Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V.
Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid-dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506-FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.
Heeb
M J
MJ
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
Kojima
Y
Y
Greengard
J S
JS
Griffin
J H
JH
D000328:Adult; D005165:Factor V / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005190:Family; D005998:Glycine / Q000235:genetics; D006720:Homozygote; D006801:Humans; D008297:Male; D017354:Point Mutation; D011486:Protein C / Q000378:metabolism*
D011486:Protein C; D005165:Factor V; D005998:Glycine