Clinical Significance of Arg306 Mutations of Factor V Gene

To the Editor:

Human coagulation factor V is a cofactor involved in the conversion of prothrombin to thrombin. Activated factor V is normally inactivated by activated protein C (APC) in the process of hemostasis.1Activated factor V is initially cleaved at Arg506 and then at Arg306 and Arg679. Peptide bond cleavage at Arg506 is essential for optimal exposure of cleavage sites at Arg306 and Arg679. Cleavage at Arg306 accounts for a 70% loss of activated factor V activity and the subsequent Arg 679 cleavage is responsible for the loss of the remaining. Therefore, any defect on one or more of these three cleavage sites at Arg506, Arg306, and Arg679 may potentially affect APC inactivation.

Two novel DNA sequence mutations in exon 7 of the factor V gene have recently been identified and they are both reported in the February 15, 1998 issue of Blood.^2,3 Both of them affect Arg306, an important APC cleavage site. The first one is a new factor V mutation associated with APC resistance (factor V Cambridge, G1091 → C mutation, Arg306 → Thr substitution). This mutation was found in a family with a strong history of thrombosis. Our group has independently described another novel mutation (A1090 → G) that results in Arg306 → Gly substitution but it is, however, not associated with APC resistance.2 The mutation was found in both thrombotic and nonthrombotic subjects. Although both new mutations affect similarly the Arg306 cleavage site, only the Arg306 → Thr substitution results in both APC resistance and increased risk of thrombosis. The clinical significance of Arg306 → Gly substitution remains uncertain.

Our group has studied further the prevalence of the Arg306 → Gly substitution in Hong Kong Chinese. High-molecular-weight DNA was extracted from the blood samples obtained from 89 healthy blood donors and 260 diabetic patients. The mutation is detected by a restriction enzyme digest of the amplified exon 7 of factor V gene withBstNI (New England Biolabs, Beverly, MA), which has a restriction site of CC^∇AGG. The DNA fragments were studied by 6% polyacrylamide gel electrophoresis. The undigested DNA fragment was a 240-bp polymerase chain reaction product. A complete digestion of this 240-bp DNA fragment with BstNI was expected to yield two shorter fragments of 100 and 140 bp in length. The presence of an A1090 → G mutation resulted in the loss of the cleavage site for BstNI.3 The mutation was found in 4 of the 89 (4.5%) healthy blood donors and 8 of the 260 (3.1%) diabetic subjects. There is no statistically significant difference between these two figures and the incidence rate of 2 of 43 (4.7%) previously reported in thrombotic patients.3 

It appears that there is a difference in the APC capacity to cleave Thr306 and Gly306. Thr306 seems to confer APC resistance to the factor V molecule. On the other hand, susceptibility to APC cleavage appears to persist for Gly306. Although, like Arg 306 → Thr, the Arg306 → Gly substitution affects similarly the Arg306 APC cleavage site of factor V gene, Arg306 → Gly may not have any clinical significance. The current data do not suggest that it predisposes to clinical thrombosis.