Antithrombotic potential of a single-domain antibody enhancing the activated protein C-cofactor activity of protein S.

BACKGROUND: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa). OBJECTIVE: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo. METHODS: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay. RESULTS: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model. DISCUSSION: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy.

Measuring beta-galactose exposure on platelets: Standardization and healthy reference values.

BACKGROUND: Correct diagnosis of the cause of thrombocytopenia is crucial for the appropriate management of patients. Hyposialylation/desialylation (characterized by abnormally high ß-galactose exposure) accelerates platelet clearance and can lead to thrombocytopenia. However, the reference range for ß-galactose exposure in healthy individuals has not been defined previously. OBJECTIVE: The objective of the present study was to develop a standardized assay of platelet ß-galactose exposure for implementation in a clinical laboratory. METHODS: ß-Galactose exposure was measured in platelet-rich plasma by using flow cytometry and Ricinus communis agglutinin (RCA). A population of 120 healthy adults was recruited to study variability. RESULTS: We determined an optimal RCA concentration of 12.5 µg/mL. The measure was stable for up to 4 hours (mean fluorescence intensity [MFI]-RCA: 1233 ± 329 at 0 hour and 1480 ± 410 at 4 hours). The platelet count did not induce a variation of RCA and the measure of RCA was stable when tested up to 24 hours after blood collection (MFI-RCA: 1252 ± 434 at day 0 and 1140 ± 297 24 hours after blood sampling). To take into account the platelet size, results should be expressed as RCA/forward scatter ratio. We used the assay to study variability in 120 healthy adults, and we found that the ratio is independent of sex and blood group. CONCLUSION: We defined a normal range in a healthy population and several preanalytical and analytical variables were evaluated, together with positive and negative controls. This assay may assist in the diagnosis of thrombocytopenic diseases linked to changes in ß-galactose exposure.