Date Published: July 6, 2017
Publisher: Public Library of Science
Author(s): Behnaz Pezeshkpoor, Ursula Schreck, Arijit Biswas, Julia Driesen, Ann-Cristin Berkemeier, Anna Pavlova, Jens Müller, Johannes Oldenburg, Christopher B Doering.
Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg759> Arg1708>Arg391, indicating the flanking residues of the latter to be less optimal. This study aimed to investigate, in silico and in vitro, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by in silico analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our in silico results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the in vitro analysis shows that the sequences flanking the Arg1708 cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´ residues surrounding Arg1708 of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.
Blood clotting factor VIII (FVIII) is a nonenzymatic cofactor of activated factor IX (FIXa). Activated FVIII (FVIIIa) binds on a membrane surface to FIXa and activates factor X (FX) in the tenase complex . The FVIII protein is synthesized as a ~330 kDa single-chain molecule with six distinct domains. It consists of three homologous A-domains, a unique B-domain and two C-domains . Two short acidic segments, a1 and a2, follow the A1 and A2 domains, respectively, and a similar short a3 segment precedes the A3 domain. Due to cleavage at the B-a3 junction, followed by a number of additional cleavages within the B domain, a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2) are generated .
In this study we investigated the impact of the residues flanking the Arginine cleavage sites in FVIII protein by performing in silico and in vitro analysis of FVIII variants harboring a hypothetical optimized thrombin cleavage sequence (L-V-P-R-G-S) [13, 14, 19].