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R T Barrow

Publications and source records attributed to R T Barrow.

10 recordsLinked to original sources
PubMed2006

The humoral response to human factor VIII in hemophilia A mice.

BACKGROUND: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. OBJECTIVES: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. METHODS: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. RESULTS: The robust immune response resulted in the production of approximately 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG(1) and IgG(2a) isotypes, respectively. Because the IgG(2a) isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. CONCLUSION: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.

Animals↗
PubMed2006

Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII.

BACKGROUND: Inhibitory antibodies to factor (F) VIII (FVIII inhibitors) present a major clinical challenge as a complication of hemophilia A and as acquired autoantibodies in non-hemophiliacs. Porcine FVIII is a potentially useful therapeutic agent because of its low crossreactivity with many inhibitors. Recombinant porcine FVIII (rpFVIII) is undergoing clinical trials in inhibitor patients. OBJECTIVES: The goals of this study were to neutralize human FVIII inhibitors in vitro with rpFVIII and to characterize the relationship between recovery of FVIII activity and the antiporcine FVIII inhibitor titer. METHODS: rpFVIII was added to 28 antihuman FVIII inhibitor plasmas and assayed either immediately or after a 2 h preincubation at 37 degrees C. The concentration of rpFVIII required for recovery of FVIII activity to 50% of normal (EC(50)) was determined by polynomial regression analysis and compared with the antiporcine FVIII inhibitor titer measured by Bethesda assay. RESULTS: Six of nine plasmas classified as non-crossreactive by Bethesda assay had detectable inhibitory activity in the FVIII recovery assay. Recovery was decreased following a 2 h preincubation compared with immediate assay for the 19 plasmas with detectable antiporcine FVIII inhibitors. There was a linear relationship between EC(50) and inhibitor titer for plasmas with antiporcine FVIII titers ranging from 0.6 to 10 BU per milliliter in both the immediate and the 2 h preincubation assays. CONCLUSION: A quantitative method for analysis of inhibitor neutralization in vitro has been developed, which may be useful for comparison with pharmacodynamic studies of rpFVIII in FVIII inhibitor patients and subsequent rational dosing in this patient population.

Animals↗
PubMed2004

Comparative immunogenicity of recombinant B domain-deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII.

Hyate is a commercial plasma-derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI-1 is a recombinant B domain-deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI-1 or Hyate:C in a comparative immunogenicity trial. OBI-1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg(-1). Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI-1 or Hyate:C at doses of 1 or 10 U kg(-1), but were identified in 81% and 94% of mice given 100 U kg(-1) of OBI-1 or Hyate:C, respectively. There was no significant difference between OBI-1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI-1-treated mice at 10 U kg(-1) (P = 0.09). Total anti-FVIII antibodies to Hyate:C and OBI-1 were also measured by ELISA using immobilized purified plasma-derived porcine FVIII and OBI-1, respectively, as antigens. At the 10 and 100 U kg(-1) doses, the mean anti-FVIII response was higher in Hyate:C-treated-mice than in OBI-1-treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI-1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.

Animals↗
PubMed2001

Antigenicity of putative phospholipid membrane-binding residues in factor VIII.

Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 beta-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

Amino Acid Sequence↗
PubMed2000

Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules.

Factor VIII (fVIII) circulates as a heavy chain/light chain (A1-A2-B/ap-A3-C1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cleaved from fVIII during proteolytic activation by thrombin or factor Xa. We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII molecules that contained combinations of porcine sequence replacements within the A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhibitory antibodies between human fVIII and the hybrids was inversely related to the degree of porcine substitution. In all plasmas, the substitution of all 3 regions yielded cross-reactivities that were not significantly different from those of porcine fVIII. To differentiate between inhibitor binding to the ap region and the A3 domain, we constructed 2 additional hybrids that contained porcine A2 and C2 domain substitutions and either porcine A3 or porcine ap substitutions. The porcine ap segment was less antigenic than the human ap segment in several plasmas that had activity against the ap-A3 region. This indicates that some inhibitor plasmas contain antibodies directed against the fVIII ap segment in addition to A2, A3, and C2 domain epitopes identified in previous studies. Substitution of porcine sequences within the A2, A3, C2, and ap regions of human fVIII is necessary and sufficient to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas. (Blood. 2000;95:564-568)

Animals↗
PubMed1998

Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.

The human blood coagulation factor VIII C2 domain (Ser2173-Tyr2332) contains an epitope recognized by most polyclonal inhibitory anti-factor VIII alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine factor VIII and mapped a major determinant of the C2 epitope by using a series of active recombinant hybrid human/porcine factor VIII molecules. A series of five C2-specific human antibodies and a murine anti-factor VIII monoclonal antibody, NMC-VIII/5, inhibited a hybrid containing a substitution of porcine sequence for Glu2181-Val2243 significantly less than human factor VIII. In contrast, four of the five patient antibodies and NMC-VIII/5 inhibited a hybrid containing a substitution of porcine sequence for Thr2253-Tyr2332 equally well as human factor VIII. Thus, a major factor VIII inhibitor epitope determinant is bounded by Glu2181-Val2243 at the NH2-terminal end of the C2 domain. Because C2 inhibitors block the binding of factor VIII to phospholipid and von Willebrand factor, for which binding sites have been localized to Thr2303-Tyr2332, these results imply that the segment bounded by Glu2181-Val2243 also is involved in these macromolecular interactions.

Amino Acid Sequence↗
PubMed1997

Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis.

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484-Ile508 (Healey, J. F. , Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D. , and Lollar, P. (1995) J. Biol. Chem. 270, 14505-14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 --> Ala, Tyr487 --> Ala, Arg489 --> Ala, and Arg492 --> Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 --> Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 --> Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 --> Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 --> Ala, Arg489 --> Ala, Pro492 --> Ala, Val495 --> Ala, Phe501 --> Ala, and Ile508 --> Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 --> Ala and Pro485 --> Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.

Alanine↗
PubMed1994

Inhibition by heparin of the human blood coagulation intrinsic pathway factor X activator.

The effect of heparin and other glycosaminoglycans on the activation of factor X by the phospholipid membrane-bound human factor IXa-factor VIIIa complex (intrinsic fXase) was studied. Standard heparin inhibited purified intrinsic fXase by 50% at approximately 0.08 unit/ml (0.4 microgram/ml), which is below the normal range of heparin concentrations achieved during antithrombotic therapy (0.2-0.7 unit/ml). Kinetic and binding experiments revealed that heparin behaves as a partial noncompetitive inhibitor. The inhibition constant of heparin with low affinity for antithrombin was indistinguishable from heparin with high affinity for antithrombin (Ki = 20 nM). Additionally, "low molecular weight" heparin, which also is used as an antithrombotic drug, was a potent inhibitor of intrinsic fXase (Ki = 60 nM). Dermatan sulfate inhibited intrinsic fXase much more weakly than standard heparin (IC50 = 80 micrograms/ml). The IC50 of the other mammalian glycosaminoglycans, chondroitin sulfate, keratan sulfate, and hyaluronic acid, were greater than 100 micrograms/ml. Purified prothrombinase and extrinsic fXase were not inhibited by heparin. We propose that part of the antithrombotic action of heparin and low molecular weight heparin is due to anti-thrombin-independent inhibition of intrinsic fXase and that heparin with low affinity for antithrombin may be useful as an antithrombotic agent.

Blood Coagulation↗
PubMed1994

Inhibition by heparin of thrombin-catalyzed activation of the factor VIII-von Willebrand factor complex.

The activation of factor VIII (fVIII) by thrombin is associated with heavy chain cleavages at Arg372 and Arg740 and light chain cleavage at Arg1689. In a defined, plasma-free assay of fVIII activation and at physiological ionic strength and pH, heparin inhibited the rate of activation of either human or porcine fVIII by thrombin in either the presence or absence of von Willebrand factor (vWf). The inhibitory effect of heparin was associated with inhibition of all three thrombin-catalyzed bond cleavages. At plasma concentrations of fVIII (approximately 1 nM) and vWf (approximately 35 nM), the rate of fVIII activation was inhibited by 50% at approximately 0.1 unit/ml heparin, which is below the normal range of heparin concentrations in plasma during therapeutic anticoagulation (0.2-0.7 unit/ml). We propose that, in addition to catalyzing the inhibition of thrombin and other intrinsic pathway coagulation proteases by antithrombin, heparin functions as an anticoagulant by direct inhibition of the activation of the fVIII-vWf complex by thrombin.

Animals↗