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

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 Zn 2+ -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 Zn 2+ 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 Zn 2+ -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 l M [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 aZn 2+ -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 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: 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) Eur. J. Biochem. 270, 111–118 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03367.x 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 M r of 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 Zn 2+ binding substances. Plasma was isolated from normal blood anti- coagulated with hirudin (50 mgÆL )1 )byusingastandard 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 Na 125 I 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 · 10 3 cellsÆcm )2 in 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 37 °C in a humidified 95% air : 5% CO 2 atmosphere. The medium was changed the day after plating and subsequently every second day. At confluence (2.8 · 10 4 cellsÆcm )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 10 4 cellsÆcm )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 m M NaCl, 5.6 m M KCl, 3.6 m M NaHCO 3 ,2.3m M CaCl 2 ,1.0m M MgCl 2 ,5.6m M glucose, 5m M Hepes pH 7.4) containing 15 l M ZnCl 2 (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 n M . This preincubation period allowed FXIIa to bind to the cells without being affected by the inhibitor. After a 60 min preincubation period, 40 lLof varying concentrations of C1-INH or 0.15 M 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 lL0.8m M S-2302 in EDTA buffer (50 m M Tris/HCl, 12 m M NaCl, 10 m M 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 lL0.4m M S-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 l M Zn 2+ 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 n M 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 112 I. Schousboe (Eur. J. Biochem. 270) Ó FEBS 2003 was recorded continuously at 405 nm in mixtures of S-2302 (0.4 m M in EDTA buffer), containing FXIIa and varying concentrations of CTI or SBTI. Complete inhibition was observed at CTI concentrations > 0.005 mgÆmL )1 ( 0.5 l M ) and no inhibition was recorded at SBTI concentrations < 0.1 mgÆmL )1 . Radiolabelling of proteins FXIIa was labelled with Na 125 I by the Iodogen method as described by Salacinski et al. [30]. Following iodination, 125 I-labelled proteins were separated from 125 I 2 by gel-siev- ing on a Sephadex G-25 column equilibrated with NaCl/Pi (50 m M phosphate buffer, pH 7.4 containing 0.5 M NaCl). Specific labelling ranged from 10 · 10 9 to 19 · 10 9 c.p.m.Æmg protein )1 . Binding of 125 I-labelled proteins to HUVEC and solubilization of the cells The confluent layer of cells was incubated with 125 I-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 m M 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-n M solution was approximately fivefold higher than the than that which inactivated the activity in the 40 n M solution 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 l M Zn 2+ , 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 Zn 2+ 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 Zn 2+ 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)andinthe 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 index 100. The results are corrected for activity measured on cells incubated in the absence of FXII/FXIIa. Ó FEBS 2003 Factor XIIa activity on endothelial cells (Eur. J. Biochem. 270) 113 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 · 10 5 M )1 Æmin )1 . This is 3–10-fold lower than the constant determined analogously for the inactivation of FXIIa in the medium (2.3)4.3 · 10 5 M )1 Æmin )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 NaClandkeptat4°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 six determinations 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 + Zn 2+ 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 + Zn 2+ and no FXII + Zn 2+ , and b no FXII + Zn 2+ 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 inEDTAbuffer,incubatedfor4handacidifiedwithaceticacidbefore reading the absorption at 405 nm. The results are shown as means ± SD of six individual experiments using two individual sub- cultures of cells. SD is shown by vertical bars, when extending beyond the symbols. 114 I. Schousboe (Eur. J. Biochem. 270) Ó FEBS 2003 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 n M , and a value that did not change in the presence of C1-INH. Increased C1-INH concentrations resulted, however, in a decreased ÔmaximalÕ 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). Zn 2+ -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 Zn 2+ , which is of importance for the binding of not only FXII but also the HK/PPK complex to 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 Zn 2+ increasing its concentration by 25 l M . This concentration of Zn 2+ 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 Zn 2+ 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)or82n 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 D . Ó FEBS 2003 Factor XIIa activity on endothelial cells (Eur. J. Biochem. 270) 115 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 K D 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 FXIIageneratedonthecellsbyactivationofFXIIinthe presence of Zn 2+ was significantly higher than that generated in the absence of Zn 2+ , 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 Zn 2+ [3,5] and binding of FXIIa to the cells is Zn 2+ -dependent [16]. A conformational change of FXIIa could, however, not by itself explain the protection against inactivation, as the presence of Zn 2+ 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 inactivation of thefreeFXIIainthemediumabovethecells.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 K D for 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 Zn 2+ 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,0.05mgÆmL )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. 116 I. Schousboe (Eur. J. Biochem. 270) Ó FEBS 2003 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 Zn 2+ - 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 complex no 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 n M ), which is 4–6-fold higher than the dissociation constant for binding to HUVEC. The normal concentration of C1-INH is 2.5 l M [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 Zn 2+ 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 Zn 2+ - dependent enhanced FXIIa/kallikrein-like amidolytic activity on HUVEC measured in the absence of FXIIa may be the initiating factor. 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