High molecular weight kininogen binds to laminin – characterization and kinetic analysis Inger Schousboe and Birthe Nystrøm Department of Biomedical Sciences, The Panum Institute, University of Copenhagen, Denmark Keywords extracellular matrix; high molecular weight kininogen; kininostatin; laminin; Zn2+-independent Correspondence I Schousboe, Department of Biomedical Sciences, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark Fax: +45 3536 7980 Tel: +45 3532 7800 E-mail: schousboe@sund.ku.dk (Received 11 March 2009, revised July 2009, accepted 16 July 2009) doi:10.1111/j.1742-4658.2009.07218.x High molecular weight kininogen (HK) is an abundant plasma protein that plays a central role for the function of the kallikrein ⁄ kinin ⁄ kininogen system Thus, cleavage of HK by kallikrein liberates bradykinin, which stimulates vascular repair and a two-chain protein, activated HK (HKa), which induces apoptosis in proliferating endothelial cells The localization of these events remains obscure, although the basement membrane may be of importance Analyzing the interaction between HK and HKa and selected basement membrane proteins, we observed that they bound to the major noncollageneous proteins laminin, but not to vitronectin or fibronectin coated on microtiter plates The binding to laminin was Zn2+ independent However, at low but not at high concentrations of albumin, Zn2+ increased the affinity for the binding by abolishing an inhibitory effect of Ca2+ Recombinant human kininostatin encompassing the amino acid sequence, Arg439-Ser532 but not the endothelial cell binding peptide sequence (His479-His498; HKH20) within kininostatin inhibited the binding of HKa to laminin This established that the amino acid sequence Arg439-Lys478 in domain of HK is of importance for its binding to laminin Extensive proteolytic cleavage of HK and HKa with kallikrein abolished the binding to laminin, releasing a 12 kDa anti-kininostatin reacting peptide On the basis of these results, we propose that the binding of HK to laminin is a primary event, which secures proper localization of the cleavage products for subsequent interaction with the endothelium to promote inflammatory and pro- and anti-angiogenic activities Structured digital abstract l MINT-7218019: Laminin alpha (uniprotkb:Q61001), Laminin beta (uniprotkb:P02469), Laminin gamma (uniprotkb:P02468), Laminin alpha (uniprotkb:Q61001), Laminin beta (uniprotkb:Q61292) and Laminin gamma (uniprotkb:P02468) physically interact (MI:0915) with HK (uniprotkb:P01042) by solid phase assay (MI:0892) l MINT-7219326: Laminin alpha (uniprotkb:P19137), Laminin beta (uniprotkb:P02469) and Laminin gamma (uniprotkb:P02468) physically interact (MI:0915) with HK (uniprotkb: P01042) by solid phase assay (MI:0892) Introduction The extracellular matrix (ECM) controls a variety of cellular functions by interacting with a vast array of macromolecules, exhibiting a wide range of activities [1] Recent in vitro investigations have indicated that Abbreviations ECM, extracellular matrix; FN, fibronectin; HK, high molecular weight kininogen; HKa, activated HK; HRP, horseradish peroxidase; HUVEC, human umbilical vein endothelial cells; LM, laminin; uPAR, urokinase plasminogen activator receptor; VN, vitronectin 5228 FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS I Schousboe and B Nystrøm this array of macromolecules includes the surface binding proteins in the contact activation system of the blood coagulation system This system consists of factor XII, high molecular weight kininogen (HK), prekallikrein and factor XI, but only factor XII and HK interact directly with the ECM However, in the plasma, prekallikrein and factor XI form complexes with HK, enabling the assembly of the entire contact activation system on the vascular wall [2–5] After being assembled, the system functions locally dependent on the demand for activation Thus, as a result of factor XI activation by activated factor XII, the rate of fibrin formation becomes enhanced [6], whereas activation of prekallikrein by activated factor XII enhances the proteolytic cleavage of HK by plasma kallikrein [7] However, the localization of the activation remains to be revealed, although factor XII recently was shown to bind to fibronectin (FN) [8], and several endothelial cell membrane proteins have been suggested as receptors for HK, including the globular C1q receptor [9,10] and cytokeratin-1 [10–12] The binding of HK to these receptors is strictly dependent upon the free Zn2+ concentration Cleavage of HK releases a short-lived strong inflammatory nonapeptide, bradykinin, leaving behind a two-chain activated HK (HKa), which Zn2+-dependently binds to the urokinase plasminogen activator receptor (uPAR) [13] This binding is considered to inhibit angiogenesis [14] The process of angiogenesis is a complex event requiring signals from both plasma and the extracellular basement membrane, and adhesive interactions of endothelial cells with the underlying basement membrane are instrumental in regulating the development and maintenance of the vascular wall The basement membrane contains a network of collagen and laminin (LM) [15] Formation of new vessels involves the migration and proliferation of cells To assist the cells in their migration, the extravascular matrix provides an environment consisting of hyaluronic acid, vitronectin (VN) and FN [16] LM plays an important role in cell adhesion to the basement membrane by interacting in the endothelium with a series of integrins, including a3b1, a6b1, avb1, avb3 and avb5 [16–18] The latter three of these integrins are activated and engaged by VN [19] when VN interacts with uPAR [20–22] The induction of apoptosis in proliferating endothelial cells by HKa [23] has been suggested to be the result of the binding of HKa not only to uPAR, but also to VN [24–26], preventing the interaction between VN and uPAR However, the apoptotic effect of HKa is apparently regulated by several ECM proteins [11,24] Thus, Guo et al [24] observed that the adhesion of endothe- HK binding to laminin lial cells cultured on VN, but not that of cells cultured on FN, was inhibited by HKa, whereas Sun and McCrae [14] demonstrated that HKa induced apoptosis of endothelial cells cultured on not only VN, but also on FN and LM Whether this inhibition is the result of the binding of HKa to the ECM proteins remains to be revealed LMs are a family of glycoprotein heterotrimers composed of an a, b and c chain To date, five a, four b and three c LM chains have been identified that can combine to form 15 different isoforms [15,27] The prototype of LMs is LM The LM isoform is characterized by the presence of one LM a1 chain, which combines with one LM b1 chain and one LM c1 chain LMs expressed in endothelial cells are characterized by the presence of LM a4 and a5 chains, which combine with LM b1 and c1 chains to form LM and LM 10, respectively In LM 11, which is also present in the endothelium, one a5 chain combines with b2 and c1 chains [15] In the present study, using a solid phase binding assay, we analyzed the binding of HK and HKa to LM, FN and VN, and showed that both HK and HKa bind with high affinity to LM The LMs used in the study comprised LM and LM 10 ⁄ 11, the latter of which are characteristic of the endothelium Results HK and HKa binding to proteins of the extracellular membrane To analyze the ability of HK to bind to selected proteins present in the extracellular membrane, freshly drawn citrate anti-coagulated plasma was incubated on LM, VN or FN coated on microtiter plates The amount of HK absorbed from the plasma by these matrix proteins was subsequently analyzed by immunoreactions using a monoclonal antibody to the heavy chain of HK (mAb 2B5) as the primary antibody This demonstrated that HK apparently could be extracted from plasma by binding to all three matrix proteins However, only the amount extracted by LM was higher than the amount extracted by the noncoated surface (Fig 1A) Analyzing the binding of purified HK and HKa to LM, VN and FN showed that, overall, a larger amount of HKa than of HK bound when incubated at the same concentration However, the amount of HKa bound to VN was identical to the amount bound to the noncoated plate, whereas the amount bound to LM and FN was lower By contrast, relative to the amount bound to the noncoated plate, more HK bound to LM and VN than to FN (Fig 1B) FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS 5229 HK binding to laminin A I Schousboe and B Nystrøm problem of nonspecific binding This was achieved by increasing the concentration of the LM and VN coated on the microtiter plate As indicated above, HK did not bind to the noncoated plate With increasing LM concentrations up to lgỈmL)1, increasing amounts of HK bound to LM By contrast, the amount of HKa that bound to the noncoated plate decreased as the concentration of LM increased to lgỈmL)1 A constant amount of HK and HKa bound to LM at concentrations higher than mgỈmL)1 (Fig 2A) The binding of HKa to LM was inhibited by the presence of soluble LM, but not by the presence of soluble VN or FN (data not shown) Zn2+ has been shown to play a determining role for the interaction of HK and HKa with all previously identified receptors and ligands The presence of Zn2+ increased the amount of HK as well as HKa bound to the noncoated plate but, analogous to the binding in Plasma VN Ligand coated on the microtiter plate FN LM None 0.2 0.4 0.6 0.8 1.2 HK/HKa bound (absorbance units) B Purified HK and HKa VN FN LM 0.2 0.4 0.6 0.8 1.2 1.4 1.6 HK/HKa bound (absorbance units) Fig Extraction of HK from plasma (A) and of HK and HKa (B) from solutions of purified proteins by immobilized LM, VN and FN (A) Blood was drawn by the syringe method using one volume of 3.8% (w ⁄ v) sodium citrate to nine volumes of blood and centrifuged at 1180 g for Aliquots of 100 lL of the supernatant (citrate anti-coagulated plasma) were then added to wells coated overnight with LM (10 lgỈmL)1), VN (5 lgỈmL)1) and FN (10 lgỈmL)1), respectively, and blocked with Locke’s buffer containing 1% (w ⁄ v) BSA (B) Alternatively, the wells were incubated with purified solutions of 20 nM HK (hatched columns) or 20 nM HKa (grey columns) in Locke’s buffer containing 0.35% (w ⁄ v) BSA After h of incubation, the content in the wells was removed and the plates were washed and incubated with primary and secondary antibodies, as described in the Experimental procedures Binding to the surface coated with buffer alone was used as a measure of nonspecific binding The results are the mean ± SD (n = 3) as indicated by vertical bars Attempts to reduce the binding to the noncoated plate by increasing the BSA concentration from the standard concentration of 3.5 mgỈmL)1 to as much as 75 mgỈmL)1 in the block buffer gradually decreased the amount of HKa bound nonspecifically, but had no influence on the amount of HKa bound to LM, VN and FN relative to the amount bound to the noncoated surface (results not shown) Because a considerable amount of HKa bound to the noncoated microtiter plate, we next analyzed whether the concentration of LM and VN was sufficiently high to saturate the surface of the microtiter plate, thereby preventing the 5230 4.0 HK HK + Zn HKa HKa + Zn 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 10 12 LM concentration (µg·mL–1) B HKa bound (absorbance units) None HKa bound (absorbance units) A 4.5 HK HK + Zn HKa HKa + Zn 3.5 2.5 1.5 0.5 0 10 15 VN concentration (µg·mL–1) 20 25 Fig Binding of HK and HKa to wells coated with increasing concentrations of LM (A) and VN (B) Wells were coated overnight with increasing concentrations of LM (A) and VN (B) in coating buffer as indicated and subsequently blocked with Locke’s buffer containing 1% (w ⁄ v) BSA Next, the wells were incubated for h with 20 nM HK or 20 nM HKa in Locke’s buffer containing 0.35% (w ⁄ v) BSA in the presence (open symbols) or absence of 50 lM Zn2+ (closed symbols) The amount of HK (squares) and HKa (circles) bound after extensive washing was determined by incubation with mAb 2B5 The results are the mean ± SD (n = 3) as indicated by vertical bars when extending beyond the symbols FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS I Schousboe and B Nystrøm HK binding to laminin the absence of Zn2+, the binding to LM decreased with increasing concentrations of LM and became constant at concentrations higher than lgỈmL)1 (Fig 2A) Therefore, a standard concentration of 10 lgỈmL)1 LM was used throughout the study A similar analysis, performed with VN as the immobilized ligand, showed no binding of HK at any concentration of VN in the absence of Zn2+ (Fig 2B) The high amount of HKa that bound to the noncoated plate (Fig 1B), particularly in the presence of Zn2+, decreased exponentially with increasing VN concentrations over the whole VN concentration range (0–20 lgỈmL)1) (Fig 2B) This indicated that any concentration of VN lower than 20 lgỈmL)1 was insufficient to saturate completely the surface in the microtiter plate Thus, the binding of HK and HKa to VN shown in Fig 1B was most likely nonspecific The effect of Zn2+ on the binding of HK and HKa to LM Further investigations of the effect of Zn2+ on the binding of HKa to LM showed that the amount of bound HKa increased with increasing Zn2+ concentrations up to 15–20 lm and then decreased (Fig 3) Because the enhancement was more pronounced at 200 3.000 150 100 50 0 20 40 60 80 100 120 Concentration of Zn2+ (µM) Fig Binding of HKa to LM as a function of the concentration of Zn2+ A microtiter plate was coated overnight with LM (10 lgỈmL)1) and blocked in Locke’s buffer containing 1% (w ⁄ v) BSA Next, it was incubated with either 10 nM HKa (open squares) or 30 nM HKa (open circles) diluted in Locke’s buffer containing 0.35% (w ⁄ v) BSA and supplemented with increasing concentrations of Zn2+ The amount of HKa bound to the LM after incubation for h was determined using mAb 2B5, as described in the Experimental procedures Using the amount of HKa bound to LM in the absence of Zn2+ as the reference (100%), the enhancement of the binding at the varying Zn2+ concentrations is shown as a percentage The results are the mean ± SD (n = 3) as indicated by vertical bars when extending beyond the symbols Hka bound (absorbance units) Relative amount of Hka bound (%) 250 lower than at higher HKa concentrations, this indicated that the effect of Zn2+ might be caused by a change in the affinity for the binding of HKa to LM Titration of LM with increasing concentrations of HKa in the presence and absence of 20 lm Zn2+ showed that the presence of Zn2+ enhanced the affinity of the binding, but apparently had no or only little effect on the maximum amount of bound HKa (Fig 4) Moreover, in the presence of Zn2+, the amount of HKa bound at high HKa concentrations was constant, indicating that HKa did not bind nonspecifically to LM The LM isoform used in the above measurements was the prototype, LM To determine whether HK and HKa would bind also to LM 10 ⁄ 11, the dissociation constants, KD, for the binding were determined in a series of identical experiments using LM as well as LM 10 ⁄ 11 This revealed that, in the absence of Zn2+, HK and HKa bound with the same affinity, regardless of whether LM or LM 10 ⁄ 11 had been coated on the microtiter plate (Table 1) The presence of Zn2+ affected more the affinity of the binding of HKa than HK Thus, a five- to seven-fold increase was observed in the affinity for the binding of HKa to both LM and LM 10 ⁄ 11, whereas only a three-fold increase was observed for the binding of HK to LM (Table 1) The presence of Zn2+ had no or only a minimal effect on the maximal amount bound in each individual experiment (data not shown) 2.500 2.000 1.500 1.000 0.500 0.000 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 Concentration of HKa (nM) Fig Concentration dependent binding of HKa to LM in the presence or absence of Zn2+ A microtiter plate was coated overnight with LM (10 lgỈmL)1) and blocked in Locke’s buffer containing 1% (w ⁄ v) BSA, as described in the legend to Fig Next, it was incubated with increasing concentrations of HKa diluted in Locke’s buffer containing 0.35% (w ⁄ v) BSA in the presence (open circles) or absence (closed circles) of 20 lM Zn2+ The amount of HKa bound to the LM after incubation for h was subsequently determined using mAb 2B5, as described in the Experimental procedures The results are the mean ± SD (n = 3) as indicated by vertical bars when extending beyond the symbols FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS 5231 HK binding to laminin I Schousboe and B Nystrøm Table The effect of Zn2+ on KD for the binding of HK and HKa to LM and LM 10 ⁄ 11 The concentration of Zn2+ was 20 lM Data are the mean ± SD (n) KD (nM) LM Hka None +Zn2+ HK None +Zn2+ LM 10 ⁄ 11 35.8 ± 9.8 (5) 7.1 ± 3.0 (5) 34.3 ± 0.9 (3) 4.6 ± 1.7 (4) 27.0 ± 4.6 (6) 8.7 ± 1.6 (4) 22.2 ± 6.1 (4) 16.6 ± 5.7 (3) The nonhyperbolic shape of the Zn2+-dependency curve (Fig 3) indicated further that the effect of Zn2+ might be explained by the presence of other divalent cations in the incubation mixture To verify this, the standard constituents, Ca2+ and Mg2+ in the incubation buffer (Locke’s buffer) were excluded This enhanced the amount of HKa bound to LM in the absence, but not in the presence, of Zn2+ and indicated that the effect of Zn2+ was to abolish an inhibitory effect of Mg2+ or Ca2+ or both Excluding one of these ions at a time showed that the inhibition was mainly the result of the presence of Ca2+ The amount of HKa bound to LM in the presence of Zn2+ was not or only slightly affected by the depletion of Ca2+ and Mg2+, as long as the BSA concentration was low (3.5 mgỈmL)1) At a higher BSA concentration, the presence of 20 lm Zn2+ was unable to abolish the inhibitory effect of Mg2+ and Ca2+ (Table 2) Identification of the binding region in HKa The amino acid sequence Arg439-Ser532 in domain of HK (Fig 5) [28,29], and particularly the His479His498 sequence, have been mapped as the endothelial cell-, ECM- and surface-binding site [11,13,30] To define the binding site within HK for the binding to LM, we determined whether a recombinant human kininostatin peptide (rhkininostatin; Arg439-Ser532) and a synthetic dodeca-peptide (His479-His498; HKH20) competitively inhibited the binding of HKa to LM We also tested whether a synthetic sequence of amino acids copying the Ser372-Arg419 sequence N-terminally to kininostatin affected the binding This sequence is cleaved off secondary to bradykinin Only rhkininostatin inhibited the binding and, both in the presence and absence of Zn2+, a 50% inhibition was observed at a 500 molar excess of rhkininostatin compared to the concentration of HKa (Fig 6) However, the presence of HKH20, even at a 5000 molar excess 5232 Table The effect of Ca2+, Mg2+ and Zn2+ on the binding of HKa to LM The complete binding buffer (Locke’s buffer) contained physiological concentrations of Ca2+ (2.3 mM) and Mg2+ (1 mM) The experiment was performed as described using the buffer composition of Locke’s buffer, but lacking the divalent cations indicated Some of the experiments were performed at a 10-fold higher BSA concentration, as indicated The results are representative of one of three experiments performed in triplicate and are shown as the mean ± SD A statistical analysis was performed using one-way analysis of variance followed by the Bonferoni post-hoc test Locke’s buffer HKa bound in the absence of Zn2+ [BSA] (absorbance (mgỈmL)1) units) Complete (control) 3.5 Lacking 2.3 mM Ca2+ 3.5 and mM Mg2+ Lacking mM Mg2+ 3.5 Lacking 2.3 mM Ca2+ 3.5 Complete (control) 35.0 Lacking 2.3 mM Ca2+ 35.0 and mM Mg2+ HKa bound in the presence of Zn2+ (20 lM) (absorbance units) 0.806 ± 0.014 2.133 ± 0.005 1.580 ± 0.044* 1.929 ± 0.032** 0.963 1.538 0.835 1.470 Statistically significant difference *P < 0.001; **P < 0.005 ± ± ± ± 0.017 0.027* 0.036 0.018* from 1.795 2.190 0.879 1.675 respective ± ± ± ± 0.058** 0.042 0.024 0.072* control: of HKa, had no effect This indicates that the N-terminal region of kininostatin encompassing the Arg439- Lys478 sequence might be of importance for the binding of HKa to LM Further investigations reveled that the binding to LM was abolished if HK and HKa had been pre-incubated at increasing lengths of time up to 60 with kallikrein at a : molar concentration ratio (Fig 7A) This was not the result of a time-dependent binding of kallikrein to HK and HKa because no inhibition was observed when HK and HKa were incubated with kallikrein for the same length of time in the presence of protease inhibitors (0 time data point) The use of western blotting at reduced conditions to follow the progression of the kallikrein catalyzed cleavage of HK revealed the generation of a 55 kDa heavy chain fragment as visualized by the mAb 2B5 antibody Complete cleavage was seen only at equimolar concentrations of HK and kallikrein (Fig 7B) Visualization of the cleavage products using anti-rhkininostatin IgG revealed that the 45 kDa light chain generated after 60 of incubation of HK with a : 10 molar concentration of kallikrein became partially cleaved, generating a 12 kDa anti-rhkininostatin reacting peptide, when HK was incubated for 60 with an equimolar concentration of kallikrein (Fig 7C) FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS I Schousboe and B Nystrøm HK binding to laminin Domain LM binding sequence Fig Functionally identified fragments of domain Numbering of the N- and C- terminal amino acids of indicated fragments and the generally accepted cleavage sites of kallikrein are based on the previously reported sequence [28] Bradykinin Kininostatin (1 µM) None N499 – S 532 rhKininostatin Plus zink Minus zink 0.5 H479 – H498 Surface binding sequence S372-R419 (10 µM) R 439 – K478 Kallikrein HKH20 (50 µM) Kininostatin (5 µM) S372 –R419 K420 – Q438 1.5 HKa bound (absorbance units) Fig Specificity of HKa binding to LM in the presence or absence of Zn2+ A LM coated microtiter plate (Fig 2) was incubated with HKa diluted in Locke’s buffer containing 0.35% (w ⁄ v) BSA in the presence (closed columns) or absence (open columns) of 20 lM Zn2+ In the experiments indicated, binding was measured in the presence of rhkininostatin (1 and lM), the amino terminal sequence of the light chain (Ser372-Arg419; 10 lM) and the surface binding peptide, HKH20 (50 lM) The amount of HKa bound to LM after incubation for h in the presence or absence of the effectors was determined using mAb 2B5, as described in the Experimental procedures The antibodies did not react with any of the effectors The results are the mean ± SD (n = 3) as indicated by vertical bars Binding of HK to ECM Although HK has been shown to bind Zn2+-dependently to the ECM generated during the growth of human umbilical vein endothelial cells (HUVEC) and ECV304 cells of a carcinoma cell line [5], the target for the binding was not identified Because LMs are being deposited in vivo in the basement membrane by proliferating cells, it was next determined whether LM was present in ECM generated during growth of HUVEC Immunostaining of ECM with anti-LM IgG verified the presence of LM in this matrix (data not shown) Analysing the binding of HK to ECM, the influence from binding to the cell-free surface in the culture dish had to be taken into account because a confluent layer of cells covers only the bottom of the cell culture dish Thus, the cell-free surface would be expected to account for approximately two-thirds of the surface in a 96-well cell culture plate incubated with 100 lL per well, and hence be accessible for nonspecific binding Because a considerably higher amount of HKa than HK bound nonspecifically to the noncoated surface (Figs and 2), and this could not be prevented by increasing the concentration of BSA, only the binding of HK was used in this part of the study However, at conditions in which cell-free areas were blocked with a 0.2% (w ⁄ v) gelatin or a 0.35% (w ⁄ v) BSA, no HK bound to ECM in the absence of Zn2+ and, in its presence, a higher amount of HK bound to the cellfree surface than to ECM (Fig 8) Increasing the BSA concentration increased the optimal concentration of Zn2+ for the binding of HK without blocking the nonspecific binding (data not shown) This excludes the possibility of measuring the binding of HK to ECM and suggests that HK might bind to one of the compounds in the cell culture medium that was absorbed on the surface of the well when the cells were growing Discussion There are numerous investigations showing that HK ⁄ HKa binds Zn2+-dependently to different receptors on the surface of endothelial cells, and only a few analyzing the possibility of the interaction of the kininogens with the proteins in the basal membrane, which is of equal importance when explaining the in vivo effect of HKa Therefore, the binding of HK and HKa to selected noncollageneous proteins could be important for the function of the kallikrein ⁄ kinin ⁄ kininogen system on the vascular wall During the present study, it was shown that both HK and HKa bind to LM, which is the most abundant noncollageneous protein in the basal membrane [15] The binding was inhibited by rhkininostatin, but not by the surface binding peptide sequence (His479-His498; HKH20) within kininostatin, which has been identified as the sequence that is responsible for the Zn2+-dependent binding of HK ⁄ HKa to endothelial cell [11] Equimolar concentrations of HK ⁄ HKa and kallikrein cleaved off an anti-rhkininostatin reacting peptide from HKa, abol- FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS 5233 HK binding to laminin I Schousboe and B Nystrøm HK/HKa bound (absorbance units) A 2.5 HK: PK (10 : 1) HK: PK (1 : 1) HKa: PK (10 : 1) HKa: PK (1 : 1) 1.5 0.5 C 10 20 30 40 50 Incubation period (min) B b/0.5 b/30 b/60 60 HK b/60 c/60 70 c/0.5 c/30 c/60 120 kDa 120 kDa 45 kDa 55 kDa 12 kDa Fig Incubation with kallikrein abolished the binding of HKa and HK to LM (A) by cleaving the light chain of HKa (B, C) In Locke’s buffer containing 0.35% (w ⁄ v) BSA, one volume of HK and HKa, respectively, at a concentration of 60 nM was incubated for varying lengths of time with one volume of either nM (10 : 1) or 60 nM (1 : 1) kallikrein (A) At the times indicated, cleavage was stopped by adding one volume of a protease inhibitor cocktail consisting of 40 lgỈmL)1 each of leupeptin, aprotenin, benzamidin and soy bean trypsin inhibitor, giving a final concentration of 20 nM of HK and HKa, respectively The binding of the proteolysed HK (squares) and HKa (circles) to LM coated on a microtiter plate at a concentration of 10 lgỈmL)1 (Fig 2) are shown after incubation at high (closed symbols) and low (open symbols) concentrations of kallikrein Western blots of incubation mixtures of HK and kallikrein followed the proteolysis (B) Using the mAb 2B5 towards the heavy chain showed that, at a low kallikrein concentration, a higher amount of HK was cleaved after 60 (b ⁄ 60) than after 30 (b ⁄ 30) of incubation, whereas, at equimolar concentrations of HK and kallikrein, HK was completely cleaved after 30 (c ⁄ 30) incubation and a considerable amount was already cleaved after 0.5 (c ⁄ 0.5) (C) Using the anti-rhkininostatin IgG specific for the light chain, it was found that 60 of incubation of equimolar concentrations of HK with kallikrein (c ⁄ 60) apparently cleaved the light chain generating a 12 kDa anti-rhkininostatin recognizable peptide ishing the binding of HK ⁄ HKa to LM This, when combined with the inhibition of the binding by rhkininostatin, indicates that the binding of HK ⁄ HKa to LM is mediated via the N-terminal region in kininostatin encompassing the amino acid sequence Arg439-Lys478 (Fig 5) Zn2+ has been assumed to have a decisive role with respect to the binding and function of HK and HKa In the present study, we show that Zn2+ is not required for the binding of either HK or HKa to LM However, it enhanced the affinity of the binding of both HK and HKa to LM, although the total amount bound was not affected However, this was without significance because the binding affinity, even in the absence of Zn2+, was approximately 15-fold higher than the plasma concentration of HK, demonstrating 5234 no obvious need for the presence of Zn2+ Furthermore, the effect of Zn2+ on the binding to LM was only evident at low concentrations of BSA, at which it abolished the inhibitory effect of Ca2+ At a higher BSA concentration, the effect of Ca2+ remained, whereas the effect of Zn2+ was eliminated by reducing its free concentration by binding to BSA Because the total plasma concentration of Zn2+ is 25 lm [31], the free Zn2+ concentration in vivo may never be sufficiently high to influence either the function of HKa or the binding of HK and HKa to LM Therefore, the present study, showing that HK and HKa bind Zn2+independently to LM, must be physiologically relevant Experimentally, there was a considerable difference in the minimal amount of matrix protein required to cover the microtiter plate The concentration of LM FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS I Schousboe and B Nystrøm HK binding to laminin HK bound (absorbance units) 1.2 0.8 0.6 0.4 0.2 0 50 100 150 200 Concentrarion of Zn2+ (µM) Fig Binding of HK to ECM and cell-free wells Cell-free wells, which had been exposed to growth medium, exactly as for the cultures of HUVEC were, similarly to a confluent layer of the cells, rinsed with NaCl ⁄ Pi and incubated with EDTA Next, the wells (ECM and cell-free) were blocked for 30 with Locke’s buffer containing 0.2% (w ⁄ v) or 0.35% (w ⁄ v) BSA (blocking solutions) and subsequently incubated for h with 20 nM HK diluted in the respective blocking solutions containing varying concentrations of Zn2+ The amount of HK bound was measured by immunoreactions, as described in the Experimental procedures A higher amount of HK bound Zn2+-dependently to cell-free wells (open symbols) than to ECM (closed symbols), regardless of whether the surface had been blocked with gelatin (circles) or BSA (triangles) The results are shown as the mean ± SD (n = 3) when extending beyond the symbols was lgỈmL)1, whereas more than 20 lgỈmL)1 was required for VN, and almost no HKa bound to VN at this concentration, even in the presence of Zn2+ A previous study reported that HKa binds Zn2+-dependently to VN coated at a concentration of lgỈmL)1 [26] At present, this discrepancy cannot be explained because the use of a considerably higher BSA concentration for blocking did not prevent Zn2+-dependent nonspecific binding However, the nonspecific binding, particularly that of HKa, in the presence of Zn2+ may explain the controversial results reported on the effect of matrix proteins on regulation of the apoptotic effect of HKa on HUVEC grown in culture HKa induced Zn2+-dependent apoptosis of the cells, regardless of the cells being cultured on lgỈmL)1 VN or LM [14,24,26,32], although not on 10 lgỈmL)1 FN [24,26,32] It was not possible to measure binding of HK to LM in ECM generated during the growth of HUVEC This may likewise be the result of too low a concentration of the LM deposited during the 2–4 days of cell growth The addition of Zn2+ enhanced the nonspecific binding and indicated that the previously shown Zn2+-dependent binding of HK to ECM [5] might have been nonspecific The strongest evidence for this was that a lower amount of HK bound Zn2+-depen- dently to ECM than to the cell-free wells, implying that ECM blocked the binding of HK to the cell-free area of the well Furthermore, the Zn2+ optimum for the binding of HK to the surface of the microtiter plate was exactly the same as those reported for the binding of HK to ECM and HUVEC [4,5,33,34], regardless of whether the binding assay was performed in buffer supplemented with gelatin or albumin Accordingly, and because the correction for nonspecific binding in these previous studies was performed by the subtraction of binding in the absence of Zn2+, a series of investigations investigating the significance of Zn2+ for the interaction between HK and endothelial cells in cell culture systems [4,5,13,23,34,35] needs to be revisited In vivo, LM might be a matrix protein of significance with respect to the function of HKa on the activity of the endothelial cells in as much as LM plays an important role in cell adhesion to the basement membrane [16–18] Furthermore, both HK and HKa bind to LM, suggesting that this may be a prerequisite for the kallikrein ⁄ kinin ⁄ kininogen system to be assembled and activated to enable the local liberation of the short-lived bradykinin and its participation in inflammatory and pro- and anti-angiogenic reactions [25,36,37] Thus, the binding of HK to LM in the basement membrane may be followed by activation of prekallikrein bound in a : molecular complex to HK The activation of prekallikrein is accomplished either by factor XIIa, heat shock protein 90 [38] or a prolylcarboxypeptidase [39] The observation that kallikrein cleaves off a 12 kDa anti-rhkininostatin reacting peptide shows that kallikrein, secondary to bradykinin, releases kininostatin from HK, as previously anticipated [36] This fits neatly with the observation that the HKa signaling of an apoptotic effect is promoted as a result of extensive cleavage by kallikrein [37,40] Experimental procedures Materials One-chain HK, two-chain HKa and kallikrein delivered lyophilized from Enzyme Research Laboratories Ltd (Swansea, UK) were dissolved, aliquoted and stored in siliconized test tubes at –80 °C All dilutions of HK, HKa and kallikrein were likewise performed in siliconized test tubes and excess dilutions were discarded VN, FN, LM from Engelbreth-Holm-Swarm murine sarcoma basement membrane (LM 1) and LM from placenta (LM 10 ⁄ 11) were obtained from Sigma Chemicals (St Louis, MO, USA) The surface binding peptide sequence within the light chain of FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS 5235 HK binding to laminin I Schousboe and B Nystrøm HK, His479-Lys-His-Gly-His-Gly-His-Gly-Lys-His-Lys-AsnLys-Gly-Lys-Lys-Asn-Gly-Lys-His498 (HKH20), was a kind gift from Dr Alvin Schmaier (Case Western Reserve University, Cleveland, OH, USA) The sequence of amino acids copying the Ser372-Arg419 region [28] was synthe˚ sized at Novo Nordisk (Maløv, Denmark) Recombinant human kininostatin (rhkininostatin) and goat anti-(human rhkininostatin) were obtained from R&D Systems (Abingdon, UK) The monoclonal antibody to the heavy chain of human HK (mAb 2B5) was obtained from Abcam (Cambridge, UK) Horseradish peroxidase (HRP) conjugated rabbit anti-(mouse IgG) (P-0260), HRP-conjugated rabbit anti-(goat IgG) (P-0449) and ortophenylenediamine were obtained from Dako (Glostrup, Denmark) SuperSignalÒ West Femto Maximum Sensitivity Substrate was from Pierce Biotechnology, Inc (Rockford, IL, USA) Essentially fatty acid free BSA (A7030) was obtained from Sigma Chemicals All other reagents were of the purest grade commercially available Solid phase binding assay Maxisorp microtiter plates (high binding capacity; Nunc, Roskilde, Denmark) were coated overnight at °C with 150 lL of lgỈmL)1 VN or 10 lgỈmL)1 of either FN, LM or LM 10 ⁄ 11 in coating buffer (0.1 m potassium phosphate, pH 7.4), or at the concentrations indicated After blocking for 30 with 200 lL of 1% (w ⁄ v) BSA in Locke’s buffer (154 mm NaCl, 5.6 mm KCl, 3.6 mm NaHCO3, 2.3 mm CaCl2, mm MgCl2, 5.6 mm glucose, mm Hepes, pH 7.4), 100 lL of HK or HKa (20 nm or as indicated) diluted in Locke’s buffer containing 0.35% (w ⁄ v) BSA was added After incubation for h at room temperature, the wells were washed three times with Locke’s buffer, one time with ice-cold methanol and five times with wash buffer [50 mm Tris, 0.15 mm NaCl, pH 8.0, containing 0.05% (v ⁄ v) Tween 20] This was followed by h of incubation with mAb 2B5 diluted 5000-fold in wash buffer containing 1% (w ⁄ v) dry skim milk After a further washing cycle with wash buffer (five times), the plate was incubated for h with HRP-conjugated rabbit anti-(mouse IgG) diluted 5000-fold in wash buffer Finally, the plates were incubated for 10–30 with ortophenylenediamine, dissolved in water according to the manufacturer’s instructions The peroxidase reaction was stopped by a two-fold dilution with 0.5 m H2SO4 and the relative amount of HK bound to the wells determined as absorbance units (A) at 490 nm All experiments were performed in triplicates and repeated at least twice To obtain estimates of affinity constants, the data were analyzed according to the isotherm: A = Amax · [B] ⁄ (KD + [B]) where [B] is the molar concentration of the analyt, which is either HK or HKa; A is the absorbance of the oxidized HRP substrate, which is assumed to be proportional to the 5236 amount of bound analyt; and Amax represents the absorbance at saturating concentrations of the analyt Western blotting Proteolytic cleavage of HK was visualized by western blotting of aliquots of HK incubated with kallikrein in Locke’s buffer Reduced samples were separated on 4–12% SDS-PAGE simultaneously with a standard sample of a mixture of Mr markers After blotting to a poly (vinylidene difluoride) membrane according to standard procedures, the membrane was incubated for 10 in Tween-BSA block-buffer [50 mm Tris, 0.15 mm NaCl, pH 8.0, containing 0.1% (v ⁄ v) Tween 20 and 0.1% (w ⁄ v) BSA], and subsequently overnight with the primary antibody; either mAb 2B5 or goat anti-(human rhkininostatin), diluted 5000-fold in 1% (w ⁄ v) dry skim milk in the Tween-BSA block-buffer The positions of the light and the heavy chains of HK were visualized by incubation with HRP conjugated rabbit anti-(mouse IgG) (P-0260) and HRP-conjugated rabbit anti-(goat IgG) (P-0449), respectively, followed by SuperSignalÒ West Femto Maximum Sensitivity Substrate as recommended by the manufacturer The results were monitored using a chemiluminator Endothelial cell culture HUVEC (Clonetics, Cambrex Bio Science, Verviers, Belgium) were prepared as previously described [41] Briefly, the cells were grown to confluence under standard conditions, cryopreserved and sub-cultured in half of a 96-well microtiter plate (Nunc) The microtiter plate was not pre-coated The other half of the plate (without cells) was used as a control The wells in this half were incubated with growth medium in absence of cells, and the medium was changed in accordance with the exact same schedule as for the exchange of medium in the wells with cells HK binding to ECM At confluence, all wells including the cell-free wells were washed with NaCl ⁄ Pi (50 mm phosphate, 0.1 m NaCl, pH 7.5), and incubated with mm EDTA in NaCl ⁄ Pi for 30 at room temperature, as previously described [41] This treatment detached the cells from the surface of the wells as visualized microscopically and as analyzed by the absence of anti-actin binding components [ELISA using mouse-anti actin IgG (M-0635); Dako] The wells were next washed twice with Locke’s buffer and incubated for minimum of 30 at room temperature with 200 lL per well of 1% (w ⁄ v) BSA or otherwise as noted LM deposited in the matrix during growth of the cells was detected using rabbit anti-LM (Z-0097; Dako) Binding of HK was determined as described above FEBS Journal 276 (2009) 5228–5238 ª 2009 The Authors Journal compilation ª 2009 FEBS I Schousboe and B Nystrøm HK binding to laminin Statistical analysis The results are shown as the mean ± SD and statistically significant differences were estimated using analysis of variance and the Bonferoni post-hoc test P < 0.05 was considered 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Journal compilation ª 2009 FEBS ... 361 5–3 620 Mahdi F, Shariat-Madar Z, Kuo A, Carinato M, Cines DB & Schmaier AH (2004) Mapping the interaction between high molecular weight kininogen and the urokinase plasminogen activator receptor... [8], and several endothelial cell membrane proteins have been suggested as receptors for HK, including the globular C1q receptor [9,10] and cytokeratin-1 [1 0–1 2] The binding of HK to these receptors... b2 and c1 chains [15] In the present study, using a solid phase binding assay, we analyzed the binding of HK and HKa to LM, FN and VN, and showed that both HK and HKa bind with high affinity to