Premium
Factor VIII A2 domain residue Asp721 facilitates thrombin–catalyzed cleavage at Arg372
Author(s) -
Newell Jennifer,
Zhou Qian,
Fay Philip J.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a49
Subject(s) - cleavage (geology) , factor ixa , chemistry , thrombin , mutant , factor v , stereochemistry , cofactor , wild type , factor x , biophysics , biochemistry , enzyme , platelet , gene , biology , medicine , paleontology , fracture (geology) , surgery , thrombosis , immunology
Blood coagulation factor VIIIa serves as an essential cofactor for the factor IXa‐catalyzed activation of factor X. Factor VIIIa is generated from the procofactor, factor VIII, a heterodimeric protein comprised of a heavy chain (A1‐A2‐B domains) and a light chain (a3‐A3‐C1‐C2 domains) following cleavage by thrombin at Arg372 (A1‐A2 junction), Arg740 (A2‐B junction), and Arg1689 (a3‐A3 junction). Both N‐ and C‐termini of the A2 domain (residues 373–740) are rich in acidic residues. Results from a previous study revealed that the N‐terminal acidic region modulated cleavage at the A2‐B domain junction (Nogami et. al., J. Biol. Chem. 280:18476, 2005). We now show a role for the A2 C‐terminal acidic region (residues 717–725) in thrombin‐catalyzed cleavage at the A1‐A2 junction. In this study, Asp721Ala Factor VIII was constructed, expressed, and purified from stably‐transfected BHK cells as a B‐domainless protein. The cleavage rate of Asp721Ala factor VIII by thrombin was reduced ~5‐fold at Arg372 compared with wild type (WT) factor VIII as judged by SDS‐PAGE and western blotting, whereas cleavage rates for the mutant at Arg740 and Arg1649 were equivalent to the WT values. Furthermore, the mutant protein possessed an ~2‐fold reduced activity in generating factor Xa, consistent with its reduced activation rate. These results suggest that Asp721, and possibly other residues within this C‐terminal acidic cluster, selectively modulate thrombin interaction with factor VIII facilitating cleavage at Arg372 during procofactor activation. This work is supported by NIH grants HL38199 and HL76213.