Case Report: A 19-Years Old Patient with Haemophilia a with High Response Inhibitor Screening and Quantitation and the Bethesda Method

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4956-4956
Author(s):  
Marianela Trejos Herrera ◽  
Alicia Lopez Campos
Keyword(s):  
Factor Viii ◽  
Conflicts Of Interest ◽  
Current Knowledge ◽  
High Titer ◽  
Laboratory Diagnosis ◽  
Factor Ix ◽  
High Response ◽  
Patient Specific ◽  
Aptt Reagent ◽  
Low Sensitivity

Abstract A severe haemophiliac patient, high response inhibitors, 19 years, who was diagnosed at 8 months old and since then he begins to administer factor VIII concentrates. However, a year later after the start of treatment, they were detected inhibitors that behave as high response inhibitors from the start. He discontinues treatment with Factor VIII concentrate and instead he begins to administer Factor IX concentrate and prothrombin complex as an alternative treatment. Through his illness, the patient has made significant bleeding at the level of joint and other muscles such as the psoas. It is a bleeding patient at rest up for what is currently administered prophylactically Prothrombin Concentrate 3 times a week. This case, the comment will be directed towards laboratory diagnosis and its evolution since he was diagnosed in 1994 to date. From 2013, screening protocols and quantification of both factors and inhibitors were modified in the Specialized Hematology laboratory of Hospital México, due to problems in the sensitivity and specificity of the method and reagents we were using. The results from these patient specific inhibitors are described in the following paragraphs. Observations With the experience and current knowledge of the following it is concluded, according to a literature review that was performed (see Table 1): 1) This time period has persisted inhibitor high title, which is evidenced of the study of mixtures which do not clearly show a potentiation by incubating 2 hours at 37 ° C, since the values of the Control Mix and patient give very similar high values. 2) We were shown only on the date of 14.02.2013, there was a real interference Lupic Anticoagulant (LA), which is confirmed by the method of Russel viper venom. 3) The dates high titer inhibitors were reported against factor VIII (from 6 November 2013 to date), both screening and for the Bethesda, aPTT reagent was used with Kaolin activator which is low sensitivity to LA. 4) On November 20, 2015 there was an error in the interpretation and anti factor VIII inhibitors as negative were reported as potentiation at 37 ° C is not evidenced, but rather was interpreted as interference of an LA, which was communicated to the medical and preventive measures cited in paragraph corrections were made. 5) The last date that the sample was processed, on February 1, 2016, the inhibitor against factor VIII did not affect dilution of silica APTT, so the index ROSNER was not affected (<12) and it was not necessary to mount the LA test, according to the request in identifying protocols inhibitors. 6) Factor VIII deficient plasma is currently being used, which contains von Willebrand factor, as recommended by international guidelines quantization factors. Conclusions According to an experience as support center Reference Center in our country, we conclude and recommend the following: 1) Registration of haemophiliac patients with high antibody titer is essential as the description of the protocol to be followed in these patients. 2) The behavior of this inhibitor in the screening test of time and temperature dependence, it was decided to directly mount the Bethesda assay, following the recommendations of the literature on when the use of a reagent with low sensitivity to lupus inhibitor. 3) Within the protocol and as far as we can, we will process the purchase for quantification of factor VIII chromogenic by ELISA methodology, as a confirmatory method. Disclosures No relevant conflicts of interest to declare.

Pharmacokinetics of Continuous Infusion Therapy of Factor VIII Concentrates in Hemophilia A Patients with Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3320-3320
Author(s):  
Katsumi Nishiya ◽  
Ichiro Tanaka ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Koji Yada ◽  
...  
Keyword(s):  
Factor Viii ◽  
Continuous Infusion ◽  
Infusion Rate ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Infusion Therapy ◽  
Target Level ◽  
Ci Therapy ◽  
The Difference

Abstract Abstract 3320 Continuous infusion (CI) of factor VIII (FVIII) concentrates is aimed at maintaining a steady hemostatic level of FVIII activity (FVIII:C) in hemophilia A patients during various surgeries. However, there are few reports that mentioned the difference of pharmacokinetics of CI therapy in hemophilia A patients with inhibitors. We investigated the relationship between the FVIII:C levels and the rate of CI, and the difference of clearance (CL) and volume of distribution (Vd) of FVIII in hemophilia A patients with/without inhibitors. 8 severe hemophilia A patients without inhibitors (arthroscopic synovectomy; 4 cases, total knee arthroplasty; 2 cases, total nephrectomy; 1 case, partial hepatectomy; 1 case), 3 patients with low-titer (2.0–2.9 BU) inhibitors and 3 patients with high-titer (6.0–9.0 BU) inhibitors (insertion or removal of a central venous access device), were enrolled in this study between 2005 and 2010. According to the Japanese guideline for hemophilia treatment, we should do CI therapy to keep target level 80–100% for 5–10 days for joint surgery and other major surgeries. An initial bolus infusion (BI) of FVIII concentrates was administered to achieve this level prior to CI. In addition, we have to neutralize the inhibitors by FVIII concentrates in case of the patients with inhibitors. FVIII:C was measured using one-stage clotting assays and FVIII inhibitor assays were performed using the Bethesda method. All therapy was conducted after obtaining fully informed consent. The median FVIII:C level after BI was 120.2% (range: 90–150) in the patients without inhibitors, 72.0% (range: 68–160) with low-titer inhibitors, and 20.0% (range: 9.4–30) with high-titer inhibitors, respectively. The target level of FVIII:C was adjusted to approximately 100%. The initial infusion rate was 3.7 U/kg/hr (range: 2.2–5.0), 8.3 U/kg/hr (range: 8.0–8.5) and 18.5 U/kg/hr (range: 15–22), respectively. After adjustment for the target level, the final infusion rate decreased to 2.6 U/kg/hr (range: 1.5–5.4), 4.7 U/kg/hr (range: 3.0–5.6) and 8.0 U/kg/hr (range: 7.0–9.0), respectively. CL was 2.3 ml/hr/kg (range: 1.5–3.9), 4.0 ml/hr/kg (range: 2.3–5.1) and 9.3 ml/hr/kg (range: 9.0–9.6), respectively. Vd was 0.04 L/kg (range: 0.031–0.047), 0.18 L/kg (range: 0.12–0.29) and 1.54 L/kg (range: 0.95–2.43), respectively. No unexpected safety concerns associated with CI, such as thrombosis, was identified during the study. On CI therapy, we could keep target level of the patients without inhibitors and with low-titer inhibitors easier than those with high-titer inhibitors. One of the reason is that CL and Vd in patients with inhibitors are higher than those in patients without inhibitors. CI with appropriate monitoring of FVIII:C level and concerning CL and Vd forms a safe method for perioperative care in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

Use of Prothrombin Complex Concentrates in Hemophiliacs with Inhibitors: Clinical and Laboratory Studies

PEDIATRICS ◽  
1978 ◽  
Vol 62 (5) ◽  
pp. 767-774
Author(s):  
George R. Buchanan ◽  
Sherwin V. Kevy
Keyword(s):  
Factor Viii ◽  
High Titer ◽  
Factor Ix ◽  
Laboratory Studies ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Prolonged Incubation ◽  
Plasma Factor ◽  
Bleeding Episodes ◽  
Proven Benefit

Nine patients with severe classic hemophilia and inhibitors against factor VIII were treated for 156 bleeding episodes with 503 infusions of Proplex, Konyne, or Auto-Factor IX, three preparations of prothrombin complex concentrates (PCCs). Approximately two thirds of the bleeding episodes were managed successfully. Although the prothrombin time (PT) and partial thromboplastin time (PTT) were shortened after most PCC infusions, there was no evidence of disseminated intravascular coagulation. The degree of shortening of PT or PTT was not related to the particular PCC preparation used, dose, or cessation of hemorrhage. All PCC preparations contained activated clotting factors, as manifested by their ability to shorten the PTT of normal plasma, factor-VIII-deficient plasma, and factor-IX-deficient plasma. Shortening, which was greater with Auto-Factor IX than with the other products, was inhibited partially by a factor IX antibody and blocked completely by prolonged incubation with plasma. Although the nature of the procoagulant material in PCCs is uncertain, these products are of proven benefit to hemophilic patients with high-titer inhibitors. Side effects have been minimal and inhibitor titers have not risen.

Identification Of The Thrombin-Binding Site On Factor VIII Regulating Arg372 Cleavage In The Factor VIII Heavy Chain

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3570-3570
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Koji Yada ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Solid Phase ◽  
Factor Ix ◽  
Cross Linking ◽  
Dependent Manner ◽  
Factor X ◽  
Dose Dependent

Abstract Factor VIII is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin. Activated factor VIII (VIIIa) forms the tenase complex and markedly amplifies the activation of factor X as a cofactor of factor IX. We had demonstrated that thrombin interacts with factor VIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008). The A2 residues 484-509 partially contribute to cleavage at Arg372 by thrombin, however, the major thrombin binding-site(s) regulating cleavage at Arg372 is unclear. Thrombin recognizes macromolecular substrates and cofactors through either or both of two anion-binding exosite I and II (ABE-I and -II), which are characterized by a high density of solvent-exposed basic residues. ABE-I binds to fibrinogen and hirudin (residues 54-65), whilst ABE-II is primarily characterized as the heparin-binding exosite. The A1 domain of factor VIII also binds to thrombin through the ABE-I (Nogami K. JBC 2005). In this study, we attempted to identify the thrombin-binding region on A1, and focused on the A1 residues 340-350, involving the clustered acidic residues and similar sequences of hirudin (residues 54-65). A synthetic peptide corresponding to the A1 residues 340-350 with sulfated Tyr346 (340-350-S(+)) was prepared to investigate factor VIII interaction with thrombin. Activation of factor VIII (100 nM) by thrombin (0.4 nM) with various concentrations of peptide was evaluated by measurement of factor VIIIa activity in a one-stage clotting assay. A 340-350-S(+) peptide showed a dose-dependent inhibition (by ∼60%) of thrombin-catalyzed activation, and the IC50 was 75 µM. A non-sulfated peptide also showed a modest inhibition by ∼40% (IC50 >400 µM), however. An experiment using thrombin substrate S-2238 demonstrated that P340-350-S(+) did not affect the thrombin activity. The effect of 340-350-S(+) peptide on the thrombin-catalyzed cleavage of heavy chain was further examined by SDS-PAGE/western blotting.The peptide significantly blocked the cleavage at Arg372 in a timed- and dose-dependent manner (IC50; 150 µM), whilst of interest the cleavage at Arg740 was little affected. A non-sulfated peptide also delayed the cleavage at Arg372, with a modest fast cleavage compared to sulfated one. The peptide did not inhibit factor FXa-catalyzed reaction to factor VIII. Direct binding of 340-350-S(+) peptide to thrombin was examined by a surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based solid phase assay, thrombin bound to immobilized 340-350-S(+) peptide with high affinity (Kd; 1.13 nM). EDC cross-linking fluid phase assay similarly revealed that formation of EDC cross-linking product between the biotinylated 337-350-S(+) peptide and thrombin were observed, and this cross-linking was completely inhibited by non-labeled 340-350-S(+) peptide (IC50; 1.0 µM). Taken together, we demonstrated that the A1 residues 340-350 (NEEAED(sY)DDDL) involving sulfated Tyr346 contained the thrombin binding-site responsible for the proteolytic cleavage at Arg372 in factor VIII. Disclosures: No relevant conflicts of interest to declare.

Laboratory Diagnosis of von Willebrand Disease

2009 ◽  
Vol 03 (01) ◽  
pp. 33 ◽  
Author(s):  
Muriel Meiring ◽  
Philip N Badenhorst ◽  
Mareli Kelderman ◽  
◽  
◽  
...  
Keyword(s):  
Factor Viii ◽  
Correct Diagnosis ◽  
Laboratory Diagnosis ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Coagulant Activity ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (VWD) is a bleeding disorder caused by either quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). No single available test provides appropriate information about the various functions of VWF, and the laboratory diagnosis of VWD is based on a panel of tests, including the measurement of factor VIII coagulant activity (FVIIIC), VWF antigen levels (VWF:Ag), VWF activity as measured by the ristocetin co-factor activity (VWF:RCo), the collagen-binding activity of VWF (VWF:CB), VWF multimer analysis, ristocetininduced platelet agglutination (RIPA), the factor-VIII-binding assay of plasma VWF and VWF propeptide levels. Due to the heterogeneity of VWF defects and the variables that interfere with VWF levels, a correct diagnosis of types and subtypes may sometimes be difficult, but is very important for therapy. Furthermore, the RCo assay and the RIPA test are based on platelet agglutination in reaction with the non-physiological antibiotic ristocetin. These tests also have low sensitivity and are difficult to standardise. Therefore, several analyses (tests) are required to diagnose VWD and it is important to be aware of the pitfalls to which these tests are subjected in terms of the diagnosis. In this article, the laboratory diagnosis of patients with type 1, 2A, 2B, 2M, 2N and 3 VWD will be explained by using a modified algorithm that was first proposed by the guidelines for diagnosis and treatment of VWD in Italy.

Factor VIII Region 1811–1818 Is Involved in Factor IXa Binding and Factor VIIIa Stability

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3357-3357
Author(s):  
Esther Bloem ◽  
Henriet Meems ◽  
Maartje van den Biggelaar ◽  
Koen Mertens ◽  
Alexander B Meijer
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Factor Ix ◽  
The Other ◽  
Factor V ◽  
Fluorescent Intensity ◽  
A2 Domain

Abstract Abstract 3357 Previously, we identified a role for the lysine residue couple 1967/1968 in the stability of activated factor VIII (FVIIIa). Using tandem mass tags (TMT 126/127) in combination with mass spectrometry, we identified lysine residues involved in the interaction between the A2 domain and the rest of heterodimer (A1/A3-C1-C2) of FVIIIa (Bloem et al., J Biol Chem 2012;287:5575–83). Upon FVIII activation and A2 domain dissociation, the highest increase in surface exposure occurred for the lysine couple 1967/1968, and functional studies confirmed the role thereof in FVIIIa stability. In addition to lysines 1967/1968 also other lysines displayed an increased surface exposure, including those in positions 1804, 1808, 1813 and 1818. The A3 domain region 1803–1818 has previously been implicated in interactions with ligands such as activated factor IX (FIXa). As such, one might expect increased surface exposure due to FVIII activation. On the other hand, A2 domain dissociation may have rearranged the A3 domain surface in this region. The relation between FIXa assembly and A2 domain retention was therefore explored in the present study. To unravel the role of region 1803–1818 in FVIIIa stability and FIXa binding, either region 1803–1810 or 1811–1818 was replaced by the corresponding regions of the homologous factor V. Additionally, as Asn1810 is N-linked glycosylated and this glycan is maintained in both chimeras, a variant that lacks this glycan (N1810C) was investigated. Factor × activation kinetics were used to investigate the apparent FIXa binding affinity of the FVIII variants. FXa generation was assessed on 15% phosphatidyl serine (PS) containing membranes. FIXa titration experiments showed that the affinity for the 1811–1818 variant is reduced (apparent Kd from 1.3 nM to 2.4 nM). Removal of the glycan and substitution of 1803–1810 has little or no effect on the apparent FIXa binding. FVIIIa-FIXa assembly on membranes containing 15% PS was studied using lipid- coated glass beads (lipospheres). Lipospheres were incubated with fluorescein-labeled FIXa and different FVIIIa concentrations. FIXa did only display liposphere binding in the presence of FVIIIa. Therefore, the mean fluorescent intensity on the lipospheres at increasing FVIIIa concentration could be used as a measure for FVIIIa-FIXa assembly on lipids. Results from these experiments showed that the 1811–1818 variant fails to promote FVIIIa-FIXa assembly, whereas the other variants behave like WT. To investigate FVIIIa stability, and thereby the role of the mutations on A2 domain dissociation, the activity of the variants was followed over time. Results showed that the 1811–1818 variant has a decreased half life of 1.5 min, compared to 6.9 min for WT. Also substitution of region 1803–1810 results in a lower half life, although to a lesser extent (2.8 min), whereas the glycan lacking variant behaves like WT (6.8 min). Incubation of FVIIIa variants with FIXa is known to stabilize FVIIIa and leads to an increased half life for all variants. However, the 1803–1810 variant is most efficiently stabilized by FIXa, shown by a 3-fold increase in half life, instead of 1.6-fold as seen for both WT and N1810C. The 1811–1818 variant is stabilized by 2.2-fold and therefore remains significantly less stable than WT. Together these results show that the 1811–1818 region is not only involved in FIXa binding, but additionally plays a major role in A2 domain retention. Region 1803–1810 also plays a role in FVIIIa stability, although to a lesser extent. Remarkably, the glycan at position Asn1810 does not influence neither FIXa binding nor FVIIIa stability, and apparently serves some other function. Disclosures: No relevant conflicts of interest to declare.

Immunologic aberrations, HIV seropositivity and seroconversion rates in patients with hemophilia B

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 276-281
Author(s):  
DB Brettler ◽  
F Brewster ◽  
PH Levine ◽  
A Forsberg ◽  
S Baker ◽  
...  
Keyword(s):  
Factor Viii ◽  
Male Volunteer ◽  
Hemophilia A ◽  
High Titer ◽  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Immunodeficiency Virus ◽  
And Function ◽  
Factor Concentrate

Because there have been reports that factor IX concentrate is less immunosuppressive and therefore factor IX users have less immunologic aberrations, we have studied a group of 22 patients with hemophilia B and six patients with factor VIII deficiency and high titer inhibitors with respect to lymphocyte numbers and function, human immunodeficiency virus (HIV) serology, and factor usage. This group was compared to 111 patients with hemophilia A and a group of 28 healthy male volunteer controls. When the study began in 1983, the majority of patients with hemophilia B and with higher titer factor VIII inhibitors were seronegative, 77% and 83% respectively, as compared to only 30% of patients with hemophilia A. At that time the factor IX users also had milder immune aberrations than the hemophilia A group. However, with time and increasing clotting factor concentrate usage, seroconversion and more striking abnormalities in immune function have occurred in the hemophilia B group. In a subgroup of 16 patients with hemophilia B studied twice, the incidence of seropositivity increased from 31% in 1983 to 69% in 1985. We thus conclude that factor IX concentrate in itself is not less immunosuppressive than factor VIII concentrate. Seroconversion in factor IX concentrate users appears to be lagging behind seroconversion in factor VIII concentrate users, perhaps secondary to the lower cumulative dosage of concentrate that patients with hemophilia B utilize.

scholarly journals Application of Turbidity Curve Analysis to New Coagulation Factor VIII and IX Assays with Superior Analytical Resolution in Low-Level Range

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5015-5015
Author(s):  
Jaewoo Song ◽  
Juwon Kim ◽  
Jungwoo Han ◽  
Jin Seok Kim ◽  
Jihye Ha
Keyword(s):  
Factor Viii ◽  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Analytical Sensitivity ◽  
Low Level ◽  
Assay Result ◽  
Viii And Ix ◽  
Analytical Resolution

Abstract Background: The needs for sensitive coagulation factor assays able to measure factor VIII (FVIII) and factor IX (FIX) in the range of 0.0 to 1.0 %, are continuously growing with diversification of hemophilia management. However, practical methods with sufficient analytical sensitivity available in clinical laboratory have not yet been introduced. We developed new coagulation factor assays applying various parameters derived from a turbidity based coagulometer and examined their ability to measure low-level FVIII and FIX and analytical resolution in that range. Method: We prepared 12 spiked samples with FVIII and FIX levels from 0.0 to 2.4 % and conducted conventional one-stage coagulation factor assays in repeat. We collected measured values of APTT, velocity and acceleration peaks of coagulation (peak 1 and peak 2) from each measurement. We also calculated values of peak 1 and peak 2 from the mathematical model of turbidity curves. From the measured values of these parameters we derived calibration formulae for coagulation factor assays, FVIIICT, FVIIIpeak1, FVIIIpeak2, FVIIIcalc1, FVIIIcalc2, FIXCT, FIXpeak1, FIXpeak2, FIXcalc1, and FIXcalc2. Results: The reliability interval (range of FVIII levels producing unequivocal results) of FVIIICT (the conventional FVIII assay) covered only 9 % of 0.0 to 1.0 % range. For new assays, the coverages were 54, 31, 55, and 65 % for FVIIIpeak1, FVIIIpeak2, FVIIIcalc1, and FVIIIcalc2 respectively. The resolution between immediate levels of spiked samples could be determined from modeled distributions or be checked simply by inspecting the actual assay result distributions. For FVIIIpeak1, 0.2 % and 0.6 % results stood apart from each other. For FVIIIcalc1 and FVIIIcalc2, 0.2, 0.4, and 0.6 % were distinguished from each other. When we measured recombinant human (rh) FVIII, the coverages were 7, 64, 52, 73, and 79 % for rhFVIIICT, rhFVIIIpeak1, rhFVIIIpeak2, rhFVIIIcalc1, and rhFVIIIcalc2 respectively. (rh)FVIIIpeak1, (rh)FVIIIcalc1, and (rh)FVIIIcalc2 particularly showed wide measurable ranges of guarantee. For FVIIIpeak1, 0.2 % and 0.6 % results stood apart from each other. For FVIIIcalc1 and FVIIIcalc2, 0.2, 0.4, and 0.6 % were distinguished from each other. rhFVIIIpeak1 and rhFVIIIcalc1 showed slightly better resolution than the former. rhFVIIIcalc2 was notable in that every 0.1, 0.2, 0.4, 0.6, 0.8 % result stood apart from each immediate level result. We could not determine certainty interval (the range of unequivocal values) of FIXCT and FIXpeak2 because the 0.0 % and 1.0 % ranges overlapped. Thus, the conventional FIX assay cannot measure between 0.0 and 1.0 %. FIXpeak1, FIXcalc1, and FIXcalc2 worked better and the certainty interval of unequivocal results could be determined between 0.0 and 1.0 %. The reliability interval was not available for any FIX assay. Results from rhFIX measurements were similar those of plasma FIX assays. Conclusion: We introduce new FVIII and FIX assays with superior analytical resolution in the range of 0.0 to 1.0 % in comparison to the conventional assays. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3171-3171
Author(s):  
Wensheng Wang ◽  
Amy E Griffiths ◽  
Philip Fay
Keyword(s):  
Mass Spectrometry ◽  
Factor Viii ◽  
Light Chain ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Direct Sequencing ◽  
Neutralizing Antibody ◽  
Factor Viii Inhibitor ◽  
Amino Terminal

Abstract Abstract 3171 Poster Board III-111 Hemophilia 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. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immunologic aberrations, HIV seropositivity and seroconversion rates in patients with hemophilia B

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 276-281 ◽  
Author(s):  
DB Brettler ◽  
F Brewster ◽  
PH Levine ◽  
A Forsberg ◽  
S Baker ◽  
...  
Keyword(s):  
Factor Viii ◽  
Male Volunteer ◽  
Hemophilia A ◽  
High Titer ◽  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Immunodeficiency Virus ◽  
And Function ◽  
Factor Concentrate

Abstract Because there have been reports that factor IX concentrate is less immunosuppressive and therefore factor IX users have less immunologic aberrations, we have studied a group of 22 patients with hemophilia B and six patients with factor VIII deficiency and high titer inhibitors with respect to lymphocyte numbers and function, human immunodeficiency virus (HIV) serology, and factor usage. This group was compared to 111 patients with hemophilia A and a group of 28 healthy male volunteer controls. When the study began in 1983, the majority of patients with hemophilia B and with higher titer factor VIII inhibitors were seronegative, 77% and 83% respectively, as compared to only 30% of patients with hemophilia A. At that time the factor IX users also had milder immune aberrations than the hemophilia A group. However, with time and increasing clotting factor concentrate usage, seroconversion and more striking abnormalities in immune function have occurred in the hemophilia B group. In a subgroup of 16 patients with hemophilia B studied twice, the incidence of seropositivity increased from 31% in 1983 to 69% in 1985. We thus conclude that factor IX concentrate in itself is not less immunosuppressive than factor VIII concentrate. Seroconversion in factor IX concentrate users appears to be lagging behind seroconversion in factor VIII concentrate users, perhaps secondary to the lower cumulative dosage of concentrate that patients with hemophilia B utilize.

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