The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII by Kagehiro Amano, Rita Sarkar, Susan Pemberton, Geoffrey Kemball-Cook, Haig H. Kazazian, and Randal J. Kaufman Blood Volume 91(2):538-548 January 15, 1998 ©1998 by American Society of Hematology

Synthesis of FVIII WT and mutants in COS-1 cells. Synthesis of FVIII WT and mutants in COS-1 cells. WT and mutant expression plasmids were transfected into COS-1 monkey cells. At 60 hours posttransfection, cells were pulse-labeled with [35S]-methionine for 20 minutes and cell extracts (CE) were harvested. Duplicate plates were chased for 4 hours in medium containing excess unlabeled methionine and then CE were harvested. Equal volumes of CE were immunoprecipitated with anti-FVIII antibody and equal aliquots were analyzed by SDS-PAGE. Mock indicates cells that did not receive plasmid DNA. The migration of FVIII in the CE is indicated at the right as FVIII. Molecular weight size markers are shown on the left. Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Secretion and thrombin cleavage of WT and mutant FVIII Secretion and thrombin cleavage of WT and mutant FVIII. The secretion of each mutant was analyzed by immunoprecipitation of conditioned medium from [35S]-methionine pulse-labeled transfected cells chased for 4 hours in medium containing excess unlabeled met... Secretion and thrombin cleavage of WT and mutant FVIII. The secretion of each mutant was analyzed by immunoprecipitation of conditioned medium from [35S]-methionine pulse-labeled transfected cells chased for 4 hours in medium containing excess unlabeled methionine. Immunoprecipitated FVIII molecules were analyzed by SDS-PAGE before (−) and after (+) thrombin (IIa) digestion. Mock indicates cells that did not receive plasmid DNA. Molecular weight size markers are shown on the left. Single chain (Single), heavy chain (Heavy), and light chain (Light) are indicated for undigested samples. A3-C1-C2, A1, and A2 fragments are indicated for digested samples. The symbol * represents the A2 fragment with reduced mobility. Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

The specific activity for WT and mutant FVIII The specific activity for WT and mutant FVIII. CM of WT and mutant FVIII were harvested at 60 hours posttransfection for FVIII assay. The specific activity for WT and mutant FVIII. CM of WT and mutant FVIII were harvested at 60 hours posttransfection for FVIII assay. The activity and antigen in the CM were measured by COAMATIC chromogenic assay and ELISA using an anti-FVIII light chain antibody, respectively. Specific activity is expressed as percent of WT. Bars are expressed as a mean ± standard deviation (SD) of three independent transfection experiments. Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Effect of N-glycanase for I566T mutant FVIII Effect of N-glycanase for I566T mutant FVIII. (A) Immunoprecipitated WT and I566T mutant from radiolabeled CM were treated with nothing (IIa−, N-Gly−) or thrombin (IIa+, N-Gly−) or N-glycanase after thrombin (IIa+, N-Gly+) before SDS-PAGE as described in th... Effect of N-glycanase for I566T mutant FVIII. (A) Immunoprecipitated WT and I566T mutant from radiolabeled CM were treated with nothing (IIa−, N-Gly−) or thrombin (IIa+, N-Gly−) or N-glycanase after thrombin (IIa+, N-Gly+) before SDS-PAGE as described in the Materials and Methods. Molecular weight size markers are shown on the left. A3-C1-C2, A1, A2, and mutant A2 fragments are indicated at the right. (B) FVIII in conditioned medium as well as in patient's plasma were incubated with 10 U/mL of N-glycanase at 37°C. FVIII activity after increasing times was determined by one stage clotting assay. Data are plotted as the percent activity of the mutant compared with the WT-recombinant (○) or plasma-derived (•) FVIII, respectively. Data represent the average of two independent experiments. Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Inhibition of intracellular degradation causes intracellular accumulation of DeltaF652/3. Inhibition of intracellular degradation causes intracellular accumulation of DeltaF652/3. Parallel plates of transfected COS-1 monkey cells were labeled at 60 hours posttransfection with [35S]methionine for 30 minutes, chased for 4 hours in the absence (lanes 2, 6, 10, 13, 16, and 19) or presence of increasing amounts of ALLN (lanes 3, 4, 7, 8, 11, 12, 14, 15, 17, 18, 20, and 21). CE and conditioned medium (CM) were harvested and equal proportionate volumes of CE and CM were immunoprecipitated with anti-FVIII specific antibody for analysis by SDS-PAGE. Mock indicates cells that did not receive plasmid DNA. Molecular weight size markers are shown on the left of each CE and CM. Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology

Structural model of FVIII mutations I566T and S558F. Structural model of FVIII mutations I566T and S558F. (A) Homology model of the triplicated A domains of FVIII is shown as viewed perpendicular to threefold axis with “top” of molecule to top of Fig Red, blue, and green represent A1, A2, and A3 subunit, respectively. Binding loop of factor IXa is shown as magenta CPK spheres. N564 (new N-glycosylation site) and T566 are colored by atom (carbon, green; hydrogen, white; oxygen, red; nitrogen, blue). (B) Factor IXa binding loop 558 to 565 shown in CPK spheres: residue 558 colored by atom, 559 to 565 in gray. Blue ribbons represent the alpha-carbon trace of neighboring residues in the A2 domain. Left, WT S558; Right, variant F558 (reminimized). Kagehiro Amano et al. Blood 1998;91:538-548 ©1998 by American Society of Hematology