Protein Interactions Involving the Laminin G-type Domains of Vitamin K-dependent Protein S

Abstract: The studies described in this thesis focus on the involvement of the two laminin G-type domains of vitamin K-dependent protein S in various biological contexts. The G-type domains are located in the C-terminal sex-hormone-binding globulin (SHBG)-like region. In human plasma, the majority of protein S circulates in a high-affinity complex with the complement regulatory protein C4b-binding protein (C4BP) while the free form has an anticoagulant potential as it functions as a cofactor to activated protein C (APC). In addition, bovine protein S has the ability to stimulate human Sky, a member of a specific subfamily of receptor tyrosine kinases (RTKs). The extravascular protein Gas6, highly homologous to protein S, has been shown to be the genuine ligand for all members of this RTK subfamily in man. Nevertheless, Gas6 expresses no APC-cofactor activity and does not form any complex with C4BP. Chimeras of human protein S were constructed by replacing parts of the SHBG-like region by the corresponding parts of either human Gas6 or bovine protein S and these recombinants became important tools in the different characterisations. Protein S accelerates the APC-mediated degradation of the procoagulants, activated factor VIII (FVIIIa) and factor V (FVa). Protein S/Gas6 chimeras were used to investigate the role of the SHBG-like region in these processes. For the inactivation of FVa, the results showed unambiguously that only the second G-type domain is directly involved. In contrast, in the FVIIIa inactivation, where FV and protein S act in a synergistic manner, both domains of protein S are required to obtain optimal performance. Surface-plasmon resonance experiments and micro-titre based assays demonstrated that both G-type domains of protein S are critical for the interaction with C4BP. These results, obtained using the same chimeras as in the coagulation studies, offer an explanation for the apparent inconsistency between the previous reports, in which very different sequences in the SHBG-like region were suggested. Finally, in the receptor studies we used both kinds of chimeric molecules. The SHBG-like region was shown to be essential for the interaction with Sky and sequential differences in this domain explain the human-bovine difference in the activation of human Sky. Although both G-type domains seem to be involved in the interaction, the first domain is indispensable for the stimulation of Sky. Moreover, the critical residues within this domain do not appear to be sequentially clustered.