We describe five families presenting with type II hereditary
protein C deficiency characterized by normal
antigen and amidolytic activity levels but low
anticoagulant activity. All the exons and intron/exon junctions of the
protein C gene were studied using a strategy combining amplification by the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis of the amplified fragments, and direct sequencing of fragments displaying altered melting behavior. We detected five novel mutations. Three were located in the C-terminal part of the propeptide encoded by exon III:
Arginine (Arg)-5 to
tryptophan (Trp), Arg-1 to
histidine (His), and Arg-1 to
cysteine (Cys) mutations. The two others, located in exon IX, affected Arg 229 and
serine (Ser) 252, which were respectively replaced by
glutamine (Gln) and
asparagine (Asn).
DNA studies of the other exons from affected individuals showed no other abnormalities. These novel mutations provide further insight into the importance of the affected
amino acids located close to the active site, near Asp 257, one of the three
amino acids of the catalytic triad. The low
anticoagulant activity of the abnormal
protein C indicated that Arg 229 and Ser 252 play a key role during the interaction between
protein C and its
cofactor protein S,
phospholipids, or factors Va and VIIIa. The Arg-1 to Cys mutation led to the dimerization of
protein C with another plasmatic component, as evidenced by the presence in the plasma of a high molecular weight form of
protein C that disappeared after reduction. No molecular mass abnormalities were observed in heavy and light chains of all other
protein C mutants. In the five families explored, 9 (64%) of the 14 subjects bearing the mutations reported thrombotic events. This suggests that the
protein C amino acids affected by the mutations are very important for the in vivo expression of the antithrombotic properties of
protein C.