Factor VIII S373L: Mutation at P1’ Site Confers Thrombin Cleavage Resistance, Causing Mild Haemophilia A

Johnson, Dwayne; Pemberton, S.; Acquila, Maura; Mori, Pier Giorgio; Tuddenham, E G D; O’Brien, David

doi:10.1055/s-0038-1642455

Cited by 18 publications

(23 citation statements)

References 5 publications

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“…This result was also observed in the low activation rates for a factor VIII R372C mutant 17 and the P 1 Ј S373L mutant 31 in which no proteolysis at residue 372 was detected. This plateau of activity may derive from a partially activated form of factor VIIIa wherein light chain is cleaved and heavy chain is not, thus the basis for inherent cofactor instability, dissociation of A2 subunit, 19,32 is not relevant.…”

Section: Discussionsupporting

confidence: 62%

Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Nogami

Zhou

Wakabayashi

et al. 2005

Blood

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Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type.N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar MichaelisMenten constant (K m , apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V max ) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P 1 Arg, and this property likely reflects the severity of the hemophilia A phenotype. IntroductionFactor VIII, a plasma protein deficient or defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa. 1 Factor VIII is synthesized as a multidomain, single-chain molecule (A1-A2-B-A3-C1-C2) consisting of 2332 amino acid residues with a molecular mass of approximately 300 kDa. 2,3 Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a variable heavy chain (90-210 kDa) consisting of the A1, A2, and heterogeneous fragments of partially proteolyzed B domains, together with a light chain (80 kDa) consisting of the A3, C1, and C2 domains. [2][3][4] Activation of factor VIII proceeds by limited proteolysis catalyzed by thrombin or factor Xa, with the former likely representing the physiologic activator. 5 Both proteases convert factor VIII into the active cofactor form, factor VIIIa, following cleavage of 3 P 1 positions at Arg372 between the A1-A2 domainal junction and at Arg740 between the A2-B domainal junction to generate the 50-kDa A1 and 40-kDa A2 subunits, and at Arg1689 to release a 40 acidic residue-rich peptide from the 80-kDa light chain to generate the 70-kDa A3-C1-C2 fragment. 6 Proteolysis at Arg372 and Arg1689 appears essential for generating factor VIIIa cofactor activity (see Fay 7 for review). Cleavage at the former site exposes a functional factor IXainteractive site within the A2 domain that is cryptic in the unactivated molecule. 8 Cleavage at the latter site libera...

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Section: Discussionsupporting

confidence: 62%

Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Nogami

Zhou

Wakabayashi

et al. 2005

Blood

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“…These results support the importance of A2 subunit dissociation as a mechanism for inactivation of FVIIIa, as originally proposed by Lollar and Parker (45). The importance of cleavage at residue 372 previously was demonstrated by identification of mutants in Arg-372 that result in hemophilia A (16) and by analysis of site-directed mutants of Arg-372 (13). Interestingly there was a Ϸ25% loss of IR8 activity within the first 30 sec to 2.5 min after thrombin activation followed by only a very gradual loss of activity occurring over hours.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies demonstrated that cleavages after Arg-372 and Arg-1689 are the most critical for efficient functional activation, whereas cleavage after Arg-740 was not required (13,(15)(16)(17)(18). Cleavage after Arg-1689 removes an acidic amino acidrich region from Arg-1648 to Arg-1689, and is necessary for dissociation of FVIIIa from vWF and makes FVIIIa available for interaction with phospholipids (4,(19)(20)(21).…”

mentioning

confidence: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

Proc. Natl. Acad. Sci. U.S.A.

126

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Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII), necessitating exogenous replacement by either plasma-or recombinant-derived FVIII preparations. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-aa single-chain glycoprotein of 280 kDa from which a signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (1-3). Whereas the A and C domains exhibit 35-40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein. Intracellular proteolytic processing within the B domain after residue Arg-1648 generates an 80-kDa light chain (domains A3-C1-C2) and a heterogeneous-sized heavy chain of 90-200 kDa (domains A1-A2-B). The heavy and light chains are associated as a heterodimer through a divalent metal-ion-dependent linkage between the A1 and A3 domains.In plasma, FVIII circulates in an inacti...

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“…Previously, several mutations of the critical P 1 residue Arg372 have been reported [57][58][59][60][61] with phenotypes ranging from mild to severe. Replacement of the P 1 Ј (p.Ser373Leu and p.Ser373Pro) 62,63 and P 2 residues (p.Ile371Thr) 16 also compromises cleavage at this site and results in mild bleeding tendencies. Inspection of the thrombin structure, 64,65 together with work conducted using synthetic peptide libraries 66 and phage display, 67 suggests a minor influence of positions on the nonprimed side of the P 1 -P 1 Ј scissile peptide bond.…”

Section: Type II Mutationsmentioning

confidence: 99%

Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

Venceslá¹,

Corral-Rodríguez

Baena³

et al. 2008

Blood

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Factor VIII S373L: Mutation at P1’ Site Confers Thrombin Cleavage Resistance, Causing Mild Haemophilia A

Cited by 18 publications

References 5 publications

Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

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