Functional properties of recombinant factor V mutated in a potential calcium-binding site.
Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation. FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner. We constructed a recombinant FV Asp111Asn/Asp112Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity. Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity, incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity, which was rapidly lost upon prolonged incubation. Loss of FVa activity was most likely due to dissociation of HC and LC since, upon chromatography of rFVa-NN on a SP-Sepharose column, the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength. In contrast, rFVa-wt adhered to the column, and both the HC and LC coeluted at high ionic strength. In the presence of phospholipid vesicles, the loss of rFVa-NN activity was partially prevented by FXa, active site inhibited FXa, and prothombin in a dose-dependent manner. We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa. We propose that the introduced substitutions disrupt the calcium-binding site in FV, thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.
Sørensen
Kristoffer W
KW
Department of Clinical Chemistry, Lund University, Malmö University Hospital, Malmö, Sweden.
Nicolaes
Gerry A F
GA
Villoutreix
Bruno O
BO
Yamazaki
Tomio
T
Tans
Guido
G
Rosing
Jan
J
Dahlbäck
Björn
B
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001780:Blood Coagulation Tests; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D002522:Chlorocebus aethiops; D019281:Dimerization; D005165:Factor V / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015943:Factor Va / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions
D002135:Calcium-Binding Proteins; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D012996:Solutions; D015943:Factor Va; D001216:Asparagine; D005165:Factor V; D011516:Prothrombin