Page 263 - Haematologica - Vol. 105 n. 6 - June 2020

APC-I73N variant
ing to this site makes a key contribution to APC interac- tion with protein S.39 Due to close proximity of residue 73 to this functionally important metal ion binding-site, it is possible a glycosylated Asn73 impairs the affinity of APC- I73N for protein S by altering the affinity of EGF1 for Ca2+. Since this site has a much higher affinity for Ca2+ than the Gla-domain of APC (Kd low mM range), evaluating the effect of mutagenesis on the affinity of EGF1-domain for Ca2+ was not feasible by functional assays.
In summary, our results strongly suggest that substitu- tion of Ile73 with Asn introduces a new N-linked glycosy- lation site on EGF1-domain of APC. This modification in the compound heterozygote patient leads to a weaker affinity of APC-I73N for protein S, thereby causing antico- agulant defect and recurrent DVT. Noting the anticoagu- lant defect is observed only in the presence of protein S, results further suggest the I73N mutation would be most harmful under conditions where the co-factor level is low (i.e. pregnancy, oral contraceptive use, etc.).40,41 This hypothesis is consistent with the observation that recur- rent DVT in the affected patient was associated with preg- nancy (28th week, left lower limb DVT), during the postpar-
tum period (3rd and 4th weeks, lower limb DVT and mesen- teric venous thrombosis, respectively) and when taking oral contraceptives (mesenteric venous thrombosis after finishing one year of anticoagulant therapy). The I73N mutation does not adversely affect the anti-inflammatory signaling function of APC. Molecular modeling predicts the newly attached N-linked glycan on Asn73 can impede with the proper interaction of APC EGF1-domain with Gla-TSR-EGF1 domains of protein S on the membrane sur- face. This modification appears to not only weaken the affinity of APC for protein S but also adversely affect func- tionally important protein S-dependent topographical changes in the active-site of APC, thereby impairing its anticoagulant function on the membrane surface.
Acknowledgments
This study was supported by an institutional fund from OMRF and grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health HL101917 and HL062565 to ARR; and The General Program of National Natural Science Foundation of China (81570114) to QD and (81870107) to YL.
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