Phenotype Of Severe Hemophilia Research Articles

Bleeding phenotype in nonsevere hemophilia by International Society on Thrombosis and Haemostasis bleeding assessment tool, bleeding frequency, and the joint status

BackgroundAlthough the phenotype of severe hemophilia has been well studied, there are still knowledge gaps in nonsevere hemophilia. ObjectivesThe objective of this study was to characterize the clinical bleeding phenotype in nonsevere hemophilia and its association with different factor VIII/IX assessments. MethodsThis was a cross-sectional, multicenter study to investigate the bleeding phenotype in adults with nonsevere hemophilia by the number of bleeding and joint bleeding in the past 5 years, a joint score, and the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT). Factor levels were analyzed by 1-stage (lowest in history and at study inclusion) and chromogenic assay (at study inclusion). Patients were enrolled between March 2015 and May 2019. ResultsOf the 111 patients (86 with mild and 25 with moderate hemophilia), 57 patients (54.8%) reported any bleeding and 24 (23.1%) any joint bleeding in the past 5 years. A joint score ≥1 was found in 44 patients (41.9%), an ISTH-BAT ≥4 in 100 patients (90.1%), and an ISTH-BAT joint item ≥1 in 50 patients (45.0%). Within the ISTH-BAT, muscle and joint bleeds showed the largest difference between mild and moderate hemophilia. The lowest factor VIII/IX level in patients’ history was best associated with bleeding outcomes. Factor was inversely associated with joint bleeds (incidence rate ratio 0.88; 95% CI, 0.79-0.98), joint score, and ISTH-BAT (odds ratios from proportional odds ordinal logistic regression 0.92; 95% CI, 0.87-0.97; and 0.89; 95% CI, 0.86-0.93, respectively). ConclusionThe occurrence of joint bleeding differentiated persons with mild and moderate hemophilia. The ISTH-BAT and lowest factor in patients’ history provided valuable information of the bleeding phenotype in nonsevere hemophilia.

Interfering with Protein Z Dependent Inhibition of Factor Xa Improves Coagulation in Hemophilia

Blood coagulation involves a highly regulated balance between pro-coagulant and anticoagulant activities, allowing for the abatement of bleeding while limiting clot formation, which, if unchecked, could result in thrombosis. Hemostasis is triggered upon vascular injury when extravascular tissue factor (TF) binds plasma coagulation factor VIIa (FVIIa) forming a FVIIa/TF complex that generates limited amounts of activated factors IX (FIXa) and X (FXa). In normal hemostasis, FIXa with FVIIIa dramatically amplifies the production of FXa; FXa with FVa cleaves prothrombin to thrombin, and thrombin cleaves fibrinogen to form a fibrin clot. FXa generated by FVIIa/TF alone is insufficient for normal coagulation as FVIIa/TF activity is limited due to tissue factor pathway inhibitor (TFPI) and TFPI and other plasma protease inhibitors suppress the action of the initially generated FXa (and thrombin). Lacking FVIII or FIX, hemophiliacs fail to generate the additional FXa that is critical for normal hemostasis. Several methods designed to reduce the activity of endogenous inhibitors of coagulation are currently being investigated as a means to ameliorate the bleeding in hemophilia. These include antibody-mediated reduction in the activities of TFPI and protein S, and siRNA-mediated reduction in antithrombin (AT).Protein Z (PZ) is a vitamin K-dependent coagulation factor that serves as the cofactor for FXa inhibition by the serpin, PZ-dependent protease inhibitor (ZPI), at phospholipid surfaces. If inhibition of the action of PZ/ZPI significantly enhanced coagulation in hemophilia, it might be associated with a reduced risk of thrombosis than other approaches. In the latter regard it is of interest that TFPI, PS and AT deficiency are associated with clinical thrombotic disease and mice lacking these proteins die in utero or perinatally of thromboembolic disease. In contrast, PZ and ZPI KO mice appear healthy.Re-bleeding is a hallmark of clinical hemophilia. A tail vein re-bleeding (TVRB) assay, adapted from the saphenous vein re-bleeding assay of Whinna, was developed to assess the effects of PZ and ZPI deficiency on re-bleeding in the hemophilia A mouse model. In this assay, the lateral vein of the tail (diameter 2.25 mm) of an anesthetized mouse is lacerated to a depth of 1 mm with scalpel and the tail placed in warm saline. When bleeding stops, the wound is wiped with a balled Kimwipe to re-induce bleeding and the tail replaced in warm saline. This process is repeated and the number of wound disruptions occurring within 15 minutes is recorded. In normal mice, the period of bleeding following each wound disruption remains relatively constant, whereas in hemophilia mice the bleeding periods tend to progressively prolong with each subsequent disruption leading to a reduced number of total disruptions over 15 minutes.Disruptions in the mouse TVRB assay were: for C57Bl/6 25.8+1.7 (mean+SEM); for FVIII KO 4.2+1.3; for PZ/FVIII double KOs 15.2+3.2; for ZPI/FVIII doubles KOs 14.6+3.5; for WT/KO littermates 4.0+2.6; and for FVIII KO with 100% human FVIII replacement 25.4+3.7. Based on FVIII dose-response experiments, the results in PZ/FVIII and ZPI/FVIII double KOs were equivalent to ~15% FVIII replacement. This outcome of the TVRB assays was confirmed in tests of the survival of sibling WT/FVIII KO (0%, n=5) and ZPI/FVIII double KO (75%, n=12) mice following tail transection 10 mm from the tip.To assess the translation of the PZ/ZPI results in mice to humans, the effect of an anti-PZ monoclonal antibody (Mab) was compared to FVIII replacement on TF (1 pM) induced thrombin generation in human hemophilia A plasma. The Mab enhanced thrombin generation in hemophilia plasma to a similar degree as ~15% FVIII.These studies provide proof of concept, both in vivo in mice and in vitro in human plasma, that blockade of the PZ/ZPI system may be sufficient to alter the phenotype of severe hemophilia to that of mild hemophilia, perhaps with limited thrombotic risk. DisclosuresNo relevant conflicts of interest to declare.

Innate Immune Mechanism of Hemarthrosis in Hemophilia-a Mice

Hemarthrosis is a chronic arthropathy responsible for irreversible joint damage, disability, and acute joint pain in hemophilia patients. Hemarthrosis is caused by spontaneous or traumatic bleeding into joints, which when recurrent, leads to synovial inflammation and cartilage degeneration. Although release of erythrocyte-derived damage associated molecular pattern molecules (eDAMPs) is believed to promote sterile inflammation in the synovium, the innate immune mechanism of hemarthrosis remains poorly understood and the current therapy is limited to factor replacement and pain management. Here, we use factor 8 total knock-out (F8TKO) hemophilia A mice (C57BL/6J background) that manifest a complete deletion of the F8 coding region, expressing no detectable F8 mRNA and exhibiting a severe hemophilia phenotype. Right knee joint capsules of F8TKO mice were punctured with a 30-g needle below the patella between the anterior portions of the femur and tibia, followed by assessment of bleeding severity score and histological analysis one-week post injury. Intravital multiphoton excitation fluorescence microscopy of injured synovium was performed to assess the role of thrombo-inflammatory events in promoting hemarthrosis. Neutrophil, platelets, and blood vessels were visualized by intravenous administration of fluorescent anti-Ly6G mAb, anti-CD49b mAb, and dextran, respectively. Protein and mRNA levels of proinflammatory cytokines were measured in plasma, joint, synovium, and cartilage tissue using Real-Time PCR and ELISA, respectively. F8TKO but not wild-type (WT) control (C57BL/6J) mice manifested unresolved joint bleeding, cartilage degeneration and synovitis leading to impaired mobility and high bleeding severity scores. Significantly more recruitment of neutrophils and neutrophil-platelet aggregates as well as elevated IL-1b levels were observed in the synovium of F8TKO compared to WT mice. These results are the first to suggest that sterile inflammation contributing to hemarthrosis in hemophilia involves enhanced neutrophil-platelet recruitment to the synovium. Currently, experiments are underway to identify the role of eDAMPs (heme and hemoglobin) mediated activation of TLR4 and inflammasome pathway in promoting IL-1b generation, neutrophil-platelet aggregation, and progression of joint injury in F8TKO mice Disclosures Ragni: OPKO: Research Funding; Sangamo: Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases .

Suppressing protein Z‐dependent inhibition of factor Xa improves coagulation in hemophilia A

Essentials Protein Z (PZ) catalyzes PZ-dependent proteinase inhibitor (ZPI) inactivation of factor (F)Xa. Gene-deletion of PZ or ZPI improves coagulation in hemophilia (FVIII knockout) mice. A PZ blocking antibody enhances thrombin generation in human hemophilia plasma. Suppression of the PZ/ZPI pathway may ameliorate the phenotype of severe hemophilia. SUMMARY: Background Hemostasis requires a balance between procoagulant and anticoagulant factors. Hemophiliacs bleed because of a procoagulant deficiency. Targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a cofactor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of activated factor X at phospholipid surfaces. Objectives To evaluate the effects of PZ or ZPI gene deletion in hemophilic mice, and of blocking PZ in human hemophilic plasma. Methods A tail vein rebleeding assay (TVRB) was developed on the basis of the serial disruption of clots forming over a period of 15 min following tail vein laceration in an anesthetized mouse. Wild-type (WT)/FVIII knockout FVIIIKO, PZ knockout PZKO/FVIIIKO and ZPI knockout ZPIKO/FVIIIKO mice were evaluated in this model, and their plasmas were tested in thrombin generation assays. A mAb against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results The numbers of clot disruptions (mean ± standard error of the mean) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice supplemented with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to that in FVIIIKO plasma supplemented with ~ 15% recombinant FVIII. A mAb against PZ added to human hemophilic plasma enhanced thrombin generation to an extent similar to the addition of ~ 15% FVIII. Conclusions Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia.

Bleeding Risk Reduction in Relation to Predicted Factor IX Levels in Hemophilia B Patients Receiving Idelvion (rIX-FP)

Factor IX (FIX) replacement is an effective therapy for hemophilia B patients. The current clinical rationale for maintaining higher trough levels of FIX is to transition patients from a severe hemophilia phenotype (eg. FIX <1% of normal) to a moderate (FIX of 1-5%) or mild disease state (FIX >5%), thereby reducing the total number of bleeding events. Although there is a tentative linkage between trough FIX levels and bleeding tendency which guides hemophilia B management, a thorough quantitative analysis was undertaken for rIX-FP (Idelvion) to robustly define the FIX exposure versus clinical outcome relationship.Pooled adult hemophilia B data from the rIX-FP Phase III development program was utilized for the analysis. A Cox proportional hazards model was employed to relate time-to-bleed event data and various individual measures of exposure (FIX activity) and dosing regimen, while testing the influence of covariates (e.g. baseline FIX). FIX activity levels were simulated using a validated rIX-FP population pharmacokinetic (PK) model. The analysis included all recorded bleeding episodes in subjects receiving either prophylaxis and/or on-demand regimens. Dose amounts and intervals included 50 IU/kg (every 1 week) and 75 IU/kg (every 2 weeks, or every 10 days). A total of 478 bleeding episodes (267 spontaneous bleeds, 190 traumatic bleeds, 21 other bleeds) from 57 adult patients were included in the primary analysis. The highest proportion of bleeds occurred during the on-demand portion of the clinical studies.Patients maintaining cumulative FIX activity trough levels above 5% and 10% were predicted to have significant reductions in bleeding risk of 83% and 81%, respectively, per 1 year above these levels, compared to patients not maintaining these FIX activity trough levels. Cumulatively, maintaining FIX levels above 2% was not statistically significant for risk reduction. Furthermore, a time-direct evaluation relating daily FIX trough activity to daily bleeding risk found that >2%, >5% and >10% FIX activity thresholds were significant predictors of bleeding events with 69% (95% CI, 53 to 80) 77% (95% CI, 67 to 84) and 78% (95% CI, 69 to 85) risk reductions, respectively. Although >2% was significant, the wider confidence intervals suggest a more reliable risk reduction at the >5% and >10% levels.This in-depth exposure-response analysis of rIX-FP determined a strong relationship between FIX activity levels and hemophilia B bleeding risk reduction. Trough FIX activity above 5% (either cumulatively or on any given day) was identified as a statistically significant and overall sufficient pharmacological threshold for reduction of bleeding risk in adult hemophilia B patients receiving rIX-FP. Trough FIX activity > 10% was also a predictor of significant risk reduction, and the magnitude of risk reduction was similar to that of achieving levels > 5%. These results are aligned with existing World Hemophilia Foundation (WFH) hemophilia severity definitions where patients with a range of observed FIX activity 5-40% are categorized as a single mild hemophilia phenotype. Post-approval clinical exposure versus response relationships may also be affected by an individual’s level of physical activity which was not assessed in the current analysis. Overall, this analysis demonstrates the quantitative clinical rationale for optimization of adult rIX-FP dose regimens through targeting and maintaining FIX activity trough levels above 5% to 10%. DisclosuresRoberts:CSL Behring: Employment. Fosser:CSL Behring: Consultancy; Cytel: Employment. Tortorici:CSL Behring: Employment. Veldman:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Sidhu:CSL Behring: Employment.

Does sickle cell trait reduce the frequency of spontaneous bleeds in severe haemophilia?

Abstract Haemophilia A is an X-linked recessive disorder associated with deficiency of coagulation factor VIII and lifelong bleeding diathesis. Sickle cell trait (SCT) is the heterozygous state for the sickle β-globin gene. The frequency of SCT is up to 30% in Africa, wherein it confers survival advantage by providing resistance against severe malaria. SCT does not cause vaso-occlusive crisis, but is associated with high risk of venous thromboembolism as variously reported in the literature. We consider SCT as a hypercoagulable prothrombotic state and hypothesise that coinheritance of SCT may ameliorate the clinical phenotype of severe haemophilia. We conducted a retrospective analysis of frequencies of spontaneous bleeding among severe haemophiliacs with SCT (Hb AS phenotype) and their counterparts with normal Hb phenotype (Hb AA phenotype) in order to determine the possible ameliorating effect of SCT on spontaneous bleeding rates in severe haemophilia A. If our hypothesis is correct, severe haemophiliacs with SCT will have lower frequencies of spontaneous bleeding than their counterparts with normal Hb phenotype. Our results revealed that severe haemophiliacs with normal Hb phenotype had significantly higher mean annual bleeding episodes per patient in comparison with their counterparts with SCT (45±7 vs 31±5, p=0.033), suggesting that severe haemophiliacs with SCT had lower frequencies of spontaneous bleeding episodes. The result of this study indicates that coinheritance of SCT in patients with severe haemophilia may be associated with reduced frequency of spontaneous bleeding, which may imply better overall prognosis. However, the study has important limitations, which include its retrospective nature and the very low number of subjects. The findings should therefore be validated by a larger and prospective study.

Impact of Prothrombotic Risk Factors in a Cohort of Egyptian Hemophilia A Patients.

Hemophilias are a group of related bleeding disorders that show an X-linked pattern of inheritance. The clinical phenotype of severe hemophilia may vary markedly among patients as a result of many factors, including genetic prothrombotic risk factors. Our objective was to study the incidence of the most common prothrombotic risk factors for additive effects among Egyptian patients with hemophilia A and their impact on clinical phenotype; annual bleeding frequency and severity of hemophilic arthropathy, as well as the effect of a single variation in these patients. This study was carried out in 100 patients with hemophilia A. Genotyping for factor V Leiden (FVL) G1691A, prothrombin G20210A, MTHFR C677T, and A1298C mutations was conducted using a real time-polymerase chain reaction (RT-PCR) assay. Our study revealed mutations in hemophilia patients as follows: prothrombin G20210A (3 %), FVL (14 %), MTHFR C677T (42 %), and A1298C (59 %). Despite a lack of statistical significance when each gene was analysed separately, heterozygosity of prothrombin G20210A or FVL was always associated with either a mild or moderate, but never a severe, clinical presentation. The lowest bleeding frequency (less than once per month) was identified among patients with two heterozygous variants irrespective of the involved genes. In addition, the incidence of hemarthrosis was significantly higher among patients with a wild genotype of the prothrombin gene and FVL, and the average number of affected joints was significantly higher among patients with wild-type prothrombin and FVL genes than among heterozygous patients. These prothrombotic mutations have a cumulative effect in amelioration of the severity of bleeding in hemophiliacs. The most prominent effect is that of prothrombin G20210A and FVL, while MTHFR C677A and A1298C gene mutations are less conclusive.

Analysis of Bleeding Phenotype of Severe Hemophilia a Carriers and Comparation with a Working Population Group. Impact on Quality of Life

INTRODUCTIONWe have little information about if there is a particular bleeding phenotype of severe haemophilia A female carriers. In the literature, an increased bleeding tendency is described in this group, justified even by levels of factor VIII (FVIII) very close to normal values, in absens of primary hemostasis evaluation in many cases. There is no published evidence about quality of life (QoL) of these women and the relationship between QoL and this bleeding tendency. OBJECTIVES• To evalute the hemorrhagic phenotype of severe haemophilia A carrier and to compare them with the general population.• To identify symptomatic carriers and to study from laboratory point of view the reasons for that bleeding tendency.• To define the bleeding profile of symptomatic carriers.• To analyze the quality of life and state of anxiety/depression in carriers and to compare them with the general population. PATIENTS AND METHODSThis is a descriptive cross-sectional, non-interventional, single center study. Ethics Committee evaluation and written informed consent are requested to be included for carriers and controls. The target population are severe Haemophilia A carriers from our area aged between 18 and 70 years old. The control group are women from regular health laboral checkings. We will evaluate family bleeding, ischemic and thrombotic personal and familiar antecedents, bleeding profile (ISTH/SSC bleeding assessment tool, ISTH BAT, and pictorial blood assessment chart, PBAC), carrier genetic study, complete blood count, basic biochemistry, haemostasis (aPTT, PT, fibrinogen, platelet function tests, FVIIIc, FvWAg and FvWRCo, FXIII). QoL has been studied trough SF-36 (Spanish version 1,4 1999) and anxiety and depression states using Goldberg questionary (1998, Spanish version Gzempp, 1993).The controls have been studied in the same way except for laboratory studies of hemostasis. Only in controls with pathological ISTH BAT (greater than 3) basic and primary hemostasis have been studied. RESULTSOut of a total of 81 carriers identified between August 2012 to December 2013. We have evaluated 69 carriers. To achieve a confidence level of 95% with 50% heterogeneity we have recruited 138 controls. The mean and standard deviation (SD) age of the carrier and controls was 43.7+/-15 and 41.5+/-11.7 years old (p 0.308). In the population of carriers, the mean and SD of FVIII levels are 87.2+/-35.7%. We found no relationship between levels of FVIII:c and haemophilia genetic defect (34.8% substitutions, 34.8% intron 22, 27.5% mutations). 20.3% of carriers and 2.2% of controls present a pathologic ISTH BAT score (p 0.001). There are 14 carriers with a pathologic ISTH BAT score (median 5.5, range 4-11). In this group we found FVIII levels less than 40% in 4 carriers, 1 thrombopathy and levels of VWF:RCo and VWF:Ag compatible with von Willebrand disease in other 4 carriers. The remaining 5 present ISTH BAT score between 4 and 6 (low), FVIII levels above 60% and no other laboratory abnormal results to justify bleeding tendency. In the 3 controls with pathologic ISTH BAT score (4-5), we have not found any hemostatic disorder from laboratory point of view. The carrier group has a higher incidence of abnormal bleeding at wounds (1.5% vs. 14.5% p 0.000), tooth extraction (3.6% vs. 11.6%, p 0.028), surgery (0% vs. 11.6 %, p 0.028) and methrorragia (13.8% vs 43.5%, p 0.000). However when methrorrragia is analysed with an objective scale of menstrual bleeding as PBAC, methrorragia incidence in carriers is similar to controls (23.2% carriers vs 31.5% controls, p 0.071). Regarding quality of life, the population of carriers presented worse scores in the domains of social activity (p 0.01) and mental health (p 0.014). In the group of symptomatic carriers there are no differences in quality of life with the control group. The control population has a higher incidence of anxiety (42.3% vs 21.7%, p 0.002), with no differences in the presence of depression. CONCLUSIONSIn our series, women with symptomatic severe hemophilia A are those with FVIII lower than 40% or another disorder of hemostasis associated to carrier condition. The hemorrhagic symtoms able to define a severe haemophilia A carrier could be abnormal bleedings from wounds (surgical or traumatic) and in dental extractions. Quality of life in women with hemophilia is worse in the mental health and social activity fields, but for reasons other than their own bleeding phenotype. DisclosuresNo relevant conflicts of interest to declare.

Developments in the treatment of hemophilia B: focus on emerging gene therapy

Hemophilia B is a genetic disorder that is characterized by a deficiency of clotting factor IX (FIX) and excessive bleeding. Advanced understanding of the pathophysiology of the disease has led to the development of improved treatment strategies that aim to minimize the acute and long-term complications of the disease. Patients with hemophilia B are ideal candidates for gene therapy, mostly because a small increase in protein production can lead to significantly decreased bleeding diathesis. Although human clotting FIX was cloned and sequenced over 30 years ago, progress toward achieving real success in human clinical trials has been slow, with long-term, therapeutically relevant gene expression only achieved in one trial published in 2011. The history of this extensive research effort has revealed the importance of the interactions between gene therapy vectors and multiple arms of the host immune system at multiple stages of the transduction process. Different viral vector systems each have unique properties that influence their ability to deliver genes to different tissues, and the data generated in several clinical trials testing different vectors for hemophilia have guided our understanding toward development of optimal configurations for treating hemophilia B. The recent clinical success implementing a novel adeno-associated virus vector demonstrated sufficient FIX expression in patients to convert a severe hemophilia phenotype to mild, an achievement which has the potential to profoundly alter the impact of this disease on human society. Continued research should lead to vector designs that result in higher FIX activity at lower vector doses and with reduced host immune responses to the vector and the transgene product.

Influence of Factor V G1691A or Prothrombin G20210A Mutation On Inhibitor Development in Patients with Severe Hemophilia A - an Israeli-German Database Study.

AbstractAbstract 2234It has been reported that the clinical phenotype of severe hemophilia A (HA) is influenced by co-inheritance with the factor V (FV) G1691A mutation and that the first symptomatic bleeding onset in children with severe HA carrying prothrombotic risk factors, namely the FV or the factor II (FII) G20210A variant, is significantly later in life than in non-carriers. Until today many factors, such as a positive family history, the use of recombinant FVIII [rFVIII] concentrates, or high dose FVIII administration, are included as potential risk factors for inhibitor development. The present non-concurrent Israeli-German database study was performed to investigate the impact of FV and FII mutations on clinical meaningful inhibitor development (outcome variable) in patients with severe HA. 272 patients with HA < 1% consecutively ascertained from Israel (n=88) and four different German catchment areas (n=184) born between 1980 and 2011 were followed after initial HA diagnosis over the first 200 exposure days. To avoid family cluster effects [inhibitor; IT] in both countries only the first HA patient who presented for diagnosis at the treatment center was included in the present study. From the entire cohort 80% were tested for thrombophilia [IT]. 32 of 216 children (14.8%) carried either the FV or the FII variant. During the follow-up period of 200 exposure days 54 of 216 (26%) developed a clinical meaningful inhibitor: 14 out of 32 carriers of IT (44%) compared with 40 of 184 (22%) without IT [Hazard ratio [HR]/95% confidence intervals [CIs] 2.1/1.16–3.9]. In addition, univariate Cox regression analysis for patients treated with rFVIII products compared with plasma-derived [pdFVIII] showed a significant increased risk for inhibitor development [HR/CI: 2.1/1.3–3.6]. Multivariate analysis adjusted for patient’s individual median single FVIII dose, administered during the first three months, first-line use of pdFVIII (n=82) versus rFVIII concentrations (n=134) and treatment periods [1980–2011] revealed that the presence of IT independently increases the risk of inhibitor development to a hazard of 2.5 [CI: 1.1–5.8]. In addition, the increase of FVIII per one IU/kgbw was significantly associated with the risk to develop a high responding inhibitor [HR/CI: 1.2/1.04–1.1]. In multivariate analysis neither rFVIII products, nor treatment periods showed a statistically significant influence on clinical important inhibitor development. Data presented here suggest that clinical meaningful inhibitor development is of multifactorial origin and that apart from a positive family history also FV and FII mutations should be included in the aetiology research. Disclosures:No relevant conflicts of interest to declare.

Epitope Mapping of a Human Inhibitor Plasma by Affinity-Directed Mass Spectrometry.

Abstract 3171Poster Board III-111Hemophilia A results from a defect or deficiency in the blood clotting protein, factor VIII. Treatment of patients with factor VIII results in the development of neutralizing antibodies in a significant fraction (∼30%) of patients with a severe hemophilia phenotype. We employed an affinity-directed mass spectrometry (MS) technique using immunoprecipitation coupled with protein fragmentation to map the epitopes recognized by the IgG fraction from a factor VIII inhibitor plasma. To accomplish this aim, antibody (IgG fraction) was purified from 10 ml of plasma obtained from a high titer (533 Bethesda Units/ml) inhibitor patient (obtained from George King BioMedical, Inc., GK1838-1156). This IgG fraction was immobilized onto Protein G agarose beads and then reacted with limited tryptic or chymotryptic digests of heavy chain and light chain purified from recombinant factor VIII. The factor VIII heavy and light chain digests showed peptide maps that covered approximately 20% and 40%, respectively, of the factor VIII sequence. The immunoprecipitated complexes were washed and directly applied to analysis by MALDI-TOF MS. Five peptides with mass values (m/z ratio) of 1105.6, 1291.7, 1309.7, 1362.7, and 1681.2 were identified. Database searches and direct sequencing showed these peaks corresponded to factor VIII peptides HYFIAAVER (residues 1697-1705), HNIFNPPIIAR (residues 2137-2147), SWYLTENIQR (residues 584-593), TRHYFIAAVER (1695-1705), and IRAEVEDNIMVTF (residues 1763-1775), respectively. This method of epitope identification does not discriminate neutralizing from non-neutralizing antibody binding. However, inspection of the sequence contained in these epitopes reveals that residues 1695-1705 occur near the amino terminal region of the A3 domain in close proximity to the thrombin cleavage site at Arg1689 and may contribute to facilitating factor VIII activation. Residues 2137-2147 (C1 domain) are of interest since residue Arg2150 contributes to binding von Willebrand factor. While no macromolecular interactions have been attributed to A2 domain residues 584-593 or the A3 domain residues 1763-1775, the former sequence appears on the same face of A2 as the 558-loop which is an important interactive site for factor IXa in the assembly of factor Xase. Thus, these observations suggest that several of the above sequences may possess functional attributes that, if bound by antibody, could lead to inhibition of factor VIII activity. To further test this hypothesis, synthetic peptides were employed in an attempt to reduce the potency of the inhibitor plasma. Two peptides, corresponding to residues 1697-1705 and 2137-2147 have been examined to date. Addition of either peptide (100 nM) to the inhibitor plasma reduced its titer by approximately 4-fold, suggesting these sequences represent sites in factor VIII that when bound by antibody, block cofactor function. Taken together, these results identify in part, the ensemble of epitopes recognized by the inhibitor antibody fraction derived from a single patient. DisclosuresNo relevant conflicts of interest to declare.

Polymorphism in factor VII gene modifies phenotype of severe haemophilia

The basis for 10-15% of patients with severe haemophilia having clinically mild disease is not fully understood. We hypothesized that polymorphisms in various coagulant factors may affect frequency of bleeding while functionally significant polymorphisms in inflammatory and immunoregulatory genes may also contribute to variations in the extent of joint damage. These variables were studied in patients with severe haemophilia, who were categorized as 'mild' (<5 bleeds in the preceding year, <10 World Federation of Haemophilia clinical and <10 Pettersson scores, n = 14) or 'severe' (all others, n = 100). A total of 53 parameters were studied in each individual for their association with the clinical severity. Age, F8:c activity and the incidence of thrombotic markers were comparable between the groups while the median number of bleeds, number of affected joints, clinical, radiological and functional joint scores (P < or = 0.001) and life-time clotting factor use (P < or = 0.007) were different. Patients with severe molecular defects had a 4.1-fold increased risk for a severe phenotype (95% CI: 1.18-14.42, P = 0.026) compared with other mutations. Of the polymorphisms studied, the FVII353Q (RR = 3.5, 95% CI: 1.04-12.05, P = 0.044) allele was associated with a severe phenotype. This data shows that apart from the F8/F9 genotype, functional polymorphisms in FVII gene affect the phenotype of patients with severe haemophilia.

Interpatient Phenotypic Inconsistency in Severe Congenital Hemophilia: A Systematic Review of the Role of Inherited Thrombophilia

It is well known that the clinical phenotype of hemophilia may vary greatly among patients with the same apparent level of coagulation factor and the same genetic mutation. Thus, patients with severe hemophilia may experience a severe phenotype or only a milder bleeding tendency, suggesting some other moderating influence. To elucidate the mechanism of this heterogeneity, some investigators have recently suggested that inherited thrombophilic factors may play a role in the milder clinical presentation of severe hemophilia. In this review, we summarize current knowledge with respect to the modulation of the clinical phenotype of severe hemophilia by prothrombotic genetic risk factors. Although the published literature seems to indicate a protective effect for the coinheritance of factor V Leiden, the limited data available do not permit any firm conclusions. Further trials on a large population of patients are needed to establish the role of genetic thrombophilia in the phenotypic expression of severe hemophilia.

Presence of Factor V Leiden or Prothrombin Mutation Influence Inhibitor Development in Children with Severe Hemophilia A: Data of a Multicenter Cohort Study

It has been recently suggested that the clinical phenotype of severe hemophilia A (HA) is influenced by co-inheritance with the factor V (FV) G1691A mutation. In addition, we have recently demonstrated that the first symptomatic bleeding onset in children with severe HA carrying prothrombotic risk factors, namely the FV Leiden or the factor II (FII) G20210A variant, is significantly later in life than in non-carriers. The course of bleeding episodes, however, is also controversially discussed with respect to individual therapeutic regimens performed, and recently a protective effect of early use of prophylaxis on inhibitor development in hemophilic children compared with on-demand therapy has been reported. Thus, the present non-concurrent cohort study was performed to investigate the impact of FV and FII (prothrombin) mutations on clinical meaningful inhibitor development (outcome variable) in white children with severe HA. 122 patients with HA consecutively ascertained from four German catchment areas were followed over a median (min-max) period of 15 years (1–24) from HA onset. 19 of 122 children (15.6%) additionally carried either the FV or the FII variant. During the follow-up period 28 of 122 children (22.9%) developed a clinical meaningful inhibitor: 9 out of 19 in carriers of thrombophilia (47.4%) compared with 19 of 103 (18.44%) in the non-thrombophilia affected cohort [p=0.01]. Multivariate analysis (Cox regression), adjusted for age at first bleeding onset, substitution regimen (start of prophylaxis latest at second joint bleed versus more than two joint bleeds), FVIII dosage (≤ 30 IU/kgbw versus > 30 IU/kgbw), and first-line use of plasma-derived versus recombinant FVIII concentrations revealed that the presence of thrombophilia independently increases the risk of clinical meaningful inhibitor development to a hazard of 2.4 [95%CI: 1.1–5.5]. The cumulative high-responder-free survival with respect to FVIII exposure days was significantly reduced in carriers of the factor V or II mutation (logrank p=0.009). None of the confounders adjusted for, showed a statistically significant influence on clinical important inhibitor development. Data presented here support the hypothesis that clinical meaningful inhibitor development is of multifactorial origin and that FV and FII mutations should be included in the aetiology research in future studies.

Polymorphisms in Coagulant and Inflammatory Genes Modify the Phenotype of Severe Hemophilia A and B.

Among patients with severe hemophilia (<1% factor level), 10–15% are known to have a clinically mild phenotype. The basis for this phenomenon is unclear. We hypothesized that functionally significant polymorphisms in the coagulant, inflammatory and immunoregulatory genes may affect the phenotype of severe hemophilia. A total 114 patients with hemophilia A (n=95) and hemophilia B (n=19) were studied. All these patients were on minimal on-demand treatment. Patients were evaluated for the frequency and site of hemorrhage. Their clinical and radiological joint scores were documented. They were categorized as ‘mild' (<1 affected joint and < 5 bleeds in the preceding year, n=15) or ‘severe' (>1 affected joint and >5bleeds, n=99). Functional polymorphisms in the coagulant system (human platelet alloantigen; tissue factor; fibrinogen; factors II; V; VII; XIIIA; thrombin activable fibrinolysis inhibitor (TAFI); endothelial protein C receptor; endothelial nitric oxide synthase 3; tissue plasminogen activator; plasminogen activator inhibitor; tissue factor pathway inhibitor; protein C and S; thrombomodulin), known procoagulant factors (methylene tetrahydrofolate reductase gene), inflammatory cytokine genes (tumor necrosis factor alpha; transforming growth factor beta; interleukin (IL) 10; IL 6; IL 1beta; IL 1 beta receptor antagonist; tumor necrosis factor beta), immunoregulatory cytokine genes (interferon gamma; HLA B27; FC gamma receptor), MDM2, angiotensin converting enzyme and HFE genes were genotyped. The mean age in the two groups was 18.5 & 14.85, p=0.124. The clinical features showing significant difference are shown in the table. Of the polymorphisms studied, the FVII RQ/QQ (lower levels) (RR-3.99, p=0.022, 95% CI 1.2–13.4), TNF alpha-308AA/AG (pro-inflammatory) (RR-3.4, p=0.037, 95% CI, 1.07–10.7), TGF beta Codon 10 CC/CT (pro-inflammatory) (RR-2.8, p=0.07, 95% CI, 0.91–8.3), have been associated with a severe phenotype while MDM2GG (anti-inflammatory, RR-0.3, p=0.038, 95% CI, 0.1–0.93) was associated with a milder phenotype. We hypothesize that the bleeding frequency in severe hemophilia may be increased due to relatively lower FVII levels and a combination of cytokine driven pro-inflammatory state involving TNF alpha, TGF beta and MDM2 would cause destruction of the cartilage resulting in elaboration of metalloproteinases from chondrocytes leading to the development of arthropathy.ParameterSevere, n=99 Median (Range)Mild, n=15 Median (Range)p ValueNumber of bleeds /yr15(3–74)2(0–5)0.000Number of joints /yr3 (1–6)1 (0–1)0.000Age at first clinical symptom (months)21(1–300)60(6–90)0.056WFH clinical score10 (0–27)4 (0–21)0.000Pettersson score13 (0–57)6 (0–20)0.001

Coated-Platelet Levels May Explain Some Variability in Clinical Phenotype of Severe Hemophilia.

Objective: To determine if variability in clinical phenotype of severe hemophilia patients is influenced by the percentage of coated-platelets.Background: Co-activation of platelets with thrombin and collagen results in a unique subset of platelets, with high levels of alpha granule proteins on their surface, such as Factor V, fibrinogen, von Willebrand factor, and thrombospondin. This subset of activated platelets is referred to as coated-platelets (J. Thromb. Haemostasis 3:2185, 2005). The high concentration of adhesive and prohemostatic proteins observed on coated-platelets provides a unique procoagulant locus that may influence the number of bleeding episodes in patients with severe hemophilia.Methods: After informed consent, 3–5 ml of blood was drawn from patients with severe hemophilia (Factor VIII <1%) and healthy controls. The number of bleeding episodes reported in the last 6 months was taken as an indicator of clinical phenotype.Results: In 6 patients with more than 3 bleeds reported in the last 6 months (14 ± 4.9 bleeds; mean ± 1SD), the average coated-platelets percentage was 22.3 ± 11.2%. In 17 patients with 3 or less bleeds (1.6 ± 1.3) in the last 6 months, the average coated-platelets percentage was 37.6 ± 12.0%, a difference that was statistically significant (p=0.012). In healthy controls (n=12), the mean coated-platelets were 30.2 ± 9.5%.Conclusion: A higher percentage of coated-platelets may provide a better procoagulant locus for residual Factor VIII, thereby reducing the number of clinical bleeding episodes and partially explaining some variability observed in clinical phenotype of severe hemophilia.

Factor V Leiden improves in vivo hemostasis in murine hemophilia models

The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real-time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser-induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.

Variability in clinical phenotype of severe haemophilia: the role of the first joint bleed

To quantify variation in clinical phenotype of severe haemophilia we performed a single centre cohort study among 171 severe haemophilia patients. Age at first joint bleed, treatment requirement (i.e. annual clotting factor use), annual bleeding frequency and arthropathy were documented. Because treatment strategies intensified during follow-up, patients were stratified in two age groups: patients born 1968-1985 (n = 91), or 1985-2002 (n = 80). A total of 2166 patient-years of follow-up were available (median 12.0 years per patient). Age at first joint bleed ranged from 0.2 to 5.8 years. Patients who had their first joint bleed later needed less treatment and developed less arthropathy. In patients born 1968-1985 during both on-demand and prophylactic treatment, the 75th percentile of annual joint bleed frequency was consistently four times as high as the 25th percentile. In both age groups variation in annual clotting factor use between 25th and 75th percentiles was 1.4-1.5 times for prophylaxis and 3.8 times for on-demand treatment. To conclude, the onset of joint bleeding is inversely related with treatment requirement and arthropathy and may serve as an indicator of clinical phenotype. Thus, providing a starting point for aetiological research and individualization of treatment.

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.

In Vivo Evidence of Modulation of the Hemophilia Phenotype by the Factor V Leiden.

The amelioration of hemophilia phenotype or delayed onset of the bleeding episodes in subjects with severe hemophilia A (FVIII deficiency) has been associated with inherited resistance to activated protein C due to factor V Leiden (R506Q), FVL. These observations were confirmed by in vitro systems in which homozygous phenotype of FVL increased thrombin formation in presence of < 1% FVIII by nearly 5-fold (Blood 90:3067). Here we provide in vivo evidence of the beneficial interaction of FVL on the hemophilia phenotype in mice. Animals with severe deficiency of factor VIII due to deletion of intron 16 of factor VIII gene (HA) or large gene deletion of factor IX (HB) were crossed with FVL homozygous mice [+/+] on C57Bl6 strain [Cui et al. (Blood 96:4222)] We used a modified activated partial thromboplastin time (aPTT) assay to compared clotting times among male HA (n=30), HA/FVL [+/+] (n=7), FVL [+/+] (n=5), and litermate WT mice (n=6) with age ranging from 6–12 weeks. Blood samples were collected by tail vein transection into 3.8% sodium citrate. Values for the aPTT in male HA were 67.3 ± 4 sec, and among WT or FVL [+/+] values were 37 ± 4 sec or 31± 2 sec, respectively. Whereas intermediate aPTT values of 53 ± 3.7 sec were determined in HA/FVL [+/+], which differs from HA mice (p<0.0001) but also from WT or FVL (p<0.0001). A similar shortening of aPTT was also determined among HB/FVL[+/+] which were compared to HB, 55 ± 7 sec vs. 64 ± 0.9. Hemostatic challenge by tail clipping assay failed to revealed differences in bleeding times/blood loss among hemophilia animals with or without FVL mutation. To test whether a more sensitive technique would provide further evidence of the improved hemostasis in HA/FVL mice, we assessed real-time in vivo thrombus formation by confocal and widefield microscopy. Mice were anesthetized and the cremaster muscle was exposed for intravital microscopy. Infusion of fluorescently labeled antibody to murine platelet glycoprotein IIb/IIIa complex via the jugular vein allowed monitoring of platelet deposition upon laser-mediated endothelial injury at several sites of the arterial vessel wall. No thrombus formation was observed in severe HA mice following successive vascular injuries, a finding also common in severe HB mice. However, infusion of factor VIII concentrated clearly induced the thrombi formation upon vascular injury. HA/FVL mice tested presented thrombus formation in a comparable fashion of HA-FVIII transfused mice. These in vivo data provide support to the hypothesis that the FVL mutation has the potential to improve the phenotype of severe hemophilia and may offer a novel therapeutic target for hemophilia.