Abstract
Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621-635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K(i) approximately 2 microM). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621-635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action.
Highlights
Protein S is a vitamin K-dependent protein that can act as a cofactor for the anticoagulant functions of activated protein C [1,2,3]
These results suggest that residues 621– 635 of protein S are essential for its inhibition of Factor Va (FVa) cofactor activity in the proby adding 200 M PSP14 or 560 nM anti-PSP14 antibodies
This report explores the interaction of protein S with FVa and identifies an essential binding site for FVa on protein S involving the region of residues 621– 635
Summary
Proteins and Peptides—Human protein S, FVa, prothrombin, monoclonal antibody S7 to protein S, and phospholipid vesicles (80% phosphatidylcholine and 20% phosphatidylserine) were prepared as described [16, 17, 24]. Prothrombinase Assays—Prothrombinase activity assays were described in detail elsewhere [16, 24] and were performed in the same binding buffer as described under “Binding Assays” below except that bovine serum albumin was substituted for gelatin and NaCl was 0.1 M. Prothrombinase assays contained 1 nM FXa, 20 pM FVa, 0.6 M prothrombin, and 50 M phospholipid vesicles (80% phosphatidyl choline/20% phosphatidylserine). Model for the Competitive Inhibition of Prothrombinase by Peptide PSP14 —Because the data suggested that peptide PSP14 competes with FXa for binding to FVa, rates of prothrombin activation (which are directly proportional to the amount of FXaFVa complexes formed) obtained at varying [FXa] and [PSP14] were analyzed according to the following model. Binding of biotin-FXa to immobilized protein S was detected with streptavidin-alkaline phosphatase and phosphatase substrate as for other binding assays. After 5 min of preincubation at 37 °C, 40 l of 30 mM CaCl2 at 37 °C was added, and clotting time was recorded using an ST4 coagulometer (Stago, Asnieres, France)
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