section.

Prevalence, clinical characteristics and treatment outcome of factor X deficiency in a consecutive cohort of primary light-chain amyloidosis

Factor X (FX) deficiency is prevalent in light-chain (AL) amyloidosis but its clinical significance was not investigated deeply. We conducted a retrospective analysis of a consecutive cohort with 207 primary AL amyloidosis patients. FX deficiency was present in 129 patients (62.3%). Those with FX deficiency had higher dFLC (299.6 mg/L vs. 102.3 mg/L, P < 0.001), higher cardiac troponin I (0.05 μg/L vs. 0.02 μg/L, P < 0.001) and N-terminal pro-brain natriuretic peptide (3115 ng/L vs. 392 ng/L, P < 0.001), and more patients with bone marrow plasma cells > 10% (18.0% vs. 4.3%, P = 0.008). The prevalence of FX deficiency increased with the Mayo 2004 stage. FX-deficient patients exhibited inferior overall survival (P < 0.001) and progression-free survival (P < 0.001) than others. Fifty-five patients with FX deficiency received retesting of FX activity after anti-plasma cell therapy. The median variation in FX activity was + 6.8% (range, −24.5% ~ +73.4%). Better improvement of FX activity was observed in patients with complete hematologic response (+18.2% vs. +4.0%, P = 0.036) and at least one organ response (+14.4% vs. +3.4%, P = 0.024). FX deficiency is associated with a heavier disease burden and poorer survival in primary AL amyloidosis. Improvement of FX activity tends to appear in patients with better hematologic and organ responses after chemotherapy.

Congenital factor X deficiency: a retrospective analysis of 11 cases

目的探讨遗传性凝血因子Ⅹ（FⅩ）缺乏症的临床特征、实验室检查、诊断、治疗及转归。方法回顾性分析2009年7月至2021年2月期间就诊于中国医学科学院血液病医院的11例遗传性FⅩ缺乏症患者的临床资料。结果11例患者中，男3例，女8例，中位初诊年龄39（5～55）岁。1例有家族史。10例（90.9％）存在出血事件，包括皮肤磕碰后瘀斑或出血（7例）、鼻出血（7例）、齿龈出血（6例）和肌肉血肿（1例）。8例女性患者中，6例有月经增多，1例正常分娩后发生出血。8例患者有手术史，4例发生术后出血。实验室检查见活化部分凝血活酶时间（APTT）延长、凝血酶原时间（PT）延长、FⅩ促凝活性（FⅩ∶C）减低。4例患者接受F10基因检测，发现5个新突变位点。11例患者中，4例输注凝血酶原复合物浓缩物（PCC），7例输注新鲜冰冻血浆（FFP），1例女性患者应用PCC预防性治疗后月经量明显减少，另有1例患者手术前预防性输注FFP后未发生术中、术后出血。结论多数遗传性FⅩ缺乏症患者有出血倾向，F10基因突变检测对疾病的诊断和预后判断具有一定意义。

Characterization of a Missense Mutation in the Catalytic Domain and a Splicing Mutation of Coagulation Factor X Compound Heterozygous in a Chinese Pedigree.

Background: Congenital coagulation factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million caused by mutations in the FX-coding gene(F10), leading to abnormal coagulation activity and a tendency for severe hemorrhage. Therefore, identifying mutations in FX is important for diagnosing congenital FX deficiency. Results: Genetic analysis of the proband identified two single-base substitutions: c.794T > C: p.Ile265Thr and c.865 + 5G > A: IVS7 + 5G > A. His FX activity and antigen levels were < 1% and 49.7%, respectively; aPTT and PT were prolonged to 65.3 and 80.5 s, respectively. Bioinformatics analysis predicted the two novel variants to be pathogenic. In-vitro expression study of the missense mutation c.794T > C: p.Ile265Thr showed normal synthesis and secretion. Activation of FXs by RVV, FVII/TF, and FVIII/FIX all showed no obvious difference between the variant and the reference. However, clotting activity by PT and aPTT assays and activity of thrombin generation in a TGA assay all indicated reduced activity of the mutant FX-Ile265Thr compared to FX-WT. Minigene assay showed a normal splicing mode c.865 + 5G > A: IVS7 + 5G > A, which is inconsistent with clinical phenotype. Conclusions: The heterozygous variants c.794T > C: p.Ile265Thr or c.865 + 5G > A: IVS7 + 5G > A indicate mild FX deficiency, but the compound heterozygous mutation of the two causes severe congenital FX deficiency. Genetic analysis of these two mutations may help characterize the bleeding tendency and confirm congenital FX deficiency. In-vitro expression and functional study showed that the low activity of the mutant FX-Ile265Thr is caused by decrease in its enzyme activity rather than self-activation. The minigene assay help us explore possible mechanisms of the splicing mutation. However, more in-depth mechanism research is needed in the future.

A Multicenter, Retrospective Data Collection Study on the Compassionate Use of a Plasma-Derived Factor X Concentrate to Treat Patients with Hereditary Factor X Deficiency

A Multicenter, Retrospective Data Collection Study on the Compassionate Use of a Plasma-Derived Factor X Concentrate to Treat Patients with Hereditary Factor X Deficiency

Splenectomy in a Severe Case of Acquired Factor X Deficiency Secondary to Amyloidosis: Case Discussion and Review of the Literature

Amyloidosis is a protein deposition disorder wherein amyloid fibrils affect soft tissues and visceral organs. One subtype is light-chain (AL) amyloidosis, which is characterized by amyloidogenic immunoglobulins produced generally by clonal plasma cells. Kidney and heart are the most commonly involved organs by AL amyloidosis while liver and sensory/autonomic nervous system can be clinically prominent. In a minority, acquired Factor X (FX) deficiency (related to amyloid binding this factor) can provoke a profound bleeding diathesis.We present a 62-year-old black male who was referred to our institution in May 2016 by a community hematologist after diagnoses of FX deficiency and monoclonal gammopathy. The patient initially presented with ecchymosis, hemearthroses, and blood per rectum. Workup revealed a hemoglobin 7.7g/dL, platelets 62x10³/µL and prolonged PT (35.4s) and aPTT (53.9s). Echocardiogram revealed concentric LV ‘hypertrophy’ and grade 2 diastolic dysfunction. Abdominal ultrasound identified an enlarged spleen of 18 cm. Fibrinogen level was 295mg/dL, PT and PTT mixing studies normalized, and FX activity was measured at 0.02U/mL (normal 0.2-0.4U/mL). Bone marrow biopsy showed plasma cell myeloma involving 50% of the marrow. He was started on therapy with cyclophosphamide, bortezomib, and dexamethasone.Unfortunately, the patient had no measurable response after 3 cycles of therapy at which time he presented with severe and progressive hemarthrosis, mucocutaneous bleeding, symptomatic anemia, thrombocytopenia, and elevations in PT/PTT. He received transfusions with fresh frozen plasma (FFP) around the clock, aminocaproic acid, and supportive transfusion of packed RBC and platelets. Due to active mucosal bleeding, the patient was administered vitamin K and transfused FFP every 4 hours. His coagulation parameters did not improve. Interestingly, despite his mixing studies correcting “in vitro,” he failed to demonstrate any “in vivo” correction despite FFP transfusion and three plasmapheresis treatments. Given his refractory bleeding, the surgical service was consulted regarding the prospect of a splenectomy. In an effort to optimize the patient for surgery, he received prothrombin complex concentrate (PCC) without significant improvement (Table 1). Ultimately, the patient underwent splenectomy (estimated blood loss 200cc) and was managed intra-operatively with FFP and aminocaproic acid infusion. His coagulation studies and platelet count improved immediately post-operatively (Table 1), and repeat FX level the following day demonstrated a normal value of 0.48 U/mL. Surgical pathology of the spleen was consistent with extensive deposition of amyloid (lambda subtype). To date, the patient has had a complete normalization of coagulation studies and FX levels and is clinically responding to daratumumab therapy.The incidence of coagulopathy in AL amyloidosis has been estimated to approach one third of all patients, largely due to acquired coagulation factor deficiency, and most commonly FX. Splenomegaly is present in fewer than 15% of patients with AL amyloidosis, however it is much more frequent in patients with acquired FX deficiency. Amyloid deposition in the spleen is thought to bind FX as well as sequesters platelets leading to potentially life threatening bleeding. Our review of the literature identified five previously reported cases of acquired FX deficiency secondary to AL amyloidosis which completely reversed after splenectomy (Kapoor et. al. American Journal of Hematology 2006), but our patient's presentation was particularly severe. Previous reports have also described the improvement of acquired FX deficiency in AL amyloidosis patients with high-dose melphalan (Choufani et. al. Blood 2001), although it is unclear what degree of clinically significant bleeding these patients were experiencing. Splenectomy is frequently reserved as last-line management in these patients, when more conservative measures such as FFP and plasmapheresis are not effective. This is due to the short half-life of the infused FX (Furie et. al. NEJM 1977), as well as its expedited clearance in the spleen, where it is bound to amyloid. While some cases using recombinant Factor VIIa and PCC have worked, they are more often ineffective. In patients with severe bleeding, splenectomy can produce rapid improvement as evidenced here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pediatric split liver transplantation for congenital factor X deficiency: first 10-year follow-up of a case with portal vein stenting

Congenital factor X (FX) deficiency is a rare autosomal-recessive disease that induces bleeding disorder. Herein, we present the 10-year posttransplant course of a pediatric patient who underwent liver transplantation (LT) with portal vein (PV) stenting for correction of severe congenital FX deficiency, with focus on long-term maintenance of coagulation function and patency of PV stenting. A 17-month-old infant with recurrent hemorrhagic episodes due to FX deficiency underwent split LT using a left lateral section graft. The graft-recipient weight ratio was 2.2%. The graft implantation procedures were performed by following the standard pediatric split LT procedure. Nevertheless, a wall stent was inserted due to PV anastomotic stenosis on posttransplant day 1. Graft function recovered slowly because of partial parenchyma infarct, and the patient was discharged at 46 days after LT operation. The FX activity started to increase soon after LT and gradually normalized; the coagulation profiles have been maintained well for the past 10 years. The patient has been doing well for the past 10 years after LT without any episodes of abnormal bleeding. Due to the risk of vascular complications owed to PV stenting, life-long follow-up is mandatory with special attention until attainment of complete physical growth to adolescent and adulthood.

Paroxysmal atrial fibrillation is associated with early coagulation activity regardless of risk factors for embolism.

Paroxysmal atrial fibrillation (PAF) is associated with an increased incidence of embolic events, even in patients with no embologenic risk factors. This fact raises the question for the hypercoagulability in PAF as a state closely related to the arrhythmia itself, independent of other well established embologenic risk factors. The scarce data on that topic predisposed our aim that was to study coagulation activity in the early hours (up to the twenty-fourth hour) of the disease. Fifty-one non-anticoagulated patients (26 men, 25 women; mean age 59.84±1.60 years) and 52 controls (26 men, 26 women; mean age 59.50±1.46 years) were consequently selected for the study. Plasma coagulation activity of factor II (FII), factor V (FV) and factor X (FX) was examined. In the PAF group, there was a higher activity of FII (167.81±9.12% vs. 100.43±5.77%, P<0.001), FV (198.47±10.88% vs. 121.53±4.79%, P<0.001) and FX (193.20±11.85 vs. 116.20±5.86, P<0.001). The deviations were independent of age, sex, body mass index, presence of hypertension and CHA<inf>2</inf>DS<inf>2</inf>-VASc risk score (P>0.05). PAF duration was a significant predictor of FII activity (r=0.83, P<0.001) but no correlation was established between FV and FX activity and the arrhythmia duration (r=0.10, P>0.05; r=0.11, P>0.05, respectively). We established high coagulation activity during the first twenty-four hours of PAF. The observed deviations in the studied parameters give us reason to consider PAF a procoagulant state independent of the well-established prothrombotic risk factors, even in its early clinical manifestation.

A Compound Heterozygosis of Two Novel Mutations Causes Factor X Deficiency in a Chinese Pedigree

Background: Mutations in the F10-coding gene can cause factor X (FX) deficiency, leading to abnormal coagulation activity and severe tendency for hemorrhage. Therefore, identifying mutations in F10 is important for diagnosing congenital FX deficiency. Methods: We studied a 63-year-old male patient with FX deficiency and 10 of his family members. Clotting and immunological methods were used to determine activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen levels, FX activity, and FX antigen levels. The platelet count was determined. A mixing study was performed to eliminate the presence of coagulation factor inhibitors and lupus anticoagulant. Mutations were searched using whole-exome sequencing and certified by Sanger sequencing. Results: Genetic analysis of the proband identified two single-base substitutions: c.1085G>A (p.Ser362Asn) and c.1152C>A (p.Tyr384Ter, termination codon, caused by the DNA sequence TAA). His FX activity and antigen levels were 1.7% and 408.53 pg/mL, respectively; aPTT and PT were 52.3 and 48.0 s, respectively. One brother had the same compound heterozygous mutations, and his FX activity and antigen levels were 1.3% and 465.47 pg/mL, respectively; his aPTT and PT were 65.2 and 54.5 s, respectively. His mother, another brother, and one sister were heterozygous for c.1085G>A (p.Ser362Asn), and his daughter and grandson (6 years old) were heterozygous for c.1152C>A (p.Tyr384Ter). Conclusion: The heterozygous variants p.Ser362Asn or p.Tyr384Ter indicate mild FX deficiency, but the compound heterozygous mutation of the two causes severe congenital FX deficiency and bleeding. Genetic analysis of these two mutations may help characterize the bleeding tendency and confirm congenital FX deficiency.

Clinical, Laboratory and Molecular Characterization of Factor VII, Factor X and Their Combined Deficiencies in a Tertiary Care Center

Background. Inherited factor VII (FVII) and factor X (FX) deficiencies are rare coagulopathies with heterogeneous genetic background. Combined FVII-FX deficiency was also described. However, genotype-laboratory and clinical phenotype correlations have rarely been studied, in particular in pediatric patients. Methods. We investigated adult and pediatric patients with FVII and FX deficiency in a tertiary care center and analyzed the genotype-phenotype relationship and consequences of novel mutations by in silico methods. The study was approved by the National Ethics Committee (No. 58523-4/2017/EKU) and conducted according to the Declaration of Helsinki. All patient-related data were anonymized and represent results of routine clinical and laboratory investigations so as to establish the exact diagnoses and the optimal treatment of patients. Activity levels of coagulation factors including FVII and FX were determined by PT-based one-stage clotting assays. FVII antigen concentration (FVII:Ag) was measured by ELISA. The whole coding region, the exon-intron boundaries and the promoter region of F10 and F7 were amplified by PCR under standard conditions. Purified PCR products were directly sequenced with the Big Dye Terminator Cycle Sequencing and resolved on an automated ABI PRISM 3130 DNA sequencer. Sequencing data were analyzed by the Sequencing Analysis 5.4 software. UniProtKB/Swiss-Prot database was used for investigating the conservative or un-conservative nature of the mutated amino acids and surroundings in case of novel missense variants. The novel missense variants found in F7 or F10 genes were analyzed by free bioinformatics tools, MutPred2, PolyPhen2, PredictSNP, and Mutation Taster for pathogenicity. Results. Between 2003 and 2018, 18 individuals with FX deficiency (1-41 yrs) and 31 patients with FVII deficiency (5-85 yrs) were identified. Sixteen different pathogenic mutations were found in F7. Novel variants were p.Glu67Gly, p.Cys428Phe and p.Gly435Arg. Most common combinations were p.Arg413Gln with p.Ala354Val and p.Arg413Gln with IVS1A+9G&gt;A. Eight mutations, including four novel ones (p.Ala67Val, p.Gly406Ala, p.Ther273Met and p.Trp348Leu), were found in F10. p.Gly406Ala and p.Thr273Met proved pathogenic by all prediction tools and 2/4 methods suggested pathogenicity of p.Ala67Val and p.Trp348Leu. Similar mutation pattern was observed in four unrelated families with combined FX and FVII deficiency suggesting founder effect. Our cases supported the possibility of independent occurrence of small genetic alterations, like missense mutations and small deletions, affecting both genes. Regarding genotype-phenotype relationships, FVII activity values in patients with symptoms (most frequently epistaxis, menorrhagia and bleeding upon minor surgery) were between undetectable and 48 U/dL. Interestingly, patients without any bleeding showed a wide range of FVII activity (between undetectable and 61 U/dL). There were no associations between F7 genotype, FVII activity, and clinical symptoms in our population. There were 8 surgical interventions performed in rFVIIa coverage with excellent bleeding control. All patients with FX activity &lt;1U/dL were homozygous or compound heterozygous for F10 mutations and had severe bleeding manifestation symptoms requiring PCC prophylaxis. The heterozygous carriers had FX activity levels between 45 and 67% and only one patient with isolated mild FX deficiency had bleeding symptoms. Six persons belonging to four families had combined FX and FVII deficiency. All of them were heterozygous for FX deficiency having FX activity between 47 and 66%. One patient had FVII activity below 1% caused by homozygous mutations in F7, which was associated with umbilical stump bleeding. Conclusions. There are no large clinical studies available in FX and in combined FVII-FX deficiencies. Similarly, no large studies investigated the genotype- clinical and laboratory phenotype relationships in patients with FVII deficiency. Consequences of mutations in the F7 and F10 gene should be investigated at the molecular level. As novel treatment strategies are rapidly emerging in the field of rare bleeding disorders, clinical, laboratory and molecular investigations of larger cohorts, as the present one, are deemed important to guide optimal management of patients. Disclosures No relevant conflicts of interest to declare.

Does Low-Intensity Anticoagulation Using Vitamin K Antagonists Correlate with Factor X Activity Inpatients Implanted with the HeartMate 3 Pump? A Validation Analysis of the MAGENTUM 1 Study

Purpose The MAGENTUM 1 Trial targeted an INR of 1.50-1.90 in patients implanted with HeartMate 3 (HM3) pump. Anticoagulation was maintained in the low intensity range with high efficiency (INR 1.50-1.90, TTR, 75.2 ± 8.6% n=15 patients). None developed thromboembolic events. Factor X activity (FXa) has been suggested as a biologically relevant pharmacodynamic surrogate measure of anticoagulation intensity. We sought to correlate suppression of FXa in MAGENTUM 1 patients to demonstrate the effect of low intensity anticoagulation in HM3 patients. Methods Samples for chromogenic factor X (CFX) assays (Hyphen BioMed) were obtained simultaneously with INR values at routine intervals and stored for batch analysis and not made available to the investigators. Normal reference range for the CFX assay is 71-97%. INR was correlated with CFX; median values and interquartile ranges were determined for INR values >1.50, 1.50-1.90 and Results The correlation of INR range with CFX is shown in Figure 1A. The median CFX value for INR range 1.50-1.90 was 40%. When the INRs were above 1.90 the median CFX value was 30%, when the INRs were below 1.50 the median CFX was 53%. Conclusion We demonstrate that a measured INR range of 1.50-1.90 pharmacodynamically corresponds to less suppression of FX activity than at an INR >1.90 but greater than with INR

On-Demand Treatment of Bleeding Episodes in Patients with Hereditary Factor X Deficiency Using a High-Purity Factor X Concentrate: Data from 2 Clinical Trials and a Data-Collection Study

Introduction: Hereditary factor X (FX) deficiency is a rare bleeding disorder characterized by spontaneous joint, mucosal, and muscle bleeds as well as intracranial hemorrhage. Women and girls may also experience menorrhagia. In the past, hereditary FX deficiency has been treated with fresh frozen plasma or a prothrombin complex concentrate, both of which contain variable amounts of FX. Plasma-derived FX (pdFX) is a high-purity FX concentrate approved in the United States for on-demand treatment of patients with hereditary FX deficiency aged 12 years and older and for perioperative management of bleeding in patients with mild hereditary FX deficiency. pdFX is approved in the European Union for treatment and prophylaxis of bleeding episodes as well as perioperative management in patients with hereditary FX deficiency.Objectives: To present efficacy and safety data from 2 phase 3 clinical studies and 1 data-collection study evaluating pdFX for on-demand treatment of bleeds in patients with hereditary FX deficiency.Methods: Results from 2 phase 3 clinical studies and 1 data-collection study of patients with severe (FX activity [FX:C] <1 IU/dL) or moderate (FX:C 1-5 IU/dL) FX deficiency are included. TEN01 was a prospective, open-label, multicenter, nonrandomized study of subjects aged ≥12 years. Bleeds were treated with an initial dose of 25 IU/kg pdFX followed by additional doses as needed. TEN02 was a prospective, open-label, multicenter, nonrandomized study in children less than 12 years old. In TEN01 and TEN02, pdFX efficacy was assessed as excellent, good, poor, or unassessable. TEN05 was a retrospective, multicenter data-collection study in patients of any age who received ≥1 dose of pdFX on a compassionate-use basis. Investigators retrospectively rated pdFX treatment of bleeds as effective (with hemostasis achieved with the expected number of doses for the severity of the bleed), not effective, or unknown. In TEN05, dosing was at the discretion of the investigator. All protocols were approved by each center's local or national independent ethics committee. All subjects provided written informed consent prior to enrollment.Results: A total of 19 subjects were analyzed from the 2 prospective clinical trials; 12 of these subjects were also included in the retrospective data-collection study. Ages ranged from 1 to 58; 16 of 19 patients were aged ≥12 years. FX deficiency was severe in 17 patients and moderate in 2 patients. During the studies, 326 bleeds were reported, with a median of 12 (range 1-59) bleeds per patient in TEN01, 4 (1-5) bleeds per patient in TEN02, and 5.5 (2-26) bleeds per patient in TEN05. Of these 326 bleeds, 270 bleeds in 18 subjects were treated with pdFX and evaluated. Severity was major for 108 bleeds, minor for 116 bleeds, and not rated for 46 bleeds. Reported bleed causes were menorrhagia (n=97), spontaneous (n=93), trauma or injury (n=75), other (n=3), and unknown (n=2). Bleed locations were mucosal (n=117), joint (n=79), muscle (n=38), other (n=30), and unknown (n=6). Most bleeds (n=230, 85.5%) were treated with a single pdFX infusion (the maximum number of infusions for a single bleed was 12). The total factor dose per bleed per subject ranged from 7.92 to 82.0 IU/kg, with a median of 25.0 IU/kg in TEN01, 31.7 IU/kg in TEN02, and 23.8 IU/kg in TEN05. Treatment effectiveness was assessed by investigators as excellent or good in 43 of 44 rated bleeds (97.7%) in TEN01 and TEN02. One was rated poor in TEN01. In TEN05 pdFX was reported as effective in all 79 rated bleeds (100%). In TEN01, 6 adverse events in 2 patients were reported as related or possibly related to pdFX treatment; all were mild or moderate. No adverse events in TEN02 were considered related or possibly related to pdFX treatment, and no adverse drug reactions or safety concerns were reported in TEN05. No thromboembolic events, inhibitor development, or hypersensitivity reactions were reported in the 3 studies.Conclusions: Across 3 studies, pdFX was rated as effective at treating a variety of bleeds in patients with FX deficiency. Only one dose of pdFX was needed to treat most (85.5%) bleeds. At the doses used in these studies, on-demand pdFX treatment was well tolerated in pediatric and adult subjects. DisclosuresHuang:Baxter: Research Funding; Biogen: Research Funding; Pfizer: Research Funding; Novartis: Other: support to attend meeting; Bio Products Laboratory: Other: Study Investigator. Akanezi:Bio Products Laboratory: Employment. Shapiro:BioMarin: Research Funding; Bayer Healthcare: Other: International Network of Pediatric Hemophilia; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Bio Products Laboratory: Consultancy; Bioverativ, a Sanofi Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion Biopharma: Consultancy, Research Funding; Prometic Life Sciences: Consultancy, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo Biosciences: Consultancy; Octapharma: Research Funding; OPKO: Research Funding.

Dosing-time-dependent effect of rivaroxaban on coagulation activity in rats

The anticoagulant effect of rivaroxaban, a direct inhibitor of activated factor X (FX), might be influenced by its dosing time because the activity of the coagulofibrinolytic system exhibits daily rhythmicity. In rats, FX activity follows a 24-h rhythm with a peak in the middle of the light phase and a trough at the beginning of the dark phase. Consistent with these findings, a single dose of rivaroxaban had a stronger inhibitory effect on FX activity after dosing at the beginning of the light phase than after dosing at the beginning of the dark phase. A similar chronopharmacological effect was seen in a quantitative model of venous stasis thrombosis. In comparison, the dosing time had minimal influence on the pharmacokinetics of rivaroxaban. These data indicate that the anticoagulant effect of rivaroxaban is influenced by the dosing time. Further studies should confirm this finding in a clinical setting.

MG1113, Anti-TFPI Antibody, Efficiently Recovers Coagulation Efficacy through Blocking the Kunitz 2 Domain of Human TFPI

Extrinsic coagulation pathway is an initial phase of clotting when tissue damage occurs. Activation of extrinsic pathway by tissue factor (TF) triggers the coagulation cascade through factor VII (FVII), and factor X (FX). Tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of the extrinsic coagulation pathway, blocks the coagulation signal. TFPI is a single-chain polypeptide which can reversibly inhibit Factor Xa (FXa). While FXa is inhibited, the FXa-TFPI complex can also inhibit the FVIIa-TF complex. TFPI consists of three tandem linked Kunitz domains (KD). Among the three kunitz domains, KD1 is well known for binding to FVII directly and KD2 is for FXa or FX. MG1113 is a human IgG4 type antibody targeting KD2 of human TFPI. Inhibiting TFPI can trigger extrinsic pathway and lead to coagulation independent of intrinsic pathway.To verify the coagulation efficacy of MG1113, we performed several in vitro and ex vivo assays. Through FXase activity assay, we measured the recovery of FX activity by blocking TFPI. Since TFPI can bind and inhibit TF/FVIIa/FX complex, we also measured how much FX is activated through the TF/FVII/FX complex in the presence of MG1113. MG1113 targets the KD2 of TFPI. To confirm blocking KD2 can activate the FVII, we checked FVII activity in the presence of MG1113. Modified prothrombin time (PT) assay under a diluted condition was conducted to measure the efficacy of MG1113. Additionally, thrombin is a key factor of coagulation and therefore, thrombin generation was also determined.Most of all, ex vivo whole blood coagulation test is one of the best ways to experimentally confirm the clotting efficacy. We recruited healthy subjects and hemophilia patients and collected their blood samples. Healthy donor-derived blood was pretreated with FVIII-neutralizing antibody to make it to hemophilia condition. This condition is similar to that of hemophilia patients with inhibitors. After spiking MG1113 to their blood, clotting time is measured by ROTEM.In FXase assay, FXa was generated with increasing concentrations of MG1113. FXa was also increased by MG1113 treatment through the formation of TF/FVIIa/FX complex. Next, FVIIa activity was measured in the presence of MG1113 or anti-TFPI KD1 antibody. Although FVII directly binds to KD1 of TFPI, blocking KD2 by MG1113 was much more efficient to recover FVIIa activity compared with blocking KD1 by anti-TFPI KD1 antibody.Furthermore, prothrombin time was significantly shortened by increasing concentrations of MG1113 under the modified PT assay condition. The results reached normal prothrombin time when 2.5 nM of MG1113 was treated. Thrombin also increased meaningfully to the normal level at increasing concentrations of MG1113.Finally, MG1113 shortened clotting time in a dose-dependent manner in the hemophilia mimicking condition produced by the pretreatment of FVIII-neutralizing antibody in healthy blood. MG1113 also shortened significantly the clotting time for both types of hemophilia, A and B.In conclusion, treatment of MG1113 increased coagulation efficacy in all of the assays used. Moreover, hemophilia patients' blood efficiently clotted by the spiking of MG1113. Our results suggest the potential of anti-TFPI antibody as one of the alternative therapeutics for hemophilia patients. DisclosuresChoi:Greencross: Research Funding. Lee:Greencross: Research Funding. Kwak:Greencross: Research Funding. Hwang:Greencross: Research Funding.

Homozygous missense mutation p.Val298Met of F10 gene causing hereditary coagulation factor X deficiency in a Chinese pedigree

To identify potential mutation underlying coagulation factor X (FX) deficiency in a consanguineous Chinese pedigree. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FX activity (FX:C) and other coagulant parameters were determined with a one-stage clotting assay. The FX antigen (FX:Ag) was determined with an ELISA assay. All coding exons and exon-intron boundaries of the F10 gene were amplified with PCR and subjected to direct sequencing. Suspected mutation was confirmed by reverse sequencing and analyzed with CLC Genomics Workbench 7.5 software. The PT and APTT in the proband were prolonged to 67.2 s and 102.9 s, respectively. Further study showed that her FX:C and FX:Ag were reduced by 1% and 8%, respectively. The PT of her father, mother, and little brother were slightly prolonged to 14.5 s, 14.4 s and 14.4 s, respectively. The FX:C and FX:Ag in her father, mother and little brother were all slightly reduced. Genetic analysis of the proband has revealed a homozygous G>A change at nucleotide 27881 in exon 8 of the F10 gene, which predicted a p.Val298Met substitution. The proband's father, mother, and little brother were all heterozygous for the p.Val298Met mutation. The proband has inherited the homozygous mutation from her parents by consanguineous marriage. Other family members were all normal. Bioinformatics analysis has indicated that this mutation may result in changes in the secondary structure of the FX protein. A homozygous mutation g.27881G>A(p.Val298Met) of the F10 gene has been identified, which probably accounts for the low FX concentrations in this pedigree.

CRISPR/Cas9 Mediated Gene Repair in Inherited Bleeding Disorders

[Introduction] Inherited bleeding disorders (IBD), such as coagulation factor deficiencies, Von Willebrand disease and Glanzmann thrombasthenia, are caused by various gene abnormalities of coagulation proteins, blood vessels, and platelets. IBD have been considered to be suited for gene therapy and clinical trials are ongoing. However, the safety and effectiveness of viral vectors has not been established. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, originates from the archaeal and bacterial adaptive immunity system, provides an efficient genome-editing tool in various organisms including the mammalian genome and holds potential for gene therapy. Here, we report an application of this system to gene repair using induced pluripotent stem cells (iPSCs) derived from patients of three types of IBD.[Case1] Hemophilia B (63-year-old male). Factor IX (FIX) activity was less than 1% (normal range (NR) 70-130%) and antigen level was 2.37μg/ml (average 5.0μg/ml). Molecular analysis of the FIX gene revealed an in-frame deletion in exon 2.[Case2] Factor V (FV) deficiency (55-years-old female). FV activity was less than 3% (NR 70-135%) and antigen level was less than 2% (NR 60-150%). A homozygous missense mutation was detected in FV gene of exon 14.[Case3] Factor X (FX) deficiency (4-years-old male). FX activity was less than 2.84 IU/dl (NR 50-150 IU/dl) and antigen level was 0.567 IU/dl (NR 50-150 IU/dl). A compound heterozygous missense mutation was found in FX gene of exon 6 and 8 respectively.[Methods and results] The CRISPR/Cas system comprises of a Cas9 nuclease and a sequence-specific guide RNA (gRNA). We designed gRNAs close to gene mutations.We transfected both expression vectors into HT-1080 or 293T cells, and assessed the editing activity by SURVEYOR nuclease assay. In order to repair the mutations by homology-directed repair (HDR), we prepared targeting constructs with homology arms (1.0 kbp in length) containing the corrected sequence. After introduction of Cas9, gRNA and targeting plasmid into each iPSCs generated from peripheral blood mononuclear cells (PBMCs) using Sendai virus vector expressing the Yamanaka 4-factor genes (Oct3/4, Klf4, Sox2 and c-Myc), we could obtain iPSC clones with corrected genes by HDR from all of three IBD patients. Successful HDR events were verified by PCR amplification using integration site- and targeting construct-specific primers. Locus-specific knock-in events were confirmed by Southern blot analysis.[Conclusion] We observed the cleavage of the target genome by using our designed gRNAs. Furthermore, the CRISPR/Cas system induced successful gene repair of iPSCs from three IBD patients. We are preparing hepatocytes induced from repaired iPSCs to confirm corrected coagulation factor synthesis. Gene-corrected iPSCs hold great promise as a cell source for autologous cell transplantation. DisclosuresNo relevant conflicts of interest to declare.