A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

Toole, John J.; Pittman, Debra D.; Orr, Elizabeth C.; Murtha, Patricia E.; Wasley, L C; Kaufman, Randal J.

doi:10.1073/pnas.83.16.5939

Cited by 300 publications

(190 citation statements)

References 18 publications

(16 reference statements)

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“…Previous studies on the requirements for functional activity of FVIII demonstrated that cleavage after Arg residues 372 and 1689 both were required for activation of factor VIII and that the B domain was not required for functional activity (13,(22)(23)(24). Deletion of residues 741-1689 yielded a functional molecule (90͞73) that displayed WT thrombin cleavage and activation, resulting in a heterotrimer similar to WT FVIIIa that was susceptible to rapid dissociation of the A2 subunit.…”

Section: Discussionmentioning

confidence: 99%

“…Cleavage after residue Arg-740 releases the heavily glycosylated B domain. Previous studies demonstrated that the B domain of FVIII is dispensable for FVIII cofactor activity (22)(23)(24). Genetically engineered FVIII molecules that have varying degrees of B domain deletion yield functional FVIII molecules that are more efficiently expressed in mammalian cells (22,23,25,26).…”

mentioning

confidence: 99%

“…Previous studies demonstrated that the B domain of FVIII is dispensable for FVIII cofactor activity (22)(23)(24). Genetically engineered FVIII molecules that have varying degrees of B domain deletion yield functional FVIII molecules that are more efficiently expressed in mammalian cells (22,23,25,26). These deletion molecules exhibit typical thrombin activation that correlates with cleavage after Arg-372, Arg-740, and Arg-1689, generating a FVIIIa heterotrimer that is indistinguishable from wild-type FVIII (FVIII WT) and also is subject to rapid inactivation though dissociation of the A2 domain subunit.…”

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confidence: 99%

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Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

125

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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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

125

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“…The ΔΦMF8IZ and control ΔΦMIZ vectors were generated using standard molecular cloning techniques (sequences available on request) using the complementary DNA encoding full-length hFVIII from the PMT2-VIII vector, 39,40 and the IRES-Zeo fragment from the MIZV vector (a gift from Robert Hawley). Vector stocks were generated as previously described.…”

Section: Methodsmentioning

confidence: 99%

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

Epp

Latchman

et al. 2010

Molecular Therapy

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“…For these purposes genetically engineered FVIII-derivatives (e.g. B-domain-deleted FVIII (12,13)) and FIX-derivatives (e.g. pointmutated FIX (14) and chimeric FIX (15)) have been produced.…”

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confidence: 99%

An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex

Kerschbaumer

Riedrich

Kral

et al. 2004

Journal of Biological Chemistry

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During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (k cat ) of the enzyme complex increased 2-to 3-fold whereas the K m for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.One of the key events during normal hemostasis is the conversion of the zymogen factor X (FX) 1 into its enzymatically active form factor Xa (FXa) via the intrinsic coagulation pathway, a process that subsequently leads to activation of prothrombin and the formation of a fibrin clot (1). The FX-activating protease factor IXa (FIXa) assembles (together with FVIIIa as well as Ca 2ϩ ions) on a phospholipid surface to form the intrinsic FX-activating complex (2). In this complex FVIIIa serves as a cofactor for the enzyme FIXa and increases the turnover number (k cat ) of FIXa by 4 to 5 orders of magnitude (3, 4). Binding of the stoichiometric FVIIIa-FIXa complex and the substrate FX to a phospholipid surface strongly reduces the K m for the substrate (3, 4), probably because the protein-protein interactions are limited to two dimensions (5).The physiological importance of the intrinsic FX-activating complex is well defined because the severe bleeding disorders hemophilia A and hemophilia B are characterized by very low plasma levels (Ͻ 5%) or by the absence of the pro-cofactor FVIII and the pro-enzyme FIX, respectively. Treatment of hemophilia is successfully achieved by supplementing the blood of patients with either human FVIII or human FIX. Both proteins can be derived from human plasma or produced as recombinant protein in mammalian cell culture (6, 7). Although progress in the production of coagulation factors to ensure their purity, efficacy, and viral safety has been made over the last 20 years, treatment of hemophilia is still beset with several difficulties. First, production of recombinant and plasma-derived, therapeutic coagulation factors is expensive, an...

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A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

Cited by 300 publications

References 18 publications

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex

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