scholarly journals Diagnosis and Treatment of Acquired Hemophilia: One Single-Center Experience from PR. China

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4250-4250
Author(s):  
Rong-Fu Zhou ◽  
Yueyi Xu ◽  
Wenjin Gao
Keyword(s):  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Clinical Manifestations ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Coagulation Factor Vii ◽  
Prolonged Aptt ◽  
Inhibitor Titer

Abstract Objective: To deepen the understanding of the clinical manifestations of acquired hemophilia A for timely and correctly treatment. Methods: The clinical data of the acquired hemophilia A patients diagnosed in the hospital from Jan 2006 to Mar 2021 were retrospectively analyzed, and the relevant literature was reviewed. Results: 17 patients with acquired hemophilia A, male: female =10: 7, median age 61 years (19 to 78 years), were diagnosed and treated in the hospital with the median time from the onset to diagnosis 21 days (2 days to 6 months). Six patients had comorbidity, including hepatitis B carrying, chronic myelomonocytic leukemia, diabetes, hypertension and positive autoantibodies, pemphigoid and gastric cancer, respectively. Other 11 patients were healthy before the onset. All patients had large large ecchymosis of skin, and one case was combined with hematuria, and one case with retroperitoneal hematoma. All patients had APTT extension (45s-144.7s) and the prolonged APTT could not be corrected with normal mixed plasma with and without incubation at 37℃ for 2 hours. FVIII activity was 1% - 8.9% and inhibitor titer 2 - 128 Bu/ml. All patients with bleeding were with prothrombin complex/recombinant activated coagulation factor VII, some of them with pd-coagulation factor FVIII preparations. Inhibitors were removed with prednisone acetate (1 case) + chemotherapy (1 case), prednisone acetate / + CTX (11 cases) + chemotherapy (1 case), prednisone acetate/prednisolone + mabthera (2 cases) + CTX (1 case), respectively. The removal time of inhibitor was from 8 days to 4 years. During the treatment process, two patients developed lower extremity venous thrombosis, and one patient was complicated with lung infection. Conclusion: Patients with unexplained bleeding and prolonged APTT should be conducted normal mixed plasma correction test, coagulation factor activity and inhibitor titer examination. After correctly diagnosis, bypass agents /coagulation factor VIII preparations should be given timely for hemostasis, protocol based on glucocorticoid + CTX/mabthera to remove the inhibitor and symptomatic treatment for patients with primary comorbidity disease at the same time. Disclosures No relevant conflicts of interest to declare.

Improved Prognosis of Acquired Hemophilia A: A 10-Year-Experience

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4348-4348 ◽  
Author(s):  
Rudiger E. Scharf ◽  
Barbara Bomke ◽  
Holger Seidel ◽  
Roya Gheisari ◽  
Marie Antonia Scharf ◽  
...  
Keyword(s):  
Immune Tolerance ◽  
Respiratory Diseases ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Treatment Algorithm ◽  
Factor Vii ◽  
Laboratory Evaluation ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Life Threatening

Abstract Abstract 4348 Background: Acquired hemophilia A (AHA) is a rare but significant hemostatic disorder caused by inhibitory autoantibodies against coagulation factor VIII (FVIII:C). The annual incidence of AHA is low with about 1 to 4 cases per million individuals. However, the mortality rate due to severe hemorrhages and comorbidity is high reaching 22% in several series. In the past, only a few patients were reported in whom an association of AHA with respiratory disorders was observed. Patients, Methods, and Study Protocol: We have performed a monocenter study on 35 consecutive patients with AHA A who were referred for diagnosis and treatment to the Düsseldorf Hemophilia Comprehensive Care Center between March 2001 and June 2011. The cohort included 24 males (age: 44–86 years) and 11 females (age: 20–83 years). For laboratory evaluation, a standardized staged protocol of APTT, FVIII:C activity and concentration, mixing studies with patient and normal plasma, and quantitation of inhibitor titers (Nijmegen modification of the Bethesda assay) was used. Diagnostic work-up for any underlying disease was performed according to a standardized protocol of clinical examinations and imaging procedures (including X-ray examination of thorax, sonography of abdomen, retroperitoneum and thyreoidea and, whenever indicated, computerized tomography of thorax, abdomen, or pelvis). Therapy was performed according to a treatment algorithm consisting of (a) acute antihemorrhagic therapy (irrespective of residual FVIII:C activity and inhibitor titer), (b) immediate immunosuppression (individually tailored to the patients’ risks with regard to age and comorbidity), and, if life-threatening bleedings persisted, (c) inhibitor elimination by immunoadsorption or plasmapheresis, and (d) concomitant immunotolerance regimens. Predefined clinical endpoints were control of bleeding, eradication of the inhibitor, complete or partial remission (CR, PR), relapse, or early death (< 30 days). CR was defined as no inhibitor detectable, FVIII:C activity > 80%, and withdrawal of immunosuppressive therapy. Results: In 21 (60%) of the 35 patients with AHA, an underlying disorder was identified, including 9 patients with respiratory diseases (26%), 8 patients with autoimmune disorders (23%), 3 with malignancies, and one with postpartum state, while in 14 patients (40%) AHA remained idiopathic. Upon admission, 16 of the 35 patients presented with life-threatening hemorrhages. In 13 of these 16 patients, control of bleeding was achieved by high doses of recombinant activated factor VII (rFVIIa; 90–120 μ g/kg every 2–3 h), while 3 patients required combined FVIII bypassing agents (rFVIIa plus bolus injections of activated prothrombin complex concentrates, aPCC; 100 IU/kg every 8–12 h). In the other 19 patients, bleeding also subsided in response to rFVIIa. Concurrent immunosuppression with prednisone alone (2 mg/kg/day) was performed in 11 patients, while 24 patients received cyclophosphamide (2 mg/kg/day) sequentially in combination with prednisone. In 5 patients in whom this first-line immunosuppression failed, 4 doses of rituximab (375 mg/m2) were administered as second-line therapy. Of the 35 patients, 13 required extracorporeal inhibitor elimination procedures due to persisting life-threatening bleeds. Exchange plasmapheresis was performed in 4, daily large-volume immunoadsorption (Ig-Therasorb) for up to 4 weeks in 9 patients. In 3 of them, immune tolerance was concomitantly induced by exogenous FVIII (100 IU/kg/day). Of the 35 patients in total, 28 individuals achieved CR (80%), 3 had PR, one relapsed, and 3 died within 30 days (one of acute myocardial infarction while on antihemorrhagic treatment, one of sepsis while on immunosuppression due to active AHA, one of lung bleeding in assocociation with pre-existing pulmonary sarcoidosis). Conclusions: This monocenter study demonstrates that control of life-threatening bleeding, eradication of the inhibitor, and induction of immune tolerance to FVIII have clearly improved the clinical outcome of AHA. Our data also suggest a shift in underlying disorders associated with AHA, whereby, in comparison to published studies, a relative increase in the proportion of patients with respiratory diseases is observed. Large controlled multicenter studies are required to confirm these findings. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acquired Hemophilia A Presenting as Massive Postoperative Bleeding in a Patient with Oral Squamous Cell Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Susumu Oba ◽  
Mitsuhiko Nakahira ◽  
Yasunao Kogashiwa ◽  
Yasuhiro Ebihara ◽  
Masashi Sugasawa
Keyword(s):  
Hemophilia A ◽  
Postoperative Bleeding ◽  
Laboratory Data ◽  
Tumor Resection ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Spontaneous Bleeding ◽  
Recombinant Activated Factor Vii

Acquired hemophilia A (AHA) is an extremely rare and serious bleeding disorder caused by autoantibodies against coagulation factor VIII (FVIII). Approximately, 10% of patients with AHA have an underlying malignancy. We report on a 46-year-old man with AHA and advanced oral cancer who presented with massive bleeding after surgery. Preoperative blood coagulation tests showed no abnormalities. He underwent radical tumor resection followed by reconstruction using a free rectus abdominal musculocutaneous flap. Massive subcutaneous hemorrhage developed in his neck and abdomen on the first postoperative day. The hemorrhage remained uncontrolled, despite embolization of the responsible vessels. Subsequent laboratory data showed prolonged activated partial thromboplastin time and decreased FVIII levels. On the basis of his clinical course and the presence of the FVIII inhibitor, we speculated that the patient suffered from AHA. We administered recombinant activated factor VII and prednisolone, after which the spontaneous bleeding stopped and the subcutaneous hemorrhage resolved. A review of the literature identified only three previous documented cases of AHA associated with head and neck cancer. This case indicates that AHA should not be ruled out in patients with uncontrolled postoperative bleeding, while attempting to ensure bleeding control and preventing potentially catastrophic fatal consequences.

Reduced Antigenicity of Recombinant Ovine Factor VIII in Hemophilia A Inhibitor Patient Plasmas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1124-1124
Author(s):  
Philip M Zakas ◽  
Shannon L. Meeks ◽  
Christopher B Doering
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Amino Acid Level ◽  
Coagulation Factor ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Inhibitor Patient

Abstract Abstract 1124 Hemophilia A is an X-linked recessive disorder caused by deficiencies or functional defects in coagulation factor VIII (fVIII). Approximately 20–30% of patients with severe hemophilia A develop antibodies against fVIII (inhibitors) following fVIII replacement therapy, which presents significant complication to the control of subsequent bleeding episodes. State of the art treatment options for patients with inhibitors include fVIII-bypassing agents such as recombinant factor VIIa or activated prothrombin-complex concentrate. Previously, plasma-derived porcine fVIII was a treatment option for inhibitor patients and was effective due to the reduced antigenicity of porcine fVIII toward anti-human fVIII inhibitors. However due to concerns regarding viral contamination, the plasma-derived porcine fVIII products were discontinued and no alternative fVIII products have been made available to patients with inhibitors. Presently, a recombinant porcine fVIII product (OBI-1, Inspiration Biopharmaceuticals) is being investigated in two phase 3 clinical trials for congenital and acquired hemophilia A. Rationale for the development of such a product consists of the prior success of plasma-derived porcine fVIII and the concept that the most effective and lowest risk treatment for fVIII deficiency, even in the presence of inhibitors, remains a fVIII product. Recently, a line of hemophilia A sheep was reestablished from banked frozen sperm and the ovine fVIII (ofVIII) gene, causal mutation, and protein were genetically and biochemically characterized. B-domain deleted (BDD) ovine fVIII shares 86% identity to human fVIII at the amino acid level and confers phenotypic correction, in vivo, to hemophilia A mice using a tail transaction bleeding model. Recombinant ofVIII was expressed in baby-hamster kidney cells and purified to > 95% homogeneity using a two-step ion exchange chromatography procedure. Highly purified ofVIII displays a specific activity of 18,300 units/mg, which is approximately twice that of recombinant BDD human fVIII. Furthermore, the decay of ofVIII activity following thrombin activation is slower than BDD human fVIII suggesting prolonged activity in vivo. Lastly, ofVIII demonstrates equivalent binding to human von Willebrand factor at physiological concentrations in vitro. A translational aim of the present study was to test the hypothesis that unique sequences within ofVIII confer differential antigenicity compared to human and/or porcine fVIII in congenital and acquired inhibitor patient plasmas. To address this hypothesis, the reactivity of 28 samples (22 congenital patient samples designated 1–22, and 6 acquired hemophilia A patient samples designated A1-A6) from the Emory IRB approved inhibitor bank towards recombinant BDD human, porcine, and ovine fVIII were assessed by enzyme-linked immunosorbant assay (Figure 1). When normalized to the reactivity towards human fVIII, the data revealed reduced reactivity towards ofVIII in 27 of 28 total samples. In only one patient was the reactivity towards ofVIII greater than that towards human fVIII and, in this sample, the reactivity towards porcine fVIII also was greater than 100%. Furthermore, plasma reactivity to ovine fVIII was significantly reduced compared to porcine fVIII (P = 0.025; Mann-Whitney U Test). Median values of the relative cross reactivity towards porcine and ovine fVIII were 54 and 38%, respectively. Preliminary inhibitor analysis (Bethesda assay) of three samples shown to contain titers against human fVIII of 25, 19, and 68 BU/ml, revealed undetectable inhibitor titers towards ofVIII in 2 samples, and a titer of 5 BU/ml in the third, respectively. These results suggest that additional orthologous recombinant fVIII molecules may be enabling to the treatment of patients harboring pathogenic inhibitors to human fVIII. Disclosures: No relevant conflicts of interest to declare.

Acquired Hemophilia A. Experience Of a Single Center

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4781-4781 ◽  
Author(s):  
Mauricio A Alzate ◽  
Susana S Meschengieser ◽  
Alicia Blanco ◽  
Silvia Grosso ◽  
María A Lazzari ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Initial Symptoms ◽  
Bleeding Episodes ◽  
The Mean ◽  
Inhibitor Titer

Introduction Acquired hemophilia A is a rare and serious autoimmune disease. Morbidity and mortality are associated with advanced age, comorbidities, toxicity of treatment and bleeding severity. Treatment goals are control of the bleeding and eradication of the inhibitor, while treating the underlying condition if it is present. Objective To describe the baseline characteristics of acquired hemophilia A patients and to assess the response to treatment. Patients and Methods Between November 1991 and April 2013, 27 patients were diagnosed with acquired hemophilia A (mean age 59, range 21-86; 18 women - 66%) in the Departamento de Hemostasia y Trombosis. Five patients were lost from follow-up. APTT mixing studies with normal plasma (1:1) and time-temperature dependent effect on the APTT were performed for a-FVIII diagnosis. Whenever possible, inhibitor activity was titrated by Bethesda method at diagnosis (BU/mL). Medical records were reviewed to evaluate the initial symptoms, underlying diseases, treatments and outcome. Results The mean follow-up was 86 weeks (range 1-640). Underlying etiologies included: idiopathic 70.4%, postpartum 14.8%, malignancy 11.1%, autoimmune disease 3.7%. All patients had bleeding at diagnosis. The most frequent sites of bleeding were: muscular 32%, soft tissue 18%, urinary tract 9%, gastrointestinal tract 6%; being from multiple sites in 9%. At diagnosis, the mean value for FVIII was 6% (range 1-40), and inhibitor titer 220 BU/mL (range 2.2-1173). Initial therapeutic scheme included glucocorticoids in 97% of the patients, 13 in monotherapy (mean age 53 years ± 19), 13 with cyclophosphamide (63 years ± 18) (p= ns), and human immunoglobulin in 1 patient. This last patient died after 1 week of diagnosis due to uncontrolled gastrointestinal bleeding (previous to the era of rVIIa). As a second-line therapy, rituximab was used in 3 patients. Sixty-three (63%) patients achieved complete response (CR) (inhibitor titer < 0.6 BU/mL without bleeding episodes), and 23% achieved partial response (PR) (reduction in inhibitor titer > 50% without bleeding episodes), without differences between monotherapy or combined. Overall, women responded more frequently than men (93.3% vs. 71.4%, p= ns). All patients that received rituximab achieved CR. Conclusions In this study, the overall response rate was higher than 80%. In most cases, the disease has a prolonged course like other autoimmune diseases, with remissions and relapses. Disclosures: No relevant conflicts of interest to declare.

512 A CASE OF AN IDIOPATHIC ACQUIRED HEMOPHILIA A IN AN ELDERLY WOMAN

2021 ◽  
Vol 50 (Supplement_2) ◽  
pp. ii14-ii18
Author(s):  
E E Phyu ◽  
H P Than ◽  
M Hayward
Keyword(s):  
Hemophilia A ◽  
Bleeding Episode ◽  
Coagulation Factor ◽  
Oral Prednisolone ◽  
The Elderly ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Fviii Inhibitor ◽  
Prolonged Aptt ◽  
The Uk

Abstract Introduction Acquired Hemophilia is a bleeding diathesis caused by autoantibodies that interfere with factor VIII (FVIII). Reasons for autoantibodies production are not clear but may be related to gene polymorphisms and/or CD4+ T lymphocytes. 1.3 to 1.5 cases per million population per year are reported in the UK. Half of the cases are secondary to malignancy, pregnancy related conditions, connective tissue disorders or drug reactions while the rest are idiopathic. Case Report We report a case of an acquired hemophilia A in an 86-year-old lady with underlying type 2 diabetes, hypertension, and cognitive impairment, being treated as the left lower limb cellulitis with antibiotics. She was found to have a sudden hemoglobin drop and her CT (Abdomen) confirmed a spontaneous intra-abdominal hematoma. Clotting profile showed prolonged APTT to 168.5 seconds, being not corrected at mixing study, with normal PT and INR. The FVIII assay was reduced to 18.4 iU/dL with FVIII inhibitor concentration of 0.7 Bu. Viral and autoimmune screenings were negative. The idiopathic acquired hemophilia A was diagnosed. Red blood cell transfusions, bypassing agents (FEIBA) and oral tranexamic acid were given for acute bleeding episode. Concomitantly, oral prednisolone was used to reduce the inhibitor levels. Repeated FVIII assay showed 121 iU/dL and 199iU/dL on day 6 and 12, respectively. Steroid was continued for the next 4 weeks and then gradually tapered. No further bleeding episode was noted. Conclusion The diagnosis of acquired hemophilia should be considered in any elderly patient with prolonged APTT. Mixing study is to measure the presence of inhibitors of coagulation or to detect coagulation factor deficiency. Quantitative coagulation factor assays and Bethesda Assays are performed for definitive diagnosis. Immunosuppressive regimens are the mainstay treatment. However, premorbid conditions and co-morbidities should be taken into consideration before initiating the aggressive immunosuppressive therapy in the elderly patients.

scholarly journals Postpartum Acquired Hemophilia: A Rare Cause of Postpartum Hemorrhage

2013 ◽  
Vol 2013 ◽  
pp. 1-2 ◽  
Author(s):  
Srikanth Seethala ◽  
Sumit Gaur ◽  
Elizabeth Enderton ◽  
Javier Corral
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Vii ◽  
Normal Vaginal Delivery ◽  
Factor Viii Inhibitor ◽  
Activity Levels ◽  
Factor Viii Activity ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Viii Inhibitor

A 36-year-old female started having postpartum vaginal bleeding after normal vaginal delivery. She underwent hysterectomy for persistent bleeding and was referred to our institution. An elevation of PTT and normal PT made us suspect postpartum acquired hemophilia (PAH), and it was confirmed by low factor VIII activity levels and an elevated factor VIII inhibitor. Hemostasis was achieved with recombinant factor VII concentrates and desmopressin, and factor eradication was achieved with cytoxan, methylprednisolone, and plasmapheresis.

Emicizumab for the treatment of acquired hemophilia A

Blood ◽  
2020 ◽  
Author(s):  
Paul Knoebl ◽  
Johannes Thaler ◽  
Petra Jilma ◽  
Peter Quehenberger ◽  
Karoline Veronika Gleixner ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Coagulation Factor ◽  
Standard Of Care ◽  
Early Discharge ◽  
Severe Bleeding ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Breakthrough Bleeding ◽  
Hemostatic Efficacy ◽  
Bypassing Agents

Acquired hemophilia A (AHA) is a severe bleeding disorder caused by inhibiting autoantibodies to coagulation factor VIII (FVIII). For hemostatic treatment, bypassing agents, human or porcine FVIII are currently standard of care. Emicizumab is a bispecific, FVIII-mimetic therapeutic antibody, that reduced the annualized bleeding rates in congenital hemophiliacs. Here we report on 12 patients with AHA, 6 male, 6 female, age 74 yrs (64/80) (all data medians and IQR), treated with emicizumab. Initial FVIII was &lt;1%, inhibitor 22.3BU/mL (range 3-2000). Eight patients had severe bleeding. Emicizumab was started with 3mg/kg sc. weekly for 2-3 doses, followed by 1.5mg/kg every 3 weeks to keep the lowest effective FVIII levels. For FVIII monitoring, chromogenic assays with human and bovine reagents were used. All patients received immunosuppression with steroids and/or rituximab. After the first dose of emicizumab, APTT normalized in 1-3 days, FVIII (human reagents) exceeded 10% after 11 (7.5/12) days. Hemostatic efficacy was obtained and bypassing therapy stopped after 1.5 (1/4) days. FVIII (bovine reagents) exceeded 50%, indicating complete remission, after 115 (67/185), and emicizumab was stopped after 31 days (15/79), in median 5 injections (range 3-9) were given. No patient died from bleeding or thromboembolism, and no breakthrough bleeding was observed after the first dose of emicizumab. In conclusion, emicizumab seems to be an effective hemostatic therapy for AHA, with the advantages of sc. therapy (every 1-3 weeks), good hemostatic efficacy, early discharge, reduction of immunosuppression and adverse events.

scholarly journals Acquired hemophilia A and plasma cell neoplasms: a case report and review of the literature

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Katarzyna A. Jalowiec ◽  
Martin Andres ◽  
Behrouz Mansouri Taleghani ◽  
Albulena Musa ◽  
Martina Dickenmann ◽  
...  
Keyword(s):  
Factor Viii ◽  
Plasma Cell ◽  
Hemophilia A ◽  
High Titer ◽  
Coagulation Factor ◽  
Laboratory Diagnostics ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Plasma Cell Neoplasm ◽  
Cell Neoplasm

Abstract Background Acquired hemophilia A is a rare autoimmune disease with clinically often significant bleeding diathesis resulting from circulating autoantibodies inhibiting coagulation factor VIII. Half of acquired hemophilia A cases are associated with an underlying disorder, such as autoimmune diseases, cancer, or use of certain drugs, or occur during pregnancy and in the postpartum period. In the other half, no underlying cause is identified. An association of acquired hemophilia A with plasma cell neoplasm seems to be extremely rare. Case presentation We describe a case of a 77-year-old Swiss Caucasian man who was diagnosed with acquired hemophilia A and smoldering multiple myeloma as an underlying cause. Acquired hemophilia A was treated with prednisolone, cyclophosphamide, and immunoadsorption. Extensive workup revealed a plasma cell neoplasm as the only disorder associated with or underlying the acquired hemophilia A. For long-term control of acquired hemophilia A, we considered treatment of the plasma cell neoplasm necessary, and a VRD (bortezomib, lenalidomide, and dexamethasone) regimen was initiated. Due to multiple complications, VRD was reduced to VRD-lite after two cycles. After nine cycles of induction therapy and five cycles of consolidation therapy, the patient is in complete remission of his acquired hemophilia A and very good partial remission of the plasma cell neoplasm. We conducted a literature review to identify additional cases of this rare association and identified 15 other cases. Case descriptions, including the sequence of occurrence of acquired hemophilia A and plasma cell neoplasm , treatment, evolution, and outcome are presented. Discussion and conclusions Our case, together with 15 other cases described in the literature, underscore the possibility of plasma cell neoplasm as an underlying cause of acquired hemophilia A. Physicians should consider including protein electrophoresis, immunofixation, and analysis of free light chains in laboratory diagnostics when treating a patient with acquired hemophilia A. The occurrence of excessive and unexplained bleeding in patients diagnosed with plasma cell neoplasm should raise suspicion of secondary acquired hemophilia A and trigger the request for coagulation tests, particularly in patients treated with immunomodulatory drugs such as thalidomide or lenalidomide. Additionally, early intervention with immunoadsorption can be lifesaving in cases with high-titer factor VIII inhibitors, especially when surgical interventions are necessary.

Novel Sites of Gamma Carboxylation in Human Factor VII

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3759-3759
Author(s):  
Emily M Wilkerson ◽  
Barbara Bates ◽  
Kraig T Kumfer ◽  
Nicholas M Riley ◽  
Brad S Schwartz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Protein Function ◽  
Synthetic Peptides ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Post Translational Modification ◽  
Coagulation Factor Vii ◽  
Gla Domain

Abstract Introduction Gamma- (γ-) carboxylation of glutamate residues is a vitamin K-dependent post-translational modification critical to the function of several plasma proteins. Found in the N-terminal domains of specific proteins, most of which are involved in hemostasis, these γ-carboxyglutamate residues (Gla) help mediate binding of divalent cations and are essential to protein function. Coagulation factor VII(a) bears 10 known Gla residues as characterized by N-terminal sequencing, yet Thim et al.( Biochemistry 27:7785 1988) used amino acid analysis to quantify 11.0 mol of Gla/mol of protein for plasma-derived factor VII(a). We used mass spectrometry to map and validate Gla residues of coagulation factor VII(a) to potentially identify Gla residues outside of the Gla domain. Methods Four sources of factor VII(a) (2 plasma-derived and 2 recombinant) were extracted, digested, and analyzed by tandem mass spectrometry (LC-MS/MS). Each sample was digested with trypsin and chymotrypsin to provide orthogonal coverage. Peptides derived from factor VII(a) proteolysis were analyzed on a nanoLC coupled to a quadrupole-Orbitrap-quadrupole linear ion trap mass spectrometer (Orbitrap Fusion Lumos Thermo Scientific). Multiple fragmentation methods were used to map and validate the sites including collisional based dissociation (CAD), higher energy collisional activated dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer/higher-energy electron transfer activation (EThcD). Tandem MS spectra were collected at resolution 30K at 200 m/z, and data were processed using MaxQuant, COMPASS, and Proteome Discoverer. All identified sites were validated through manual annotation of spectra. We estimate that any site with >1% occupancy will be identified as Gla with this method. To validate select sites of novel gamma carboxylation, synthetic peptides were made for 4 different sites that were consistently identified in factor VII(a) from all 4 sources. Synthetic peptides were analyzed using MS methods described above, generating "true positives" to match with peptides identified from the factor VII(a) sources. Spectra from the synthetic peptides and factor VII from each source were compared using manual spectral annotation. Results In addition to identifying known Gla residues at positions 6, 7, 19, 20, 29, and 35 of factor VII(a), we detected and validated 9 novel Gla residues outside of the N-terminal Gla domain. Novel sites include residues 94, 116, 132, 219, 215, 229, 265, 196 and 385. Four of these residues (210, 220, 296 and 385) were identified as Gla in all 4 sources of factor VII(a) and were validated with synthetic peptides using a combination of fragmentation methods, providing high confidence in their characterization. Published crystallographic data suggest that residues 210 and 220 of factor VIIa-tissue factor are closely approximated to a Ca2+ ion complexed to the C-terminal protease domain; this is not the case for residues 296 and 385. We continue to refine the technique to map the Gla residues (novel and known), and to quantify the fraction of factor VII(a) molecules from each source that contain the modification at each site, in order to better incorporate our data with established studies showing >90% occupancy at each of the 10 Gla domain sites. These data suggest there is room to expand our understanding of how carboxylation contributes to specific protein function, in order to provide more comprehensive understanding of this post-translational modification, and refine our understanding of hemostatic mechanisms. Disclosures No relevant conflicts of interest to declare.

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