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Biomedical subjects

E T Parker

Publications and source records attributed to E T Parker.

14 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↗
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↗
PubMed1997

Subunit structure and function of porcine factor Xa-activated factor VIII.

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.

Amino Acid Sequence↗
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

Isolation and characterization of thrombin-activated human factor VIII.

Recombinant human factor VIII (fVIII) was activated by thrombin at pH 7.4, followed by CM-Sepharose chromatography at pH values ranging from 3.5 to 7.4. Optimal coagulant activity was recovered at pH 5.5 and was associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, but decayed slowly (t1/2 approximately 1 week) and nonproteolytically at room temperature or 4 degrees C. The coagulant activity of the pH 5.5 fVIIIa preparation assayed in human hemophilia A plasma was only 20% that of porcine factor VIIIa. However, its activity was approximately 75% that of porcine fVIIIa in a plasma-free assay, indicating that human fVIIIa is unstable relative to porcine fVIIIa during the coagulation assay. The first-order rate constant for spontaneous, nonproteolytic loss of activity of human fVIIIa at pH 7.4 was decreased 8-fold by fIXa and phospholipid, indicating that human fVIIIa is stabilized when incorporated into the intrinsic pathway factor X activation complex.

Animals↗
PubMed1994

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

Animals↗
PubMed1992

Coagulant properties of hybrid human/porcine factor VIII molecules.

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.

Animals↗
PubMed1992

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs

Animals↗
PubMed1991

Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog.

The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.

Animals↗
PubMed1990

Production of monoclonal antibodies that detect Hodgkin's high molecular weight transforming growth factor-beta.

High molecular weight transforming growth factor-beta (TGF beta) is a physiologically active TGF secreted by nodular sclerosing Reed-Sternberg cells. Five monoclonal murine antibodies were prepared that distinguished Hodgkin's TGF beta from platelet-derived TGF beta using an enzyme-linked immunosorbent assay, neutralization of biologic activity, and Western blotting. These monoclonal antibodies directed at unique antigenic determinants (epitopes) of Hodgkin's TGF beta will allow further characterization of the role of Hodgkin's TGF beta in Hodgkin's disease and related entities.

Antibodies, Monoclonal↗
PubMed1979

Host treatments affecting artificial pulmonary metastases: interpretation of loss of radioactively labelled cells from lungs.

The effect has been examined of various host treatments (C. parvum injection, immunization, thoracic irradiation, cyclophosphamide injection, and anticoagulation) on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.

Animals↗