We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inac-tivation.. We performed a detailed kinetic analysis of the influe
Trang 1Altered 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¨ck2and Bruno O Villoutreix4
1
Department of Biochemistry, Cardiovascular Research Institute Maastricht, the Netherlands;2Department of Clinical Chemistry, University Hospital, Malmo¨, Sweden;3Immunochemistry Department, Novo Nordisk A/S, Gentofte, Denmark;4INSERM 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
k506by 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
FVLeidenmutation, 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: G.Nicolaes@Bioch.Unimaas.nl
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; FVa 2 , 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)
Trang 2promotes cleavage at Arg306, whereas FXa specifically
blocks the cleavage at Arg506 [14]
It has recently been shown that basic residues in two
surface loops (37 and 70) in the SP domain of APC
(chymotrypsinogen nomenclature) form an extended
bind-ingsite for FVa [15–17] In addition, APC loop 148 also
plays a role in FVa degradation [18,19] Loop 60 is probably
less important as mutagenesis of positive residues in this
loop did not affect inactivation of FVa by APC [15,16]
Heparin is an important regulator of APC activity,
promotingthe interaction between APC and one of its
inhibitors, the serpin protein C inhibitor (PCI) [17] This is
probably mediated via a template mechanism, for which
bindingof heparin to basic residues in three of the four
surface loops in APC (60, 37 and 70) is crucial [17,20,21]
Interestingly, heparin has also been reported to stimulate
APC-catalyzed inactivation of intact FV, but not FVa [22,23]
We performed a detailed kinetic analysis of the influence
of heparin on the inactivation of FVa by APC and found a
specific heparin-mediated inhibition of the cleavage at
Arg506, whereas cleavage at Arg306 was mildly stimulated
Structural analysis strongly suggested that the
heparin-bindingloops 37, 60 and 70 in APC could complement
electronegative areas surrounding the Arg506 site of FVa
indicatingthat electrostatic interactions between regions of
FVa and APC could be critical for the formation of the
APC–FVa complex which is involved in the cleavage at
position Arg506 These electrostatic interactions are
inhib-ited by heparin when heparin is bound to the electropositive
cluster on loops 37, 60 and 70 located at one edge of the SP
domain of APC Our data further suggest that heparin can
potentially bridge APC to exosites around Arg306, thereby
facilitating cleavage at position Arg306
Materials and Methods
Proteins and reagents
Human FV and FVLeiden were purified from the plasma
of a normal individual and an individual homozygous for
the FV Arg506Gln mutation, and FVa2 was prepared as
described [24] Throughout the work presented here FVa2
was used Factor Xa, a-thrombin, protein S, human
activated protein C and prothrombin were purchased
from Kordia Laboratory Supplies (Leiden, the
Nether-lands) All coagulation factors were of human origin
unless otherwise stated 1,5-DNS-GGACK-factor Xa
(DEGR-FXa) was prepared as described previously [14]
The monoclonal antibody AHV 5146 was purchased from
Haematologic Technologies (Essex Junction, VT, USA)
Unfractionated heparin (UFH) and low molecular weight
heparin (Fragmin) were obtained from Leo (Ballerup,
Denmark); 1 IUÆmL)1 UFH contains 5.7
lgUF-HÆmL)1[23] Pentasaccharide was from Sanofi-Re´cherche
(Montpellier, France) Phospholipid vesicles [10% 1,2
dioleoyl-sn-glycero-3-phosphoserine (DOPS), 90% 1,2
dioleoyl-sn-glycero-3-phosphocholine (DOPC), mol/mol]
were prepared as described [25] The chromogenic
sub-strates S-2366 and S-2238 were obtained from
Chromo-genix (Milano, Italy), and biotraceTM poly(vinylidene
difluoride) transfer membranes from Pall Gelman
Labor-atory (Ann Arbor, MI, USA)
Expression and purification of recombinant human protein C
Recombinant protein C variants K37S/K38Q/K39Q (37-loop mutant), K62N/K63D (60-loop mutant), K37S/K38Q/K39Q/K62N/K63D (37+60-loop mutant) were created by PCR-based site-directed mutagenesis of the eukaryotic expression vector pGT-hyg(Eli Lilly), expressed in 293 cells (CRL-1573; ATCC), purified, and characterized as described previously [21,26]
Assay of FVa FVa activity was determined by quantification of the rate of FXa-catalyzed prothrombin activation, as described previ-ously [10] Briefly, in a reaction mixture that contained 0.5 lM prothrombin, a limitingamount of FVa (83 pM FVa), 5 nM FXa, 40 lM phospholipids (10 : 90 DOPS/ DOPC, mol/mol), 0.5 mgÆmL)1 ovalbumin, and 2 mM CaCl2, prothrombin activation was allowed for 1 min at
37C The amount of prothrombin activated was then determined usingS-2238 [2]
APC-catalyzed inactivation of FVa Time courses of FVa inactivation by APC were determined
by followingthe loss of FXa cofactor activity of FVa in the prothrombinase complex as a function of time Routinely, 0.8 nMplasma-derived human FVa or FVaLeidenwas prein-cubated with 25 lMphospholipid vesicles (10 : 90 DOPS/ DOPC, mol/mol) in the absence or presence of protein S (200 nM) and/or heparin (0.01–25 IUÆmL)1) in 25 mM Hepes buffer (pH 7.5), containing150 mM NaCl, 3 mM CaCl2, and 5 mgÆmL)1BSA, for 5 min at 37C Inactivation was started by addingwild-type APC or APC variants, and the progressive loss of FVa was monitored for up to 20 min
by transfer of aliquots to the FVa assay described above Analysis of kinetic data
Rate constants for APC-catalyzed Arg506 and Arg306 cleavage were obtained as described previously by fitting the time courses of FVa inactivation to a random-order, two-cleavage model using nonlinear least-squares analysis [10] In this model, FVa can be randomly cleaved at either Arg306 or Arg506 Cleavage at Arg306, with an apparent second-order rate constant of k¢306, results in complete loss of FVa cofactor activity (FVai; pathway 1, Eqn 1) Alternatively, initial cleavage at Arg506, with a correspondingrate constant of k506, results in a reaction intermediate (FVaint) with 40% residual cofactor activ-ity, which must be further cleaved at Arg306 (k306) in order to completely abolish FVa cofactor activity (path-way 2, Eqn 2)
Factor Vak
0 306
Factor Vak! Factor Va506 int
k306
! Factor Vai ð2Þ
In wild-type FVa, in which cleavage at Arg506 is 20-fold faster than cleavage at Arg306, the major part ( 95%) of
Trang 3FVa is inactivated via pathway 2 To reliably determine
k¢306, single exponential inactivation time courses were
determined for FVaLeiden, representingcleavage at Arg306
only and the k¢306values obtained were used in the fits for
normal FVa We have verified our previous findings [10]
that the APC-catalysed FVa inactivation time courses were
second-order throughout, i.e were directly proportional to
FVa and APC concentrations between 0 and 1.5 nMFVa
and between 0.05 and 5 nMAPC Thus, the second-order
rate constants obtained by this method can be directly
compared within the given ranges of FVa and APC
Statistical analysis usingthe StatGraphics Plus for Windows
package was performed to determine kinetic parameter
significance
Western blot analysis of FVa inactivation by APC
Human FVa (10 nM), phospholipid vesicles (25 lM) and
wild-type APC were incubated at 37C in 25 mM Hepes
(pH 7.5), containing150 mM NaCl, 3 mM CaCl2, and
5 mgÆmL)1BSA in the absence and presence of 25 IUÆmL)1
UFH Aliquots of 20 lL were removed at various time
points, and subjected to SDS/PAGE (7.5% gel) under
reducingconditions After transfer to poly(vinylidene
diflu-oride) membranes, heavy chain fragments were visualized
usinga monoclonal antibody (AHV 5146) directed against
the FVa heavy chain
Electrostatic potentials for APC and FVa A domains
The 3D structure of Gla-domainless APC [27] and the
homology model for the three A domains of FVa [28]
(co-ordinate file at http://www.klkemi.mas.lu.se/dahlback) were
investigated using the programs INSIGHTII, BIOPOLYMER
AND DELPHI(Accelrys, San Diego, CA, USA) Electrostatic
potentials were computed with DelPhi (reviewed in [29])
usinga standard set of formal charges The standard
protocol was applied The volume inside the APC or FVa
molecular surface was assigned a dielectric constant of 4 and
the outside volume was given a value of 80
Docking heparin-like molecules on to APC
Three different methods were used to dock heparin and
APC The first method follows the script reported by
Fernandez-Recio and coworkers [30] as integrated in the
modelingpackage ICM (Molsoft LLC, San Diego, CA,
USA) This first protocol included a pseudo-Brownian
rigid-body docking, an extended force field, and a soft
interaction energy function precalculated on a grid To
validate the dockingprotocol, it was first applied to three
different experimental protein–heparin complexes deposited
at the Protein Data Bank (PDB) files [31], PDB code 1bfc,
1azx, 1e0o [32–34] These proteins were energy-minimized
to allow relaxation of side chains, and to simplify
calcula-tions, we used a negatively charged polypeptide to mimic
heparin Negatively charged groups were moved with the
simulation software Discover (Accelrys) in order to
repro-duce the overall positioningin space of charges and overall
shape, as observed on the NMR/modeled structure of
heparin (file 1hpn [35])
Amino acids known to be part of the heparin-bindingsite
in these three crystallized protein–heparin complexes were given as startingpoint for the dockingsearch [30] At least one conformation of the simulated protein–peptide com-plexes amongthe lowest five dockingenergy scores repro-duced accurately the X-ray crystal structures of the equivalent complexes Therefore, the above dockingproto-col combined with partial knowledge of the binding site for heparin at the surface of APC as defined by several mutagenesis studies [17,18,21] was deemed appropriate to reasonably predict the overall orientation of a peptide mimickingheparin at the surface of APC
The second approach involved flexible dockingof heparin, PDB files 1hpn [35] and 1e0o [34], onto a rigid APC structure usingthe ICM package
Finally, a structure-based virtual screeningapproach (reviewed in [36]) was used to dock short sugar molecules in the APC loop 37 area The heparin (PDB file 1hpn) was shortened, and 10 trisaccharides were generated These sugar molecules were considered rigid during the docking but in order to consider some conformational flexibility, 13 conformers for each molecule were generated and all structures stored in a single data file A shape-based Gaussian docking function as integrated in the program FREDwas used to position the short sugar molecules at the surface of APC [37]
Partial docking of APC on to FVa The X-ray crystal structure of APC (with modifications in loop 148, see below) and a model structure for the three A domains of FVa (with some modifications at the Arg506 and Arg306 sites, see below) were used in two different automated dockingprocedures Rigid-body-dockingcalcu-lations with soft potentials were performed with the ICM package, as described [30] Alternatively, we used the approach reported by Norel et al [38] In dockingwith ICM, FVa Arg506 or Arg306 was given as starting point for the search, whereas for the method of Norel et al the entire surfaces of both interactingmolecules were investigated and
as such the search was not restricted to known binding regions (i.e at the Arg306 or Arg506 site)
In all experimentally known complexes of serine prote-ases/macromolecular inhibitors/substrates, the peptide bond to be cleaved tends to be located on a loop structure that protrudes far outside the molecular surface, either because this loop is indeed ill-structured or because its conformation has to change during the interaction with proteases A loop structure in FVa includingArg506 was predicted from the X-ray crystal structure of ceruloplasmin and does not project significantly outside the surface of FVa Numerous initial conformations (2000–20 000) were generated for residues 500–510 using the loop prediction program of Xiang et al [39] Several runs were performed, and the 10 best-energy conformations were kept From these structures, two residues before and after Arg506 were template-forced to partially adopt the conformation of the serpin reactive loop as present in the PDB file 1l99 [40] Similarly, the FVa loop that contains Arg306 was built again (residues 302–320) and several conformations were generated Because it is known that APC loop 148 plays a
Trang 4role in FVa inactivation [19,26] and as this loop is not
well defined in the PDB file 1aut, the APC loop
includ-ingresidues 145–153 was rebuilt usingthe approach of
Xiang et al., and the 10 lowest-energy conformations were
selected
One structure of FVa with the Arg506 loop sufficiently
solvent-exposed to allow interactions with APC was selected
for the dockingprocedures Dockingsimulations were
performed with the two best ranked structures for the FVa
Arg306 loop and an APC model in which the loop 148 tends
to be coveringthe active site (one of the lowest-energy
conformations)
For final optimization of the dockingprocedure, it was
necessary to include experimentally obtained data
Inter-active dockingwas consequently performed startingfrom
the best theoretical complexes (i.e the complexes that have
orientation compatible with key experimental/structural/
theoretical data) usingas guidelines the relative orientation
of a protease associated with a substrate/inhibitor as seen in
the X-ray crystal structure of a Michaelis serpin–protease
complex (file 1l99 [40]) to optimize further the positioningof
the molecules
Results
APC-catalyzed inactivation of FVa in the presence
of heparin
Heparin strongly influenced the time course of FVa
inactivation by wild-type APC (Fig 1) In the absence of
heparin (Fig 1A, open circles) the typical biphasic
inacti-vation curve was obtained for normal FVa (see also [10])
This is indicative of rapid cleavage at Arg506, which yields a
partially active reaction intermediate that is subsequently
fully inactivated on cleavage at Arg306 In the presence of
25 IUÆmL)1 heparin, FVa inactivation was significantly
reduced (Fig 1A, closed circles) The shape of the curve
suggested strong impairment of the fast first phase (Arg506
cleavage) of the reaction without influence on the second
phase (Arg306 cleavage) A direct effect of heparin in the
FVa assay was excluded because preincubation of FVa with
25 IUÆmL)1UFH or direct addition of heparin (1 IUÆmL)1)
to the FVa assay mixture resulted in assay outcomes that
were identical with those obtained in the absence of heparin
(data not shown)
To gain insights into which rate constants (k506and k¢306
in intact FVa and k306in FVaint) were influenced by heparin,
two additional experiments were performed In the first,
shown in Fig 1B, FVa was incubated for 5 min with APC
to allow completion of the first phase of the reaction, i.e
cleavage at Arg506 with all FVa activity converted into
FVaint Thereafter, the reaction volume was divided into
two equal parts and transferred into new reaction tubes
containingeither heparin or buffer Monitoringof FVa
activity in these two tubes was continued for another
25 min, duringwhich time the loss of FVa activity in the
absence of heparin (open circles) did not differ from that
obtained in the presence of heparin (closed circles) This
indicates that the presence of heparin did not influence the
k306in the partially active FVa intermediate In a second
experiment, the effect of heparin on the direct cleavage at
Arg306 (k¢ ) in FVa was determined from a time course of
Fig 1 Effect of heparin on the APC-catalyzed inactivation of FVa Plasma purified human FVa was inactivated by recombinant wild-type APC in the absence and presence of UFH as described in Materials and Methods (A) Time courses of inactivation of 1.5 n M FVa by 0.32 n M APC in the absence (s) or presence (d) of 25 IUÆmL)1UFH (B) FVa (1.5 n M ) was inactivated with 0.32 n M wild-type APC After
5 min of incubation, indicated by the arrow, the reaction volume was split into two equal volumes and transferred to two new tubes con-tainingeither 25 IUÆmL)1UFH (final concentration, d) or an amount
of compensation buffer (s), and the monitoringof FVa activity in the two reaction mixtures was continued (C) Time courses of inactivation
of 0.70 n M FVa Leiden by 1.0 n M APC in the absence (s) or presence (d) of 25 IUÆmL)1UFH The mean of two experiments is given Inactivation time courses were very reproducible with variations per time point < 7% From time courses like these, apparent second-order rate constants k 506 , k¢ 306 and k 306 were calculated as described in Materials and methods.
Trang 5FVaLeiden inactivation in the absence and presence of
heparin From these data, an approximately 2–3-fold (2.48;
standard error: 0.47) stimulation of the rate of inactivation
(k¢306) by heparin was calculated (Fig 1C) by fitting to a
single exponential Table 1 summarizes all rate constants
obtained by fittingall time courses to the random-order
cleavage model as described in Materials and methods,
under the constraints that k306 is minimally influenced by
heparin and using k¢306 obtained from the FVaLeiden
inactivation
Concentration dependence of heparin effect onk506
To further characterize the influence of heparin on the
APC-catalyzed inactivation of FVa and to confirm the specificity
of the effects observed in Fig 1, time courses of FVa
inactivation by wild-type APC were determined in the
presence of various concentrations of UFH (0.1–
55 IUÆmL)1), and rate constants for the inactivation were
calculated The effect of heparin on k506was dose-dependent
and saturable, with 50% inhibition observed at
2 IUÆmL)1UFH (Fig 2)
The inhibitory effect was not specifically mediated by
unfractionated heparin because we found that 25 IUÆmL)1
low molecular weight heparin (which is less than 18
saccharide residues in length) displayed similar inhibitory
activity to UFH In contrast, equimolar amounts of
pentasaccharide did not influence the inactivation of FVa
by APC
To relate the inactivation curves of FVa to specific
cleavages in FVa, Western blotting was performed (Fig 3)
A monoclonal antibody (AHV 5146) against the FVa heavy
chain was used to visualize the inactivation fragments In
the absence of heparin, the transient 75 kDa fragment
representingthe heavy chain fragment 1–506, typical for the
partially active reaction intermediate (FVaint), can be seen
(pathway 2) The 30-kDa band represents the 307–506
fragment formed on cleavage at Arg306 in FVaint In the
presence of UFH, the pattern of fragment generation was
considerably different from that obtained in its absence The
amount of 75-kDa fragment was greatly reduced, and
instead a 60/62-kDa fragment accumulated, representing
the 307–709 fragment, which is the result of cleavage at
Arg306 in FVa These data indicate that heparin strongly
influences cleavage at Arg506, and shifts the pathway towards initial cleavage at Arg306 (pathway 1), after which Arg506 can be cleaved, resulting in the formation of a
30 kDa fragment
Kinetic analysis of cleavage of FVa at Arg506 by APC
To further characterize the inhibitory effect of heparin on the Arg506 cleavage, we performed a kinetic analysis by usingnonlinear regression analysis of initial rates of FVa inactivation by APC (initial rates representingalmost exclusively cleavage at Arg506) at FVa concentrations of 0.2–30 nM, in both the absence and presence of 25 IUÆmL)1 heparin (Fig 4) In the FVa assay, a lower concentration of FXa (0.5 nM) was used than in the standard FVa assay, in order to minimize the FXa cofactor activity of the reaction intermediate, FVaint This facilitated the determination of loss of FVa cofactor activity duringthe initial stage of inactivation and minimized the influence of the k
Table 1 Apparent second-order rate constants for the APC-catalyzed inactivation of FVa and FVaLeiden in the presence and absence of heparin Rate constants ( M )1 Æs)1) for inactivation of normal FVa (0.50–1.5 n M ) and FVa Leiden (0.50–1.5 n M ) catalyzed by recombinant wild-type APC (0.037– 1.0 n M ) obtained by fittingtime courses of inactivation, such as presented in Fig 1, to an integral time course equation as described in Materials and Methods Reactions were performed in 25 mm Hepes (pH 7.5), containing150 m M NaCl, 3 m M CaCl 2 and 5 mgÆmL)1BSA in the presence of
25 l M phospholipid vesicles (10 : 90 DOPS/DOPC, mol/mol) at 37 C in the absence or presence of 25 IUÆmL)1UFH FVa activity was assayed as described in Materials and Methods Rate constants given are means from at least three experiments, with SEM being < 20% Values for k¢ 306 for inactivation of normal FVa by APC were obtained from time courses of inactivation for FVa Leiden as described in Materials and methods ND, Not determined; k 506 and k 306 could not be determined in FVa Leiden because of the absence of the Arg506 cleavage site.
Rate constant
2.17 · 10 6
6.83 · 10 5
2.17 · 10 6
Fig 2 Effect of varying heparin concentration on APC-mediated clea-vage at Arg506 in FVa Rate constants for cleaclea-vage at Arg506 in FVa
by wild-type APC were calculated for inactivations in the presence of various concentrations of UFH Inactivations were performed and analyzed as described in Materials and methods Data points represent means ± SEM from three independent time courses of inactivation.
Trang 6cleavage The kinetic parameters obtained were Km-app¼
2.13 ± 0.49 nM (mean ± SEM) and kcat-app¼
0.68 ± 0.048 s)1in the absence of UFH, and Km-app¼
11.4 ± 1.49 nM and kcat-app¼ 0.26 ± 0.016 s)1 in the
presence of UFH, which correspond to second-order rate
constants for cleavage at Arg506 (kcat/Km) of
3.2· 108M )1Æs)1and 2.3· 107M )1Æs)1, respectively These
values are in reasonable agreement with the rate constants
obtained from fittingthe time courses of FVa inactivation
(cf Figure 1, Table 1) These data suggest that, under the
conditions tested, heparin increases the Km of APC for
cleavage of FVa at Arg506 5.5-fold and at the same time
decreases the kcat 2.6-fold, thus actingas a mixed-type
inhibitor of APC As this analysis was performed using
initial rates for inactivation of FVa that are almost
completely due to cleavage at Arg506, no individual kinetic
parameters can be deduced for the second cleavage at
Arg306
Effects of heparin on FVa inactivation in the presence
of DEGR-FXa and protein S
To further verify that heparin inhibits cleavage at Arg506
and stimulates initial cleavage at Arg306, inactivation of
FVa by APC with and without heparin was performed
in the presence and absence of 20 nM DEGR-FXa, which is known to completely and specifically block APC cleavage at Arg506 [14] The presence of DEGR-FXa resulted in abrogation of the fast phase of the reaction,
as compared with inactivation in the absence of DEGR-FXa Addition of 25 IUÆmL)1 UFH in the presence of DEGR-FXa stimulated the APC-mediated inactivation approximately twofold (data not shown), indicatingthat the stimulatory effect of heparin on the cleavage at Arg306 is also observed in the presence of FXa Theoretically, the addition of heparin may induce the formation of a binary FXa–FXa complex, thus abolish-ingthe inhibitory effect of DEGR-FXa on the Arg506 cleavage This possibility was excluded, however, because time courses of FVa inactivation in the presence of DEGR-FXa could be fitted, both with and without heparin, to a mono-exponential equation, indicating single cleavage at Arg306 only The effect of UFH on FVa inactivation by APC was also investigated in the presence of 200 nM protein S The resultingtime course
of inactivation of FVa by APC became monophasic, an effect known to be due to stimulation of the cleavage at Arg306 [14] Addition of 10 IUÆmL)1heparin resulted in
a somewhat slower inactivation rate (data not shown), which presumably is due to the inhibitory effect of UFH
on the cleavage at Arg506
Fig 4 Kinetic analysis of the inactivation of FVa by APC in the pres-ence and abspres-ence of heparin Initial rates of FVa inactivation were determined at various concentrations of FVa in the presence (d) or absence (d) of 25 IUÆmL)1UFH, after incubation with 0.14 n M APC (d) or 0.04 n M APC (s) The incubation was performed in 25 m M Hepes (pH 7.5), containing150 m M NaCl, 3 m M CaCl 2 and
5 mgÆmL)1 BSA in the presence of 25 l M phospholipid vesicles (10 : 90 DOPS/DOPC, mol/mol) at 37 C After different time inter-vals the FVa activity was determined as described in Materials and methods Initial rates of FVa inactivation are expressed as n M FVa inactivatedÆmin)1Æ(n M APC))1 The solid lines represent a fit of the data accordingto the Michaelis–Menten equation with K m-app ¼ 11.4 n M and k cat-app ¼ 0.26 s)1in the presence of UFH (d) and K m-app ¼ 2.1 n M and k cat-app ¼ 0.68 s)1in the absence of UFH (s).
Fig 3 Western blotting of FVa degradation To identify fragment
generation during APC-catalyzed inactivation of FVa, plasma purified
human FVa (10 n M ) was incubated with 25 l M phospholipid vesicles
(10 : 90 DOPS/DOPC, mol/mol) at 37 C in 25 m M Hepes (pH 7.5),
containing150 m M NaCl, 3 m M CaCl 2 and 5 mgÆmL)1BSA In the
absence of heparin (lanes 1–5), inactivation was started by the addition
of 0.33 n M wild-type APC In the presence of 25 IUÆmL)1UFH (lanes
6–10), FVa inactivation was started by the addition of 3.9 n M
wild-type APC Samples were withdrawn from the inactivation mixture and
subjected to SDS/PAGE Subsequently, proteins were blotted on a
poly(vinylidene difluoride) membrane usinga semidry blotting
apparatus After transfer to poly(vinylidene difluoride) membranes,
heavy chain fragments were visualized using a monoclonal antibody
(AHV 5146) directed against the FVa heavy chain Lanes 1–5 and 6–10
describe reaction samples of similar FVa activity with the percentages
of residual FVa cofactor activity being(no heparin, lanes 1–5) 100, 37,
20, 10 and 2 and (25 IUÆmL)1UFH, lanes 6–10) 100, 36, 17, 9 and 2,
respectively.
Trang 7Lack of heparin effect on FVa inactivation by
APC mutants deficient in heparin binding
To verify that the inhibition of APC-mediated FVa
inactivation by heparin is related to the ability of heparin
to bind APC, we used recombinant mutants of APC
harboringmutations in the heparin-bindingsite The APC
variants chosen were: K37S/K38Q/K39Q with strongly
reduced affinity for heparin; K62N/K63D which has a
modest effect on the APC–heparin interaction; K37S/
K38Q/K39Q/K62N/K63D which shows no detectable
bindingto heparin [21] Table 2 shows the apparent
second-order rate constants obtained from time courses of
inactivation of FVa by the various APC variants Results
obtained in the absence of heparin were consistent with
those on record [15] APC variants carryingmutations in
the 37 loop had much lower rate constants for cleavage at
Arg506, whereas the rate constants for cleavage at Arg306
were similar to those obtained with wild-type APC The
mutations in loop 60 did not affect k506 and showed a
modestly increased k¢306 (Table 2) In the presence of
heparin, the abilities of wild-type APC and the 60-loop
variant to cleave the Arg506 site in FVa were strongly
inhibited, with reduction of k506of 11.7-fold and 8.4-fold,
respectively, whereas only minor effects were seen on the
cleavage at Arg306 The addition of heparin during
inactivation of normal FVa by the loop-37 mutant
resulted in a further decrease in k506 and a small
stimulation in the secondary cleavage at Arg306 (k306)
However, heparin did not influence Arg506 cleavage in
FVa by the APC variant that completely lacked the
heparin-bindingcapacity (37+60 loop), but a small
(inhibitory) effect on k¢306and k306was noted
These observations suggest that, for heparin to exert its
inhibitory effect on the cleavage at Arg506, a normal
interaction with APC is required It is likely that the
heparin-bindingloop 37 of APC interacts directly with FVa
in an area adjacent to the Arg506 cleavage site and that this
interaction is severely hampered by heparin in the ternary
heparin–APC–FVa complex
Electrostatic potentials of APC and the A domains of FVa
Loops 37, 60, 70 and 148 in APC form an electropositive
surface, while the catalytic triad is located in an
electronegative environment (Fig 5) In FVa, both Arg306 and Arg506 are in electropositive regions How-ever, movingtowards the C-terminus, the prime side of Arg306 is first positive then neutral and finally positive, while the prime side of Arg506 is electronegative (Fig 5)
At this site, two electronegative zones that possibly interact with APC involve residues Asp513, Asp578 and Asp577 (site 1) and Glu323, Glu374, Asp373, and Glu372 (site 2) A very negatively charged segment following Cys656, which is the last residue in the homology model based on ceruloplasmin, formed by Asp659-Asp660-Asp661-Glu662-Asp663 may also play a role duringthe dockingof APC on to the FVa Arg506 site In contrast, the segment containing Arg306 protrudes outside the surface of FVa, and, besides the residues directly fitting into the catalytic groove, there are no obvious interacting regions for APC
Docking heparin-like molecule onto APC
As numerous problems have been noticed when tryingto dock heparin at the surface of a protein [41], we used three different methods To facilitate calculations, a negatively charged peptide mimicking the overall charge distribution and shape (see Materials and Methods) of a heparin molecule was created and the validity of this approach was tested The validated protocol was next applied to dock the heparin-like peptide on to APC The lowest conformation energies positioned the long axis of the peptide along a small electropositive groove formed by loops 37 and 70 (Fig 6) These orientations are compatible with known experimental data suggesting that APC loops 37, 60 and 70 are directly or indirectly involved in heparin binding[21] In our structural model, loop 60 had no direct contact with the negatively charged peptide, but the distance between Lys62 and Lys63 and negative groups on the peptide (6–8 A˚) was compatible with electrostatic interactions and preorientation of heparin
on the APC surface duringformation of an encounter complex However, it is important to note that direct contact could occur between the heparin-like peptide and loop 60 if flexibility had been allowed
In the second dockingapproach, APC was maintained rigid during the simulation but a real heparin molecule (10 sugar units, length 40 A˚) was used and flexibility was tolerated (Fig 6, inset) The top ranking conformation
Table 2 Apparent second-order rate constants for the inactivation of FVa and FVa Leiden catalyzed by several recombinant variants of APC in the presence and absence heparin Rate constants ( M )1 Æs)1) for inactivation of normal FVa (0.80 n M ) and FVa Leiden (0.80 n M ) catalyzed by recombinant wild-type APC (0.090–0.60 n M ) obtained by fittingtime courses of inactivation, such as presented in Fig 1, to an integral time course equation as described in Materials and Methods Reactions were performed in 25 m M Hepes (pH 7.5), containing150 m M NaCl, 3 m M CaCl 2 and 5 mgÆmL)1 BSA in the presence of 25 l M phospholipid vesicles (10 : 90 DOPS/DOPC, mol/mol) at 37 C in the absence or presence of 25 IUÆmL)1UFH FVa activity was assayed as described in Materials and Methods Rate constants given are means from at least two experiments.
APC variant
1.65 · 10 8
3.86 · 10 6
3.38 · 10 6
1.97 · 10 7
2.48 · 10 6
a
The value for k¢ 306 in the presence of heparin in FVa Leiden , could not be reliably measured, implyingit is < 4 · 10 5
M )1 Æs)1.
Trang 8positioned heparin against loops 37, 60 and 70 In this case,
heparin also had direct contact with positively charged
residues located on loop 60 The last approach followed a
protocol used for virtual ligand screening, and also
positioned the short sugar molecules in between loop 37
and loop 70 of APC
Docking of APC on to FVa
To elucidate molecular interactions between APC and FVa
at cleavage sites Arg306 and Arg506, two theoretical dockingprotocols were used [30,38] Because the segment containingArg306 and Arg506 had to fit into the APC
Fig 5 Molecular models of FVa and APC demonstrating positions of potentially important residues for the APC–FVa interaction Top left: 3D structure of Gla-domainless APC [27] shown with a view down the active site The catalytic triad (from left to right), D102, H57 and S195, is colored red The light chain structure (in white) only includes epidermal growth factor (EGF)1 and EGF2 domains (residues 49–146) The SP domain (yellow) runs from residues 16–244 (chymotrypsinogen nomenclature) Positively charged residues in loops 60, 37 and 70 play a key role in heparin binding Only the loops 37, 70 and 148 have been shown to form a binding exosite for FVa important for cleavage at Arg506 but not Arg306 Other regions may be important for interactions with FVa but are not defined at present.Top right: molecular surface of APC color-coded according to its electrostatic potentials (red, regions of negative potentials; blue, regions of positive potentials; white, neutral potential; a linear interpolation was used to produce the color for surface potentials between )3 and +3 kTÆe )1 ) Bottom left: 3D model for the three A domains of FVa The A1 domain is colored yellow, the A2 white, and the A3 green The position of the C domains is not clearly defined at present, and a negatively charged segment at the end of the A2 domain is missing Two cleavage sites for APC (Arg306 and Arg506) are highlighted Bottom right: molecular surface
of FVa domains A1, A2, and A3 color-coded accordingto electrostatic potentials Preliminary dockingof FVa Arg506 into the catalytic cleft of APC suggests that the 37 loop may interact with the FVa D578 area (see text) Docking of FVa Arg306 into APC does not suggest significant contact between the 37 and 148 loop exosites, and the 37 loop may point towards relatively neutral regions (see text).
Trang 9active site, and because we used essentially rigid-body
docking, several conformations of the loops displaying
Arg306 and Arg506 of FVa as well as the APC loop 148
were generated before docking computations Test dockings
were performed, and a theoretical FVa model with
struc-tural changes at the level of loop 306 and 506 was selected
for further dockingsimulation (data not shown) Similarly,
a model of APC in which the loop 148 partially covers the
active site as compared with the structure present in the
PDB file 1aut was selected (Fig 6)
When usingthe dockingmethod of Norel et al [38], we
noticed that, in the best-ranked complex, APC was
positioned very close to the Arg506 site in a conformation
basically suitable for cleavage at this site Some interactive
reorientations of APC were needed in order to remove steric
clashes This was performed usingthe orientation of a serpin
reactive loop crystallized into a serine protease as guideline
[40] Interestingly, in none of the predicted complexes was
APC positioned next to the Arg306 site, suggesting that the
shape complementarity in this region is not optimal With
the ICM method, models of the complex with APC docked
at FVa position 506 or 306 could be generated that are in
agreement with published experimental data To develop
our two final models of the FVa–APC complex, we merged
the two sets of computations reported above and performed
limited interactive reorientations of the two proteins to remove minor steric clashes (Fig 7)
When APC is docked at position 506, the electropositive loops 37, 70 and 148 of APC seem to have contact with the electronegative area in FVa formed by residues Asp513, Asp578 and Asp577 APC could also interact with FVa residues Asp659-Asp660-Asp661-Glu662-Asp663, but these residues are not present in the model as it was only possible
to predict the structure of the FVa A2 domain up to residue
656 FVa residues Glu323, Glu374, Asp373 and Glu372 could facilitate the dockingprocess, but the role of these residues is unknown In the present model, APC loop 60 does not seem to have significant contact with FVa When superimposingthe APC–heparin complex on to the APC– FVa complex, we observed that heparin clashes against FVa and/or could be very close to negatively charged FVa residues Asp513, Asp578 and Asp577 and/or Asp659-Asp660-Asp661-Glu662-Asp663 (Fig 7A) Con-tact between FVa and APC loop 148 only occurs when the 148 loop is in a partially closed conformation In the PDB file 1aut, the 148 loop is open and with this conformation, very limited direct contacts with FVa were noted
When APC is docked at position Arg306, only the electropositive segment containing FVa Arg306 seems to
Fig 6 APC–heparin docking and loop 148 predictions APC is shown with an orientation similar to the one presented in Fig 5 A neg-atively charged peptide mimicking heparin was docked on to the APC X-ray crystal structure, and the lowest-energy conforma-tions positioned the negatively charged ribbon
in direct contact with loops 37 and 70 This positioningis compatible with known experi-mental data Loop 148 is not well defined in the X-ray crystal structure, and different con-formations were generated The loops that tend to be closed above the active site have lower energies The very open conformation present in the APC PDB file represents only one possible structure; the loop probably oscillates between open and closed confor-mations as observed for the equivalent loop of thrombin Inset: APC is presented with the peptide mimickingheparin (ribbon) docked
on to its surface and with one of the lowest conformation energy obtained from the dockingof a flexible heparin molecule.
Trang 10Fig 7 Proposed models of APC docked at
Arg506 and Arg306 The 3D structure of APC
was docked on to FVa at position Arg 506 (A)
or at position Arg306 (B) FVa is shown as a
solid surface with the different A domains
color coded as in Fig 6 (bottom left) APC is
presented as a ribbon Different loops are
colored and labeled for orientation Some
residues expected to play a role in the
inter-action are mentioned (see text) The peptide–
heparin-like 3D structure was extracted from
our dockingsimulations and positioned on
top of APC When APC is docked at Arg506,
heparin seems to disturb the interaction,
whereas when APC is at Arg306, heparin
has enough room and could bridge the two
molecules (see text).