Summary
We studied the molecular basis of protein C deficiency in a family with a history
of thromboembolic disease. An approximately 50% reduction in anticoagulant activity
despite normal levels of protein C amidolytic activity and antigen was detected in
plasma from the proband. All the exons and intron/exon junctions of the protein C
gene were studied using a strategy that combined polymerase chain reaction amplification
with DNA sequencing of the amplified fragments. We identified a C-to-A change at nucleotide
number 1387 of the protein C gene in the proband and his mother, and this mutant was
designated protein C Osaka 10. The C-to-A change resulted in the substitution of Ser
for Arg at position -1, which is the processing protease cleavage site. The mutant
protein C was partially purified from plasma of the patient’s mother using barium
adsorption followed by ion-exchange column chromatography. It eluted at the same sodium
chloride concentration as normal protein C, and thus γ-carboxylation of the mutant
protein appeared to be normal. The apparent molecular weight of this mutant protein
C was the same as that of the normal protein on immunoblotting. Amino-terminal sequence
analysis showed that the light chain of the mutant protein C had an additional Ser
at position -1. Thus, the loss of anticoagulant activity of protein C Osaka 10 can
be explained by alteration of the conformation of the Gla domain by the additional
Ser in the mutant molecule.
