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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2209.)
© 2005 American Heart Association, Inc.

Thrombosis

In Vivo Clearance of Human Protein S in a Mouse Model

Influence of C4b-Binding Protein and the Heerlen Polymorphism

Cécile V. Denis; Sarah J. Roberts; Tilman M. Hackeng; Peter J. Lenting

From INSERM U.143 (C.V.D.), Kremlin-Bicêtre, France; the Laboratory for Thrombosis and Haemostasis (S.J.R., P.J.L.), Department of Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; and the Department of Biochemistry (T.M.H.), University of Maastricht, Maastricht, the Netherlands.

Correspondence to Dr P.J. Lenting, Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. E-mail p.j.lenting{at}lab.azu.nl

Objective— To explore the effect of the Heerlen polymorphism and C4b-binding protein (C4BP) on protein S catabolism in vitro and in vivo.

Methods and Results— Radiolabeled protein S was efficiently bound and intracellularly degraded by THP-1 macrophages, and both processes were strongly reduced in the presence of the protein S-carrier protein C4BP. To test whether C4BP displays a similar protective effect in vivo, survival experiments were performed in mice. In the absence of C4BP, radiolabeled human protein S disappeared in a biphasic manner (mean residence time [MRT] 2 hours). However, the presence of C4BP resulted in a 4-fold prolonged survival of protein S (MRT 8 hours; P<0.0001). We also applied this experimental model to recombinant protein S-Heerlen, a naturally occurring variant that contains a Ser460Pro substitution. These clearance experiments revealed a strongly decreased survival of recombinant protein S-S460P (MRT 0.6 hours; P=0.021), which could be compensated partially by C4BP (MRT 1.4 hours; P=0.012 compared with protein S-S460P).

Conclusion— Protein S-S460P has a reduced survival in vivo, which may explain the low levels of free protein S in individuals carrying this polymorphism. Furthermore, C4BP prevents premature clearance of protein S and uses this ability to compensate the increased clearance of protein S-S460P.

We established that binding and degradation of protein S by macrophages was partially inhibited by C4BP. C4BP mimicked this effect in vivo by prolonging the half-life of protein S from 2 to 4 hours in a mouse model. The presence of the Heerlen polymorphism reduced survival of protein S 3-fold to 0.6 hours.

Key Words: protein S • C4b-binding • clearance

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