Tài liệu Báo cáo khoa học: Mouse recombinant protein C variants with enhanced membrane affinity and

Mouse recombinant protein C variants with enhanced

membrane affinity and hyper-anticoagulant activity in

mouse plasma

Michael J. Krisinger1

, Li Jun Guo1

, Gian Luca Salvagno2

, Gian Cesare Guidi2

, Giuseppe Lippi2

and Bjo¨rn Dahlba¨ ck1

1 Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmo¨, Sweden

2 Clinical Chemistry Section, Department of Morphological-Biomedical Sciences, University Hospital of Verona, Italy

Introduction

Protein C is a vitamin K-dependent c-carboxyglutamic

acid-containing protein (Gla protein) found in human

and mouse plasma at a concentration of approximately

70 nm [1]. This zymogen is efficiently converted by the

thrombin–thrombomodulin complex to the multifunc￾tional serine protease activated protein C (APC). With

its cofactor, protein S, APC degrades factors Va and

VIIIa on anionic phospholipid membranes, thereby

Keywords

anticoagulation; Gla domain; mouse protein C;

mouse plasma; protein–membrane

interactions

Correspondence

B. Dahlba¨ck, Department of Laboratory

Medicine, Division of Clinical Chemistry,

Wallenberg Laboratory, Entrance 46, Floor

6, Lund University, University Hospital,

S-20502 Malmo¨, Sweden

Fax: +46 40 337044

Tel: +46 40 331501

E-mail: [email protected]

(Received 1 July 2009, revised 4 September

2009, accepted 9 September 2009)

doi:10.1111/j.1742-4658.2009.07371.x

Mouse anticoagulant protein C (461 residues) shares 69% sequence identity

with its human ortholog. Interspecies experiments suggest that there is an

incompatibility between mouse and human protein C, such that human

protein C does not function efficiently in mouse plasma, nor does mouse

protein C function efficiently in human plasma. Previously, we described a

series of human activated protein C (APC) Gla domain mutants (e.g.

QGNSEDY-APC), with enhanced membrane affinity that also served as

superior anticoagulants. To characterize these Gla mutants further in

mouse models of diseases, the analogous mutations were now made in

mouse protein C. In total, seven mutants (mutated at one or more of

positions P10S12D23Q32N33) and wild-type protein C were expressed and

purified to homogeneity. In a surface plasmon resonance-based membrane￾binding assay, several high affinity protein C mutants were identified. In

Ca2+ titration experiments, the high affinity variants had a significantly

reduced (four-fold) Ca2+ requirement for half-maximum binding. In a

tissue factor-initiated thrombin generation assay using mouse plasma, all

mouse APC variants, including wild-type, could completely inhibit throm￾bin generation; however, one of the variants denoted mutant III (P10Q⁄

S12N ⁄ D23S ⁄Q32E⁄ N33D) was found to be a 30- to 50-fold better anti￾coagulant compared to the wild-type protein. This mouse APC variant will

be attractive to use in mouse models aiming to elucidate the in vivo effects

of APC variants with enhanced anticoagulant activity.

Abbreviations

APC, activated protein C; Cmax, maximal concentration of thrombin; ETP, endogenous thrombin potential; Gla protein, c-carboxyglutamic

acid-containing protein; DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]; FU, fluorescence units; PE, phosphatidylethanolamine; POPC,

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS, phosphatidylserine;

Rmax, maximum surface coverage; RU, response units; SPR, surface plasmon resonance; Tmax, time required to reach maximum thrombin

generation.

6586 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS