Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function

Summary. Objective: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. Patients: 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. Results: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C-independent activities (24–38%) when compared with wild-type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 ± 30.8 nm, rPSwt; Kd 15.2 ± 0.9 nm) as well as to Ca2+-dependent conformation-specific monoclonal antibodies for GLA domain was significantly reduced. Conclusions: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.