Tài liệu Báo cáo khoa học: Altered inactivation pathway of factor Va by activated protein C in the

Altered inactivation pathway of factor Va by activated protein C

in the presence of heparin

Gerry A. F. Nicolaes1

, Kristoffer W. Sørensen1,2, Ute Friedrich2,3, Guido Tans1

, Jan Rosing1

, Ludovic Autin4

,

Bjo¨ rn Dahlba¨ ck2 and Bruno O. Villoutreix4

1

Department of Biochemistry, Cardiovascular Research Institute Maastricht, the Netherlands; 2

Department of Clinical Chemistry,

University Hospital, Malmo¨, Sweden; 3

Immunochemistry Department, Novo Nordisk A/S, Gentofte, Denmark; 4

INSERM U428,

University of Paris V, France

Inactivation of factor Va (FVa) by activated protein C

(APC) is a predominant mechanism in the down-regulation

of thrombin generation. In normal FVa, APC-mediated

inactivation occurs after cleavage at Arg306 (with corres￾pondingrate constant k¢306) or after cleavage at Arg506

(k506) and subsequent cleavage at Arg306 (k306). We have

studied the influence of heparin on APC-catalyzed FVa

inactivation by kinetic analysis of the time courses of inac￾tivation. Peptide bond cleavage was identified by Western

blottingusingFV-specific antibodies. In normal FVa, un￾fractionated heparin (UFH) was found to inhibit cleavage at

Arg506 in a dose-dependent manner. Maximal inhibition of

k506 by UFH was 12-fold, with the secondary cleavage at

Arg306 (k306) beingvirtually unaffected. In contrast, UFH

stimulated the initial cleavage at Arg306 (k¢306) two- to

threefold. Low molecular weight heparin (Fragmin) had

the same effects on the rate constants of FVa inactivation as

UFH, but pentasaccharide did not inhibit FVa inactivation.

Analysis of these data in the context of the 3D structures of

APC and FVa and of simulated APC–heparin and FVa–

APC complexes suggests that the heparin-binding loops 37

and 70 in APC complement electronegative areas sur￾roundingthe Arg506 site, with additional contributions

from APC loop 148. Fewer contacts are observed between

APC and the region around the Arg306 site in FVa. The

modeling and experimental data suggest that heparin, when

bound to APC, prevents optimal dockingof APC at Arg506

and promotes association between FVa and APC at position

Arg306.

Keywords: coagulation; factor V; heparin; protein C; protein

docking.

Activated factor V (FVa) is an essential cofactor in the

prothrombin-activatingcomplex, stimulatingthe activity

of membrane-bound factor Xa (FXa) more than

100 000-fold [1,2]. Hence, FVa is an ideal target for

the regulation of thrombin formation [3]. Downregula￾tion of FVa activity is achieved through proteolysis

mainly mediated by the anticoagulant protein C pathway

(reviewed in [4,5]). Protein C is composed of a heavy and

a light chain held together by a single disulfide bond [6].

The light chain contains the c-carboxyglutamic acid

(Gla)-rich domain and two epidermal growth factor-like

domains [7]. The heavy chain comprises a short activa￾tion peptide and a serine protease (SP) domain which

contains the active site of the enzyme. Activated protein

C (APC), the product of a thrombin–thrombomodulin￾catalyzed activation of the zymogen protein C, proteo￾lytically inactivates the coagulation cofactors, FVa and

FVIIIa [8], in reactions stimulated by the APC cofactor

protein S.

FVa consists of a 105 kDa heavy (A1 and A2 domains)

and a 71–74-kDa light (A3, C1, and C2 domains) chain

which are noncovalently associated.

DuringAPC-catalyzed inactivation of FVa, the heavy

chain of FVa is cleaved at three sites: Arg306, Arg506 and

Arg679 [9]. The cleavages at Arg306 and Arg506 appear to

be crucial for inactivation, but the cleavage at Arg679 is

probably less important [10]. The cleavage at Arg506 is

kinetically favored over that at Arg306 and results in the

formation of an inactivation intermediate (FVaint), which

retains partial FVa cofactor activity owingto its ability to

bind FXa, albeit with lower affinity [10]. The FVa activity is

lost after cleavage at Arg306. In carriers of the common

FVLeiden mutation, in whom the Arg506 has been replaced

by a Gln, inactivation occurs via the slow Arg306 cleavage

(reviewed in [11]). Cleavage at Arg306 results in a large

reduction in FXa affinity and also the dissociation of

the A2 domain, the two processes ultimately rendering

FVa inactive as a cofactor of FXa [12,13]. APC￾catalyzed inactivation of FVa is modulated by other plasma

components. Thus, the nonenzymatic cofactor protein S

Correspondence to G. A. F. Nicolaes, Department of Biochemistry,

Cardiovascular Research Institute Maastricht, Maastricht,

the Netherlands. Fax: + 31 43 3884159, Tel.: + 31 43 3881539,

E-mail: [email protected]

Abbreviations: APC, activated protein C; DEGR-FXa, 1,5-DNS￾GGACK-factor Xa; DOPS, 1,2 dioleoyl-sn-glycero-3-phosphoserine;

DOPC, 1,2 dioleoyl-sn-glycero-3-phosphocholine; FV, coagulation

factor V; FVa, activated FV; FVa2, the FVa isoform lackingglyco￾sylation at Asn2181; FVIII, factor VIII; Gla, c-carboxyglutamic acid;

SP, serine protease; UFH, unfractionated heparin.

Note: Numberingof amino-acid positions in protein C corresponds to

the chymotrypsinogen nomenclature.

(Received 29 January 2004, revised 30 March 2004,

accepted 4 May 2004)

Eur. J. Biochem. 271, 2724–2736 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04201.x