1. Protein S Gla-domain mutations causing impaired Ca2+-induced phospholipid binding and severe functional protein S deficiency
by Suely M. Rezende, David A. Lane, Blandine Mille-Baker, Michel M. Samama, Jacqueline Conard, and Rachel E. Simmonds
Blood
Volume 100(8):
October 15, 2002
©2002 by American Society of Hematology

2. Pedigree of the family under investigation
Pedigree of the family under investigation.Males are shown as squares, females as circles, and deceased family members are crossed through.
Pedigree of the family under investigation.Males are shown as squares, females as circles, and deceased family members are crossed through. The proposita is indicated with an arrow, and family members who had experienced venous thrombosis are indicated with a T above and to the right of the symbol. Information regarding protein S levels is shown for all 6 family members for whom it was available, as indicated in the legend. For one individual, protein S levels were assessed on 2 separate occasions. DNA was available only from 2 family members. The presence of the Gly11Asp and Thr37Met mutations are indicated by a left-hand half-filled symbol and by a right-hand filled symbol, respectively (see legend). nt indicates family members for whom the genetic status was not tested; PS, protein S.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

3. Relative transient expression levels of recombinant wild-type and variant protein S in COS-1 cells.Mean, relative concentration of protein S expressed after correction for transfection efficiency.
Relative transient expression levels of recombinant wild-type and variant protein S in COS-1 cells.Mean, relative concentration of protein S expressed after correction for transfection efficiency. A value of 100% was assigned to wild-type protein S. Each experiment was performed at least 3 times in duplicate. Unpaired t test was used to compare the levels between wild-type protein S and the variants; *P = .62; **P < .0001.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

4. Western blotting of wild-type protein S and variants expressed in HEK293 cells.SDS-PAGE of 30 ng protein S under reducing conditions and immunoblotting with a polyclonal protein S antibody were performed as described in the text.
Western blotting of wild-type protein S and variants expressed in HEK293 cells.SDS-PAGE of 30 ng protein S under reducing conditions and immunoblotting with a polyclonal protein S antibody were performed as described in the text. The positions to which protein size markers migrated are indicated to the left, along with their molecular weight in kDa.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Enhancement of APC-dependent factor Va inactivation by wild-type protein S and variants.A range of recombinant protein S concentrations (0 to 20 nM) was incubated with 0.1 nM APC, 3 nM factor Va, and 25 μM phospholipid vesicles for 2 minutes at 37°C.
Enhancement of APC-dependent factor Va inactivation by wild-type protein S and variants.A range of recombinant protein S concentrations (0 to 20 nM) was incubated with 0.1 nM APC, 3 nM factor Va, and 25 μM phospholipid vesicles for 2 minutes at 37°C. Remaining factor Va activity was then quantified using a prothrombinase assay, assessing thrombin generation with the chromogenic substrate S2238 (see text). Results are expressed relative to the remaining activity, with no protein S present (100%), as mean of 3 experiments ± SD. ♦ indicates commercial plasma purified human protein S; ▪, wild-type recombinant protein S; ●, protein S with Gly11Asp; ▴, protein S with Thr37Met; ⋄, dialyzed conditioned medium from cells transfected with the vector pcDNA6 diluted to the same extent as that for 20 nM wild-type protein S; ▵, 20 nM wild-type recombinant protein S added in the absence of APC.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Binding of recombinant human protein S to phospholipid vesicles
Binding of recombinant human protein S to phospholipid vesicles.PE/PC/PS 40:40:20 phospholipid vesicles were coated onto the wells of microtiter plates and blocked with 3% (wt/vol) BSA. (A) Dialyzed recombinant protein S (0 to 26 nM) was incubated in the we...
Binding of recombinant human protein S to phospholipid vesicles.PE/PC/PS 40:40:20 phospholipid vesicles were coated onto the wells of microtiter plates and blocked with 3% (wt/vol) BSA. (A) Dialyzed recombinant protein S (0 to 26 nM) was incubated in the wells for 2 hours at 37°C in a buffer containing 3 mM CaCl2, and bound protein S was detected with a polyclonal anti–human protein S peroxidase-conjugated antibody (see text). (B) Dialyzed recombinant protein S (10 nM) was incubated in the wells for 2 hours at 37°C in a buffer containing various concentrations of CaCl2 (0 to 5mM), and bound protein S was detected with a polyclonal anti–human protein S peroxidase-conjugated antibody (see text). In both panels, results are expressed as mean A492nm of 3 experiments ± SD (in many cases the SDs are so small they cannot be visualized) as a proportion of the maximum (designated 100%). ▪ indicates wild-type protein S; ●, protein S with Gly11Asp; ▴, protein S with Thr37Met; ⋄, wild-type protein S assessed in a buffer that contained no Ca2+ and was instead supplemented with 5 mM EDTA.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Interaction of recombinant human protein S with Ca2+-dependent monoclonal antibodies.Antibodies were coated into the wells of microtiter plates at 10 μg/mL and blocked with casein.
Interaction of recombinant human protein S with Ca2+-dependent monoclonal antibodies.Antibodies were coated into the wells of microtiter plates at 10 μg/mL and blocked with casein. Dialyzed recombinant protein S at a range of concentrations was incubated for 1 hour at 37°C in the presence of 3 mM CaCl2. Bound protein S was detected by means of a rabbit anti–human protein S peroxidase-conjugated antibody. (A) HPS21; (B) HPS54. In both panels, results are expressed as mean A492nm of duplicates as a proportion of the maximum (designated 100%). Each of the duplicates gave essentially identical results. ▪ indicates wild-type protein S; ●, protein S with Gly11Asp; ▴, protein S with Thr37Met.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology

8. Predicted effect of the Gly11Asp mutation on the conformation of the protein S Gla domain.The Gla domain of protein S shown was derived by homology modeling and refinement.36 In order to predict the effects of the Gly11Asp amino acid substitution on protein...
Predicted effect of the Gly11Asp mutation on the conformation of the protein S Gla domain.The Gla domain of protein S shown was derived by homology modeling and refinement.36 In order to predict the effects of the Gly11Asp amino acid substitution on protein S structure and function, the Insight II 3D Graphics Program (Accelrys, San Diego, CA) was used. The alpha-carbon backbone trace is shown in dark gray ribbon representation. The side-chain of Asp11 (mutated from Gly11) is depicted in thick stick representation shaded by atom type. The side-chain of Gla29 is depicted in ball-and-stick style, also colored by atom (carbon, pale gray; oxygen, mid-gray). Two Ca2+ions that are coordinated (black lines) by the Gla29 side-chain are shown as very pale spheres. Replacement of wild-type Gly11 by Asp causes multiple bad contacts with atoms of the neighboring Gla29 side-chain, likely to result in loss of Ca2+ ion coordination, Ca2+ ion affinity, and destabilization of the Ca2+-dependent conformation of the Gla domain that is required for interaction with phospholipids.
Suely M. Rezende et al. Blood 2002;100:
©2002 by American Society of Hematology