Functional mapping of the A2 domain from human factor VIII

Abstract

Coagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine. The mutants were expressed in COS-1 cells and their antigen levels and procoagulant activities were measured. The residues were classified in three categories: those with a non-detrimental alteration of their activities (activity >50 % of control FVIII; n=98), those with a moderate alteration (15 %<activity<50%; n=45) and those that were severely affected (activity<15%; n=63). The mutants sensitive to mutation were retrieved in the HAMSTeRS database with a higher percentage than those that were not affected (58.8% vs. 9.2%). The results revealed the existence of clusters of residues that are sensitive (Arg418-Phe436, Thr459-Ile475, Ser535-Gly549, Asn618-Ala635) or not (Leu398-Arg418, Pro485-Asp500, Gly506-Gly520, Pro596-Asp605) to mutations. The stretches of residues sensitive to mutations were buried within the molecule suggesting that these amino acids participate in the maintenance of the A2 domain structure. In contrast, residues resistant to mutations formed external loops without well- defined structures suggesting that these loops were not crucial for the process of factor X activation. This study provided a detailed map of the FVIII A2 domain between residues 371 and 649, identifying residues crucial for maintaining FVIII function and residues that can be mutated without jeopardising the coagulant activity.

Prediction of hemophilia A severity using a small-input machine-learning framework

“…We used an in vitro mutagenesis screening where almost all of the residues of the A2 and the C2 domains were mutated to alanine 30 , 31 . The A2 domain is important for the FVIII activity because in addition to its role in stabilizing the protein structure 32 , it contains a binding site for FIXa (ref.…”

“…The ELISA assay was used to assess the efficiency of FVIII mutant constructs expression and secretion (often referred as antigen assay because they measure both functional and nonfunctional FVIII proteins by the amount of FVIII antigen (protein) immobilized on the ELISA plate. Two commercial kits were used to apply the “sandwich” method, where the antigen (FVIII construct) is captured between two layers of antibodies 30 , 31 .…”

“…2 c, d). For instance, Lys444Ala and Phe447Ala in the A2 domain (Severity Scores 0.97 and 0.91, respectively) had 0% of the wild-type chromogenic activity 31 . These encouraging results gave us confidence that Hema-Class captured FVIII properties that were previously only observable using in vitro assays.…”

Protein residue network analysis reveals fundamental properties of the human coagulation factor VIII

“…To answer this question, we used measurements of the FVIII chromogenic activity, expression and secretion. These measurements were obtained from massive mutagenesis experiments 6 , 7 where almost all residues of the A2 and C2 domains were individually mutated to alanine, and the in vitro chromogenic activity and the secretion/expression levels (measuring thrombin formation and ELISA antigen binding, respectively). After close inspection of the distribution of the activity and secretion values of the mutant FVIII constructs, we divided the data into two groups, (i) low-chromogenic/low-expression (< 50% of wild-type), and (ii) high-chromogenic/high-expression (> 50% of wild-type).…”

“…Previous studies determined the structure of FVIII (Refs. 3 – 5 ), performed massive alanine mutagenesis experiments 6 , 7 , and made point mutations that increased the half-life of FVIII in circulation 5 . However, determining which residue substitutions are beneficial or detrimental to the FVIII activity remains a laborious and costly trial-and-error approach.…”