A Single-Domain Antibody that Exerts an Anticoagulant Effect Dependent on Protein S

J.-C. Sedzro¹, F. Adam¹, A. De Carvalho¹, I. Peyron¹, E. Bianchini¹, S. Gandrille², S. Thomassen³, T.M. Hackeng³, O.D. Christophe¹, P.J. Lenting¹, C.V. Denis¹, D. Borgel^1,4, F. Saller¹

¹HITh, UMR_S 1176, INSERM, Université Paris-Saclay, Le Kremlin-Bicêtre, France, ²UMR_S 1140, INSERM, Université Paris Descartes, Université Sorbonne Paris Cité, Paris, France, ³Cardiovascular Research Institute Maastricht (CARIM), University Maastricht, Maastricht, the Netherlands, ⁴Service d'Hématologie Biologique, Hôpital Necker Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France

Abstract Number: PB0382

Meeting: ISTH 2020 Congress

Theme: Coagulation and Natural Anticoagulants » Protein C Pathway

Background: Protein S (ProS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) and tissue-factor pathway inhibitor (TFPI). An immune library of single-domain antibodies (sdAb) generated from a llama immunized with recombinant human ProS (rhProS) was screened by phage display, allowing us to identify a strong ProS binder (PS003) after two rounds of biopanning on immobilized rhProS.

Aims: The sdAb PS003 was further characterized in vitro and in vivo.

Methods: Monovalent (PS003) and bivalent (PS003biv) forms of PS003 were produced in T7 SHuffle E. coli and purified. APC-cofactor activity of ProS was investigated in a commercial plasma clotting assay. The cofactor activity of ProS on TFPI-mediated FXa inhibition was evaluated in a purified system. The in vivo effects of sdAbs were evaluated in a FeCl₃-induced thrombosis mouse model on mesenteric vessels.

Results: In ELISA, PS003 was found to bind to rhProS and plasma-derived human ProS and not to other vitamin K-dependent proteins, including Gas6. Very unexpectedly, PS003 potentiated the APC-cofactor activity of ProS in a plasma clotting assay on ProS-deficient plasma supplemented with ProS, while having minor effects in the absence of ProS. To increase the potency of PS003, we generated a bivalent form of PS003 (PS003biv) that bound to immobilized rhProS with a 2-fold higher affinity than PS003 and again stimulated the APC-cofactor activity of rhProS. Neither PS003 nor PS003biv had any effect in the TFPI-cofactor activity of ProS.
PS003biv bound to recombinant murine ProS in ELISA and prolonged the occlusion times in mesenteric vessels, especially venules, in a murine thrombosis model, with thrombi that were very unstable and highly prone to embolization.

Conclusions: These results suggested that PS003biv, and possibly PS003, might exert antithrombotic effects in vivo through ProS-dependent anticoagulant mechanisms that need to be clarified.

To cite this abstract in AMA style:

Sedzro J-, Adam F, De Carvalho A, Peyron I, Bianchini E, Gandrille S, Thomassen S, Hackeng TM, Christophe OD, Lenting PJ, Denis CV, Borgel D, Saller F. A Single-Domain Antibody that Exerts an Anticoagulant Effect Dependent on Protein S [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-single-domain-antibody-that-exerts-an-anticoagulant-effect-dependent-on-protein-s/. Accessed September 22, 2023.

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