Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Abstract

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.

Resistance to activated protein C caused by the R506Q mutation in the gene for factor V is a common risk factor for venous thrombosis

“…The FVR 506 Q mutation, which does not affect the procoagulant properties of Fv. leads to loss of one of three APC-cleavage sites in the heavy chain of normal FVa (FVa:R 506 ) [34][35][36][37][38][39]. The cleavage at Arg506 has favourable kinetics as compared to those at Arg306 and Arg679 [34][35][36].…”

“…At physiological concentrations of normal FVa, the 506 cleavage is approximately 1 0-fold faster than the cleavage at Arg306. As a consequence, the activity of FVa:Q 506 is inhibited at approximately 10-fold lower rate than FVa:R 506 [35,36,38,39]. This leads to increased generation of thrombin and a hypercoagulable state reflected by increased levels of prothrombin activation fragments in plasma of individuals with inherited APC-resistance [40,41].…”

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala‐activated protein C mediated by factor Va

“…The plasma-derived APC and rwt APC reactions both showed a decrease in the intensity of the FVa heavy chain band over time. Furthermore, fragments with the apparent molecular weights of 76 kDa, 44 kDa, and 28 kDa attributable to known APC cleavage products of the FVa heavy chain (Kalafatis et al, 1994;Heeb et al, 1995) were visible.…”

“…FVa and FVa cleavage products were monitored by Western blotting with a polyclonal antibody against the human FVa heavy chain (generous gift from Drs. Jan Rosing and Guido Tans, University of Limburg, Maastricht, The Netherlands) and a monoclonal antibody against the human FVa heavy chain (Enzyme Research Laboratory, Inc., South Bend, IN) (Heeb et al, 1995).…”

Search for mutations in the genes for coagulation factors V and VIII with a possible predisposition to activated protein C resistance

“…Keeping in mind that the recognition site for APC consists not only of the two amino acids forming the cleavage site, but also includes the three closest amino acids from each side of the bond [15] and the importance of those closest amino acids for the APC digestion of the Arg-506-Gly bond [16], we continued the investigation of the APC cleavage sites by sequencing the DNA fragments of exon 2 (Arg-306-Asn cleavage site) and exon 12 (Arg-679-Lys cleavage site) of the factor V gene and exon 8 (Arg-336-Met cleavage site) and exon 11 (Arg-562-Gly cleavage site) of the factor VIII gene in 29 selected patients (Table 3). These selected patients represented the four possible combinations of APC response: eight individuals were APC resistant in both clotting and amidolytic assays, whereas seven and eight were resistant either in the clotting or in the amidolytic assay, respectively.…”