An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII

PLOS ONE, Dec 2019

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.

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An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII

July An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII Behnaz Pezeshkpoor 0 1 2 Ursula Schreck 0 1 2 Arijit Biswas 0 1 2 Julia Driesen 1 2 Ann- Cristin Berkemeier 0 1 2 Anna Pavlova 0 1 2 Jens MuÈ ller 0 1 2 Johannes Oldenburg 0 1 2 0 Center for Rare Diseases Bonn (ZSEB), University Clinic Bonn , Bonn , Germany 1 Institute of Experimental Hematology and Transfusion Medicine, University of Bonn , Bonn , Germany 2 Editor: Christopher B Doering, Emory University School of Medicine , UNITED STATES 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-P2Â 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. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction 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 [ 1 ]. 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 [ 2 ]. 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 Ba3 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 [ 3 ]. FVIII protein is activated by limited proteolysis by either thrombin or FXa [4±6] via cleavage of three peptide bonds at Arg391 (a1-A2 junction), Arg759 (a2-B junction) and Arg1708 (a3-A3 junction) [ 4 ]. Thrombin is the physiological activator of FVIII since von Willebrand factor (VWF) inhibits the membrane dependent profactor activation catalyzed by FXa [ 7 ]. Upon activation the B domain is released and an active heterotrimer (A1/A2/A3-C1-C2) is formed. FVIII activation occurs in an ordered sequence with an initial cleavage at Arg759 followed by cleavages at Arg1708 and Arg391 [ 4 ]. The initial proteolysis at Arg759 facilitates subsequent proteolysis at the latter two sites [ 8 ], but both proteolysis at Arg1708 and Arg391 are the critical steps in activation of FVIII via liberating FVIII from the associated VWF [ 9 ], and exposing the cryptic functional FIXa-interactive site in the A2 domain, respectively [ 10 ]. Previous studies have shown that both thrombin anion binding exosites contribute to the proteolytic activation of FVIII [ 11, 12 ], with the binding to the heavy chain being preferred [ 8, 9 ]. The biochemical and biophysical characteristics of several thrombin cleavage sites have been studied and analyzed in detail. A theoretical thrombin cleavage-optimized proposed peptide, P(osition)3-P2-P1-P1Â-P2Â, would exhibit the following amino acid sequence: P2-Pro, P1-Arg, P1Â-Ser/Ala/Gly/Thr and P2Â-not acidic. Thus the amino acid residues surrounding all three Arg (P1) cleavage sites influence the (...truncated)


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Behnaz Pezeshkpoor, Ursula Schreck, Arijit Biswas, Julia Driesen, Ann-Cristin Berkemeier, Anna Pavlova, Jens Müller, Johannes Oldenburg. An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII, PLOS ONE, 2017, Volume 12, Issue 7, DOI: 10.1371/journal.pone.0180456