Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S variants generated by site-directed mutagenesis

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of APC and protein S on thrombin generation. Thrombin generation was performed in protein S–deficient plasma with 100nM inhibitory antibodies against TFPI. Up to 10nM APC had no effect on thrombin generation in the absence of protein S. All concentrations generate lines that are superimposable (A). After addition of 120nM protein S (at 0-10nM APC), an APC dose-dependent effect was observed (B). The top single line represents 0 to 10nM APC in the absence of protein S. Protein S in the presence of no or 2.5nM APC generated lines that were superimposable. Conditions used are noted adjacent to the peaks to which they refer. The anticoagulant effect of 10nM APC and 120nM protein S was inhibited by polyclonal antibodies against protein S (C) or against protein C (D). PS indicates protein S; PC, protein C. Representative experiments are shown (n = 3).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Screening of protein S variants for APC cofactor activity. The APC cofactor activity of protein S was evaluated at 16nM APC and 100nM protein S by CAT. The peak height in the absence of protein S was set to 100%. A high concentration of APC, leading to almost complete inhibition of thrombin generation with 100nM WT protein S, was chosen specifically for screening purposes as this allows widening of the assay window at which mutants with reduced APC cofactor activity are visualized. Results were confirmed by evaluating protein S (at 60 and 90nM) cofactor activity toward 4 or 9nM APC.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Activity Assay, Concentration Assay, Inhibition

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of WT protein S, D95A, D95N, D78A, and Q79A variants on thrombin generation. Thrombin generation was measured in protein S–deficient plasma supplemented with 9nM APC, 100nM antibodies against TFPI, and 0 to 120nM WT protein S (A), protein S D95A (B), protein S D95N (C), or 90nM purified WT (dashed line) or purified protein S D95A (dotted line; D). Protein S concentrations are positioned adjacent to the peaks to which they refer. The cofactor activity of 60nM WT protein S and protein S variants D95A, D78A, and Q79A was compared at 9nM APC (E). Typical experiments are shown (n = 3). Whereas the cofactor activity of WT protein S is highly dependent on the APC concentration used (Figure 1B), that of protein S D95A is not, explaining the difference in fold activity between WT protein S and protein S D95A in Figures 2 and ​and3.3. Dose-response data from titrations with WT protein S, protein S D95A, and protein S D95N in the presence of 9nM APC are shown in panel F (data are expressed as mean ± SD of 2 independent experiments performed in duplicate). Inset in panel B shows recognition of WT protein S and protein S D95A in media by polyclonal antibodies and a monoclonal antibody recognizing only γ-carboxylated Gla domains. Inset in panel D shows the SeeBlue-prestained marker, plasma-purified protein S from Enzyme Research Laboratories Ltd (lane 1), purified recombinant WT protein S (lane 2), and purified protein S D95A (lane 3) visualized with silver staining.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Purification, Activity Assay, Concentration Assay, Marker, Recombinant, Silver Staining

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipid surfaces. Protein S (0-120nM) was incubated in a plate coated with 25 μg/mL phospholipids. Bound protein S was detected with an HRP-conjugated polyclonal antibody against protein S. A representative experiment is shown. The apparent Kd values, 5.69 ± 1.24 and 9.54 ± 2.26nM for WT protein S and protein S D95A, respectively, were obtained by calculating the mean ± SD of 3 independent experiments performed in duplicate. PL indicates phospholipids.

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipids and domain-specific monoclonal antibodies

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Binding Assay, Mutagenesis

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Protein S enhancement of APC-mediated cleavage of FVa in Arg306. Protein S (0-120nM) in the presence of 0.5nM APC was incubated with 0.8nM FVa R506Q/R679Q in the presence of phospholipids for 10 minutes. The remaining FVa actvity was measured with a prothrombinase assay. Results are plotted as mean ± SD from 3 independent experiments performed in duplicate (A). A time course experiment was performed to calculate the apparent pseudo–first-order rate constants of WT protein S and protein S D95A. It is observed that approximately 6-fold more APC is needed in the presence of protein S D95A to obtain a similar amount of APC-mediated FVa R506Q/R679Q inactivation as with WT protein S (B).

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Incubation

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Location of Asp78, Gln79, and Asp95 within the protein S Gla-TSR-EGF1 model. Domains are labeled on the right side of the model. Residues mutated in the GLA2 variant, Asp78, Gln79, and Asp95, are in light gray on the left side surface model. Residues Asp78, Gln79, and Asp95 are highlighted by the box to show their proximal spatial location. The model is taken from Villoutreix et al.35

Article Snippet: Protein S–deficient plasma (Affinity Biologicals), 80 μL, was incubated with 65 μg of corn trypsin inhibitor (Haematologic Technologies Inc) per milliliter of plasma to inhibit contact activation, 50μM phospholipid vesicles (DOPS/DOPC/DOPE, 20:60:20), 1pM tissue factor (Dade Innovin; Dade Behring), 4 to 16nM APC (Enzyme Research Laboratories Ltd) with 0 to 120nM protein S, in a final volume of 100 μL (all concentrations are final).

Techniques: Labeling, Variant Assay