Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

1996 ◽  
Vol 75 (01) ◽  
pp. 036-044 ◽  
Author(s):  
Thomas L Ortel ◽  
Karen D Moore ◽  
Mirella Ezban ◽  
William H Kane
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Secreted Protein ◽  
Factor V ◽  
Minimal Effect ◽  
Repetitive Domain ◽  
Human Factor Viii ◽  
A1 Domain ◽  
A2 Domain

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1618-1626 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
S de Graaf ◽  
CA Fulcher
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Factor Viii Inhibitor ◽  
Type I ◽  
Protein Fragments ◽  
Human Factor Viii ◽  
Circulating Antibodies ◽  
A1 Domain ◽  
A2 Domain

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

scholarly journals Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1618-1626 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
S de Graaf ◽  
CA Fulcher
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Factor Viii Inhibitor ◽  
Type I ◽  
Protein Fragments ◽  
Human Factor Viii ◽  
Circulating Antibodies ◽  
A1 Domain ◽  
A2 Domain

Abstract Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

scholarly journals Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 486-489 ◽  
Author(s):  
CA Fulcher ◽  
JE Gardiner ◽  
JH Griffin ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Human Factor ◽  
Activated Protein C ◽  
Light Chains ◽  
Factor V ◽  
Human Factor Viii ◽  
Thrombin Activation ◽  
A Site ◽  
Polypeptide Chains

Abstract Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

scholarly journals Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3120-3126 ◽  
Author(s):  
BJ Lamphear ◽  
PJ Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Prolonged Incubation ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
A1 Domain ◽  
Proteolytic Cleavages

Abstract Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

scholarly journals Phosphorylation of factor Va and factor VIIIa by activated platelets

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 704-719 ◽  
Author(s):  
M Kalafatis ◽  
MD Rand ◽  
RJ Jenny ◽  
YH Ehrlich ◽  
KG Mann
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor Va ◽  
Activated Platelets ◽  
Human Factor Viii ◽  
Phosphoamino Acid Analysis ◽  
Carboxyl Terminal

Abstract Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.

scholarly journals The Effects of Injection of Human Factor VIII Antibody Into Rabbits

Blood ◽  
1973 ◽  
Vol 42 (4) ◽  
pp. 509-521 ◽  
Author(s):  
S. M-C. Shen ◽  
D. I. Feinstein ◽  
S. I. Rapaport
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Rabbit Model ◽  
Kinetic Studies ◽  
Factor Vii ◽  
Factor V ◽  
Initial Values ◽  
Human Factor Viii ◽  
Hemolytic Complement Activity ◽  
Antigen Antibody

Abstract Rabbits were injected with an immunoglobulin fraction of human serum containing a factor VIII antibody. Factor VIII levels fell abruptly, persisted below 10% of a rabbit plasma standard for 12 hr, and returned to normal by 120-168 hr. The factor VIII antigen-antibody reaction did not result in Intravascular clotting as evaluated by kinetic studies with 125I-fibrinogen. However, small falls in factor V and factor VII levels were observed over a 6-hr period after the injection. Platelets fell to about one-half of initial values within 15 min, rose to 80% of initial levels over 2 hr, and subsequently declined to 65%-70% of initial levels. WBC levels fell to below 20% of initial values 2 hr after the injection but returned to about 75% of initial values by 6 hr. Total hemolytic complement activity was unaffected. Animals made granulocytopenic with nitrogen mustard and animals with hereditary C'6 deficiency behaved similarly to normal animals. One may conclude that the injection of human factor VIII antibody into rabbits produces a rabbit model with impaired intrinsic coagulation suitable for studies of the mechanism of endotoxin-induced intravascular clotting.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

1987 ◽  
Author(s):  
Richard J Jenny ◽  
Debra D Pittman ◽  
John J Toole ◽  
Ronald W Kriz ◽  
Randal J Kaufman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Human Factor ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Factor V ◽  
Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

scholarly journals Residues 484-508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII

1995 ◽  
Vol 270 (24) ◽  
pp. 14505-14509 ◽  
Author(s):  
John F. Healey ◽  
Ira M. Lubin ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Leon W. Hoyer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Major Determinant ◽  
Human Factor Viii ◽  
A2 Domain

scholarly journals Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 486-489 ◽  
Author(s):  
CA Fulcher ◽  
JE Gardiner ◽  
JH Griffin ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Human Factor ◽  
Activated Protein C ◽  
Light Chains ◽  
Factor V ◽  
Human Factor Viii ◽  
Thrombin Activation ◽  
A Site ◽  
Polypeptide Chains

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

scholarly journals Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3120-3126 ◽  
Author(s):  
BJ Lamphear ◽  
PJ Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Prolonged Incubation ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
A1 Domain ◽  
Proteolytic Cleavages

Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

Sign in / Sign up

Export Citation Format

Share Document