Protein S (PS), through its anticoagulant activities, plays a crucial role in the control of coagulation. Rare natural PS mutations responsible for dysfunctional molecules in plasma (type II deficiency) represent unique models to relate plasma phenotype with recombinant PS (rPS) expression, and to elucidate the functional role of PS domains. We characterized the first type II PS deficiency affecting the EGF4 domain, a module with a poorly defined functional role. The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. The patient was heterozygous for the IVSg-2A/T mutation that, by activating a cryptic splice site, causes the deletion of codons I203D204. Recombinant rPSwt and rPSDelI203D204 variants were expressed in BHK21 cells and immunopurified from conditioned medium. The rPSDelI203D204 showed reduced anticoagulant (<10% of rPSwt) and APC-independent activities (38% of rPSwt). Binding of the variant to phospholipid vesicles was significantly reduced (Kd=235.7±30.8 nM, rPSwt; Kd=15.2±0.9 nM) and the Kd for C4bBP was increased (Kd=107.7±14.0 nM; rPSwt, Kd=8.2±1.0 nM). The rPSDelI203D204 showed markedly reduced binding to conformation-specific monoclonal antibodies for GLA and EGF1 domains, whereas the affinity of the mutant rPS for the SHBG recognizing antibody was only slightly different from that of rPSwt. A more conservative alteration (N217S) in the EGF4, previously found, was characterized. The rPS217S showed functional (10% and 41% of anticoagulant and APC-independent activities, respectively) and binding (phospholipid vesicles, Kd=175.5±53.1; C4bBP, Kd=47.2 ±3.7) features indistinguishable from those exhibited by the micro-deleted PS. These data contribute to characterize the functional role of EGF4 domain in the anticoagulant activities of PS and to define the thrombophilic nature of type II PS deficiency. Moreover, they provide examples of extended conformational changes transmitted from the EGF4 module to the GLA and SHBG domains through intra-molecular communication.
NATURAL MUTATIONS IN THE EGF4 DOMAIN OF PROTEIN S PRODUCE EXTENDED CONFORMATIONAL CHANGES AND ALTER PHOSPHOLIPID AND C4BBP INTERACTIONS
BARONI, Marcello;MARCHETTI, Giovanna;PINOTTI, Mirko;BERNARDI, Francesco;
2006
Abstract
Protein S (PS), through its anticoagulant activities, plays a crucial role in the control of coagulation. Rare natural PS mutations responsible for dysfunctional molecules in plasma (type II deficiency) represent unique models to relate plasma phenotype with recombinant PS (rPS) expression, and to elucidate the functional role of PS domains. We characterized the first type II PS deficiency affecting the EGF4 domain, a module with a poorly defined functional role. The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. The patient was heterozygous for the IVSg-2A/T mutation that, by activating a cryptic splice site, causes the deletion of codons I203D204. Recombinant rPSwt and rPSDelI203D204 variants were expressed in BHK21 cells and immunopurified from conditioned medium. The rPSDelI203D204 showed reduced anticoagulant (<10% of rPSwt) and APC-independent activities (38% of rPSwt). Binding of the variant to phospholipid vesicles was significantly reduced (Kd=235.7±30.8 nM, rPSwt; Kd=15.2±0.9 nM) and the Kd for C4bBP was increased (Kd=107.7±14.0 nM; rPSwt, Kd=8.2±1.0 nM). The rPSDelI203D204 showed markedly reduced binding to conformation-specific monoclonal antibodies for GLA and EGF1 domains, whereas the affinity of the mutant rPS for the SHBG recognizing antibody was only slightly different from that of rPSwt. A more conservative alteration (N217S) in the EGF4, previously found, was characterized. The rPS217S showed functional (10% and 41% of anticoagulant and APC-independent activities, respectively) and binding (phospholipid vesicles, Kd=175.5±53.1; C4bBP, Kd=47.2 ±3.7) features indistinguishable from those exhibited by the micro-deleted PS. These data contribute to characterize the functional role of EGF4 domain in the anticoagulant activities of PS and to define the thrombophilic nature of type II PS deficiency. Moreover, they provide examples of extended conformational changes transmitted from the EGF4 module to the GLA and SHBG domains through intra-molecular communication.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
