Báo cáo khoa học: Factor XII binding to endothelial cells depends on caveolae ppt

To analyze whether these putative receptors accounted for the binding of FXII to human umbilical vein endothelial cells HUVEC, it was recently shown that fluorescein isothiocyanate FITC-l

Factor XII binding to endothelial cells depends on caveolae

Inger Schousboe1, Peter Thomsen2and Bo van Deurs2

1 Department of Medical Biochemistry & Genetics, and 2 Structural Cell Biology Unit, Department of Medical Anatomi,

The Panum Institute, University of Copenhagen, Denmark

It is now generally accepted that factor XII (FXII) binds to

cellular surfaces in the vascular system One of the suggested

receptors of this binding is the

glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR)

harbored in caveolae/lipid rafts However, binding of FXII

to human umbilical vein endothelial cells (HUVEC) has

never been shown to be localized to these specialized

mem-brane structures Using microscopical techniques, we here

report that FXII binds to specific patches of the HUVEC

plasma membrane with a high density of caveolae Further

investigations of FXII binding to caveolae were performed

by sucrose density-gradient centrifugations This showed

that the majority of FXII, chemically cross-linked to

HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in noncaveolae lipid rafts Accordingly, cholesterol-depleted cells were found to bind significantly reduced amounts of FXII These observations, combined with the presence of a minority of u-PAR in caveolae concomitant with FXII binding, indicate that FXII binding to u-PAR may be secondary and depends upon the structural elements within caveolae Thus, FXII binding to HUVEC depends on intact caveolae on the cellular surface Keywords: factor XII; HUVEC; lipid rafts; caveolae; u-PAR

Factor XII (FXII) is a zymogen present in plasma at a

concentration of  350 nM At local increases in the

Zn2+ concentration above the normal plasma level of

25 lM, FXII binds to endothelial cells along with plasma

prekallikrein The latter becomes attached to the cells via

complex formation with high-molecular-weight kininogen

(HK) [1] Binding of FXII, as well as plasma

prekallik-rein in complex with HK, initiates in vitro a reciprocal

activation of FXII and PK, which experimentally can

activate plasminogen, and the coagulation and

comple-ment systems So far it has not been possible to show

that FXII has an impact in any of these systems in vivo,

and its biological role remains elusive By affinity

chromatography and antibody inhibition, receptors for

HK have been identified as cytokeratin 1 [2],

urokinase-like plasminogen activator (u-PAR) [3], and the receptor

for the globular head of the subunit C1q in complement

C1 (gC1qR, also known as p33) [4,5] The binding of

FXII to immobilized, purified gC1qR in competition with

HK tentatively suggested that FXII and HK share the gC1qR as a common receptor [1,5] Incubation of FXII with prekallikrein and HK in the presence of gC1qR leads to an FXII-dependent conversion of prekallikrein to kallikrein The observation that the same conversion was observed when gC1qR was exchanged with cytokeratin 1, suggested that cytokeratin-1 could also be a receptor for FXII [6] To analyze whether these putative receptors accounted for the binding of FXII to human umbilical vein endothelial cells (HUVEC), it was recently shown that fluorescein isothiocyanate (FITC)-labeled FXII inter-acts with the multiprotein assemply of not only gC1qR and cytokeratin 1, but also u-PAR [7] u-PAR is a glycosylphosphatidylinositol (GPI)-linked glycoprotein, which plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation It is harbored in caveolae/lipid rafts [8] This prompted us to analyze, by microscopical techniques and sucrose gradient centrifugation, whether FXII binding to HUVEC is dependent on caveolae/lipid rafts

Materials and methods

Activated FXII (FXIIa) was obtained as a lyophilized powder from Enzyme Research Laboratories

Netherlands) It was stored at 4
C until dissolved and then stored in aliquots at )80
C FXII was purchased from Haematologic Technologies

USA), as a high-concentration solution in 50% (v/v) glycerol, and stored at )20
C as recommended by the manufacturer It migrated as a single band, with an

Mr of 80 000, on reducing SDS/PAGE SDS polyacryl-amide gels (4–12%) were from Invitrogen (Taa-Stru¨p, Denmark) All dilutions of FXII were performed in siliconized test tubes and excess dilutions were discarded

Correspondence to I Schousboe, Department of Medical Biochemistry

& Genetics, The Panum Institute, University of Copenhagen,

Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark.

Fax: + 45 35367980, Tel.: + 45 35327800,

E-mail: schousboe@imbg.ku.dk

Abbreviations: DTSSP, 3,3¢-dithio-bis(succinimidylpropionate); FXII,

Factor XII; FXIIa, activated FXII; GPI,

glycosylphosphatidylino-sitol; HK, high-molecular-mass kininogen; HRP, horseradish

peroxidase; HUVEC, human umbilical vein endothelial cells;

MbCD, methyl-b-cyclodextrin; u-PAR, urokinase-like

plasminogen activator receptor.

Enzyme: activated factor XII (EC 3.4.21.38).

(Received 2 December 2003, revised 5 May 2004,

accepted 27 May 2004)

Poly(vinylidene difluoride) membranes were obtained

from Amersham Biosciences AB, and the

chemiluminis-cence enhancer (SuperSignal West Femto Maximum

Sensitivity Substrate) and the crosslinking reagent

3,3¢-dithio-bis(succinimidylpropionate) (DTSSP) were obtained

from Pierce Methyl-b-cyclodextrin (MbCD) was from

Sigma The primary antibodies used were affinity-purified

goat anti-(human FXII) IgG from Affinity Biologicals

Corp

3 (Ancaster, Ontario, Canada), affinity-purified

rab-bit anti-(caveolin-1) IgG from Transduction Laboratories

4

(San Diego, CA, USA), mouse anti-(human CD31)

immunoglobulin, clone JC70A (M0823), from

Dako-Cytomation

rabbit anti-(u-PAR) immunoglobulin (399R) from

American Diagnostics

immunofluorescence, the secondary antibodies were Alexa

488-conjugated chicken anti-(goat IgG) and

TRITC-conjugated swine anti-rabbit IgG from Transduction

Laboratories For Western blotting, the secondary

anti-bodies were horseradish peroxidase (HRP)-conjugated

swine anti-rabbit

biotin-ylated rabbit anti-goat immunoglobulins (E 0466) and

biotinylated goat anti-rabbit immunoglobulins (E 0432)

from DakoCytomation Stabilized HRP-conjugated

goat anti-(mouse IgG) Ig and RestoreTM Western blot

stripping buffer were from Pierce All the biotinylated

antibodies had been solid-phase absorbed to minimize

cross-reactions with human immunoglobulins and fetal

bovine serum HRP-conjugated streptavidin (P 0397)

was from DakoCytomation and molecular mass

mark-ers from Bio-Rad H-D-Pro-Phe-Arg-para-nitroaniline

(S-2302) was from Chromogenix (Milan, Italy)

other reagents were of the purest grade commercially

available

Endothelial cell culture

Cryopreserved primary cultures of HUVEC (Clonetics

Diego, CA, USA) were subcultured as described previously

[9] Seven/eight generation cells (third passage) were used

throughout the experiments For gradient centrifugation the

cells were plated in 75 cm2flasks, and for activity

measure-ments the cells were plated in 12-well microtiter plates

Microscopic analysis was performed on cells plated on

Labtek 4-chamber slides (Nalgene Nunc International

Corp.) at a density of 104 cellsÆcm)2 The medium was

changed on day 3 and the cells used on days 4 or 5 To

eliminate any influence from FXII present in the complete

medium, cells were exposed to serum-free medium (medium

lacking fetal bovine serum)

application, when indicated

Cholesterol depletion and cholesterol determination

Cells were grown in serum-free medium for 3 h and

subsequently in the same medium containing 1% (w/v)

MbCD for 30 min or the period of time indicated The

cholesterol concentration in lysates of native and

choles-terol-depleted cells was determined

spectrophotometri-cally by a cholesterol/peroxidase assay [10] Protein

concentration was determined by the method of

Bradford [11]

Binding of FXII to cells The culture medium was aspirated and the cells were washed twice over a period of 20 min with Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, 5 mM Hepes,

pH 7.4) containing 15 lM ZnCl2 (wash buffer) followed

by a 15 min incubation in 0.1% (w/v) gelatin in wash buffer (block buffer) As judged visually by microscopic inspection, the washing procedure did not remove cells from the surface, or change the morphology of the cells The block buffer was aspirated and the cells incubated at room temperature with FXII or FXIIa diluted at least 300-fold in block buffer, giving a final concentration of 100–300 nM After 60 min of incubation, the cells were placed on ice, the incubation medium above the cells was aspirated and the cells were washed continuously for 5 s in a mild stream of ice-cold wash buffer and subsequently handled as described below

Amidolytic activity of cell-bound FXIIa

As it has previously been shown that FXIIa binds to the cells in a manner identical to FXII [9], binding of FXIIa was used to quantify relatively the amount of FXII bound to cholesterol-depleted cells Normal and cholesterol-depleted cells in 12-well microtiter plates were incubated with FXIIa,

as described above, and analyzed for the amidolytic activity

of cell-bound FXIIa, as previously described [12] To each well was added 600 lL of 0.8 mM S-2302 in 50 mMTris,

12 mM NaCl, 10 mM EDTA, pH 7.8 From previous experiments it was known that FXIIa cleavage of S-2302

on the surface of HUVEC was linear, with time, for at least

4 h After this period of time, the reaction was stopped by acidification and the absorbtion read at 405 nM

Electron microscopy HUVEC cells were fixed with 2% (v/v) formaldehyde and 0.1% (v/v) glutaraldehyde in 0.1M sodium phosphate buffer, pH 7.2 The cells were washed, scraped off the flasks, pelleted, and postfixed with OsO4, contrasted en bloc with 1% (w/v) uranyl acetate, dehydrated in a graded series of ethanols, and embedded in Epon Sections were examined

in a Philips CM 100 electron microscope (Philips, Eindh-oven, the Netherlands)

Immunofluorescence microscopy For immunofluorescence microscopy of cell-bound FXII and its colocalization with caveolin-1, the cells were fixed with)20
C methanol subsequent to washing after incuba-tion with FXII Methanol was chosen as a fixative because preliminary results had shown that glutaraldehyde fixation destroyed the immunogenicity of FXII The fixed cells were blocked in block buffer [5% (w/v) goat serum (DakoCyto-mation) in NaCl/Pi(PBS)] and incubated with primary and fluorescent secondary antibodies The primary antibodies were a mixture of goat anti-(human FXII) IgG (diluted

1 : 50) and rabbit anti-(caveolin-1) IgG (diluted 1 : 400) The secondary antibodies were a mixture of Alexa 488-conjugated chicken anti-(goat IgG) Ig (Molecular Probes)

(diluted 1 : 400) and TRITC-conjugated pig anti-(rabbit

IgG) Ig (DakoCytomation) (diluted 1 : 50), respectively

The cells were mounted with prolong anti-fade medium

from Molecular Probes Specificity analyses of the

anti-bodies showed no reaction of the secondary antianti-bodies with

cells incubated in the absence of the primary antibodies

Confocal microscopy

The microscope used was a Zeiss LSM 510 confocal

microscope The objectives were a plan-neofluor·20/0.5, a

C-apochromate ·40/1.2 W corr, and a Plan apochromat

·100/1.4 oil Iris lens The 488 nm line from an Argon laser,

and the 543 and 633 lines from two Helium/Neon lasers,

were used for excitation

Sucrose gradient centrifugation

Protein separation on a sucrose gradient was performed as

described by Spisni et al [13] Briefly, one 75 cm2 flask

containing a confluent layer of HUVEC was incubated with

FXII, and FXII adhered to the cell surface was crosslinked

to the receptor with DTSSP, as described above To avoid

disruption of caveolae/lipid rafts during lyses of the cells,

this and the following procedures were performed on ice,

unless stated otherwise After scraping off and collecting the

cells by centrifugation, the cell pellet was homogenized by

trituration (an 0.80· 80 mm needle) and the cells were

lysed for 30 min in 1 mL of ice-cold 25 mM Pi, 150 mM

NaCl, 5 mM EDTA (PNE)/TX-buffer [PNE buffer

11

containing 1% (v/v) Triton X-100, 0.1 mM

phenyl-methanesulfonyl fluoride and 0.22 mgÆmL)1 leupeptin)

Nuclei and cell debris were removed by centrifugation

(5000 g, 5 min) The supernatant was mixed (1 : 1) with

80% (w/v) sucrose in PNE buffer and placed at the bottom

of an ultracentrifuge tube A linear sucrose gradient [5–35%

(w/v) in TNE buffer) was layered on top of the lysate The

gradient was centrifuged for 20 h at 200 000 g (4
C) in an

SW 40 Ti Beckman rotor Fractions of 600 lL were

withdrawn from the bottom of the gradient and mixed 1 : 1

(v/v) with 40% ice-cold trichloroacetic acid After 2 h of

incubation (at 4
C), precipitated protein was collected by

centrifugation, the supernatant aspirated and the precipitate

washed in 1 mL of ether/ethanol (1 : 1, v/v) Precipitated

proteins were dissolved by boiling for 5 min in reducing

Laemmli buffer

SDS/PAGE and immunoblotting

For Western blot analysis, proteins were separated on

4–12% SDS/polyacrylamide gels and transferred to

poly(vinylidene difluoride) membranes according to

stand-ard procedures Standstand-ard samples of caveolin-1 and FXII

and molecular mass markers were run simultaneously After

incubation for 1 h with NaCl/Tris (TBS) block buffer

[50 mMTris, 0.15 mMNaCl, pH 8.0, containing 0.1% (v/v)

Tween 20 and 0.1% (w/v) BSA], the membranes were

probed with goat anti-FXII immunoglobulin (diluted

1 : 10 000)/biotinylated rabbit anti-goat

(diluted 1 : 10 000)/HRP-conjugated streptavidin (diluted

1 : 10 000) and rabbit anti-(caveolin-1) immunoglobulins

(diluted 1 : 10 000)/HRP-conjugated swine anti-rabbit

immunoglobulin (diluted 1 : 10 000), respectively The blot probed with antibodies against FXII was subsequently stripped using RestoreTM Western blot stripping buffer, used according to the manufacturer’s instructions, and probed against rabbit anti-(u-PAR) immunoglobulin (diluted 1 : 10 000)/HRP-conjugated swine anti-rabbit Ig (diluted 1 : 10 000) or mAb CD31 (diluted 1 : 1000)/HRP-conjugated goat anti-(mouse IgG) Ig (diluted 1 : 10 000) Dilutions of antibodies were performed in 1% nonfat skim milk in TBS block buffer Detection was carried out using the chemiluminescence enhancer, SuperSignal West Femto Maximum Sensitivity Substrate, as recommended

by the manufacturer, and the results were monitored on a Las Chemiluminator The intensity of the bands was measured using the Image Gauge, quant menu

Statistics Analysis of variance (ANOVA) with the post hoc Student’s t-test was used to determine the statistical significance of difference between sample groups

Results

FXII binds to HUVEC caveolae Caveolae, 50–100 nm invaginations of the plasma mem-brane, are a subset of sphingolipid- and cholesterol-enriched lipid rafts, characterized by the presence of the protein caveolin [14] Caveolae are abundant in endothelial cells [15] In agreement with this, HUVEC contained numerous caveolae, as revealed by electron microscopy (Fig 1A,B) Interestingly, the caveolae were not evenly distributed in the plasma membrane of these cells, but appeared regionally at very high densities, separated by caveolae-free membrane segments This concentration of caveolae at certain regions

of the HUVEC surface was also visualized by confocal microscopy using an antibody against caveolin-1 Thus, an intense immunofluorescence signal was obtained in certain stretches of the plasma membrane (Fig 2A) Double-immunofluorescence labeling for caveolin-1 and bound FXII showed a very high degree of co-localization (Fig 2B,C), indicating that bound FXII was associated with caveolae

The integrity of caveolae depends on a certain level of plasma membrane cholesterol, and cholesterol depletion achieved by incubating cells with MbCD makes caveolae disappear and allows the released caveolin to become diffusely distributed in the plasma membrane and to be internalized [16, 17] To analyze further the apparent association of FXII binding with HUVEC caveolae, cells were therefore cholesterol-depleted by incubation with MbCD Incubation of HUVEC with MbCD [1% (w/v),

60 min] caused a reduction in cellular cholesterol to 20% of the level found in untreated cells By electron microscopy it could be shown that this treatment of HUVEC resulted in

an almost complete removal of caveolae Thus, the patches with high concentrations of caveolae were never seen in cholesterol-depleted cells and, only rarely, could single, caveolae-like structures be identified (Fig 1C) This was further confirmed by confocal microscopy (Fig 2C,E) The impact of MbCD treatment on the binding of FXII was

analyzed at varying MbCD concentration and treatment

periods By confocal microscopy, only little and diffusely

distributed FXII could be identified on HUVEC after

60 min of incubation with 1% (w/v) MbCD (Fig 2D) The

ability of MbCD-treated cells to bind FXII was analyzed

quantitatively by measuring the amidolytic activity of

FXIIa, which binds to the cells in a manner identical to

that of FXII [9] This way it was shown that progressively

decreasing amounts of FXIIa adhered to the cells with

increasing periods of exposure to 1% (w/v) MbCD The

amidolytic activity of FXIIa in the wells exposed for a

longer period of time than 30 min remained constant, but

significantly lower (P < 0.001) than the activity measured

in the wells not exposed to MbCD (Fig 3) No changes in

cell number or in protein content were identified during the

treatment

FXIIa binding associated with the Triton X-100 insoluble

low-density fraction

A common means of identifying proteins associated with

caveolae is to investigate the detergent insolubility at 4
C

To examine whether the FXII/receptor complex might be

insoluble in cold Triton X-100, FXII was crosslinked to the

receptor by the cleavable disulfide cross-linking reagent,

DTSSP This prevented dissociation of FXII from the receptor during the subsequent solubilization and ultracen-trifugation To minimize contamination of the surface of the plastic with nonspecifically bound FXII, the cells were scraped off before solubilization Figure 4 shows a Western blot of an SDS polyacrylamide gel of lysates of native and MbCD-treated cells incubated with block buffer in the presence or absence of FXII and subsequently exposed to DTSSP While no visible bands were observed in the lysates

of cells incubated in the absence of FXII, FXII was present

in the reduced lysates of cells incubated with FXII, regardless of whether these were native or treated with MbCD The intensity of the anti-FXII reacting band in the lysates from MbCD-treated cells was, however, apparently weaker than that from native cells To confirm that this was not caused by analytical variations, the blot was stripped and probed with anti-CD31 CD31 is a marker protein of endothelial cells It appeared as a clear band in reduced samples and as a smear in nonreduced samples of lysates of cells treated with DTSSP Measuring the intensity of the anti-FXII and the anti-CD31 reacting bands in the reduced samples on the same blot showed, in three individual experiments, that the FXII/CD31 in lysates of cells treated with MbCD was 50 ± 10% of that measured in lysates of native cells The invisibility of FXII in nonreduced samples

Fig 1 Caveolae in human umbilical vein

endothelial cells (HUVEC) cells, detected by

electron microscopy Caveolae in control cells

are shown in (A) and (B) (A) A section

perpendicular to the plasma membrane,

revealing the typical shape of these structures,

whereas (B) is a tangential section better

showing the high density of caveolae CP,

clathrin-coated pit (C) Section through a

cholesterol-depleted cell in which the arrow

indicates a single, caveolae-like structure.

Bar: 200 nm.

indicates a poor anti-FXII immunoglobulin reactivity of

FXII crosslinked to the receptor, or formation of very high

Mrcomplexes of FXII

The solubility, in cold Triton X-100, of the FXII/receptor

in lysates of cells was analyzed by sucrose gradient

centrifugation Figure 5A shows a Western blot of a

reducing SDS polyacrylamide gel of every second fraction

of the gradient consisting of 20 fractions An 80 kDa

molecular mass anti-FXII immunoglobulin-reacting band

was present in the soluble fractions (representing one of

three fractions), with the density of a 40% sucrose solution,

as well as in the higher density fractions of the floating

fractions with sucrose densities from 40 to 23% With

decreasing intensity throughout the gradient, all of the

undiluted fractions stained heavily when probed with

anti-caveolin-1 (results not shown) This indicated that caveolae

were distributed throughout the entire gradient The intensity of these bands, however, decreased with decreasing density of the gradient This was evidenced by probing for caveolin in samples diluted fivefold, which convincingly showed the presence of the highest concentration of caveolin-1 in the higher density of the floating fractions FXII was thus identified in the same fractions as those containing the highest concentrations of caveolin-1 This indicates that FXII originating from the disulfide FXII/ receptor complex was harbored in caveolae As a possible receptor for FXII, the localization of u-PAR (Mr50 000–

60 000) was analyzed In accordance with the presence of large vesicles rich in GPI-anchored proteins (lipid rafts),

as well as smaller caveolar vesicles lacking GPI-anchored proteins in Triton X-100 insoluble membranes [18], u-PAR could be identified mainly in the very-low-density part of the

Fig 2 Colocalization of cell-bound factor XII (FXII) and caveolin-1 Human umbilical vein endothelial cells (HUVEC) were grown in serum-free medium for 4 h Then, medium in half of the wells was exchanged with the same medium containing methyl-b-cyclodextrin (MbCD) and the incubation was continued for another 60 min The cells were then washed, incubated with 100 n M FXII, and subsequently fixed with methanol The fixed cells were first incubated with a mixture of goat FXII (1 : 50) and rabbit anti-(caveolin-1) (1 : 200) and second with a mixture of Alexa 488-conjugated chicken anti-(goat IgG) Ig (1 : 400) and TRITC-labeled swine anti-(rabbit IgG) Ig (1 : 50) (B) and (D) (green) show FXII, and (A), (C) and (E) (red) show caveolin-1 The panels are representative photomicrographs of at least three independent experiments Bar: 20 lm.

gradient (Fig 5A) Upon treatment with MbCD, the

density of caveolae, as well as lipid rafts, increased As a

result of this, the presence of caveolin-1 and FXII

disap-peared from their upper density fractions, while u-PAR was

skewed towards the higher density fractions (Fig 5B) The

weakly stained 60- and 70 kDa anti-FXII reacting bands,

noticeable throughout the gradients, were present with

varying intensity in all of the three experiments performed

and were considered to be artifacts

Discussion

By a combination of microscopy and gradient

centrifuga-tion, the present study shows that HUVEC-bound FXII

localizes to caveolae, and that this localization is dependent

upon the structural integrity of these elements in the plasma

membrane

It is generally accepted that FXII binds to HUVEC and

that this binding is receptor mediated Investigation of this

binding, by immunofluorescence microscopy of monolayers

of HUVEC incubated with FXII, indicated, in contrast to a

previous study [7], that FXII does not bind diffusely to the

cells but to specific patches in the plasma membrane

corresponding to caveolae-rich domains The reasons for

the discrepancy between this and the former study may be

several In the present study, native FXII was incubated

with morphologically intact HUVEC before fixation, while

in the previous study the cells were fixed before incubation

with FXII Furthermore, the FXII binding was, in the

former study, visualized by FITC labeling, whereby Lys

residues (of possible importance for the binding of FXII to HUVEC) might have been blocked

Caveolae are highly immobile plasma membrane invag-inations [17] abundant in the cellular membrane of endo-thelial cells [14] They are enriched in glycolipids and cholesterol Depleting the cells for cholesterol by treatment with MbCD disrupts the integrity of caveolae Cholesterol-deprived cells incubated with FXII showed microscopically only little binding of FXII This confirms that FXII binds to

a receptor harbored in caveolae Previous analyses have shown that MbCD preferentially extracts cholesterol from the outside and partially solubilizes GPI-anchored and

Fig 3 Quantification of factor XII (FXII) binding to cells preincubated

with methyl-b-cyclodextrin (MbCD) for varying periods of time Human

umbilical vein endothelial cells (HUVEC), grown in serum-free

med-ium, were, for varying periods of time, pre-exposed to 1% (w/v)

MbCD and subsequently incubated with 100 n M activated FXII

(FXIIa) for 1 h After washing, the cells were incubated with S-2302.

The ordinate indicates the absorbance measured at 405 nm after 4 h of

incubation The concentration of protein was measured in the

incu-bation mixture by the method of Bradford [11] The activity was

measured on three individual setups of cells and determined as the

mean value ± SD The significance of variance of FXIIa bound to

HUVEC, pre-exposed for varying periods of time to MbCD, was

calculated by analysis of variance ( ANOVA ) Use of an asterisk

a P-value of < 0.001 relative to the control (0 min incubation period),

determined using the post-hoc Student’s t-test.

Fig 4 Western blots of factor XII (FXII) cross-linked to membranes of native and cholesterol-depleted cells One of three culture flasks of human umbilical vein endothelial cells (HUVEC), grown in serum-free medium for 15 h, was pre-exposed to 1% (v/w) methyl-b-cyclodextrin (MbCD) for 30 min This flask (+MbCD +FXII) and a second (–MbCD +FXII) of the three were subsequently incubated with

100 n M FXII, while the third flask (–MbCD –FXII) was incubated with block buffer After this incubation, the cells were washed and incubated with DTSSP After neutralization of the DTSSP, the cells were lysed Western blots of reduced (+) and nonreduced (–) samples

15

were visualized by sequential incubation with goat anti-FXII, biotin-ylated rabbit anti-(goat IgG), horseradish peroxidase (HRP)-conju-gated streptavidin and SuperSignal West Femto Maximum Sensitivity Substrate (upper part of the figure) The blot was then stripped using RestoreTMWestern blot stripping buffer and incubated sequentially with mouse anti-(human CD31), HRP-conjugated goat anti-(mouse IgG) and SuperSignal West Femto Maximum Sensi-tivity Substrate (lower part of the figure).

transmembrane proteins [18] As it has been previously

shown that no differentiation exists between the binding of

FXII and FXIIa, the change in ability of MbCD-treated

HUVEC to bind FXII was quantified by measuring the

amount of bound FXIIa colorimetrically [9] This showed that a significantly lower amount of FXII was bound to MbCD-treated cells than to untreated cells However, treatment with MbCD did not completely prevent the binding of FXII to the layer of cells in the well In fact,

 50% of the binding persisted after MbCD treatment This was confirmed by density measurements of Western blots of FXII extracted from the cells As no FXII was observed on the fluorescence microscopy of MbCD-treated cells incu-bated with FXII, a considerable amount of the cell-bound FXII might have been removed during the extensive washing of the fixed cells accompanying the immunofluo-rescence staining

Binding of FXII to lipid-rich domains in the cellular membrane was further verified by sucrose gradient centrif-ugation While confocal microscopy and the activity measurements were performed on intact cells without crosslinking, the gradient centrifugations were performed

on lysates of cells to which FXII had been chemically crosslinked to cellular proteins in its immediate vicinity by incubation with the cleavable membrane-impermeable DTSSP Sucrose gradient centrifugation of cold Triton X-100 lysates of cells showed that FXII was present in the same fractions as the marker protein for caveolae,

caveolin-1 Both FXII and the majority of caveolin-1 were distributed in the soluble fraction, as well as in the higher density floating fractions of the gradient As it has previously been suggested that FXII binds to the GPI-anchored receptor, u-PAR [7], and this receptor is organized

in caveolae and lipid rafts on the cell surface [19], it was tempting to investigate this possibility further We showed that u-PAR, in contrast to FXII, originating from the FXII/ receptor complex, appeared to be present in mainly the lower density of the floating fractions and thus not only in the fractions containing the highest concentration of caveolin I and FXII In line with this, immunofluorescence microscopy of the cellular localization of u-PAR indicated that u-PAR was equally distributed on the plasma mem-brane surface (results not shown)

The difference in fractional distribution of u-PAR and FXII in the sucrose gradient indicates that FXII binding to u-PAR may be secondary to FXII binding to caveolae This

is in accordance with previous findings showing that FXII binding to HUVEC could be inhibited by antibodies to u-PAR, but not by the much smaller ligands of u-PAR, urokinase-like

13 plasminogen activator (u-PA), and vitronec-tin [7] It may also explain the biphasic nature of FXII binding to HUVEC [9]

The binding of FXII to caveolae indicates that FXII binds to a receptor predominantly localized in these specific structural elements of the cell membrane containing a subset

of sphingolipids and cholesterol Moreover, in addition to GPI-anchored proteins, caveolae harbor GPI-anchored proteoglycans (glypicans) Glypicans are highly sulfated

by substitution with heparan sulfate and chondroitin sulfate [20], creating a binding site for polycationic molecules [21] Among several glucosaminoglycans, both heparan sulfate and chondroitin sulfate have been shown to enhance the rate of FXII activation in solution [22] Combining the established knowledge about FXII binding to negatively charged surfaces in general, and proteoglycans in particular [23], with our present findings, we tentatively propose that

Fig 5 Analysis of Triton X-100 solubility by sucrose gradient

centri-fugation Native and methyl-b-cyclodextrin (MbCD)-treated cells, to

which factor XII (FXII) had been cross-linked, were extracted with

ice-cold Triton X-100 and subjected to sucrose gradient centrifugation, as

described in the Materials and methods Every second fraction of the

gradient

16 was analyzed for the distribution of FXII, caveolin-1 and

urokinase-like plasminogen activator receptor (u-PAR) by reducing

SDS/PAGE and Western blotting The distribution of FXII and

caveolin-1 was probed on individual blots, while the distribution of

u-PAR was probed on stripped blots (Fig 4) (A) Results obtained

using native cells; (B) results obtained with MbCD-treated cells The

distribution is representative of three individual experiments.

FXII binds to a glypican in caveolae Direct analysis of the

detergent-solubilized FXII/receptor complex is ongoing but

at the moment it must be concluded that the present

investigation was unable to confirm previous studies [7],

suggesting that u-PAR is the most likely receptor of FXII

Acknowledgements

The expert technical assistance of Ms Mette Olsen and Birgit Harder is

greatly appreciated The work was supported by grants from the

Danish Medical Research Council and the Novo Nordisk foundation.

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