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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the British Heart Foundation Cardiovascular
Medicine Unit, National Heart and Lung Institute, Imperial College
School of Technology and Medicine, Hammersmith Hospital, London,
England.
There is increasing evidence for functional crosstalk between
inflammatory and thrombotic pathways in inflammatory vascular diseases
such as atherosclerosis and vasculitis. Thus, complement activation on
the endothelial cell (EC) surface during inflammation may generate
thrombin via the synthesis of tissue factor. We explored the hypothesis
that thrombin induces EC expression of the complement-regulatory proteins decay-accelerating factor (DAF), membrane cofactor protein (MCP), and CD59 and that this maintains vascular integrity during coagulation associated with complement activation. Thrombin increased DAF expression on the surface of ECs by 4-fold in a dose- and time-dependent manner as measured by flow cytometry. DAF up-regulation was first detectable at 6 hours and maximal 24 hours poststimulation, whereas no up-regulation of CD59 or MCP was seen. Thrombin-induced expression required increased DAF messenger RNA and de novo protein synthesis. The response depended on activation of protease-activated receptor 1 (PAR1) and was inhibited by pharmacologic antagonists of protein kinase C (PKC), p38 and p42/44 mitogen-activated protein kinase, and nuclear factor- The complement system consists of a large group of
plasma proteins that plays a central role in the defense against
infections and in the modulation of inflammatory
responses.1 However, by the nature of its cytolytic
effects, complement has the potential to inflict injury on bystander
host tissues. As a consequence, a variety of innate membrane-bound and
soluble protective mechanisms have evolved. The membrane-bound proteins
decay-accelerating factor (DAF, CD55), protectin (CD59), and membrane
cofactor protein (MCP, CD46) are expressed on many cell types and
provide protection against the constant low-level activation of the
alternative pathway.2,3 These factors utilize distinct
mechanisms for complement regulation and function cooperatively to
facilitate cytoprotection. DAF prevents the formation and accelerates
the decay of C3 and C5 convertases,2 while MCP binds C3b
and C4b and allows their degradation by factor I.4 In
contrast, CD59 operates at the distal end of the complement cascade,
binding to C9 and preventing its incorporation into the C5b-9 complex,
thereby inhibiting the function of the membrane attack complex
(MAC).5
Thrombin is a multifunctional serine protease that is produced at the
surface of endothelial cells (ECs) during the coagulation cascade and
induces clot formation by catalyzing the conversion of fibrinogen to
fibrin. In addition, thrombin plays an important role in both acute and
chronic inflammation through its effects on leukocytes and ECs. Thus,
thrombin is chemotactic for both neutrophils and monocytes and can also
induce leukocyte adhesion to the vessel wall via its actions on
ECs.6-9 Indeed, numerous functional effects have been
demonstrated to occur as a consequence of the stimulation of ECs by
thrombin. These include an increase in endothelial permeability to
proteins10 and the release of soluble mediators, including
platelet-activating factor and prostacyclin,6 the
chemokines interleukin-8 and MCP-1,11,12 growth
factors,13,14 and matrix
metalloproteinases.15 In addition, thrombin may induce the
expression of cellular adhesion molecules on ECs, including E- and
P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule (ICAM)-1.12,13,16-18 Thrombin exerts its
effects via a family of G-protein-coupled protease-activated receptors (PAR).19 Although both PAR1 and PAR3 are receptive to
thrombin and expressed on ECs, it appears that the effects of thrombin on EC function are mediated predominantly through the classical thrombin receptor PAR1.20-24 The binding of thrombin to
PAR1 results in receptor cleavage and exposure of a tethered ligand
that is capable of activating the receptor and inducing intracellular signaling.25
Although DAF, MCP, and CD59 are all constitutively expressed on
vascular endothelium,26 their specific roles during
inflammation, when the risk of complement-mediated injury may be
increased,27 remain to be determined. In our previous
work, we have demonstrated that DAF, but not MCP or CD59, is
up-regulated on ECs by the proinflammatory mediators tumor necrosis
factor (TNF)- Monoclonal antibodies and other reagents
Cell isolation and culture
Flow cytometry Monolayers of ECs were harvested by exposure to trypsin/EDTA and stained with the appropriate primary MoAb for 30 minutes at 4°C. After washing, ECs were stained with fluorescein isothiocynate (FITC)-labeled rabbit antimouse Ig (DAKO, Glostrup, Denmark) for 30 minutes at 4°C, followed by washing and fixation in 1% paraformaldehyde. Samples were analyzed on an Epics XL-MCL flow cytometer (Coulter, Hialeah, FL) by counting 10 000 cells per sample. In some experiments, the results are expressed as the relative fluorescent intensity (RFI), which represents the mean fluorescent intensity (MFI) with the test MoAb, divided by the MFI using an isotype-matched irrelevant MoAb. In experiments involving RO318220, SB202190, PD98059, TRAP-6, hirudin, and PSI, the RFI is expressed as a percentage of the RFI for thrombin-induced DAF expression.Northern blot analysis Northern analysis was performed as previously described.28 Briefly, confluent ECs in 75-cm2 tissue culture flasks were incubated with thrombin (10 U/mL), VEGF (25 ng/mL), or plain medium alone for up to 24 hours at 37°C. In some experiments, the ECs were pretreated for 30 minutes with CHX (1 µg/mL). At the end of the stimulation, the cells were lysed in guanidium isothiocyanate (Sigma), and RNA was extracted as described.38 The probe for DAF was released from the plasmid vector39 by incubation for 2 hours at 37°C with the SalI and XbaI restriction enzymes. Purified RNA was run on 1% formaldehyde agarose gels and blotted onto GeneScreen membrane (DuPont, Letchworth, United Kingdom). Membranes were hybridized to appropriate 32P-deoxycytidine triphosphate-labeled complementary DNA probes overnight at 42°C, washed in solutions of 0.1% sodium dodecylsulfate (wt/vol) containing successively lower concentrations of standard sodium citate buffer (0.15-mol/L NaCl, 0.015-mol/L sodium citrate, pH 7), and specific hybridization was detected by autoradiography following exposure to Kodak XOMat film. For quantification, Northern blots were scanned using an Appligene Image Analysis System (Appligene, Durham, United Kingdom), and densitometry was performed using the National Institutes of Health (Bethesda, MD) Image program 1.52 software. Values were corrected with respect to ethidium bromide-stained ribosomal RNA loading patterns, and an arbitary value of 1 was assigned to unstimulated ECs.C3-binding assay ECs were cultured overnight at 37°C prior to the addition of thrombin or plain medium alone for 24 hours. Following harvesting with trypsin/EDTA, ECs were pelleted in 96-well v-bottom plates (Costar) and incubated with the antiendoglin MoAb RMAC8 for 30 minutes at 4°C. After washing with Hank balanced salt solution (HBSS)/1% bovine serum albumin (BSA), ECs were incubated with 100 µL of 5% NHS in M199 for 2 hours at 37°C prior to washing with HBSS/1% BSA and addition of FITC-conjugated rabbit antihuman C3c (DAKO) at 1:40 dilution for 30 minutes at 4°C. After washing twice with HBSS/1% BSA, the presence of C3c was estimated using flow cytometric analysis as described above. Control samples included the replacement of NHS with heat-inactivated human serum (HIHS) and the addition of 10-mmol/L EDTA to the NHS to inhibit complement activation. In the inhibition studies, the blocking MoAbs were added to the assay with the RMAC8 MoAb to achieve a final concentration of 50 µg/mL.Cell lysis assay ECs were cultured overnight in 24-well plates at 37°C prior to the addition of thrombin or plain medium alone for 24 hours. ECs were then incubated for 30 minutes at 37°C in M199 with 10% FBS containing 7-µmol/L calcein acetoxymethyl ester (Molecular Probes, Leiden, The Netherlands). Following washing in M199/1% BSA, EC monolayers were opsonized by incubating with 250 µL of MoAb RMAC8 for 30 minutes at 37°C. After washing with HBSS/1% BSA, ECs were incubated with 250 µL of 5%-to-20% baby rabbit complement (Serotec, Oxford, United Kingdom) in M199 for 30 minutes at 37°C. The supernatant from each well was then transferred to a 96-well microtiter plate (Costar) and, after washing in M199/1% BSA, the calcein remaining in the cells was released by incubation with 250-µL M199/1% BSA/0.1% Triton X-100 (Sigma). The lysate was then transferred to a 96-well plate, and the calcein released by complement and detergent was estimated using a CytoFluor 2300 fluorescence plate reader (Millipore, Bedford, MA). Percent specific lysis in triplicate wells was calculated as [(complement-mediated release spontaneous release)/maximal release spontaneous
release)] × 100%, where maximal release = complement-mediated
release + detergent-mediated release. Spontaneous release was less than 20% in all experiments.
Statistics Differences between the results of experimental treatments were evaluated by the Mann-Whitney U test. Differences were considered significant at P < .05.
Thrombin induces DAF expression on HUVECs and DMECs To investigate the effect of thrombin on the expression of DAF, monolayers of HUVECs were treated with human thrombin at a concentration of 10 U/mL for up to 72 hours, and surface expression of DAF was measured by flow cytometry. A 4-fold increase above the constitutive expression of DAF was observed following stimulation with thrombin, reaching a maximum level at 24 hours. An increase was first detectable at 6 hours, with levels returning to baseline by 72 hours poststimulation (Figure 1). This up-regulation was dose-responsive, with increasing expression seen at concentrations of thrombin up to 10 U/mL (Figure 2A). Higher concentrations (15 and 20 U/mL) showed no significant increase in expression above that observed with 10 U/mL (data not shown). Parallel control experiments also demonstrated a similar dose-response for up-regulation of ICAM-1 expression (Figure 2B). All further experiments were therefore conducted using stimulation with 10 U/mL thrombin for 24 hours.
In contrast to DAF, no up-regulation in expression of either MCP or CD59 was observed following treatment of HUVECs with 10-U/mL thrombin for up to 48 hours. Failure to demonstrate an increase in expression of MCP and CD59 was not due to the proteolytic effects of trypsin because identical results were obtained when DAF, MCP, and CD59 were measured on ECs harvested with a nonenzymatic cell dissociation solution (not shown). It is possible that HUVECs, as large-vessel ECs of fetal origin, may not accurately represent those ECs of the microvasculature most affected during inflammatory responses. Therefore, DMECs were isolated from human skin and stimulated with thrombin for 24 hours. A significant induction of both DAF and ICAM-1 expression was observed with no up-regulation of MCP or CD59 (data not shown). Thus, in this model at least, ECs derived from large and small vessels appeared to behave similarly, and the remainder of the experiments were therefore performed with HUVECs. Thrombin up-regulates DAF via activation of PAR1 The leech product hirudin, which binds thrombin and inhibits its proteolytic effects, was used to demonstrate specificity of thrombin-induced DAF expression. The up-regulation of DAF expression by thrombin was completely blocked by preincubating ECs with hirudin while the baseline DAF expression remained unaffected (Figure 3A). Specific use of the thrombin receptor was further demonstrated using the PAR1-activating peptide TRAP-6, a short peptide containing the tethered ligand sequence of the thrombin receptor, which directly activates the thrombin receptor PAR1. As seen in Figure 3B, TRAP-6 dose-dependently increased expression of DAF, although to a lesser extent than using thrombin, a feature characteristic of the TRAP peptides.40
Role of PKC, p38 and p42/44 MAPK, and NF-  B.18,42-44
To investigate the role of these in DAF induction, we used
cell-permeable pharmacologic inhibitors of PKC (RO31-8220 and GF
109203X), MEK-1 (PD98059), p38 MAPK (SB202190), and NF-B (PSI).
Initial dose-response experiments identified the optimal concentrations
for each of the inhibitors. HUVECs were preincubated with the relevant
inhibitors for 1 hour prior to the addition of the thrombin. The role
of PKC in the up-regulation of DAF expression by thrombin was
demonstrated by complete inhibition of the response by RO31-8220
(Figure 4A) and GF 109203X (not shown).
Furthermore, preincubation of ECs with the p38 inhibitor SB202190
prevented thrombin-induced DAF up-regulation (Figure 4B). In addition,
as seen in Figure 4C, the involvement of p42/44 MAPK in the signaling pathway was suggested by inhibition of the response by PD98059. The
effects of the MAPK inhibitors observed were dose-responsive, with
inhibition first detectable with 2.5 µmol/L SB202190 and maximal at
25 µmol/L and with 5 µmol/L PD98059 maximal at 100 µmol/L.34,45 Finally, a role for NF-B was suggested
by the inhibition of DAF induction by PSI (Figure 4D). These
experiments also demonstrated that the constitutive expression of DAF
was reduced to some extent by RO31-8220, SB202190, and PD98059,
suggesting a role for these pathways in the maintenance of DAF
expression on resting ECs. The antagonists studied, and particularly
SB202190, induced EC shape change but did not lead to any demonstrable
cytotoxicity at the concentrations used, as assessed by cell counting
and trypan blue exclusion on the EC populations and also by flow
cytometric analysis of CD31 expression (data not shown).
Thrombin-induced DAF on ECs is protein synthesis-dependent The protein synthesis inhibitor CHX (1 µg/mL) was added to ECs for 30 minutes prior to the addition of thrombin and remained throughout the 24 hours of stimulation. As seen in Figure 5, the presence of CHX reduced DAF expression on resting ECs, probably due to inhibition of the normal turnover of constitutive DAF, as previously described.29 In addition, CHX inhibited thrombin-induced DAF expression, confirming a dependence on de novo protein expression. Northern blot analysis was performed using messenger RNA (mRNA) from unstimulated HUVECs and cells stimulated with thrombin in the presence and absence of CHX for up to 24 hours. As shown in Figure 6A, DAF mRNA at low levels was detectable in resting HUVECs, and following thrombin stimulation an increase was observed that was maximal at 2 to 6 hours and, although falling, remained 50% above baseline 24 hours poststimulation. Two DAF mRNA transcripts were detected at 2.4 and 1.8 kilobases, as previously reported.39,46 Quantification of mRNA levels using densitometric scanning of the 2.4-kilobase band demonstrated a 2.7-fold increase at 2 hours (Figure 6B). Moreover, preincubation of ECs with CHX prior to the addition of thrombin failed to inhibit the increase in DAF mRNA in response to thrombin (Figure 6A, lanes 7-8), while CHX alone increased DAF mRNA somewhat, an observation that has been made for other NF-B-dependent genes, including
ICAM-1.18 Comparative experiments were performed with
VEGF, which also induces DAF expression on ECs.47 Relative
to the effect of thrombin, the increase in DAF mRNA seen following
treatment with VEGF was delayed, with an increase first detectable at 3 hours and peaking at 6 to 9 hours poststimulation. In addition, the
VEGF-induced increase in DAF mRNA was completely inhibited by
preincubation with CHX (Figure 6C-D). These data suggest that the
induction of DAF by thrombin is a direct effect on gene transcription
while that in response to VEGF is indirect and dependent on the
synthesis of one or more intermediary proteins.
Thrombin-induced DAF reduces complement-mediated injury to HUVECs To investigate the potential functional role of thrombin-induced DAF, a flow cytometric assay measuring the binding of complement factor C3 to the EC surface was used as previously described.28 ECs that were unstimulated and HUVECs treated with thrombin for 24 hours were opsonized with RMAC8, an IgG2a complement-fixing antibody directed against endoglin, followed by incubation with 5% NHS for 3 hours as a source of complement proteins. C3 binding to the cell surface was then quantified using an FITC-labeled anti-C3c antibody. IgG2a is the optimal murine isotype for complement fixation, and endoglin was chosen as the target antigen because it is highly expressed on resting ECs and is not known to be inducible.48 Parallel flow cytometric analysis confirmed that the EC expression of endoglin was not altered significantly by thrombin stimulation for 24 hours (endoglin RFI ± SEM: unstimulated ECs 155.7 ± 29.0, thrombin-treated ECs 186.6 ± 53.5). As seen in Figure 7A, stimulation with thrombin reduced the deposition of C3 on the EC surface by 40% compared with the unstimulated cells (P < .05). To confirm the role of DAF in the reduction of C3 binding following thrombin stimulation, the inhibitory anti-DAF MoAb 1H4 (noncomplement fixing) was included in the assay (Figure 7B). Following exposure to 5% NHS, opsonized ECs prestimulated with thrombin demonstrated low levels of C3 binding. Moreover, the reduction in C3 binding seen in response to thrombin treatment was reversed by the presence of MoAb 1H4, with levels of C3 deposited on the cell surface becoming equivalent to those observed on unstimulated ECs in the presence of MoAb 1H4 (Figure 7B). The noncomplement-fixing antibody EN4 (anti-CD31) was used as a negative control and had no effect on the level of C3 binding.
To further assess the physiologic relevance of these findings, we determined whether thrombin can protect HUVECs against complement-mediated lysis initiated by opsonization with the antiendoglin MoAb RMAC8 and addition of baby rabbit complement. Pretreatment of ECs with thrombin for 24 hours was cytoprotective, significantly reducing cell lysis following the addition of 5%, 7.5%, and 10% baby rabbit complement (Figure 7C). At serum concentrations of 20% or above (not shown), the cytoprotective response of thrombin was overcome. These observations suggest that the increased levels of EC surface DAF induced by exposure to thrombin provide additional protection to ECs against complement-mediated injury or activation.
Although the study of thrombin has principally focused on its role
in the coagulation cascade, it has also been demonstrated to have a
significant potential role in inflammation.49 In
particular, its effects on the vascular endothelium include an increase
in vascular permeability,10 the up-regulation of cellular
adhesion molecules,12,13,16-18 and the induction of
angiogenesis.31 Situated at the blood-tissue interface,
the endothelium is continuously exposed to the risk of
complement-mediated injury,50,51 which may be
significantly enhanced during inflammation following activation of the
alternative or classical pathways.27 We have previously demonstrated that the proinflammatory mediators TNF- Initial experiments demonstrated that exposure of HUVECs to thrombin increased the expression of DAF by up to 4-fold above the basal level on resting cells. In contrast to DAF, the basal levels of MCP and CD59 were not increased by thrombin up to 72 hours poststimulation. Previous work has demonstrated heterogeneity in responsiveness to thrombin between ECs from different vascular beds.52 However, a similar pattern of DAF up-regulation was also seen when DMECs were studied parallel with HUVECs. Dose-response studies confirmed that the optimal concentration of thrombin was the same as that previously described for ICAM-1 up-regulation.17 The levels of thrombin required for DAF and adhesion molecule expression used in this and other studies are relatively high and may exceed physiologically relevant levels. However, it has previously been shown that thrombin may bind subendothelial extracellular matrix, where it is relatively protected from circulating inhibitors and remains active.53 Thus, it has been proposed that thrombin may be concentrated in this way and subsequently released and made available to the endothelium.49 It is also possible that a cofactor is required for optimal signaling via EC PAR1, analogous to the thrombin-sparing effect of PAR3 on PAR4-mediated responses recently reported for murine platelets.54 Such a cofactor might be present in vivo but absent in in vitro assay systems; hence the need for higher concentrations of thrombin. The observation that prolonged exposure to thrombin for more than 6 hours is required to induce DAF expression (data not shown) and the findings of the lesser effect of the TRAP-6 peptide suggest that binding to PAR1 may not be the sole stimulatory mechanism. To investigate the role of PKC in the up-regulation of DAF by thrombin,
we preincubated ECs with the PKC antagonists RO31-8220 and GF 109203X,
which resulted in complete inhibition of thrombin-induced DAF
expression. Thus, these data confirm the presence of 2 physiologic pathways for DAF regulation in ECs: one PKC-dependent, the other PKC-independent and utilized by TNF- Further investigation using pharmacologic antagonists of p38 MAPK
(SB202190) and p42/44 MAPK (PD98059) revealed inhibition of
thrombin-induced DAF expression by both SB202190 and PD98059 in a
dose-responsive manner. Furthermore, preincubation with the NF- The up-regulation of DAF on the EC surface by thrombin was dependent on increased steady-state mRNA and de novo protein synthesis and was first detectable 6 hours and maximal 24 hours poststimulation. The increased mRNA expression was maximal at 2 to 6 hours, falling to 1.5-fold above baseline levels after 24-hour stimulation. Moreover, experiments using CHX demonstrated that the effect of thrombin on the DAF gene was direct and independent of protein synthesis. This was in contrast to the increase in DAF mRNA seen in response to VEGF, which was maximal 6 to 9 hours poststimulation and was inhibited by the presence of CHX. In addition, VEGF-induced up-regulation of DAF on the EC surface is also delayed compared with thrombin.47,74 The demonstration of direct and indirect intracellular signaling pathways for DAF up-regulation may have important functional implications. Thus, the more rapid direct response may be important in regulating DAF expression following vascular injury,61 while the more prolonged indirect response to VEGF may have a predominant role in EC cytoprotection against complement-mediated injury during angiogenesis. To establish whether thrombin-induced DAF was functionally important in the face of complement activation, we initially used a flow cytometric assay to measure levels of surface-bound C3. Stimulation with thrombin for 24 hours significantly reduced C3 binding, and inclusion of an inhibitory anti-DAF MoAb (1H4) reversed this effect. This suggests that up-regulation of DAF on the EC surface to the levels seen following stimulation with thrombin is protective against bystander complement-mediated injury. This inhibition of C3 binding is particularly important in view of the relative inefficiency of C5 activation, which is dependent on an absolute excess of C3 activation.62 This observation was further supported by the demonstration that thrombin treatment resulted in a significant inhibition of complement-mediated EC lysis. Furthermore, during inflammation, this cytoprotective response may be amplified by a synergistic effect between thrombin and proinflammatory cytokines63 and by other inducible protective mechanisms, including synthesis of factors H and I by ECs.64,65 In chronic inflammatory disease states, including atherosclerosis,
systemic lupus erythematosus, and rheumatoid arthritis, endothelium is
continuously exposed to low levels of complement activation but without
significant EC lysis.66-68 Thrombin has been identified in
both atherosclerotic plaques and in the synovial fluid in rheumatoid
arthritis.69,70 The data presented demonstrate that, in
addition to its known roles in coagulation and inflammation, thrombin
may be important in protection against complement-mediated injury In summary, we have demonstrated that thrombin, in addition to its role
in coagulation and as a proinflammatory mediator, can induce the
expression of the complement regulatory protein DAF on the EC surface.
Thrombin is a physiologic agonist for the PKC-dependent pathway of DAF
up-regulation, which may also require p38 and p42/44 MAPK activation,
and NF-
We are grateful for the help of P. Kiely, P. Singh, Dr L. Lovat, and M. McNamara for collection of foreskins and to the staff of the Maternity Unit of Hammersmith Hospital for the provision of umbilical cords. We would like to thank Doug Lublin for the gift of MoAb 1H4 and for reviewing the manuscript. We also thank Mark Walport, Kevin Davies, Bernie Morley, Marina Botto, Jeremy Pearson, Paul Morgan, Tony d'Apice, Teizo Fujita, and John Atkinson for their help with this study.
Submitted December 28, 1999; accepted June 21, 2000.
Supported by a British Heart Foundation Professorial Award (D.O.H.).
J.C.M. is an Arthritis Research Campaign Clinician Scientist Fellow.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: J. C. Mason, BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Technology and Medicine, Hammersmith Hospital, Du Cane Rd, London W12 ONN, England; e-mail: justin.mason{at}ic.ac.uk.
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Top
Abstract
Introduction
generated at inflammatory sites in response to complement activation
and interferon (IFN)-
and also by the MAC,
resulting in enhanced cytoprotection by reducing the binding of
complement factor C3 to the EC surface.
1 and cRaf-1 in
addition to p38 MAPK.