Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Abstract

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...

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…In addition, replacing Ala108, that localized to a hydrophobic pocket at the A1-C2 domain interface, with the more bulky Ile also showed a marked increase in FVIII stability (9). Furthermore, introducing nascent disulfide bridges between FVIII subunits by double Cys mutation at A2-A3 or A1-C2 interfaces yielded selected FVIII variants with enhanced FVIII/FVIIIa stability (9)(10)(11).…”

Cutaneous gene therapy for haemophilia

“…Similarly, 'engineered' variants of human Factor VIII may provide advantages over wild-type versions. An inactivation-resistant variant of human Factor VIII, which is expressed as a single chain and has been altered at inactivation cleavage sites, has features superior to those of wild-type human Factor VIII [16]. The specific activity of this variant is reportedly five times that of wild-type human Factor VIII and its half-life is substantially longer as well.…”

Future perspective for the treatment of hemophilia A

“…Other possibilities include modifying the FVIII molecule [3,4] or developing another mode of delivery [5]. The second definition led to the development of rFVIIa [6], which stimulates thrombin generation at the site of bleeding in hemophiliac patients with FVIII inhibitors and does not itself elicit FVIII inhibitors.…”

“…von Willebrand Disease is the most common inherited disorder in humans, and its prevalence is estimated to be as high as 1% [3]. Type 1 VWD is characterized by partial quantitative deficiency of VWF.…”

“…New generations of rFVIII are designed to have increased half-lives to facilitate prophylaxis (one injection per week or less) [3]; a lower propensity to elicit inhibitors [4]; and the possibility of oral instead of intravenous administration [5].…”