Báo cáo khoa học: Binding of activated Factor XII to endothelial cells affects its inactivation by the C1-esterase inhibitor pptx

It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1-INH in medium; however, the decrease was s

Binding of activated Factor XII to endothelial cells affects

its inactivation by the C1-esterase inhibitor

Inger Schousboe

Department of Medical Biochemistry & Genetics, The Panum Institute, University of Copenhagen, Denmark

It is well known that activated Factor XII (FXIIa) and

kallikrein are rapidly inactivated in plasma as a result of

reaction with endogenous inhibitors The purpose of this

may be to prevent uncontrolled deleterious spreading and

activation of target zymogens Both FXII and the complex

plasma prekallikrein/high molecular mass kininogen

become activated when they bind, in a Zn2+-dependent

manner, to receptors on human umbilical vein endothelial

cells (HUVEC) The C1-esterase inhibitor (C1-INH) is by

far the most efficient inhibitor of FXIIa In the present study

it has been investigated whether binding of FXIIa to

HUVEC might offer protection against inactivation by

C1-INH It appeared that the relative amidolytic activity of

purified FXIIa bound to the surface of HUVEC decreased

according to the concentration of C1-INH in medium;

however, the decrease was smaller than that measured for

inactivation of FXIIa in solution The secondary rate

constant for the inactivation was 3–10-fold lower for cell-bound than for soluble FXIIa The inactivation was found

to be caused by C1-INH binding to cell-bound FXIIa Accordingly, the amidolytic activity of saturated amounts of cell-bound FXIIa was reduced in the presence of C1-INH and was theoretically nonexistent at physiological C1-INH concentrations Amidolytic activity was, however, present

on HUVEC incubated with plasma indicating that the endogenous C1-INH did not completely abolish the activity

of FXIIa generated during the incubation period This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor Hence, the function of FXII may be localized at cellular surfaces

Keywords: C1-esterase inhibitor; endothelial cells; Factor XII

Factor XII (FXII) and plasma prekallikrein (PPK) are

zymogens of proteolytically active factor XIIa (FXIIa) and

kallikrein, which constitute a linked activation system

Contact with an artificial, negatively charged surface

activates the system in the presence of Zn2+ and high

molecular mass kininogen (HK) [1] Hence, this system was

named the contact activation system Initiation of activation

of the system has been found to be a result of a slow

autodigestion and autoactivation of surface bound FXII by

FXIIa [2] Binding of FXII to a negatively charged surface

induces a Zn2+-dependent conformational change in FXII

enhancing its susceptibility to become activated [3–5] The

activated FXII then activates prekallikrein, which

recipro-cally enhances the activation of FXII and thereby the entire

system [6] In blood plasma the proteolytic efficiency of the

proteinases is attenuated by endogenous inactivation of

FXIIa and kallikrein by serine proteinase inactivators The

C1-esterase inhibitor (C1-INH), belonging to the super-family of serine proteinase inhibitors (serpins), binds covalently to the active site of its target [7] It is the major FXIIa inactivator [8] and accounts for > 90% of the inactivation of FXIIa in plasma [9,10] Its plasma concen-tration is 2.5 lM[11] Homozygous deficiency of C1-INH

is not known Patients with ineffective C1-INH caused by either a heterozygous deficiency, dysfunctional protein or the presence of autoantibodies suffer episodes of painless local swelling of the soft tissue resulting from increased vasopermeability correlated with an increased plasma concentration of activated FXII and cleavage products of

HK during attacks [12–14]

During the past decade convincing results have shown that FXII and PPK bind to receptors on endothelial cells in

a Zn2+-dependent manner FXII binds directly to the cells [15,16], whereas PPK binds via a bridge generated by the ability of HK to bind to the cells as well as to PPK [17,18] The binding results in activation of FXII and PPK by a so far controversial mechanism [15,19–21] Whatever this mechanism might be, a prerequisite for function is that the cell-bound and -generated FXIIa and kallikrein are protected from inactivation by endogenous inhibitors in the plasma Activation of PPK plays a primary role in initiating and maintaining the vascular response to injury By interacting with HK, kallikrein cleaves off the vasoactive peptide, bradykinin [21–23] Bradykinin stimulates endo-thelial cell prostaglandin synthesis, leading to inactivation of platelet function [24,25], and increased superoxide [26] and nitric oxide formation [27] Consequently bradykinin

is linked with the pathophysiological processes that

Correspondence toI 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: FXII, Factor XII; FXIIa, activated FXII; PPK,

plasma prekallikrein; HK, high molecular mass kininogen; C1-INH,

C1-esterase inhibitor; HUVEC, human umbilical vein endothelial

cells; CTI, corn trypsin inhibitor; SBTI, soybean trypsin inhibitor;

S-2302, H- D -Pro-Phe-Arg-para-nitroaniline.

(Received 3 September 2002, revised 1 November 2002,

accepted 14 November 2002)

accompany inflammation and tissue damage which is

thought to play a major role in the symptomatology of

acute attacks in patients with angiodema [12,28]

Binding of FXIIa to different negatively charged artificial

surfaces has previously been shown to offer protection

against inactivation of FXIIa by C1-INH in a surface- and

C1-INH concentration-dependent manner [29] Binding

and subsequent activation of FXII in the vascular system

may likewise offer protection of FXIIa activity against

inactivation by the C1-INH in plasma FXII and FXIIa

bind indifferently to human umbilical vein endothelial cells

(HUVEC) [16] The purpose of the present investigation

was to analyse whether binding of FXIIa to the surface of a

confluent layer of HUVEC might lead to protection against

inactivation by C1-INH

Experimental procedures

Materials

FXII was from obtained Haematologic Technologies as a

high concentration solution in 50% glycerol and stored at

)20 C as recommended by the manufacturer It migrated

as a single band with an Mrof 80 kDa on reduced SDS/

PAGE and was unable to cleave amidolytically the FXIIa

substrate, S-2302 (see below) Two-chain FXIIa was

obtained lyophilized from Enzyme Research Laboratory

It was dissolved as described by the manufacturer and

stored in aliquots at )80 C Once thawed it was never

refrozen None of the proteins, including C1-INH, corn

trypsin inhibitor (CTI; Calbiochem) or soybean trypsin

inhibitor (SBTI; Sigma) were preserved with Zn2+binding

substances Plasma was isolated from normal blood

anti-coagulated with hirudin (50 mgÆL)1) by using a standard

blood bank procedure H-D-Pro-Phe-Arg-para-nitroaniline

(S-2302) was from Chromogenix, and Sigmacoat

(chlorin-ated organo-polysiloxane in heptane) and gelatine from

porcine skin (G-8150) were from Sigma

1,3,4,6-Tetra-chloro-3a,6a-diphenyl glycoluril (Iodogene) was from

Pierce, and Na125I was from Amersham/Pharmacia All

other reagents were of the purest grade commercially

available Siliconized test tubes were used for all solutions

and dilutions

Endothelial cell culture

Cryopreserved primary cultures of HUVEC delivered from

Clonetics were plated at a density of 2.5· 103cellsÆcm)2in

tissue culture flasks containing endothelial cell growth

medium supplemented with human recombinant

endothel-ial cell growth factor, fibroblast growth factor and

insulin-like growth factor, vascular endothelial cell growth factor,

ascorbic acid, hydrocortisone, heparin, gentamicin,

ampho-tericin and 2% fetal bovine serum according to the

recommendation of Clonetics The cells were incubated at

37C in a humidified 95% air : 5% CO2atmosphere The

medium was changed the day after plating and subsequently

every second day At confluence (2.8· 104cellsÆcm)2) the

cells were subcultured After seven to eight generations (two

passages) the cells were cryopreserved During each passage,

the number of living cells was tested by Trypan blue

exclusion For experiments the cryopreserved cells were

subcultured in microtiter plates at a density of 104cellsÆcm)2 Medium was changed every second day and the cells were used 4–6 days after plating

Inactivation of amidolytic activity of cell-bound FXIIa The medium was aspirated from 12-well microtiter plates 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 mMCaCl2, 1.0 mMMgCl2, 5.6 mMglucose,

5 mMHepes pH 7.4) containing 15 lMZnCl2(wash buffer) followed by a 10-min incubation with 1 mL 0.1% (w/v) gelatine in wash buffer (block buffer) The washing proce-dure, as judged visually by microscopic inspection, neither removed cells from the surface nor changed the morphology

of the cells The block buffer was aspirated and the cells preincubated at room temperature with 400 lL FXIIa diluted at least 300-fold in block buffer giving a final concentration of 40 nM This preincubation period allowed FXIIa to bind to the cells without being affected by the inhibitor After a 60 min preincubation period, 40 lL of varying concentrations of C1-INH or 0.15M NaCl were added and mixed by horizontal circular movements and the incubation continued for a further 60 min To measure the degree of inactivation of FXIIa in the medium 250-lL aliquots of the medium above the cells were transferred to and mixed with 250 lL 0.8 mM S-2302 in EDTA buffer (50 mMTris/HCl, 12 mM NaCl, 10 mM EDTA, pH 7.8) The remaining medium was aspirated and the cells washed continuously for 5 s in a mild stream of wash buffer Subsequently, 400 lL 0.4 mMS-2302 in EDTA buffer were added The presence of EDTA destroys the cells and releases FXIIa into solution Both series of samples with S-2302 were incubated for a maximum of 4 h at room temp and the concentration of p-nitroaniline generated during the chosen incubation period was measured at 405 nm in cuvettes with a 1-cm light path after addition of acetic acid

to a final concentration of 4% (v/v)

Comparable experiments were run in parallel As deter-mined by microscopic inspection, the cells were intact until addition of the chromogenic substrate No degradation or change in specific amidolytic activity of FXIIa was observed within 6 h of incubation with the chromogenic substrate Amidolytic activity on cells incubated with plasma After washing the cells in 12-well microtiter plates as described above, the cells were incubated at room tempera-ture with 400 lL hirudin anticoagulated plasma enriched with 1/10 vol 250 lMZn2+in Locke’s buffer containing either nothing, SBTI, CTI or both SBTI and CTI After incubation for 120 min the remaining medium was aspir-ated and the cells washed continuously for 5 s in a mild stream of wash buffer Subsequently, the cells were incuba-ted with S-2302 in EDTA buffer as described above No amidolytic activity could be measured when extracts of cells were added to purified PPK and FXII

The minimal concentration of CTI for total inhibition of amidolytic activity of 40 nM FXIIa and the maximal concentration of SBTI which could be applied without affecting the activity of FXIIa at these concentrations were determined by titration The change in absorption over time

was recorded continuously at 405 nm in mixtures of S-2302

(0.4 mMin EDTA buffer), containing FXIIa and varying

concentrations of CTI or SBTI Complete inhibition

was observed at CTI concentrations > 0.005 mgÆmL)1

( 0.5 lM) and no inhibition was recorded at SBTI

concentrations < 0.1 mgÆmL)1

Radiolabelling of proteins

FXIIa was labelled with Na125I by the Iodogen method as

described by Salacinski et al [30] Following iodination,

125I-labelled proteins were separated from125I2by

gel-siev-ing on a SephadexG-25 column equilibrated with NaCl/Pi

(50 mMphosphate buffer, pH 7.4 containing 0.5MNaCl)

19· 109c.p.m.Æmg protein)1

Binding of125I-labelled proteins to HUVEC

and solubilization of the cells

The confluent layer of cells was incubated with125I-labelled

FXIIa and C1-INH added as described above At the end of

the incubation period the cells were washed and solubilized

as described previously [16] Amidolytic activity and

radio-activity were measured in 250-lL aliquots of the solubilized

cells Activity was measured as the change in absorbance at

405 nm after a 4-h incubation with S-2302 (0.4 mM final

concentration)

Statistics

Student’s t-test was used to determine the level of

signifi-cance

Results

Inactivation of the amidolytic activity of factor XIIa

bound to endothelial cells

To analyse if the presence of C1-INH would inactivate the

amidolytic activity of cell-bound FXIIa in a manner

comparable to that of inactivation of soluble FXIIa, the

following series of experiments was performed HUVEC

were preincubed with purified FXIIa for 60 min, allowing

FXIIa to bind to the cells Then C1-INH was added to the

incubation medium and the incubation continued for an

additional 60 min This showed that the ability of C1-INH

to inactivate the amidolytic activity of FXIIa was markedly

reduced when FXIIa was bound to the cells compared to

the inactivation of FXIIa in the incubation medium The

concentration of C1-INH which inactivated 50% of the

amount of FXIIa bound to the cells from a 40-nMsolution

was approximately fivefold higher than the than that which

inactivated the activity in the 40 nMsolution of FXIIa in

which the cells had been preincubated (Fig 1) This

difference may be even larger if taking into account the

difference in amounts of FXIIa bound to the cells and that

in solution

It was next analysed if amidolytic activity of FXIIa

generated on the cells from cell-bound FXII was likewise

protected from inactivation by C1-INH As autoactivation

of HUVEC-bound FXII has been widely debated [15,19–

21] it was first analysed if FXII bound to HUVEC was able

to autoactivate In the presence of 15 lMZn2+, a concen-tration previously shown to be optimal for binding of purified FXII/FXIIa to HUVEC [16], a slightly but significantly higher (P < 0.005) amidolytic activity could

be measured on the cells incubated in the presence than in the absence of FXII (Table 1) During the period of incubation with FXII, amidolytic activity had been gener-ated also in the incubation medium The total activity of this was, however, lower than the total activity on the cells, and

in the presence of Zn2+fourfold higher than the activity of FXIIa generated in the medium without cells (results not shown) The difference in activity of FXIIa in the incubation medium containing FXII may be due to equilibrium binding of FXII/FXIIa to the cells It is noticeable that the presence of Zn2+ alone increased significantly (P < 0.005) the cell-generated S-2302 amidolytic activity This indicates that the cells themselves might have expressed FXIIa- and/or kallikrein-like activity during cultivation As this activity could not be inhibited by either CTI, SBTI or C1-INH (data not shown) it is unlikely that it was due to either FXIIa or kallikrein adhering to the cells during cultivation It was subsequently shown that FXIIa gener-ated by activation of FXII bound to cells responded to increasing concentrations of the C1-INH in the incubation medium in a manner similar to that of cell-bound FXIIa (Fig 1)

By following the progress of the inactivation it was observed that the reaction between C1-INH and the cell-bound FXIIa proceeded more slowly than the reaction between C1-INH and FXIIa in the incubation medium above the cells The inactivation of cell-bound FXIIa was

Fig 1 The effect of increasing concentrations of C1-INH on the activity

of soluble and cell-bound FXIIa In the presence of Zn 2+ , the cells were preincubated with FXIIa for 1 h Then C1-INH of varying concen-trations was added in 1/10 incubation vol After incubation for another

1 h, the amidolytic activity was measured on the cells (d) and in the medium (s) as described Cells were also incubated for 2 h with a mixture of 40 n M FXII and C1-INH (m) The medium was aspirated and the amidolytic activity measured on the cells as described The medium and the cells were incubated with S-2302 in EDTA buffer for

1 h and 4 h, respectively The final concentration of the C1-INH

is shown on the abscissa The activity measured in the absence of C1-INH has been given the index100 The results are corrected for activity measured on cells incubated in the absence of FXII/FXIIa.

almost maximal after a 20-min incubation in the presence of

a high concentration of C1-INH (Fig 2) The second-order

rate constant was calculated and in five individual

experi-ments found to be in the range 0.4)0.8 · 105

M )1Æmin)1 This is 3–10-fold lower than the constant determined

analogously for the inactivation of FXIIa in the medium (2.3)4.3 · 105

M )1Æmin)1) The decrease could be explained

by either one of two mechanisms: (a) the first is based on the assumption that the cell-bound FXIIa is being pulled off the cells by the binding of soluble FXIIa to the inactivator as illustrated in Fig 3A; (b) the second mechanism assumes that the inactivator binds to FXIIa, while still bound to the cell surface (Fig 3B)

To analyse which of these mechnisms was followed, the cells were preincubated with radioactively labelled FXIIa, and the amidolytic activity as well as the amount of cell-bound FXIIa were measured in identical aliquots of lysates of the cells after incubation with varying concentra-tions of C1-INH This showed that the inactivation only slightly affected the binding while the activity decreased with increasing concentrations of the C1-INH to the level

of activity measured in the absence of FXIIa (see Table 1) (Fig 4) This suggests that C1-INH inactivates

Table 1 Amidolytic activity measured on the cells and in the incubation

medium after incubation of HUVEC with and without FXII in the

presence and absence of Zn 2+ Confluent layers of HUVEC were

incubated at room temperature with block buffer in the absence and

presence of 15 l M Zn 2+ and 40 n M factor XII [to minimize the risk of

interference from contaminating FXIIa, the stock solution of FXII

(50 l M ) was diluted twice with 0.2 m M phenyl-methyl-sulfuryl-fluoride

in 0.15 M NaCl and kept at 4 C for 20 h before start of the

experi-ment] After 120 min incubation the medium was transferred to and

diluted (1 : 1) with 0.8 m M S-2302 and incubated for 4 h As rapidly as

possible the rest of the medium was aspirated and the cells quickly

washed as described in Experimental procedures Then 0.4 m M S-2302

was added to the cells and the incubation continued for the same

period of time as that used for the incubation medium (4 h) An

ali-quot of 0.4 m M S-2302 incubated under identical conditions was used

as blank The results are obtained from sixdeterminations in three

individual subcultures of confluent cells and given as means ± SD.

Medium composition

Total S-2302 amidolytic activity, DOD/time period

On cells In medium FXII + Zn2+ 0.263 ± 0.028 0.100 ± 0.0136

FXII ) Zn 2+ 0.135 ± 0.015 0.038 ± 0.0031

No FXII + Zn 2+ 0.199 ± 0.022* a 0.000 ± 0.0015

No FXII ) Zn 2+

0.129 ± 0.026*b 0.000 ± 0.0013

* Statistically significant differences (Student’s t-test) between

a

FXII + Zn2+ and no FXII + Zn2+, andbno FXII + Zn2+

and no FXII ) Zn 2+

at P < 0.005.

Fig 2 The progress of inactivating cell-bound FXIIa by C1-INH Cells

were incubated with 40 n M FXIIa for 1 h Then C1-INH was added at

a final concentration of 100 n M (.) and in two separate experiments at

a final concentration of 400 n M (s, d), respectively, and the

incuba-tion was continued for the period of time indicated The medium was

then aspirated, cells were washed and the remaining amidolytic activity

was measured as D of the samples after a 4-h incubation with S-2302 in

EDTA buffer.

Fig 3 Possible mechanisms for C1-INH inactivation of cell-bound FXIIa (A) The C1-INH disturbs the binding of FXIIa to HUVEC by binding to soluble FXIIa (B) The C1-INH binds to receptor bound FXIIa.

Fig 4 Correlation between amidolytic activity and the amount of FXIIa bound to the cells The cells were incubated for 2 h with 40 n M radio-actively labelled FXIIa and the radioactivity (s) and amidolytic activity (d) were measured in the same aliquots of solubilized cells Following washing as described, the cells were solubilized by 10-min incubation in 0.1 M NH 4 OH, 1% (v/v) Triton X-100, and subsequently neutralized by the addition of acetic acid Aliquots of the solubilized cells were counted in a c-counter and mixed 1 : 1 with 0.8 m M S-2302

in EDTA buffer, incubated for 4 h and acidified with acetic acid before reading the absorption at 405 nm The results are shown as means ± SD of sixindividual experiments using two individual sub-cultures of cells SD is shown by vertical bars, when extending beyond the symbols.

the amidolytic activity of the cell-bound FXIIa by binding

to FXIIa while still bound to the cellular receptor

This mechanism was confirmed by analysing the effect of

C1-INH on the kinetics of FXIIa binding to the cells by

determining the S-2302 amidolytic activity of the bound

FXIIa In the absence of C1-INH the activity increased

hyperbolically with the concentration of FXIIa in the

medium (Fig 5A) The concentration of FXIIa leading to

half maximal activity – and thus half maximal binding –

was found to be 90 nM, and a value that did not change in

the presence of C1-INH Increased C1-INH concentrations

resulted, however, in a decreased maximal activity of the

bound FXIIa (Fig 5B) This result complies with a

mechanism by which C1-INH binds to the cell-bound

FXIIa (compare Fig 3B) and thus confirms the results shown in Fig 4 It is, however, not compatible with a mechanism by which the bound FXIIa is being pulled off the cells (compare Fig 3A)

Zn2+-dependent amidolytic activity generated

on the surface of cells incubated with plasma The experiments performed with purified FXIIa indicated that a C1-INH concentration identical to that in plasma would be expected to inactivate completely the expression of FXIIa amidolytic activity on the cells To verify this, HUVEC were incubated with hirudin anticoagulated plasma Hirudin anticoagulation was chosen over that based on citrate as the addition of hirudin was assumed not

to disturb the normal physiological concentration of electrolytes and minerals, as does the addition of citrate Thus citrate would disturb the endogenous balance of the concentration of free Zn2+, which is of importance for the binding of not only FXII but also the HK/PPK complexto HUVEC Cells were incubated with the plasma for 2 h, during which FXII was allowed to bind to the cells and to become activated Prior to the incubation, the plasma was enriched with Zn2+increasing its concentration by 25 lM This concentration of Zn2+was found to enhance maxi-mally the total amidolytic activity on the cells after incubation with plasma After incubation, the plasma was aspirated, the cells washed and amidolytic activity was measured according to the procedures described previously

As S-2302 is a substrate not only for FXIIa but also for kallikrein, the measured activity could be caused by an FXIIa-independent activation of PPK [20] In order to separate the amidolytic activity of FXIIa from that of kallikrein, the nonphysiological reversible inhibitors SBTI and CTI were added to the plasma CTI is a specific inhibitor of FXIIa activity [31] and SBTI inhibits the activity of several proteinases including kallikrein but not FXIIa [32] The advantage of using SBTI and CTI was that they were both removed from the surface of the cells during the washing procedure and thus did not interfere with the subsequent measurement of amidolytic activity Combined addition of SBTI and CTI was found to inhibit 82% of the totally amidolytic activity generated on the cells (Fig 6) The remaining 18% was identical to the activity generated

on the cells incubated with block buffer in the absence of plasma as well as to the activity generated on the cells after incubation with plasma to which no extra Zn2+had been added To analyse the relative magnitude of the SBTI- and CTI-insensitive activity separately, multiple sets of HUVEC were incubated in comparable series with plasma to which the inhibitors were added separately Of the 82% inhibited

by the presence of both SBTI and CTI, 65% was inhibited

by CTI alone and 91% by SBTI (Fig 6) No CTI-insensitive activity could be measured when SBTI (0.01 mgÆmL)1) was added to the chromogenic substrate This indicates that the activity inhibited by the presence of CTI may be due to reciprocal activation of FXII and PPK The difference between the activity generated in the presence

of CTI and that in the presence of SBTI, may be due to FXIIa-independent PPK activation and the difference between the activity generated in the presence of SBTI and that generated in the presence of both SBTI and CTI

Fig 5 Inhibiting effect of the C1-INH on the activity of varying

amounts of FXIIa bound to the cells (A) Cells were incubated for 1 h

with the concentrations of FXIIa shown on the abscissa, and 1/10 vol.

0.15 M NaCl (n) or C1-INH, respectively, was added leading to a final

C1-INH concentration of 206 n M (s) or 82 n M (d) After an

addi-tional 1-h incubation, the amidolytic activity on the cells was measured

as described The results are shown as means ± SD of determinations

using three individual subcultures (B) Reciprocal plots of the data

from (A) showing the amidolytic activity of bound FXIIa in the

absence (n) and presence (s, d) of the C1-INH (the slope of the lines

is 1/V max ; V max is the maximal activity of the bound FXIIa) The

intercept with the abscissa is K

may reflect a kallikrein-independent activation of FXII.

Thus both FXII and PPK became activated on the

endothelial cells incubated with plasma containing

physio-logically normal concentrations of the C1-INH but the

majority of the S-2302 amidolytic activity measured after a

2-h incubation was accounted for by kallikrein No

amidolytic activity could be measured in the plasma which

had been incubated with HUVEC

Discussion

The present study shows for the first time that the

amidolytic activity of FXIIa bound to HUVEC is

protected from inactivation by C1-INH The protection

could be explained by a decreased second-order rate

constant for inactivation of the bound FXIIa compared

to inactivating FXIIa in the medium The inactivation

only slightly affected the dissociation of FXIIa from

HUVEC indicating that C1-INH inactivates cell-bound

FXIIa by binding to FXIIa on the surface of the cells

Accordingly, C1-INH reduced the activity of saturated

amounts of bound FXIIa without changing the KD for

binding of FXIIa to the cells

The analysis of the protecting effect that the cells might

exert on FXIIa against inactivation by the C1-INH was

originally thought to be performed on HUVEC incubated

with FXII The advantage of using this set up instead of cells

preincubated with FXIIa was that the C1-INH would affect

FXIIa when generated on the cell surface while its influence

on the putative low concentrations of FXIIa in the medium

would be insignificant However, although the activity of

FXIIa generated on the cells by activation of FXII in the

presence of Zn2+ was significantly higher than that generated in the absence of Zn2+, the fraction of bound FXII that became activated was judged to be too low for further investigation A single series of experiments per-formed on cells incubated with FXII indicated, however, that the degree of inactivation of cell-bound FXIIa was independent of the cells being incubated with FXII or FXIIa That the amidolytic activity of FXIIa was protected against inactivation when bound to a cellular surface is in accordance with a previous investigation showing that incubation of FXIIa with kaolin and sulfatide protects the amidolytic activity of FXIIa against inactivation [29] Three mechanisms of protection of FXIIa activity against inacti-vation in the presence of kaolin and sulfatide have been suggested One of these was that C1-INH disturbs the reversible association of FXIIa with the binding surface either by binding to the surface or to the soluble FXIIa This mechanism was found not to be involved in the inactivation

of cell-bound FXIIa The other two mechanisms, explaining decreased inactivation, were based on either steric hindrance induced by FXIIa binding to the surface or to conforma-tional changes of FXIIa occurring when bound to the surface Both of these mechanisms may be involved in the protection against C1-INH inactivation of FXIIa bound to HUVEC Factor XIIa has been shown to change confor-mation in the presence of Zn2+[3,5] and binding of FXIIa

to the cells is Zn2+-dependent [16] A conformational change of FXIIa could, however, not by itself explain the protection against inactivation, as the presence of Zn2+in the incubation medium only slightly protected soluble FXIIa from inactivation by the C1-INH (I Schousboe, unpublished data)

It may be argued that C1-INH could bind to HUVEC

in the absence of FXII thus reducing the effective dose of the inhibitor available for inactivation, which might at least partly explain the protection However, a reduction in the effective dose of C1-INH would affect not only inactiva-tion of the cell-bound FXIIa but also the inactivainactiva-tion of the free FXIIa in the medium above the cells The consequence of this would have been that the secondary rate constant for binding the inhibitor to FXIIa would decrease but this decrease would be identical for the cell-bound FXIIa and the FXIIa in the medium above the cells and would not change the ratio between the values Thus FXIIa-independent binding of C1-INH to the cells would not have changed the overall results showing that cell-bound FXIIa is protected from inactivation by C1-INH by

a factor of 3–10 Comparison of the rate constant for inactivation of FXIIa in the medium with that previously published for inactivation of FXIIa in solution [7] indicates furthermore that it is unlikely that C1-INH should bind to HUVEC The second-order rate constant for inactivation

of soluble FXIIa was found to be unaffected by the presence of HUVEC

The gradual decrease in amidolytic activity of cell-bound FXIIa with increasing concentrations of C1-INH was not correlated with a decreased binding of radioactively labelled FXIIa This suggests that C1-INH binds to FXIIa on the cell surface consistent with a C1-INH concentration-dependent decreased amidolytic activity of saturated amounts of bound FXIIa, which did not affect the KDfor binding FXIIa to the cells

Fig 6 S-2302 amidolytic activity measured on HUVEC after

incuba-tion with hirudin anticoagulated plasma in the presence and absence of

reversible FXIIa and kallikrein inhibitors Cells were incubated for 2 h

with plasma diluted 0.9-fold with either 250 l M Zn2+in Locke’s buffer

or with this Zn 2+ -solution containing either SBTI, CTI or both [final

concentrations: SBTI of 0.1 mgÆmL)1; CTI, 0.05 mgÆmL)1 (4 l M ].

Then the plasma was aspirated and the cells washed rapidly with

Locke’s buffer containing 25 l M Zn 2+ Subsequently, S-2302 in

EDTA buffer was added for determination of cell-bound activity The

hatched parts of the columns show the measured activity as

mean ± SD of five individual subcultures The blank parts of the

columns show the calculated inhibited activity and the P-values

indi-cate the level of significance (Student’s t-test) of the change in activity

in the absence and presence of the inhibitors.

In plasma the generation of FXIIa by activation of

FXII is closely connected to activation of PPK The

S-2302 amidolytic activity measured on cells incubated

with plasma may thus be due to both FXIIa and

kallikrein activity The S-2302 amidolytic activity

meas-ured on the cells after a 2-h incubation with Zn2+

-enriched human plasma was found primarily to be due to

kallikrein (Fig 6) A considerable amount of the total

amidolytic activity of kallikrein was, however, generated

by an FXIIa enhanced activation mechanism, which was

inhibited by CTI That only a negligible amount of the

cell-bound S-2302 amidolytic activity could be measured

as FXIIa activity is in accordance with the suggested

mechanism for FXIIa inactivation on cell membranes By

binding the C1-INH to the receptor/FXIIa complexno

free receptors will be available for binding soluble FXII

from the plasma The reason why no FXIIa activity could

be measured on the surface of HUVEC after incubation

with plasma may therefore be that FXIIa had been

gradually inactivated during the 2-h incubation by binding

C1-INH to the receptor bound FXIIa All of the receptors

on the cells are saturated with FXII at the normal FXII

concentration (350 nM), which is 4–6-fold higher than the

dissociation constant for binding to HUVEC The normal

concentration of C1-INH is 2.5 lM [11] and more than

sufficient to completely inactivate any FXIIa amidolytic

activity generated on the cells The process of binding

FXII to the cellular receptor, activating the bound FXII

and inactivating the cell bound FXIIa by binding C1-INH

is a complicated time-dependent series of events It may

appear to play a significant role in regulating the function

of cell-bound FXII Disturbing the regulation by reducing

the concentration of C1-INH is known to have serious

consequences Patients with a diminished concentration of

C1-INH, having antibodies towards C1-INH or a

dys-functional C1-INH suffer from angiodema, a disease

resulting in episodic swelling of subcutaneous tissues,

bowel walls and upper airways The vasopermeability

mediator responsible for the swelling may be bradykinin,

a cleavage product of HK generated by proteolytic attack

by kallikrein generated by activation of PPK as a

consequense of vessel injury The activation of FXII

and PPK/HK may start with a local increase in the

concentration of Zn2+ as a result of leakage from

disturbed cells This leakage may lead to binding of FXII

and PKK/HK to respective receptors on endothelial cells

Localized at the site of injury, FXII and PPK become

activated and start a series of events involving generation

of bradykinin and activation of plasminogen The Zn2+

-dependent enhanced FXIIa/kallikrein-like amidolytic

activity on HUVEC measured in the absence of FXIIa

may be the initiating factor Binding of C1-INH to

activated FXII may prevent spreading of activation and

be of importance in the subsequent elimination of the

disturbed cells by macrophage invation

Acknowledgements

I thank Jørn Dalsgaard Nielsen (of The University Hospital Gentofte)

for helping to deliver the hirudin anticoagulated plasma and B Harder

for technical assistance This study was supported by grants from the

Danish Health Research Council and the Novo Nordic Foundation.

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