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J. Biol. Chem., Vol. 281, Issue 39, 28850-28857, September 29, 2006

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Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain^*

Roger J. S. Preston¹, Eva Ajzner, Cristina Razzari^¶, Stalo Karageorgi, Sonia Dua, Björn Dahlbäck, and David A. Lane

From the Department of Haematology, Division of Investigative Science, Hammersmith Hospital Campus, Imperial College London, London W12 0NN, United Kingdom, Department of Laboratory Medicine, Division of Clinical Chemistry, University of Lund, University Hospital, Malmö S-205 02, Sweden, and ^¶Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Department of Medicine and Medical Specialties, Instituto di Ricovero e Cura a Carattere Scientifico di Natura Pubblica Maggiore Hospital, Mangiagalli and Regina Elena Foundation and University of Milan, 20122 Milan, Italy

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.

Received for publication, May 24, 2006 , and in revised form, July 20, 2006.

^* This work was supported by the British Heart Foundation and by Swedish Research Council Grant 07143. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 44-20838-32297; Fax: 44-20838-32296; E-mail: roger.preston{at}imperial.ac.uk.

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