Research paper
Heparin affects the interaction of kininogen on endothelial cells
Andrezza J. Gozzo
a, Guacyara Motta
a, Ilana Cruz-Silva
a, Viviane A. Nunes
b, Nilana M.T. Barros
c,
Adriana K. Carmona
c, Misako U. Sampaio
a, Yara M.C. Michelacci
a, Kazuaki Shimamoto
d,
Helena B. Nader
a, Mariana S. Araújo
a,*aDepartment of Biochemistry, Universidade Federal de São Paulo, 04044-020 SP, Brazil bSchool of Arts, Sciences and Humanities, Universidade de São Paulo, 03828-000 SP, Brazil cDepartment of Biophysics, Universidade Federal de São Paulo, 04044-020 SP, Brazil dII Department of Internal Medicine, Sapporo Medical University, 060-0061 Sapporo, Japan
a r t i c l e
i n f o
Article history:
Received 21 December 2010 Accepted 4 July 2011 Available online 22 July 2011
Dedicated to the memory of Claudio A.M. Sampaio and Carl Peter von Dietrich for their contributions to thefields of proteases and glycosaminoglycans.
Keywords: Endothelial cells Glycosaminoglycans Kininogen Prekallikrein Zinc ions
a b s t r a c t
In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Znþ2, HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in thefluid phase, was augmented by this glycosaminoglycan in the presence of Zn2þ
. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatoryfluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.
Ó2011 Elsevier Masson SAS.
1. Introduction
The plasma kallikrein-kinin system is one of the pathways responsible for bradykinin (BK) release. Since its discovery, it has been demonstrated that BK is able to induce all four classical signals
of inflammation (heat, redness, swelling and pain) when injected into human or animal tissues [1]. In this system, when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells in a Znþ2-dependent manner[2,3], PK is activated and plasma kallikrein (huPK) is formed, which results in BK liberation from HK, NO formation[4]and factor XII (FXII) acti-vation[5].
A primary event of plasma kallikrein-kinin system activation is HK docking to the cell surface. This binding has been described on endothelial cells[2e4,6], neutrophils[7], platelets[8], astrocytes [9], vascular smooth muscle cells[10]and macrophages[11]. Just out of interest, plasma kallikrein-kinin system activation (including HK binding) has also been described in a range of epithelial cells (e.g., lung, gut and prostate epithelium)[12]. On the endothelial cell surface, some proteins have been found to contain HK binding sites; these include urokinase plasminogen activator receptor (uPAR), cytokeratin 1 and receptor for the globular heads of complement
Abbreviations:b, biotin; BDS, bovine dermatan sulfate; BK, bradykinin; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; ECM, extracellular matrix; ECV304, endothelial cell vein lineage; FXII, factor XII; Hep, heparin; HS, heparan sulfate; HK, high molecular weight kininogen; huPK, human plasma kallikrein; KS, keratan sulfate; LMWDXS, low molecular weight dextran sulfate; LMWHep, low molecular weight heparin; N-des Hep, N-desulfated heparin; PK, prekallikrein; RAEC, rabbit aorta endothelial cells; TDS, tuna dermatan sulfate.
*Corresponding author. Universidade Federal de São Paulo, Department of Biochemistry, Rua Três de Maio, 100, Vila Clementino, 04044-020 S. Paulo, SP, Brazil. Tel.:þ55 11 55764445x1085.
E-mail address:mariana.bioq@epm.br(M.S. Araújo).
Contents lists available atScienceDirect
Biochimie
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h i
0300-9084Ó2011 Elsevier Masson SAS. doi:10.1016/j.biochi.2011.07.003
Open access under the Elsevier OA license.
component C1q (gC1qR)[13]. Recently, using solid phase binding assays, Schousboe and Nystrøm[14]described the binding of HK to laminin, a basement membrane protein. Proteoglycans, comprising a protein core covalently attached to glycosaminoglycans, have also been suggested as an alternative HK binding site on endothelial cells[15,16].
Previous studies show that these cell surface proteoglycans are able to immobilize and regulate the turnover of ligands, protecting them from proteolytic and chemical inactivation[17]or resulting in their internalization into endosomes, as shown in experiments with HK [16]. In addition, the extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble glycosaminoglycans that can modulate interactions at the cell surface.
During inflammatory processes, several events occur concomi-tantly with plasma kallikrein-kinin system activation, such as the release of heparin from mastocytes, chondroitin 4-sulfate from platelets [18,19]and heparan sulfate from endothelial cells[20]. These glycosaminoglycans can interact with plasma kallikrein-kinin system proteins, modulating the inflammatory response. In this regard, heparin and heparin-like molecules have been considered to hold potential in the treatment of inflammatory diseases[21].
In our previous results, dermatan sulfate and chondroitin sulfate decreased HK hydrolysis by huPK in vitro, and dermatan sulfate reduced the rat paw edema induced by carrageenin [22]. Addi-tionally, the proteolytic activity on other substrates and inhibition of huPK were differentially modulated by glycosaminoglycans [23,24]. In the present study, we investigated the effect of glycos-aminoglycans on the interaction of HK on endothelial cell surfaces and their extracellular matrix (ECM). Our results suggest that the interaction of HK on endothelial cells with soluble glycosamino-glycans is dependent on their structural features. This interaction is particularly significant for heparin, which abolished HK binding, the initiating step for BK release.
2. Materials and methods
The rabbit aorta endothelial cell (RAEC) line was kindly provided by Dr. Vincenzo Buonassisi from the Department of Biology, University of California, San Diego, CA, USA. The endothelial cell vein (ECV304) line was obtained from American Type Culture Collection-ATCC (Manassas, VA, USA). PK and HK were purchased from EMD Biosciences (San Diego, CA, USA). Bovine lung heparin (9.5e12 kDa) was obtained from Opocrin Research Laboratory (Italy), and shark chondroitin 6-sulfate (40e60 kDa) and bovine intestinal dermatan sulfate (22 kDa) were obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Bovine chondroitin 4-sulfate (27 kDa), low molecular weight dextran sulfate (8.0 kDa) and bovine serum albumin (BSA) were purchased from SigmaeAldrich (St. Louis, MO, USA), and low molecular weight heparin (Fraxiparin, 5.6e6.4 kDa) was purchased from Sanofi-Synthelabo Grape (Paris, France). N-desulfated heparin (6.0 kDa) [25], bovine cartilage keratan sulfate[26], bovine lung heparan sulfate (30e40 kDa) and tuna dermatan sulfate (15 kDa) [27]were purified in our laboratory. Ham’s F-12 nutrient mixture medium, penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA), and fetal calf serum was purchased from Cultilab (Campinas, SP, Brazil). The biotinylation kit [NHS-LC-biotin, 2-(40-hydroxyazobenzene)
benzoic acid (HABA) and avidin], ImmunoPure streptavidin horseradish peroxidase conjugate (SA-HRP) and peroxidase-specific fast-reacting substrate, turbo-3,30,5,50
-tetramethylbenzi-dine dihydrochloride (Turbo-TMB) were obtained from Pierce (Rockford, IL, USA). H-D-Pro-Phe-Arg-pNan (S2302) was purchased from Chromogenix Instrumentation Laboratory (Milan, Italy).
2.1. Biotinylation of purified proteins
HK and PK were biotinylated as previously described[2,28]. The protein concentration in each fraction was determined by its absorbance at 280 nm using theE1%7.01 for HK and 11.7 for PK[29]. Lyophilized HK or PK was mixed with a 5-fold molar excess of NHS-LC-biotin (biotin) in 0.10 M phosphate buffer, pH 7.2 for 30 min at room temperature. The reaction mixture was transferred into a desalting column PD-10 (GE Healthcare Biosciences, Piscataway, NJ, USA) equilibrated with 10 mM phosphate buffer, pH 7.2 con-taining 0.15 M NaCl. Biotin incorporation into HK or PK was determined by addition of HABA, according to the manufacturer’s instructions. Both biotin-HK (bHK) and biotin-PK (bPK) migrated as single chain proteins under reducing conditions (SDS-PAGE).
2.2. Endothelial cell culture
ECV304 is a cell line established from primary culture of human umbilical vein endothelial cells (HUVEC) [30,31] and has been described as a derivative of the bladder carcinoma cell line T24[32]. RAEC is an established endothelial cell line derived from rabbit aorta[33]. The clones used in this work were positive for endo-thelial cell markers.
The cells were subcultured in Ham’s F-12 nutrient mixture medium supplemented with 10% heat-inactivated fetal calf serum, 10 U penicillin and 10
mg/mL streptomycin and grown to con
fluence in 60 mm dishes. For binding experiments, cells were seeded at 2.0104cells/well and grown to confluency for 24 h (ECV304) or 48 h (RAEC) at 37C in 2.5% CO2.
2.3. Preparation of extracellular matrix
ECM obtained from either ECV304 or RAEC were prepared fresh prior to the start of each experiment[34]. Briefly, cells were sub-cultured on microtiter plate wells and removed by treatment with 0.5% Triton X-100 (p/v) in 10 mM phosphate buffer, pH 7.4 con-taining 0.15 M NaCl for 15 min (37C, 2.5% CO
2). Next, cells were incubated with 25 mM NH4OH for 10 min at 37C and then washed extensively with 20 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.4 containing 0.15 M NaCl and 0.05% Tween-20 (p/v). Subsequently, no cells in the microtiter plate were detected by light microscopy. The adherent subendothelial matrix reacted with antibodies tofibronectin and perlecan (data not shown).
2.4. Binding of biotinylated proteins
Considering the experiments performed with ECV304 cell surfaces or ECM, all incubations and washes were performed in N-(2-hydroxyethyl)-piperazine-N0-2-ethanesulfonic acid
(HEPES)-Tyrode’s binding buffer (15 mM HEPES, 0.14 M NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4), pH 7.4 containing 3.5 mg/mL dextrose, 50
mM ZnCl
2, 1.0 mM MgCl2, 2.0 mM CaCl2and 0.35% BSA as protein carrier [3]. For experiments performed with RAEC surfaces or ECM, the binding buffer was HEPES-carbonate (5.0 mM HEPES, 0.15 M NaCl, 5.6 mM KCl, 3.6 mM NaHCO3), pH 7.4 con-taining 1.0 mg/mL dextrose, 25mM ZnCl
2, 1.0 mM MgCl2, 2.0 mM CaCl2and 0.10% gelatin as a protein carrier[4,16].The cell surface or ECM from both cell lines was washedfive times with the respective binding buffer before performing all binding studies. After, they were incubated with 20 nM bHK in the absence or presence of 1.0
mM glycosaminoglycans for 1 h at 37
Cin a 100
mL-reaction volume. The unbound bHK was then removed
by washing three times with the binding buffer.temperature and detected with the substrate turbo-TMB (100
mL),
as previously reported [28]. Bound bHK was quantified by measuring the absorbance at 450 nm. The bHK binding was calculated by subtracting bound bHK in the presence of Znþ2from bHK in the absence of Znþ2, and the data were expressed as a percentage with the bHK binding in absence of glycosaminogly-cans set as 100%.Bound bPK was measured on confluent monolayers of RAEC and ECV304 cells and ECM. Briefly, afterfive washes with the respective binding buffers, cells or ECM were incubated with 20 nM HK (100
mL) for 1 h at 37
C. Unbound HK was removed by aspiration,and after three washes with binding buffer, 20 nM bPK was added in the absence or presence of 1.0
mM heparin for 1 h at 37
C ina 100
mL-reaction volume. Cell or ECM-associated bPK was detected
using SA-HRP and the substrate turbo-TMB as described previously for bHK. The bPK binding was calculated by subtracting bound bPK in the presence of HK from bPK bound in the absence of HK, and the data were expressed as a percentage.2.5. PK activation on endothelial cells and their ECM in the presence of heparin
Confluent monolayers of RAEC and ECV304 cells or their matrix were washedfive times with the respective binding buffer. After, cells or ECM were incubated with 20 nM HK (100
mL) for 1 h at 37
C.Unbound HK was removed by aspiration, and after three washes with binding buffer, 20 nM PK (100
mL) was added in the absence or
presence of 1.0mM heparin for 1 h at 37
C. After this incubation, theunbound PK was removed by aspiration and the plate was washed three times with 0.10 mM phosphate buffer, pH 7.4 with 0.14 M NaCl. Kallikrein activity was measured with the hydrolysis of 0.4 mM H-D-Pro-Phe-Arg-pNan for 2 h at 37C at 405 nm[3,4].
2.6. HK hydrolysis by huPK in the presence of Znþ2and heparin
The Znþ2is an essential cofactor for assembly and activation of contact system proteins bound to endothelial cells[5]. The infl u-ence of these ions and heparin on HK hydrolysis by huPK and BK release was investigated.
HK (6.0
mM) was incubated with or without heparin (0.26
mM)
and 50mM ZnCl
2in 50 mM Tris buffer, pH 8.0 containing 0.15 M NaCl for 5 min at 37C, and then huPK (5.2 nM) was added in afinal volume of 20
mL. Aliquots were removed at various times up to
15 min, and BK was extracted in ethanol for 10 min at 70C andquantified by radioimmunoassay as described by Shimamoto et al. [35]with some modifications[22].
2.7. Statistical analysis
The results are shown as the mean SD of two different experiments performed in triplicate. Statistical analyses were per-formed using the commercial program GraphPad Prism Version 5 (GraphPad Software Inc, San Diego, CA, USA). One way analysis of variance with Dunnet post test was used.
3. Results
3.1. Effect of glycosaminoglycans on HK binding to endothelial cells
Previous studies have characterized HK binding in a Znþ2 -dependent manner with both ECV304[3]and RAEC [16, MSc. A. Gutierrez, personal communication]. Here, we investigated the binding of HK and PK on HK bound to these cells in the presence of soluble glycosaminoglycans: heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate and keratan sulfate.
To verify if the nature of the glycosaminoglycan could affect HK binding on the cell surface, two distinct cell types with different glycosaminoglycan content were used: RAEC, which presents 20% chondroitin sulfate and 80% heparan sulfate with typical heparin sequences, and ECV304 with 50% of each glycosaminoglycan [36,37].
First, experiments were performed to determine whether HK binding to RAEC or ECV304 cell surfaces and their matrix was influenced by different glycosaminoglycans. On both the RAEC surface and ECM, almost all of the glycosaminoglycans decreased HK binding compared to the control. On the RAEC surface (Fig. 1A), bHK binding was abolished in the presence of heparin, but bHK binding decreased to 57%, 61%, 68% and 75% in the presence of heparan sulfate, chondroitin 4-sulfate, tuna and bovine dermatan sulfates, respectively. On RAEC matrix (Fig. 1A), glycosaminoglycans had the same effect: decreasing bHK binding. The bHK binding to RAEC surface and their matrix was abolished in the presence of heparin and decreased to 57%, 65%, 70%, 69% and 80% with heparan sulfate, chondroitin 4-sulfate, tuna and bovine dermatan sulfates and keratan sulfate, respectively. However, binding was not affected by chondroitin 6-sulfate.
The bHK binding to the ECV304 cell surface and ECM was decreased by approximately 50% by heparin. On the cell surface, keratan and chondroitin 6-sulfate caused a discrete increase in this binding (Fig. 1B).
The results showed that in both RAEC and ECV304 cell lines, heparin was the glycosaminoglycan that had the most significant effect on disturbing HK binding to endothelial cell surface or matrix.
Fig. 1. Influence of glycosaminoglycans on HK binding to endothelial cells. bHK (20 nM) was bound to RAEC (A) or ECV304 (B) cells and their ECM in appropriate buffer for 1 h at 37
3.2. Effect of heparin on HK binding to endothelial cells
Based on the effects of heparin on HK binding to either RAEC and ECV304 cell surfaces or their ECM, its role in binding was further examined.
The bHK binding was analyzed in the presence of different heparin concentrations. Biotin-HK binding to RAEC surfaces and their ECM was completely abolished in the presence of 0.50
mM
heparin (Fig. 2A). However, on ECV304 cell surfaces and their ECM, concentrations higher than 50mM heparin were necessary to
abolish bHK binding (Fig. 2B).To determine if the effect of heparin could be related to its size or charge, heparin-like species such as low molecular weight heparin, N-desulfated heparin and low molecular weight dextran sulfate were tested. Biotin-HK binding to RAEC surfaces and their ECM was completely abolished in the presence of heparin and low molecular weight dextran sulfate, but this binding was reduced to 15e20% in the presence of low molecular weight heparin and was not affected in the presence of N-desulfated heparin (Fig. 3A). By contrast, on ECV304 cell surfaces or ECM, the binding was almost abolished in the presence of low molecular weight dextran sulfate, reduced to 50% with heparin, reduced to 55e60% with low molecular weight heparin and was unaffected by N-desulfated heparin (Fig. 3B). These data suggest that the sulfate content on glycosaminoglycans is essential for its influence on HK binding to cell surfaces and ECM of these cell lineages.
3.3. Effect of heparin on PK binding and activation
HK is a putative receptor for PK on endothelial cells, and it was observed that heparin modified HK interaction with the endothelial cell surface and ECM. Therefore, the effect of this glycosamino-glycan on the interaction between HK and PK was investigated. First, HK was incubated with endothelial cells or ECM, and then bPK with or without heparin was added (Fig. 4). Because the activation of PK on endothelial cells and ECM was previously characterized [3,4], the effect of heparin on PK activation was also investigated (Fig. 5). The results inFigs. 4 and 5show that heparin did not have any effect on bPK binding to HK or on activation of PK in these cell lineages. In this context, this glycosaminoglycan interferes with the plasma kallikrein-kinin system by specifically interacting with HK, not with PK or huPK formed on the cell surface or ECM.
3.4. Effect of heparin and Znþ2on HK hydrolysis
Despite the fact that formation of huPK on the cell surface or ECM was not modified by heparin, the influence of this glycos-aminoglycan and Znþ2on HK hydrolysis by huPK was performed in vitro. BK release in the presence or absence of heparin and/or Znþ2 was quanti
fied by radioimmunoassay. When HK was pre-incubated with Znþ2, the rate of BK release was signi
ficantly higher than in the absence of this cation (Fig. 6). Without Znþ2, heparin did not significantly affect the rate of BK formation. In the presence of the cation, heparin increased BK release when compared to the control without Znþ2, but when compared to the control with Znþ2, this effect only appeared with low concentra-tions of HK (Fig. 6). These results indicated the importance of Znþ2 within the cell environment, the presence of which enhances the
Fig. 2.Effect of heparin on HK binding to endothelial cells. RAEC (A) or ECV304 (B) cells and their ECM were incubated with bHK (20 nM) in the absence or presence of heparin (1.0 nMe50mM) in appropriate buffer for 1 h at 37
C. Thefigures show the mean SD of two different experiments performed in triplicate. *r< 0.01,
**r<0.05.
Fig. 3.Influence of different heparins and dextran sulfate on HK binding to endothelial cells. bHK (20 nM) was bound to RAEC (A) or ECV304 (B) cells and their ECM in appropriate buffer for 1 h at 37C, in the absence or presence of heparin (Hep), low molecular weight heparin (LMWHep), N-desulfated heparin (N-des Hep) or low molecular weight dextran sulfate (LMWDXS), at afinal concentration of 1.0mM. The
binding of HK to cells and HK hydrolysis by huPK. The availability of this cation in the inflammation site depends on activated platelets, which release high amounts of Znþ2and interact with endothelial cells to contribute to the inflammatory processes[38].
4. Discussion
Recent studies in glycobiology have shown that glycosamino-glycans and their related proteoglycosamino-glycans participate in a variety of cell communication events and are able to initiate and control processes associated with inflammation[39,40]. During infl amma-tory processes, free glycosaminoglycans serve as a molecular signal of injury. Higher levels of specific glycosaminoglycans and their low molecular weight fragments released by glycosaminoglycan-digesting enzymes have been observed in patients with osteoar-thritis, rheumatoid arosteoar-thritis, psoriasis, scleroderma and infl amma-tory bowel disease and may adversely influence the course of these disorders[41]. In this scenario, these released polysaccharides can interact with circulating proteins, such as plasma kallikrein-kinin system components, modulating bradykinin generation in the inflammation site.
BK release in the endothelial cell environment depends on the assembly of HK-PK complex in an ordered manner. It is already known that HK attaches to the endothelial cell surface by docking to the uPAR, gC1qR, cytokeratin 1[13], heparan sulfate and chon-droitin sulfate chains of cellular proteoglycans[15,42]. PK binds to cell-bound HK through its heavy chain and becomes activated; subsequently, huPK cleaves HK, releasing BK.
In the present work, we demonstrated that HK binding to RAEC and ECV304 was decreased or abolished, respectively, in the pres-ence of soluble heparin. Other glycosaminoglycans studied in this work had a more slight influence in this binding, showing that there is specificity in the observed effect beyond a purely electrostatic action. Corroborating this fact, chondroitin sulfate and dermatan sulfate (three times longer than heparin) were less efficacious in affecting HK binding. Additionally, using heparin-correlated mole-cules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we confirmed that the size of the glycosaminoglycan is less important for the effect on HK binding than its charge density and the N-sulfated glucosamine content. The importance of sulfate content was previously shown by Linhardt et al.[43], which described a relationship between high sulfate content of glycosaminoglycans and the enhancement of their anticoagulant and antithrombotic activities.
The decrease of this interaction with the cell surface or ECM by the addition of soluble glycosaminoglycans can be explained by the disruption of HK docking to cellular proteoglycans in a competitive manner. Similar results were previously obtained for the binding of chemokines to HUVEC, which can be affected by soluble glycos-aminoglycans such as heparin in a competitive manner [44].
Fig. 4.Effect of heparin on bPK binding to HK. RAEC (A) or ECV304 cells (B) and their ECM werefirst incubated with HK (20 nM). After washing to remove the unbound HK, bPK (20 nM) was added in the absence or presence of heparin (Hep), at afinal concentration of 1.0mM for 1 h at 37
C. Thefigures show the meanSD of two different experiments performed in triplicate.
Fig. 5.Effect of heparin on PK activation. RAEC (A) or ECV304 cells (B) and their ECM werefirst incubated with HK (20 nM). After washing to remove the unbound HK, PK (20 nM) was added in the absence or presence of heparin (Hep), at afinal concentration of 1.0mM for 1 h at 37
C. After washing to remove the unbound PK, H-D-Pro-Phe-Arg-pNan (0.4 mM) was added. The rate of hydrolysis was measured for 2 h at 37C. The
figures show the meanSD of two different experiments performed in triplicate.
Fig. 6.Effect of heparin and Znþ2on HK hydrolysis by huPK. Human plasma kallikrein
Additionally, heparin dose-dependently reduced HK binding to EA.hy926 endothelial cells[45].
The effect of heparin on the reduction of HK binding to RAEC was more prominent in comparison to ECV304 (100-fold more heparin was necessary to abolish HK binding). Considering that HK binds to the endothelium through cellular proteoglycans, the content and composition of these molecules in each cell type [36,37]may be an important factor. Another remarkable fact is that ECV304 has 2.5-times more binding sites (including proteins and proteoglycans) for HK than RAEC (unpublished data from MSc. A. Gutierrez). Together, these considerations might explain the smaller effect of heparin on ECV304 as compared to RAEC. Different patterns of glycosaminoglycan expression on cells may favor the binding of certain proteins and thereby influence the cellular composition in the inflammatory response[44,46].
The possibility that HK binds heparin has been attributed to its molecular structure composed of different well-characterized domains. In this respect, the fifth domain of HK is a histidine-glycine-rich region (amino acids 384e502), which confers to this domain the ability to bind to anionic surfaces such as heparin in the presence of Znþ2[47]. Similar binding properties were found for bHK and heparin in the presence of Znþ2(data not shown), indi-cating that the biotinylation process did not affect HK capacity to bind to anionic surfaces. The interaction between HK and heparin in the presence of Znþ2modi
fies HK binding to the endothelial cell surface or ECM. However, the structure of HK is not modified by this interaction, as verified by circular dichroism and fluorescence spectroscopy (data not shown).
Considering the activation of plasma kallikrein-kinin system on endothelial cells, our results show that if HK is already bound, PK docking to the cell-adsorbed HK and PK activation are not affected by heparin. However, Renné et al.[17]suggested that glycosaminoglycan-degrading enzymes detach HK from cell proteoglycans, promoting BK formation initiated by FXII activation. Our results seem to be different from the results shown by those authors [17]. However, here the assembly of the HKePK complex on cellular surfaces occurs in the absence of FXIIa, and PK is activated by prolylcarboxypeptidase[48,49] or heat shock protein 90[50].
Besides the effect of glycosaminoglycans on HK, which inter-feres with plasma kallikrein-kinin system activation on endothelial cells, it has been reported that these molecules can affect the action and inhibition of huPK. In this context, in vitro experiments revealed that heparin enhances huPK inhibition by antithrombin [24,51] and C1-inhibitor [24]. It was also shown that heparin interferes with huPK activity on FXII, plasminogen and synthetic substrates[23,24]and that it slightly modifies the hydrolysis of HK by huPK[22]. Here, we show that in the presence of Znþ2, heparin, at a therapeutic concentration, increased BK release. Other authors have shown that heparin added to plasma induces BK release by activation of FXII[45] and that heparan sulfate and chondroitin sulfate do not induce BK formation[17].
The fact that glycosaminoglycans interfere or augment the formation of BK can have important implications when they are used as therapeutic drugs. The contamination of heparin with over-sulfated chondroitin sulfate causes adverse reactions in patients, mainly in the form of refractory hypotension, solely in response to the high production of BK[52]. Therefore, new investigations will be necessary to understand the biological consequences of these interactions.
These results raise the possibility of a functional dichotomy in which soluble glycosaminoglycans either interfere or augment the formation of BK generated by plasma kallikrein-kinin system acti-vation. The effect of glycosaminoglycans in terms of BK release likely depends on various factors within the cell environment, namely the availability of Znþ2, the soluble glycosaminoglycans
formed, the kallikrein inhibitors and the activation of PK, among others. Together, these factors can interfere with plasma kallikrein-kinin system proteins, thereby modulating BK release and, subse-quently, the regulation of inflammatory processes.
5. Conclusion
In this work, the effect of glycosaminoglycans on the interaction of HK on the cell surface or ECM of endothelial cells was examined. Using a combination of binding, kinetic and conformational assays, we showed that heparin reduced or abolished HK binding to the endothelial cell surface or ECM. This HK binding is the initiating step for BK release, a potent mediator of the inflammatory response. However, if HK is attached to the endothelial surface or ECM, heparin does not exert any effect on PK binding or activation. However, if huPK is available in the presence of Znþ2, an increase in BK liberation is observed, which may contribute to inflammatory processes. The obtained results provide both new information on the relationship between the plasma kallikrein-kinin system and glycosaminoglycans and novel insights into the regulation of local BK generation.
Acknowledgments
This work was partially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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