thrombin-mediated impairment of fibroblast growth factor-2 activity

Thrombin-mediated impairment of fibroblast growthfactor-2 activityPierangela Totta1,*, Raimondo De Cristofaro2,*, Claudia Giampietri3, Maria S. Aguzzi1,Debora Faraone1, Maurizio C. Capogrossi1 and Antonio Facchiano1

1 Laboratorio di Patologia Vascolare, IDI-IRCCS, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico,

Rome, Italy

2 Institute of Internal Medicine, Haemostasis Research Center, Catholic University School of Medicine, Rome, Italy

3 Department of Histology and Medical Embriology, University of Rome ‘Sapienza’, Italy

Fibroblast growth factor (FGF)-2 belongs to the

23-member family of FGFs [1]. It is known as one of

the most potent angiogenic factors controlling embry-

onic development [2], tissue remodeling [3], stem cell

physiology [4] and tumor growth [5]. FGF-2 activity is

finely modulated at several levels, and recent evidence

shows that different FGF-2 concentrations may exert

opposing effects [6]. Studies have shown that the

interaction of several molecules with this growth factor

[7–9] or with its receptors [10,11] participates in the

control of FGF-2 activity. A few studies have

examined FGFs proteolytic degradation [12–15]. For

example, FGF-2 degradation by the zinc-endopro-

tease neprilysin has been recently demonstrated. This

Keywords

cell proliferation; digestion; fibroblast growth

factor-2; maturation; thrombin

Correspondence

A. Facchiano, Laboratorio di Patologia

Vascolare, Istituto Dermopatico

dell’Immacolata, IDI-IRCCS, Via Monti di

Creta 104, 00167 Rome, Italy

Fax: +39 06 6646 2430

Tel: +39 06 6646 2431

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 6 October 2008, revised 19

March 2009, accepted 6 April 2009)

doi:10.1111/j.1742-4658.2009.07042.x

Thrombin generation increases in several pathological conditions, including

cancer, thromboembolism, diabetes and myeloproliferative syndromes.

During tumor development, thrombin levels increase along with several

other molecules, including cytokines and angiogenic factors. Under such

conditions, it is reasonable to predict that thrombin may recognize new

low-affinity substrates that usually are not recognized under low-expression

levels conditions. In the present study, we hypothesized that fibroblast

growth factor (FGF)-2 may be cleaved by thrombin and that such action

may lead to an impairment of its biological activity. The evidence collected

in the present study indicates that FGF-2-induced proliferation and chemo-

taxis ⁄ invasion of SK-MEL-110 human melanoma cells were significantly

reduced when FGF-2 was pre-incubated with active thrombin. The inhibi-

tion of proliferation was not influenced by heparin. Phe-Pro-Arg-chlorom-

ethyl ketone, a specific inhibitor of the enzymatic activity of thrombin,

abolished the thrombin-induced observed effects. Accordingly, both

FGF-2-binding to cell membranes as well as FGF-2-induced extracellular

signal-regulated kinase phosphorylation were decreased in the presence of

thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by

thrombin at the peptide bond between residues Arg42 and Ile43 of the

mature human FGF-2 sequence. The apparent kcat ⁄Km of FGF-2 hydroly-

sis was 1.1 · 104 m)1Æs)1, which is comparable to other known low-affinity

thrombin substrates. Taken together, these results demonstrate that throm-

bin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity

in vitro, indicating that FGF-2 is a novel thrombin substrate.

Abbreviations

ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; HMW, high molecular weight; HUVEC, human umbilical vein

endothelial cell line; LMW, low molecular weight; PAR, protease-activated receptor; PPACK, Phe-Pro-Arg-chloromethyl ketone; TRAP,

thrombin receptor-activating peptide.

FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3277

metalloprotease was found to cleave the Leu135-

Gly136 peptide bond of FGF-2, severely inhibiting its

angiogenic activity, as demonstrated in a murine cor-

neal pocket angiogenesis model [12]. In addition, more

recently, high molecular weight (HMW) FGF-2 was

shown to be cleaved by thrombin, and its degradation

product stimulated endothelial cell migration and pro-

liferation in a similar manner to low molecular weight

(LMW) FGF-2 [13]. Moreover, fibrinogen and fibrin,

by a direct interaction, are known to protect FGF-2

from in vitro proteolytic degradation induced by tryp-

sin and chymotrypsin [16,17]. Finally, different FGF-2

fragments inhibit FGF-2 [18,19], further suggesting

that proteolytic processing of FGF-2 may represent an

endogenous way to modulate its activity.

Thrombin is a serine protease generated from its

zymogen precursor prothrombin after endothelial cell

damage and induction of the coagulation cascade [20].

Its activation is known to be increased in thrombo-

embolism [21], diabetes [22] and cancer [23]. Thrombin

pro-coagulant activity converts fibrinogen to fibrin

monomer, which then polymerizes to form the fibrous

matrix of blood clots [24]. Moreover, thrombin cleaves

protease-activated receptor (PAR)-1 and PAR-4, which

are expressed on human platelet membranes, activating

their hemostatic properties [25]. In addition to these

clot-promoting activities, thrombin down-regulates its

own generation through activation of the protein C

pathway. Activated protein C inactivates cofactors Va

and VIIIa, thereby blunting further thrombin genera-

tion [26]. Thrombin also participates directly in its

final inhibition and clearance from the circulation by

specifically recognizing the serine protease inhibitors

(serpins) antithrombin and heparin cofactor II [27].

Thrombin interaction with PAR-1 and PAR-4 does

not activate only hemostatic functions. Indeed, these

receptors are expressed on the membrane of different

cell types, including fibroblast [27], endothelial [28] and

cancer cells [29], and their activation enhances cytokine

release, cell permeability and cell growth. Furthermore,

thrombin recognizes several other membrane receptors

and substrates, such as platelet glycoprotein Ib and

glycoprotein V [30], and additional noncanonical

thrombin substrates, even in the intracellular compart-

ment, have been identified [31,32].

Taken together, these data indicate that thrombin

recognizes a complex substrates network with several

biological functions, in addition to the classical clot-

promoting effects, and prompted us to investigate

additional substrates not directly involved in blood

coagulation. In the present study, we show, for the

first time, that thrombin digests the 18 kDa LMW

isoform of human FGF-2, modulating its biological

activities such as in vitro cell proliferation and chemo-

taxis induction. In addition, we also identified the

cleavage site on FGF-2.

Results

FGF-2-induced proliferation of SK-MEL-110 is

inhibited by thrombin

Human metastatic melanoma cell line SK-MEL-110

was chosen as a model to test the FGF-2 mitogenic and

chemotactic activity in vitro. Figure 1A shows that 1 h

of thrombin pre-incubation significantly reduces FGF-

2-induced cell growth as a function of concentration in

48 h proliferation assays. The inhibitory action reached

a plateau at 0.1 nm thrombin; therefore, all the experi-

ments were carried out at this thrombin concentration,

which corresponds to 0.01 UÆmL)1. Figure 1A shows

that thrombin activity was blocked with the irreversible

selective thrombin inhibitor Phe-Pro-Arg-chloromethyl

ketone (PPACK). As a control, thrombin and PPACK,

alone or mixed, do not affect cell proliferation under

these experimental conditions (Fig. 1A, inset).

Time-course proliferation experiments were then car-

ried out. Pre-treating FGF-2 with thrombin for 1 h was

sufficient to completely inhibit the mitogenic effect of

FGF-2 at 48 and 72 h of proliferation (Fig. 1B). The

experiments shown in Fig. 1 were performed by pre-

incubating FGF-2 with active thrombin; next, before

exposing cells to this mixture, PPACK was added to

block the enzyme. This protocol was chosen to exclude

the possibility that thrombin enzymatic action may

interfere with cell growth by cleaving and activating

PARs receptors, which are known to be expressed in

SK-MEL-110 cells (data not shown) and in other mela-

noma cells [29]. To further rule out this possibility, we

exposed SK-MEL-110 cells to the action of specific

PARs agonists, namely thrombin receptor-activating

peptide (TRAP)-1 and TRAP-4, to show that specifi-

cally activating thrombin-receptors does not itself

determine any inhibition of cell-proliferation. Fig-

ure 2A shows that the specific agonists TRAP-1 alone

and TRAP-4 alone induced some proliferation of mela-

noma cells in the absence of other mitogenic stimuli,

whereas they did not affect the FGF-2-induced prolifer-

ation (Fig. 2B). These data allowed us to exclude the

possibility that thrombin agonism may per se inhibit

SK-MEL-110 proliferation in vitro. Therefore, to mimic

more closely the conditions occurring under in vivo

conditions, cells were directly exposed to the mixture

containing FGF-2 and active thrombin. Figure 3 shows

that, under these conditions, SK-MEL-110 proliferate

significantly less than cells exposed to FGF-2 alone and

FGF-2 is a thrombin substrate P. Totta et al.

3278 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS

that such inhibition was absent in the presence of inac-

tive thrombin (i.e. thrombin pre-incubated with

PPACK), further confirming that the enzymatic activity

of thrombin inhibits the mitogenic action of FGF-2.

To evaluate the influence of heparan sulfate proteogly-

cans on thrombin susceptibility of FGF-2, the effect of

heparin was investigated in proliferation assays. Hepa-

rin alone (at 10 and 50 nm) did not influence the spon-

taneous proliferation of SK-MEL-110 (Fig. 4A). Thus,

the effect of heparin (at 50 nm) was tested in the pres-

ence of FGF-2 and thrombin. Figure 4B shows that

heparin reduces the mitogenic activity of FGF-2; how-

ever, the inhibitory effect induced by thrombin was

maintained both in the absence and in the presence of

heparin, indicating that, at these doses, heparin does

not influence the observed thrombin–FGF-2 interplay.

As a specificity control, similar experiments were car-

ried out on a different cellular model. Figure 5 shows

that, similar to melanoma cells, the mitogenic effect of

FGF-2 was inhibited by thrombin (0.1 nm) in the

primary human umbilical vein endothelial cell line

(HUVEC). Taken together, these findings indicate that

the mitogenic activity of FGF-2 is controlled by throm-

bin and that FGF-2 may be a thrombin substrate.

FGF-2-induced SK-MEL-110 invasion/migration

on different matrices

We then investigated whether thrombin modulates the

ability of FGF-2 to induce cell invasion through differ-

ent matrices. Invasion assays were carried out in vitro,

in modified Boyden chambers, on filters coated either

with vitronectin, collagen IV or fibronectin. Migration

0 h

48 h25

50

75

100 A

B

Cel

l num

ber

(%)

+ + + – – – FGF-2

Thrombin (nM) 0.1

PPACK +

0.1 – – –

– – –

– –

–

0

25

50

75

100

Cel

l nu

mb

er (

%)

–

FGF-20.001Thrombin (nM) 0.01 1–

+ + + + +

0.1–

–

PPACK + + + ++

* *

** **

Cel

l nu

mb

er (

%)

0100

300

500

700

900

1100

1300

0 24 48 72 Time (h)

* ** BSA BSA + PPACK

Thrombin Thrombin + PPACK

FGF2 + PPACK FGF2 + Thrombin + PPACK

Fig. 1. Dose–response and time-course proliferation assays. (A)

Dose–response proliferation assay: SK-MEL-110 (4 · 104) cells

were seeded in six-well plates and grown for 24 h at 37 �C, 5%

CO2 in complete medium. Medium was then replaced and cells

were starved overnight with incomplete medium. Subsequently,

cells were stimulated for 48 h with medium 0.1% BSA, FGF-2

(10 ngÆmL)1, 0.6 nM) or thrombin (0.1 nM) pre-incubated for 1 h at

37 �C. Other cells were stimulated with medium 0.1% BSA con-

taining FGF-2 alone (10 ngÆmL)1, 0.6 nM), thrombin alone (0.1 nM)

or FGF-2 (10 ngÆmL)1, 0.6 nM) with different thrombin concentra-

tions (0.001, 0.01, 0.1 and 1 nM), pre-incubated for 1 h at 37 �Cand then supplemented with PPACK (50 nM) to block the enzymatic

activity of thrombin. The mitogenic effect of FGF-2 is significantly

reduced in the presence of different concentrations of thrombin

(*P < 0.005; **P < 0.001). Neither thrombin alone nor thrombin

with PPCAK nor PPACK alone influenced SK-MEL-110 growth with

respect to control (A, inset). Data are expressed as a percentage of

cell number versus cells treated with FGF-2 alone (100% corre-

sponds to 1.9 · 105 cells). Data reported are the mean ± SEM of

five independent experiments carried out in duplicate. (B) Time-

course proliferation assay: SK-MEL-110 (4 · 104) cells were seeded

and grown for 24 h in complete medium. Medium was then

replaced and cells were starved overnight with incomplete med-

ium. Subsequently, cells were stimulated for 24, 48 and 72 h with

the indicated stimuli, with doses as described in (A). FGF-2-induced

SK-MEL-110 proliferation is inhibited when FGF-2 is pre-incubated

with thrombin alone for 1 h and then thrombin is blocked with

PPACK (*P < 0.0005; **P < 0.05). Both thrombin alone and throm-

bin + PPACK do not influence SK-MEL-110 cell proliferation com-

pared to BSA and BSA + PPACK. Data are expressed as a

percentage of cell number versus cell number at t0 (100% corre-

sponds to 1.9 · 105 cells). Data reported are the mean ± SEM of

five independent experiments carried out in duplicate.

P. Totta et al. FGF-2 is a thrombin substrate


assays were also carried out using gelatin-coated filters.

The invasion ⁄ chemoattractant properties of FGF-2

were markedly and significantly reduced in the presence

of thrombin (0.1 nm, corresponding to 0.01 UÆmL)1)

on all tested matrices (Fig. 6A–D), with different

potency. We observed approximately 50% inhibition

on vitronectin (Fig. 6A), approximately 30% inhibition

on collagen IV and fibronectin (Fig. 6B,C) and approx-

imately 40% inhibition on gelatin (Fig. 6D). These data

indicate that thrombin strongly inhibits the chemotactic

and invasion properties of FGF-2.

FGF-2-dependent extracellular signal-regulated

kinase (ERK)/mitogen-activated protein kinase

phosphorylation is reduced by thrombin

Mitogen-activated protein kinases and ERK1 ⁄ 2 are

important transducers of mitogenic and differentiation

signals induced by FGF-2 and are often altered in mel-

anoma progression [33]. We therefore investigated

whether FGF-2 pre-incubation with thrombin alters the

ability to activate ERK1 ⁄ 2 in our experimental model.

Figure 7A shows that ERK1 ⁄ 2 phosporylation

induced by FGF-2 is reduced when FGF-2 is incu-

bated with thrombin. The lower band corresponding

to the 42 kDa form (i.e. ERK2) was found to be

reduced, as also revealed in densitometry analysis

(Fig. 7B), suggesting that thrombin incubation impairs

the ability of FGF-2 to signal toward one of the key

0 h48 h

–0

50

100

150

200A B

Cel

l nu

mb

er (

%)

0

50

100

150

200

Cel

l nu

mb

er (

%)

FGF-2TRAP-1TRAP-4

–+

–––

–––

–+–

*

+–+

–––

+––

++–

–––

FGF-2TRAP-1TRAP-4

0 h48 h

Fig. 2. TRAP-treatment of SK-MEL-110 cells. SK-MEL-110 (4 · 104) cells were seeded and grown for 24 h in complete medium as

described in Fig. 1. Medium was then replaced and cells were starved overnight with incomplete medium. Then, cells were directly exposed

(48 h) to FGF-2 (10 ngÆmL)1) in the presence or the absence of 5.7 lM TRAP-1 or TRAP-4. (A) TRAP-1 and TRAP-4 induced some prolifera-

tion as compared to control (*P < 0.05). The mitogenic effect of FGF-2 was not influenced by TRAP-1 or TRAP-4 (B). Data are expressed as

a percentage of cell number versus cells treated with FGF-2 (100% corresponds to 1.9 · 104 cells). The data reported are the mean ± SEM

of five independent experiments carried out in duplicate.

0

20

40

60

80

100

Cel

l nu

mb

er (

%)

FGF-2ThrombinPPACK

0 h48 h

–+

+ +++

–++

+–

+–+–

+––

–––

*

Fig. 3. Influence of PPACK-thrombin in SK-MEL-110 proliferation.

SK-MEL-110 (4 · 104) cells were seeded and grown for 24 h in

complete medium. Medium was then replaced and cells were

starved overnight with incomplete medium. Then, cells were

directly exposed for 48 h to FGF-2 (10 ngÆmL)1) in the presence or

absence of thrombin (0.1 nM) pre-incubated or not with PPACK

(50 nM) or PPACK alone (50 nM). As a control, cells were stimulated

with thrombin alone (0.1 nM) or PPACK alone (50 nM). FGF-2-

induced proliferation was measured after 48 h stimulation with

thrombin in the presence or in the absence of PPACK. PPACK

blocks thrombin enzymatic activity and reverts the thrombin anti-

mitogenic effect (*P < 0.01), suggesting that FGF-2 is degraded by

thrombin protease function. Data are expressed as a percentage of

cell number versus FGF-2 (100% corresponds to 1.9 · 105 cells).

The data reported are the mean ± SEM of five independent experi-

ments carried out in duplicate.



intracellular signal pathways mediating the biological

activity of FGF-2 [34–36].

Proteolytic degradation of FGF-2 induced by

thrombin

According to data reported above, we hypothesized

that FGF-2 may be directly cleaved by thrombin. Its

degradation was then investigated by HPLC analysis

(Fig. 8A) and by Western blotting (see, Fig. S1). Fig-

ure 8A shows the elution chromatogram of FGF-2

alone and FGF-2 incubated with thrombin for 30 min.

Incubation of FGF-2 with thrombin lowered the peak

corresponding to the full length FGF-2, eluting at

23.5 min, and induced the appearance of the peak

eluting at approximately 13.6 min, corresponding to a

proteolytic fragment of FGF-2. Area values corre-

sponding to the full length FGF-2 were then fitted

according to Eqn (1) (see Experimental procedures),

which allows calculation of the kinetic rate constant of

the peak’s area decay, equal to 4.8 ± 0.3 · 102 min)1.

This value, under our experimental conditions, reflects

an apparent kcat ⁄Km of FGF-2 hydrolysis equal to

1.1 · 104 m)1Æs)1, which is comparable to the value of

other low-affinity thrombin substrates, such as zymo-

gen protein C or thrombin-activatable fibrinolysis

inhibitor [37,38]. As shown in the (Fig. S1), FGF-2

incubated with PPACK appears as a main unique

band (arrowhead, 18 kDa), whereas one immunoreac-

tive additional band appears in the FGF-2 incubated

with thrombin for 1 h (1 : 1 molar ratio) with an

apparent molecular weight of 15 kDa. FGF-2 incu-

bated with inactive thrombin (i.e. thrombin pre-incu-

bated with PPACK) shows no additional bands

compared to basal conditions. We then confirmed the

observed biochemical degradation of FGF-2 induced

by thrombin with a biological assay. Structural degra-

dation and loss of function were then investigated as a

0 h24 h

FGF-2Thrombin

*

0

25

50

75

100

Cel

l nu

mb

er (

%)

––

++

++–

–––

Fig. 5. HUVEC proliferation assay. HUVEC (8 · 104) were seeded

and grown for 24 h in complete medium. Medium was then

replaced and cells were starved overnight with incomplete med-

ium. HUVEC growth was examined after 24 h of stimulation with

FGF-2 (10 ngÆmL)1) in the presence or in the absence of thrombin

(0.1 nM). The mitogenic effect of FGF-2 is significantly reduced in

the presence of thrombin and thrombin alone has no effect on

HUVEC proliferation. (*P < 0.01). Data are expressed as a percent-

age of cell number versus FGF-2 (100% corresponds to

12.7 · 104 cells) and are the mean ± SEM of five independent

experiments carried out in duplicate.

0

25

50

75

100

Cel

l nu

mb

er (

%)

–Thrombin+Thrombin

FGF-2 Heparin (ng·mL–1) –

– – +

50 +

* *

48 h 0 h

10 50 –

0

25

50

75

100A

B

FGF-2 Heparin (ng·mL–1)

– – – – –

Fig. 4. Heparin does not influence the effect of thrombin on FGF-2.

SK-MEL-110 (4 · 104) cells were seeded and grown for 24 h in

complete medium. Medium was then replaced and cells were

starved overnight with incomplete medium. Cells were stimulated

for 48 h with two different heparin concentrations (10 and

50 ngÆmL)1). At either dose, heparin alone does not influence spon-

taneous SK-MEL-110 growth (A); FGF-2-induced proliferation is par-

tially affected by 50 nM heparin; however, heparin does not

influence the inhibitory effect of thrombin. Indeed, thrombin pre-

incubation inhibits FGF-2 activity similarly both in the absence and

in the presence of heparin (B) (*P < 0.05). Data are expressed as a

percentage of cell number versus FGF-2 (100% corresponds to

1.9 · 105 cells) and are the mean ± SEM of five independent

experiments carried out in duplicate.



function of time of exposure to thrombin. Figure 8B

shows that increasing times of FGF-2 ⁄ thrombin incu-

bation strongly reduced both the integrity of FGF-2

(expressed as a percentage of peak area on the chro-

matogram) as well as its mitogenic action (expressed as

a percentage of proliferation). After 60 min of incuba-

tion, both the integrity and function of FGF-2 were

markedly reduced to an approximately residual 20%

of that at t0, with an almost complete loss of signal

after 120 min of incubation. The strong loss of func-

tion and loss of integrity observed after 60 min of

incubation agree with the almost complete inhibition

of mitogenic activity of FGF (Fig. 1B) carried out

under similar experimental conditions (1 h of incuba-

tion of FGF-2 and thrombin).

FGF-2 binding to cells was then investigated in a

cytofluorimetric assay. Figure 8C shows that FGF-2

binding to cells (detected via a primary antibody

recognizing FGF-2 and a secondary fluorescent anti-

body) is lowered to control levels when FGF-2 is pre-

treated with thrombin for 1 h. These data indicate that

FGF-2 pre-treated with thrombin diminishes binding

to cell membranes, explaining, at least in part, the

observed impairment of biological activity.

Cleavage site determination

Additional investigations were then carried out to iden-

tify the FGF-2 site cleaved by thrombin. The fragment

eluting at 13.6 min (Fig. 8A) was sequenced and

revealed a N-terminal sequence of I-H-P-D-G-R-V-D,

corresponding to the fragment Ile43 to Asp50 of

mature FGF-2. As expected, the N-terminal sequence

of undigested FGF-2 was sequenced as M-A-A-G-S-I-

T, corresponding to the reported N-terminal sequence

of mature human FGF-2 (accession number P09038).

These experiments show that thrombin cleaves FGF-2

at the peptide bond between Arg42 and Ile43, releasing

the N-terminal segment from Met1 to Arg42 and the

remaining C-terminal fragment from the intact mole-

cule. We then investigated whether FGF-2 shares

sequence homology with known thrombin-recognized

cleavage sites. The sequence alignment reported in

Table 1 shows structural similarities of several known

thrombin cleavage sites to that of the site found on

FGF-2 in the present study. Beside the invariant argi-

nine (R) residue at the P1 position, common residues

are present at any position, specifically the aromatic

residues phenylalanine (F), tyrosine (Y) and tryptophan

VitronectinA B

C D

0

20

40

60

80

100C

ells

/fie

ld (

%)

FGF-2Thrombin

Collagen IV

0

20

40

60

80

100

Gelatin

0

20

40

60

80

100Fibronectin

0

20

40

60

80

100

Cel

l/fie

ld (

%)

FGF-2Thrombin –

–+

+++–

– ––

++

++–

–

––

++

++–

–––

++

++–

–

Fig. 6. FGF-2-induced SK-MEL-110 inva-

sion ⁄ migration in modified Boyden cham-

bers. The invasion ⁄ migration properties of

FGF-2 is reduced in the presence of throm-

bin on different protein matrices, namely

vitronectin (A), collagen IV (B), fibronectin

(C) and gelatin (D). Data are expressed as a

percentage of cell number ⁄ field versus cells

exposed to FGF-2, (100% corresponds to 49

cells per field for vitronectin, 22 cells per

field for collagen IV, 30 cells per field for

fibronectin and 41 cells per field for gelatin).

The data reported are the mean ± SEM of

three independent experiments carried out

in duplicate.



(W) at P3; the leucine (L) and isoleucine (I) residues at

P2; and proline (P) residues at the P3¢ position.

Discussion

FGF-2 is a multi-function factor with a key role in cell

proliferation and tissue differentiation. It is mainly

bound to low-affinity receptors (heparan sulfates) and

stored on the membranes and the extracellular matrix;

low levels of FGF-2 are also found circulating in the

blood [39]. Thrombin cleaves the coagulation cascade

substrates and binds and cleaves PARs receptors

[25,29], as well as other membrane-bound substrates

such as platelet glycoprotein V [30]. It circulates either

in inactive form (prothrombin) or at low concentration

in active form, subsequent to vascular damage and

activation of the coagulation cascade. In particular,

within 45 s to 5 min after venipuncture, active

thrombin appears (�3 ngÆmL)1, 0.08 nm) and, after

2–10 min, further thrombin generation occurs, result-

ing in clotting after 15–27 min at a thrombin concen-

tration of 40–50 ngÆmL)1 (1–2.4 nm) [40]. In the

present study, we hypothesized that FGF-2 may be

cleaved by thrombin when either thrombin or FGF-2

expression levels strongly increase. This hypothesis

stems from the observation that thrombin levels

increase and coagulation is activated in several physio-

pathological conditions [41,42]. During cancer, levels

of thrombin, FGF-2 and several other factors increase

[43,44]; therefore, we hypothesized that, under such

conditions, low-affinity substrates, which usually are

only marginally hydrolyzed, may be recognized and

digested as a part of a feedback control mechanism.

FGF-2 is present in four isoforms: three HMW

forms (22, 22.5 and 24 kDa), predominantly localized

in the nucleus, and one LMW form (18 kDa), mainly

present in the cytoplasm and on the membranes. A

recent study showed that thrombin is able to cleave

HMW FGF-2, generating a fragment somewhat simi-

lar to the LMW FGF-2. Indeed, similar to LMW, the

cleaved form stimulates endothelial cell migration and

proliferation [13]. By contrast, in the present study, we

assessed whether the real LMW FGF-2 is recognized

by thrombin; indeed, the real LMW FGF-2 is the cir-

culating form and therefore the form naturally exposed

to thrombin [39]. Thrombin at a dose of 0.1 nm (i.e.

the level reached in vivo after vascular damage [45])

strongly reduced the biological functions of FGF-2

and such inhibitory effects were abolished by blocking

the enzymatic activity of thrombin with its selective

inhibitor PPACK, indicating that the enzymatic action

of trhombin was involved. The presence of heparin did

not change the observed effects. Thrombin also

decreased binding of FGF-2 to cell membranes, as well

as FGF-2-dependent ERK2 phosphorylation, one of

the main signal-pathways mediating FGF-2 activity.

Finally, HPLC analysis indicated a thrombin-depen-

dent digestion, with kinetics matching the functional

impairment of FGF-2. The inhibitory effects of throm-

bin on FGF-2, as observed in the present study on the

human metastatic melanoma cell line SK-MEL-110,

were also confirmed in additional proliferation assays

carried out using human endothelial cells (HUVEC).

The kcat ⁄Km value of FGF-2 hydrolysis revealed a

relatively low specificity for thrombin; however, the

high concentrations of active thrombin attained in vivo

during the activation of coagulation [45] may be suffi-

cient to sustain significant FGF-2 cleavage. Although

conclusive in vivo evidence is lacking, these data sug-

gest that thrombin-dependent FGF-2 cleavage may

occur under physiological ⁄pathological conditions withenhanced thrombin generation, such as in athero-

thrombosis, or with elevated levels of FGF-2 and

thrombin, such as in cancer. The cleavage site identi-

fied between Arg42 and Ile43 of the mature FGF-2

1.5

p-ERK1/2A

B

Total ERK1/2

FGF-2Thrombin –

++–

++

––

0

0.5

1.0

p-E

RK

2 / t

ota

l ER

K2

Fig. 7. FGF-2-induced SK-MEL-110 ERK ⁄ MAPK phosphorylation in

the presence of active thrombin. SK-MEL-110 (4 · 104) cells were

seeded and grown for 24 h in complete medium. Medium was

then replaced and cells were starved overnight with incomplete

medium. These cells were stimulated for 10 min with FGF-2

(10 ngÆmL)1), thrombin (0.1 nM) or FGF-2 with thrombin pre-incu-

bated for 1 h at 37 �C and then added PPACK (50 nM) to block

thrombin enzymatic activities. Next, cells were lysated and

Western blotting analysis was performed to evaluate ERK1 ⁄ 2phosporylation. (A) Active thrombin in the presence of FGF-2 (lane

FGF-2 + thrombin) significantly reduces ERK-2 (42 kDa) phosphory-

lation (one representative experiment). (B) Densitometry analysis of

three separate experiments.



sequence is close to the Asp57 residue, initiating the

FREG fragment of FGF-2, which was recently demon-

strated to strongly modulate FGF-2 activity [19]. We

hypothesize that proteolytic degradation as well as the

release of active fragments may represent, at least in

part, a feedback regulatory mechanism for modulating

the angiogenic properties of FGF-2 when FGF-2 levels

increase.

The reported proteolytic effect of thrombin on

human matrix proteins, such as vitronectin, fibronectin

Fig. 8. Thrombin-induced FGF-2 structural and functional impair-

ment. (A) FGF-2 degradation investigated by HPLC. The lower chro-

matogram reports the elution profile of FGF-2 alone; the upper

chromatogram reports the elution profile of FGF-2 incubated with

thrombin for 30 min. In the presence of thrombin, the peak eluting

at 23.5 min is reduced and an additional peak appears after

13.6 min of elution, corresponding to the degradation fragment. (B)

The structural impairment parallels the functional impairment of

FGF-2 in the presence of different time points of thrombin incuba-

tion. Structural impairment is expressed as time-dependent

decrease of the peak eluting at 23.5 min, whereas functional

impairment is expressed as the time-dependent loss of a mitogenic

effect on SK-MEL-110 cells. Structural and functional impairment

are approximately 50% at 30 min, approximately 80% at 60 min

and almost complete after 120 min of incubation. (C) FGF-2 binding

to SK-Mel-110 cells. SK-MEL-110 (2 · 105) cells were seeded and

grown for 24 h in complete medium. Medium was then replaced

and cells were starved overnight with incomplete medium. These

cells were exposed to FGF-2 (50 ngÆmL)1) or to thrombin (0.1 nM)

or to FGF-2 previously incubated with thrombin for 1 h at 37 �C(50 ngÆmL)1 and 0.1 nM respectively) FGF-2 binding to cells,

detected with a primary antibody and a secondary fluorescent anti-

body, was then measured by flow cytometry. The peak on the

right, corresponding to the peak at higher fluorescence, is

increased and enlarged toward the right-hand side (i.e. higher fluo-

rescence) in cells exposed to FGF-alone compared to control cells,

whereas it is lowered to the control level in cells exposed to FGF-2

pre-incubated with thrombin. One representative graph from three

experiments is reported.

80

100

80

100

FG

F-2

pea

k ar

ea (

%)

60

40

60

40

Cel

l nu

mb

er (

%)

Time (min)

Ab

sorb

ance

214

nm

5 10 15 20 25 30

Time (min)30

20

0

20

060 90 120

FGF-2

FGF-2 + Thrombin

No FGF-2

Co

un

ts

FL1 log100 101 1020

A

B

C

Table 1. P3–P3¢ sequence of the most common thrombin substrates. The structural similarities of FGF-2 to other known thrombin sub-

strates are indicated in bold.

Substrate

Position

Residue no.

Swiss-Prot

database entryP3 P2 P1 P1¢ P2¢ P3¢

FGF-2 F L R I H P 40–45 P09038

Fibrinogen S A R G H R 12–17 P02675

PAR-4 A P R G Y P 45–50 Q96RI0

Factor V (1) G I R S F R 737–742 Q15430

Factor V (2) Y L R S N N 1571–1576 Q15430

Factor VIII Q I R S V A 389–394 P00451

Platelet glycoproteinV G P R G P P 474–479 P40197

Carboxypeptidase B2 (TAFI) S P R A S A 112–117 Q96IY4

Vitronectin W G R T S A 302–307 P04004



and murine collagen IV [46,47], might indicate dual

effects on cell migration ⁄ invasion. Indeed, on the one

hand, thrombin digests FGF-2, reducing its angiogenic

and invasive action; on the other hand, thrombin

degrades matrix proteins, facilitating cell invasion. The

net effect measured in vitro in the present study was a

strong reduction of FGF-2-induced cell migra-

tion ⁄ invasion. Preliminary results indicate that, at simi-

lar concentrations, thrombin recognizes and degrades

at least one other angiogenic growth factor, namely

platelet-derived growth factor-BB, suggesting that

increased thrombin concentrations may modulate

angiogenesis and cell chemotaxis ⁄ invasion at different

levels. A number of studies indicate that coagulation

proteases play significant roles in cancer biology [28].

For example, melanoma is a highly metastatic cancer,

and evidence exists indicating that thrombin contrib-

utes to this aggressive pattern. Furthermore, previous

studies show that the assembly and regulation of the

prothrombinase complex on the murine melanoma cell

line B16F10 is accelerated with enhanced thrombin

formation [48]. These conditions, along with PAR-1

expression in melanoma cell lines [29], likely play a rel-

evant role in the metastatic potential of these cancer

cells. The additional effects of thrombin on growth

factors and matrix proteins reported in the present

study may further contribute to these effects, indicat-

ing that pathological conditions with increased

thrombin levels may lead to proteolytic matura-

tion ⁄degradation of both growth factors and extracel-

lular matrix proteins, affecting cell mitogenic ⁄ invasionfeatures at different levels. Cancer can activate the

coagulation cascade, as also demonstrated by the

enhanced rate of thromboembolic complications and

the beneficial effect of anticoagulant therapies in the

prevention of these disorders in cancer patients [49].

However, it is less well known whether activation of

the coagulation system may also support or inhibit

tumor progression. The findings reported in the pres-

ent study outline the functional link between thrombin

activity and the mitogenic ⁄ invasive properties of mela-

noma cells. This observation may contribute to

explaining why paradoxical pro-apoptotic effects of a

high thrombin concentration on different cell types

recently were reported [49].

Two reports available in the literature indicate that

HMW FGF-2 and FGF-1 are cleaved by thrombin,

whereas they also report that LMW FGF-2 is not

cleaved by thrombin [13,50]. In this respect, it should

be highlighted that the experimental conditions (i.e.

the amount of thrombin and FGF-2, as well as the

incubation times) are markedly different. Indeed, we

used 0.1 nm = 0.01 UÆmL)1 thrombin, whereas both

Lobb [50] and Yu et al. [13] used at least 100-fold

more thrombin. The source of thrombin was also dif-

ferent; both Lobb [50] and Yu et al. [13] used commer-

cial human thrombin, whereas we used in-house

highly-purified human thrombin [51]. Lobb [50] used

in-house purified bovine brain-derived FGF-2, whereas

both Yu et al. [13] and ourselves used commercial

human recombinant FGF-2. The incubation times

were also markedly different: Lobb [50] reports 6–20 h

of incubation of brain-derived bovine FGF-2 with

thrombin, whereas Yu et al. [13] used human recombi-

nant FGF-2 at unspecified doses incubated with

thrombin for 5–15 min. In the present study, specified

doses of human recombinant FGF-2 were incubated

for significantly longer times (60–120 min). The experi-

mental conditions employed in the present study were

designed to mimic in vitro, as much as possible, the

conditions occurring under pathological states that

show increased thrombin levels and increased FGF-2

levels, and were confirmed, in different cell models and

in the presence of heparin, to support the possible

in vivo relevance of the observed effects.

In conclusion, in the present study, we show that

thrombin is able to cleave human LMW FGF-2 in vitro,

further unravelling the complex linkage between coagu-

lation activation and cancer progression.

Experimental procedures

Cell culture and proliferation

Human metastatic melanoma cell line SK-MEL-110 [52]

(4 · 104) cells were seeded in six-well plates and grown for

24 h at 37 �C, 5% CO2 in DMEM supplemented with 1%

penicillin–streptomycin, 1% l-glutamine and 10% charcoal

stripped fetal bovine serum (Gibco, Carlsbad, CA, USA).

Medium was then replaced and cells were starved overnight

with DMEM supplemented with 1% penicillin–streptomy-

cin and 1% l-glutamine. Subsequently, dose–response and

time-course proliferation assays were performed using dif-

ferent stimuli and different time points. For dose–response

and for time-course assays (Fig. 1), human recombinant

FGF-2 (10 ngÆmL)1, 0.6 nm) (Pierce Endogen, Rockford,

IL, USA) and human a-thrombin (thrombin) [51] (0.001,

0.01, 0.1 and 1 nm or 0.1 nm) were pre-incubated for 1 h at

37 �C; enzymatic activity was then blocked with PPACK

(50 nm) (Calbiochem, BIOMOL International LP, Exeter,

UK) and cells were stimulated with these mixtures for 48

or 24 h and 48 and 72 h, respectively.

In other experiments (Figs 2–4), SK-MEL-110 cells were

directly exposed for 48 h to FGF-2 (10 ngÆmL)1, 0.6 nm) in

the presence or the absence of 5.7 lm TRAP-1 or TRAP-4

[53] (PRIMM Srl, Milan, Italy), or different mixtures of



PPACK (50 nm) or heparin 25 000 IU per 5 mL (10 and

50 ngÆmL)1 under the assumption that 170 IU corresponds

to 1 mg) (Epsoclar from Mayne Pharma Srl, Napoli, Italia)

[54] and ⁄ or thrombin (0.1 nm, 0.01 UÆmL)1). Finally, in the

experiments depicted in Fig. 8B, FGF-2 (10 ngÆmL)1,

0.6 nm) was pre-incubated at different time points with

thrombin (0.1 nm, 0.01 UÆmL)1); the enzymatic reaction

was then blocked with PPACK (50 nm) and SK-MEL-110

proliferation was stimulated for 48 h. Stimuli were resus-

pended in all cases in DMEM with 0.1% BSA (Sigma-

Aldrich, St Louis, MO, USA).

Primary HUVEC (Cambrex, Walkersville, MD, USA)

were also used. Cells were grown at 37 �C, 5% CO2. HUVEC

(8 · 104) were seeded in six-well plates and grown for 24 h in

endothelial cell basal medium 2 (EBM-2; Cambrex) supple-

mented with endothelial growth medium 2 (Cambrex), 1%

penicillin–streptomycin and 1% l-glutamine. Medium was

then replaced and cells were starved overnight with EBM-2

supplemented with 1% penicillin–streptomycin and 1%

l-glutamine. Subsequently, HUVECs were stimulated with

10 ngÆmL)1 FGF-2 resuspended in EBM-2 with 0.1% BSA in

the presence or the absence of thrombin 0.1 nm, for 24 h.

Time-course experiments were also carried out.

After treatment, cells were harvested with trypsin

(Gibco), stained with trypan blue solution (Sigma-Aldrich)

and counted in a hemocytometer (improved Neubauer

chamber) in quadruplicate.

Cell invasion assay

The SK-MEL-110 invasion assay was carried out in mod-

ified Boyden chambers as previously described [55].

Briefly, 8 lm pore-size polycarbonate filters (Costar, Cam-

bridge, MA, USA) were coated with human plasma vitro-

nectin (Calbiochem), murine collagen type IV (Becton

Dickinson, Bedford, MA, USA), human plasma fibronec-

tin (Invitrogen, Carlsbad, CA, USA) or type A gelatin

from porcine skin (10 lgÆmL)1) (Sigma-Aldrich).

SK-MEL-110 cells were grown in DMEM supplemented

with 1% penicillin–streptomycin, 1% l-glutamine and

10% fetal bovine serum and then replaced with DMEM

supplemented with 1% penicillin–streptomycin, 1% l-glu-

tamine overnight. Cells were then harvested by trypsiniza-

tion, resuspended in DMEM supplemented with 1%

penicillin–streptomycin, 1% l-glutamine and 0.1% BSA,

and 800 lL were added to the upper portion of the Boy-

den chambers containing 1 · 106 cellsÆmL)1; the lower

portion of the chambers contained either DMEM supple-

mented with 0.1% BSA or 10 ngÆmL)1 (0.6 nm) FGF-2

with 0.1% BSA, alone or mixed with 0.1 nm thrombin.

Invasion was allowed to proceed for 4 h at 37 �C in 5%

CO2; then cells were fixed in 95% ethanol and stained

with 0.5% toluidine blue in NaCl ⁄Pi (Gibco), pH 7.4, for

10 min. Migrated cells were counted by evaluation of 15

fields at · 400 magnification.

Phosphorylation and degradation analyses by

Western blotting

ERK1 ⁄ 2 phosphorylation analysis (Fig. 6): SK-MEL-110

cells were grown in DMEM supplemented with 1% penicil-

lin–streptomycin, 1% l-glutamine and 10% fetal bovine

serum, replaced with DMEM supplemented with 1% peni-

cillin–streptomycin, 1% l-glutamine overnight. FGF-2

(10 ngÆmL)1, 0.6 nm), human a-thrombin (thrombin)

(0.1 nm, 0.01 UÆmL)1) and FGF-2 with human a-thrombin

(thrombin) (0.1 nm, 0.01 UÆmL)1) were pre-incubated for

1 h at 37 �C and then PPACK (50 nm) was added to block,

where necessary, thrombin enzymatic activities. Cells were

treated with these mixtures for 10 min, rinsed with ice-cold

NaCl ⁄Pi and lysed for 15 min with 1% triton, 10% glyc-

erol, 100 mm NaCl, 5 mm EDTA, 20 mm Hepes (pH 7.4),

10 mm NaF, 2 mm phenylmethanesulfonyl fluoride, 10 lm

NaVO3 and 1% protease inhibitor cocktail (Sigma-

Aldrich). Lysates were then boiled for 7 min. After determi-

nation of protein concentration (Bio-Rad Laboratories,

Hercules, CA, USA), 30 lg of total proteins were resolved

in SDS ⁄PAGE with NuPAGE� pre-cast gels for protein

electrophoresis (10%) and NuPAGE� SDS running buffer

(Mops buffer) (Invitrogen), transferred to nitrocellulose

membrane and blocked with 5% milk in T-NaCl ⁄Pi (0.1%Tween 20 in NaCl ⁄Pi, pH 7.4). After three washes with

T-NaCl ⁄Pi, membranes were incubated with monoclonal

anti-pERK or anti-ERK sera (Cell Signaling Technology,

Beverly, MA, USA) for 1 h. For detection, secondary anti-

mouse serum (1 : 5000) (Pierce Endogen) was used followed

by chemiluminescence (ECL; Amersham, Little Chalfont,

UK) and autoradiography.

FGF-2 degradation analysis (see, Fig. S1)

FGF-2 was dissolved in a 50 mm Tris–HCl containing

150 mm NaCl, pH 8, and incubated for 1 h alone, with

PPACK, with thrombin, or with inactivated-thrombin

(thrombin pre-incubated with PPACK, PPACK-thrombin)

(1 : 1 molar ratio, 100 lgÆmL)1) at 37 �C. After incubation,

active-thrombin was blocked by PPACK (1 : 100 molar

ratio). FGF-2 (500 ng) was then resolved in SDS ⁄PAGE

with NuPAGE� pre-cast gels for protein electrophoresis

(12%) and NuPAGE� SDS running buffer (Mes buffer;

Invitrogen), transferred to nitrocellulose membrane and

blocked with 5% milk in T-NaCl ⁄Pi. After three washes

with T-NaCl ⁄Pi, membranes were incubated with poly-

clonal anti-FGF-2 (0.3 lgÆmL)1) serum (Oncogene

Research Products, Darmstad, Germany) for 1 h. For

detection, secondary anti-goat serum (1 : 5000) (Pierce

Endogen) was used followed by chemiluminescence (ECL;

Amersham) and autoradiography.

Bands were quantified using a calibrated imaging densi-

tometer (GS 710; Bio-Rad Laboratories) and analyzed

using quantity one software (Bio-Rad Laboratories).



Thrombin-induced digestion

Thrombin-induced FGF-2 digestion monitored by

HPLC analysis

FGF-2 hydrolysis was performed in 10 mm Tris–HCl,

150 mm NaCl, pH 7.5, at 37 �C containing 75 nm thrombin

and FGF-2 at 10 lgÆmL)1 (0.6 lm). The proteolytic reac-

tion was stopped at various times with PPACK (10 lm)

and the reaction products were analyzed by HPLC on a

250 · 4.6 mm reverse-phase 304 column (Bio-Rad Labora-

tories). The chromatographic run was performed with the

conditions: 5–60% acetonitrile in 0.1% trifluoroacetic acid

for 40 min at a flow rate of 1 mLÆmin)1. The peaks were

detected routinely at 214 nm. Under these conditions, the

observed velocity of the reaction did not reach saturation,

such that pseudo-first order conditions were met (concen-

tration < Km of the reaction). Hence, the FGF-2 peak area

remaining after cleavage by thrombin was fitted to the

equation:

Pt (%) ¼ 100� exp ð�kobstÞ ð1Þ

where kobs is the pseudo-first order rate of its hydrolysis,

equal to e0 kcat ⁄Km (where e0 is the concentration of

thrombin).

Sequencing of FGF-2 fragment

Fractions pertaining to the hydrolysis peaks were pooled

and dried under vacuum. Their molecular identity was

checked by N-terminal sequence performed by the Edman

reaction on an automatic analyzer (Applied Biosystems,

Foster City, CA, USA) according to standard procedures.

FGF-2 binding

Binding of FGF-2 alone or FGF-2 incubated with thrombin

was carried out on live cells and detected by flow cytometry.

SK-MEL-110 cells were exposed to FGF-2 alone

(50 ngÆmL)1) or thrombin alone (0.1 nm) or FGF-2 previ-

ously incubated with thrombin for 1 h at 37 �C (50 ngÆmL)1

and 0.1 nm, respectively). Binding was allowed to proceed

for 20 min at 37 �C. Then, cells were washed three times,

detached, and washed with NaCl ⁄Pi–1% BSA. Primary goat

anti-(human FGF-2) serum (SC-79G; Santa Cruz Biotech-

nology, Santa Cruz, CA, USA) was added for 60 min at

room temperature, and then secondary donkey anti-(goat

Alexafluor 488) (Invitrogen, Molecular Probes) serum was

added for 30 min at room temperature. After washings, cells

were analyzed at a Beckman Coulter CYAN ADP 9-colours

flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Cells were gated using forward versus side scatter to exclude

dead cells and debris. Fluorescence of 10 000 cells per sample

was acquired in logarithmic mode to quantify the binding

of the relevant molecules. summit 4.3 software (Beckman

Coulter) was used for data elaboration.

Statistical analysis

All experiments were performed at least three times in

duplicate. A Student’s t-test was carried out to evaluate

statistical significance at P < 0.05.

Acknowledgements

The present study was supported by the Italian Min-

istry of University and Research (‘PRIN-2005’) to

R.D.C.; Progetto Oncoproteomica Italia-USA (no.

527B ⁄2A ⁄5) to A.F.; and Ministry of Health

MS-RO2006, Prog. Ord. to A.F. We thank the Docto-

rate School of Scienze e Tecnologie Cellulari at

Sapienza University in Rome for the support.

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Supporting information

The following supplementary material is available:

Fig. S1. FGF-2 analysis by western blotting.

This supplementary material can be found in the

online version of this article.

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thrombin-mediated impairment of fibroblast growth factor-2 activity

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