Engagement of Glycoprotein IIb/IIIa (αIIbβ3) on Platelets Upregulates CD40L and Triggers CD40L-Dependent Matrix Degradation by Endothelial Cells
Background— CD40L-CD40 interactions induce inflammatory signals in cells of the vascular wall. We evaluated the effects of glycoprotein (GP) IIb/IIIa (αIIbβ3) engagement that occurs during platelet-endothelium interactions on CD40L surface exposure on platelets and initiation of proteolytic activity in human umbilical vein endothelial cells (HUVECs).
Methods and Results— Transient (60-minute) adhesion of thrombin-prestimulated platelets enhanced HUVEC expression of urokinase-type plasminogen activator receptor and membrane type-1 matrix metalloproteinase (MT1-MMP) (reverse transcriptase–polymerase chain reaction, flow cytometry) and secretion of urokinase-type plasminogen activator, tissue-type plasminogen activator, and MMP-1 (ELISA) and induced proteolytic activity via MMP-2 and MMP-9 (gelatin zymography). These effects were abrogated by hindrance of physical platelet-endothelial contacts using transwell systems or inhibited by GRGDSP, mAbs anti–GP IIb/IIIa (7E3), anti-αvβ3 (LM609), or anti-CD40L (TRAP1). In addition, MMP-2 and MMP-9 were inhibited by specific GP IIb/IIIa antagonists tirofiban, lamifiban, or integrelin. On endothelial cells, induction of proteolytic activity by activated platelets was mimicked by CD40 engagement using soluble CD40L but not affected by antibody clustering of αvβ3. On platelets, CD40L and CD62P exposure was enhanced on adhesion to HUVECs or immobilized fibrinogen and was abrogated by GRGDSP or LM609. In suspension, cross-linking of GP IIb/IIIa by fibrinogen plus secondary mAb upregulated CD40L surface exposure. Consistently, bivalent mAb 7E3 upregulated CD40L, whereas ligation of GP IIb/IIIa by soluble fibrinogen alone or monovalent Fab-fragment c7E3 had no effect.
Conclusions— Platelet adhesion via GP IIb/IIIa upregulates CD40L and CD62P surface exposure. Proteolytic activity of HUVEC is induced by the concerted action of β3-integrin–mediated platelet adhesion and subsequent CD40L-induced signals in HUVECs. Effective anti–GP IIb/IIIa or anti-CD40L strategies might, therefore, contribute to plaque stabilization.
Received March 15, 2002; revision received July 23, 2002; accepted July 23, 2002.
Enhanced turnover of the extracellular matrix (ECM) is thought to be involved in a variety of cardiovascular pathologies that underlie acute coronary syndromes.1–3⇓⇓ The destabilization of the vulnerable fibrous cap of the atherosclerotic plaque seems to result from an imbalance of the plasminogen and matrix metalloproteinase (MMP) activation systems.3,4⇓ In a complex cascade, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) cleave plasminogen to plasmin, a serine protease of broad specificity, capable of degrading specific components of the ECM.4 In addition, plasmin activates MMPs, such as interstitial collagenase (MMP-1) and gelatinase A (MMP-2).5 Endothelial cells secrete uPA, tPA, MMP-1, MMP-2, and MMP-9 in an activation-dependent manner1,6⇓ and restrict proteolytic activity to the cell surface via membrane-anchored protease receptors such as urokinase receptor (uPAR) and membrane type-1 MMP (MT1-MMP, MMP-14), which bind and activate uPA and MMP-2, respectively.1,4⇓ MT1-MMP not only serves as a receptor and catalyst of soluble MMPs but also directly degrades several ECM components. In line with these in vitro findings, enhanced levels of uPA, uPAR, and several MMPs have been found in atherectomy specimens from patients with unstable angina, suggesting a role in the rupture of the atherosclerotic plaque.3,7⇓
The potential role of platelets for the initiation of proteolytic activity in cells of the vascular wall has not yet been clarified. Interaction of activated platelets with the endothelium induced the expression of tissue factor,8 cytokines, and adhesion molecules,9,10⇓ leading to enhanced chemotactic and adhesive endothelial activity.11 Signals mediated by CD40 ligand (CD40L) seem to be essentially involved in these activation processes.8,10⇓ Nonetheless, the exact mechanism that upregulates CD40L on platelets and thereby allows CD40L-mediated signaling in adjacent vascular cells has not been clarified. This study provides a new concept of how platelet interactions with cells of the vascular wall induce CD40L upregulation on platelets and thereby stimulate endothelial cells to enhance matrix-degrading activity.
Blocking mAbs anti–intracellular adhesion molecule-1 (ICAM-1) (84H10), anti-GPIb (SZ2), and anti-CD40L (TRAP-1) as well as FITC-conjugated mAbs anti-CD62P and anti-CD40L were from Immunotech (Hamburg, Germany). MAb anti-MT1-MMP (114- 1F2) was from Oncogene Science (Cambridge, UK), and mAb anti-human fibrinogen (IgG1) was from Pharmingen (Heidelberg, Germany). MAb anti-uPAR (R4) was kindly provided by Dr G. Hoyer-Hansen (Copenhagen, Denmark) and blocking mAb anti-αvβ3 (LM609) by Dr D. Cheresh (La Jolla, Calif). MAb 7E3 (kind gift from Dr B. Coller, New York, NY) inhibits fibrinogen binding to glycoprotein (GP) IIb/IIIa and cross-reacts with αvβ3.12 The Fab fragment of mAb 7E3 (c7E3, Lilly Deutschland GmbH) was purified by Sepharose A chromatography. MAb anti-LIBS-1 binds and transfers GP IIb/IIIa into its fibrinogen-binding status (kind gift from Dr M. Ginsberg, La Jolla, Calif). GRGDSP and GRGESP were from Calbiochem, and soluble human fibrinogen and poly-l-lysine were from Sigma (Hamburg, Germany). Recombinant Flag-tagged CD40L and anti-Flag mAb were from Alexis (Grünberg, Germany). Human umbilical vein endothelial cell (HUVEC) supernatant was analyzed by ELISA for uPA, tPA (American Diagnostica, Pfungstadt, Germany), or MMP-1 (Amersham, Freiburg, Germany). Lamifiban was from Roche (Basel, Switzerland), tirofiban was from Merck (West Point, Pa), and integrelin was from Cor Therapeutics (San Francisco, Calif).
Coincubation of Platelets With Endothelial Cells
Washed platelets were prepared as described.9,11⇓ HUVECs were purchased (PromoCell) and cultured in medium (M199, Sigma) containing 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin until they reached confluency. Before coincubation experiments, thrombin activity (0.5 U/mL) was antagonized by hirudin (5 U/mL) to exclude potential direct thrombin-mediated effects on HUVECs. Pretreated platelets (final concentration, 2×108/mL) were resuspended and added to 24-well plates covered with confluent endothelial monolayers with or without blocking agents, as indicated in the figure legends. After 60-minute coincubation under cell culture conditions, all platelets were removed by gentle washing, which was confirmed by light microscopy. Flow cytometric analysis of CD42b immunoreactivity of HUVEC as index of platelet-endothelium adhesion proved that remaining platelet-endothelium adhesion was neglectable.13,14⇓
After an additional 6 hours of incubation of the endothelial cells, supernatant was aspirated, centrifuged at 2000g, and stored at −80°C, and the cells were processed to flow cytometry. In some experiments, platelets and HUVECs were physically separated using transwell culture plates (Nunc, 0.1-μm pore size).
Flow Cytometry, Confocal Laser Scanning Electron Microscopy, and SDS-PAGE Zymography
Surface expression of platelet or endothelial cell membrane glycoproteins was evaluated using flow cytometry as described.9 Suspensions of single platelets were analyzed as verified by direct microscopy. For confocal laser scanning microscopy, cover slips were cultivated with HUVECs or precoated with fibrinogen or poly-l-lysine (20 μg/mL).13 MMP-2–specific and MMP-9–specific gelatinolytic activity was studied by SDS-PAGE zymography15 using supernatants of cells that were kept under serum-free conditions 24 hours before the experiments, as described elsewhere.16
Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted from HUVECs using the RNeasy Mini Kit (Qiagen). Contaminating DNA was removed by DNase using Message Clean kit (Gene Hunter). DNase digested total RNA was transcribed to cDNA using Omniscript reverse transcriptase (Qiagen) and random hexamers (Gibco, Karlsruhe, FRG). Primer sequences were as follows: uPAR (forward) 5′-GCCCTGGGACAGGACCT- CTG-3, uPAR (reverse) 5′-CATTGATTCATGGGGCCTCGGC-3′, MMP2 (forward) 5′ GTGCTGAAGGACACA-CTAAAGAAGA-3′, MMP2 (reverse) 5′-TTGCCATCCTCTCAAAGTTGTAGG–3′, MT1-MMP (forward) 5′-CCCTATGCCTACATCCGTGA-3′, MT1-MMP (reverse) 5′-TCCATCCATCACTTGGTTAT-3′, Cyclophillin (forward) 5′-CATCTGCACTG-CCAAGACTG-3′, and Cyclophillin (reverse) 5′-CTGCAATCCAGCTAGGCATG-3′. Polymerase chain reaction was performed with the HotStarTaqTM DNA Polymerase (Qiagen). Annealing temperature was 59°C for MMP2 and MT1-MMP and 63°C for uPAR and Cyclophillin (internal standard).
To control for potential errors introduced by deviations from normal distribution, we validated the results by Friedman’s test followed by the Wilcoxon’s matched pairs signed-ranks test or by Mann-Whitney U test, respectively. P<0.05 was regarded as significant.
Transient Adhesion of Platelets Induces Endothelial Proteolytic Activity
HUVEC monolayers were coincubated with platelets for 60 minutes, platelets were removed, and HUVECs were additionally incubated with medium. After 6 hours, HUVEC supernatant was stored and flow cytometry was performed (Figure 1A). HUVEC incubation with α-thrombin–activated platelets resulted in a 2- to 3-fold increase in HUVEC surface expression of uPAR and MT1-MMP (P<0.001), an effect that was comparably achieved by HUVEC stimulation with interleukin-1β (IL-1β). Notably, nonstimulated platelets did not alter HUVEC expression of uPAR or MT1-MMP. These flow cytometric results were supported by immunofluorescence microscopy (Figure 1B). ADP-stimulated platelets enhanced endothelial uPAR expression in a comparable extent as α-thrombin-stimulated platelets (Figure 1C). Therefore, this activation pathway seems to require activated platelets and to be independent of the respective platelet stimulus. In addition, activated platelets enhanced endothelial secretion of uPA and tPA (≈3- to 5-fold) as well as of MMP-1 (≈10-fold) into the supernatant, whereas resting platelets had no effect (Figure 1D).
SDS-PAGE zymography using the endothelial supernatants revealed constitutive expression of 72-kD gelatinase (MMP-2) and very low expression of 92-kD gelatinase (MMP-9), which were only slightly induced by resting platelets (Figure 1E). However, adhesion of activated platelets induced secretion of the 2 MMPs to a comparable extent as achieved by IL1-β. In addition, a second, lower band (albeit weak) at 66 kDa revealed MMP-2 activation on adhesion of activated platelets. MMP activity was exclusively of endothelial origin, because all platelets had been removed by repeated washing steps after the initial 60-minute coincubation period, as verified by light microscopy and flow cytometry (not shown).
To discriminate between effects mediated by physical platelet-endothelial contact (eg, CD40L) versus effects of platelet-released soluble factors (eg, platelet factor 4), HUVECs were coincubated with activated platelets in a transwell system that prevents physical cell to cell contact. Platelet-induced HUVEC surface expression of uPAR and MT1-MMP were almost completely absent when direct platelet-endothelium interactions were hindered by transwells (data not shown).
β3-integrins and CD40L are required for platelet-induced proteolytic activity by endothelial cells. Coincubation experiments were performed in the presence of substances that block specific receptor interactions between platelets and the endothelium. Endothelial upregulation of uPAR and MT1-MMP was significantly attenuated by GRGDSP peptide that blocks both GP IIb/IIIa and αvβ3 by blocking mAbs anti–GP IIb/IIIa (7E3) and anti-αvβ3 (LM609) but also by mAb anti-CD40L (TRAP-1) (Figure 2A). No significant inhibition was achieved in the presence of control peptide GRGESP. Blocking mAbs anti-ICAM-1 or anti-GPIb, however, seemed to inhibit uPAR expression by trend, suggesting that other adhesive factors may support platelet-induced upregulation of uPAR. Similarly, platelet-induced MMP-9 was inhibited by β3-integrin antagonists, mAb anti-CD40L, and mAb anti-GPIb by trend (Figure 2B). Consistently, the presence of platelet-specific GP IIb/IIIa antagonists lamifiban, tirofiban, or integrelin reduced platelet-induced MMP-9 of HUVEC (Figure 2C). Comparable regulation (albeit less profound) of MMP-2 was noted (Figures 2B and 2C).
Whereas CD40L is known to signal inflammatory reactions within endothelial cells,10 integrins mediate platelet-endothelium adhesion13,14,17⇓⇓ and additionally transfer adhesion signals into the cell (outside in signaling).18,19⇓ Activated platelets adhere to the intact endothelial monolayer9,17⇓ mainly via GP IIb/IIIa (on platelets) and αvβ3 (on the endothelium) using fibrinogen as a bridging molecule.13,14⇓ To clarify, if platelets signal proteolytic activity of HUVEC directly via αvβ3, we used a previously published method13 and coincubated endothelial monolayers with GP IIb/IIIa–expressing CHO cells14 in the presence of soluble fibrinogen. Under these conditions (absence of platelet environment, eg, CD40L), we did not find any significant endothelial activation (data not shown). To additionally determine the role of CD40 and αvβ3 signaling for induction of endothelial proteolytic activity, direct receptor clustering was studied on HUVECs. MT1-MMP surface expression was enhanced on CD40 clustering using Flag-CD40L plus anti-Flag mAb, whereas αvβ3 engagement using mAb LM609 plus anti-mouse mAb had no effect (Figure 3A). Furthermore, CD40 clustering induced mRNA expression of MMP-2, MT1-MMP, and uPAR in HUVEC comparable with platelet-stimulated HUVEC (Figure 3B).
Adherent Platelets Upregulate CD40L and CD62P
Activated platelets were allowed to adhere to HUVEC for 60 minutes in the presence of β3-integrin antagonists (GRGDSP, anti-αvβ3) or control reagents (GRGESP, anti-ICAM-1). Adherent platelets were stained with mAbs anti-CD40L, anti-CD62P, and anti–GP IIb/IIIa in situ and were detached and evaluated by flow cytometry (Figure 4A). In fact, CD40L and CD62P expression were significantly lower on platelets that were coincubated with HUVEC in the presence of GRGDSP or anti-αvβ3 but not in the presence of GRGESP or anti-ICAM-1. Additionally, adherent platelets were stained and photographed in situ (Figure 4B). Anti–GP IIb/IIIa immunofluorescence was used to identify adherent platelets. The presence of GRGDSP mainly abrogated platelet adhesion. Nevertheless, the remaining, adherent platelets, which seemed to adhere independent of integrins (or unspecifically), did express CD40L to an obviously lesser degree compared with platelets that were allowed to adhere via β3-integrins (medium/GRGESP).
GP IIb/IIIa Engagement Upregulates CD40L and CD62P on Platelets
Receptor-specific platelet adhesion was performed (Figure 4C). Platelets, submaximally activated by thrombin, were allowed to attach to the specific ligand of GP IIb/IIIa, fibrinogen, or poly-l-lysine. After 60 minutes, nonadherent platelets were removed. Adherent cells were stained with fluorescence-conjugated mAbs, cautiously detached, and evaluated by flow cytometry. Both CD40L expression and CD62P expression were significantly enhanced on platelets that were allowed to adhere to fibrinogen compared with poly-l-lysine (Figure 4C). Integrins preferably mediate outside-in signals when cross-linked by (at least) bivalent binding partners.18,19⇓ To characterize the effects of GP IIb/IIIa ligation versus cross-linking, platelets were incubated with soluble fibrinogen in the presence or absence of bivalent mAb anti-fibrinogen (IgG), which may enhance receptor cross-linking (Figure 5A). Similarly, the effects of the monovalent Fab fragment c7E3 (anti-GP IIb/IIIa) versus the bivalent mAb 7E3 (whole IgG) were studied (Figure 5B). In fact, a significant increase in CD40L surface expression was only achieved when GP IIb/IIIa was clustered by combined fibrinogen/anti-fibrinogen treatment or by bivalent mAb 7E3. Additional clustering by anti-mouse mAb (IgG) that binds mAb 7E3 additionally increased CD40L exposure. In contrast, ligation of GP IIb/IIIa by fibrinogen alone or monovalent c7E3 (Fab) had no effect. Thus, clustering of GP IIb/IIIa as it occurs during platelet-endothelial interactions seems to effectively trigger CD40L expression on platelets. Cross-linking of GP IIb/IIIa was found to be effective on platelets regardless of whether they were preactivated with thrombin, TRAP, or ADP, all of which are agonists of the fibrinogen receptor GP IIb/IIIa (Figure 5C). Thus, GP IIb/IIIa–mediated CD40L upregulation seems to require prestimulation of GP IIb/IIIa affinity independent of the respective stimulus. The specificity of these experiments was confirmed using thrombasthenic, GP IIb/IIIa–deficient platelets, which did not reveal CD40L upregulation on treatment with fibrinogen/mAb anti-fibrinogen or 7E3/mAb anti-mouse (Figure 5D). Moreover, preincubation of platelets with human IgG did not affect GP IIb/IIIa cross-linking–induced platelet degranulation (not shown), thus excluding potential involvement of Fc receptors in this activation pathway.
This study describes a novel activation pathway on platelets that upregulate CD62P and CD40L in direct response to GP IIb/IIIa engagement and thereby allow CD40L-mediated signaling. This pathway promotes proteolytic activity on endothelial cells on transient interactions with activated platelets. Specifically, the major findings are the following: (1) transient interactions with activated platelets stimulate HUVEC to upregulate mRNA and protein expression of uPAR and MT1-MMP and to secrete uPA, tPA, and MMP-1 as well as MMP-2 and MMP-9; and (2) β3-integrin antagonists or CD40L blockade inhibit this activation pathway. CD40 clustering by soluble CD40L can mimic platelet effects on endothelial protease synthesis, whereas αvβ3 clustering did not, indicating that outside-in signals via αvβ3 in HUVEC did not play a role in this pathway. In addition, the presence of GP IIb/IIIa–specific inhibitors substantially inhibited platelet effects on the endothelium. Thus, β3-integrin interactions were essential for platelet adhesion followed by CD40L-mediated signals. Apart from β3-integrin blockade, inhibition of GPIb and anti-ICAM-1 also seemed to inhibit protease induction in HUVECs, indicating that other adhesion mechanisms may additionally contribute to adhesion-dependent CD40L signaling. Platelet adhesion via GP IIb/IIIa to the endothelium or immobilized fibrinogen upregulates CD40L and CD62P surface expression. Consistently, integrin clustering upregulates CD40L on wild-type but not on GP IIb/IIIa–deficient thrombasthenic platelets.
The finding that GP IIb/IIIa–dependent platelet adhesion and GP IIb/IIIa engagement upregulate CD40L and CD62P surface expression was proven by various approaches. Both CD40L and CD62P surface expression were substantially enhanced on platelets adherent to HUVEC or to the immobilized GP IIb/IIIa ligand fibrinogen (Figure 4). Consistently, CD40L and CD62P upregulation on platelets adherent to HUVEC was abrogated by β3-integrin blockade with GRGDSP or LM609 (Figure 4A). Moreover, cross-linking of GP IIb/IIIa on activated platelets in suspension through soluble fibrinogen plus mAb antifibrinogen or through bivalent mAb 7E3 stimulated CD40L exposure on wild-type but not on thrombasthenic platelets. In addition, ligation of GP IIb/IIIa by soluble fibrinogen or monovalent Fab fragment c7E3 had no effect in the absence of receptor clustering (Figure 5).
GP IIb/IIIa engagement transduces postoccupancy events, such as intracellular Ca2+ increase known as outside-in signaling.18,20⇓ Recently, Lindemann et al21 have shown that inhibition of GP IIb/IIIa engagement markedly attenuated the synthesis of IL-1β, supporting our finding that platelet adhesion is linked to platelet-mediated inflammatory reactions within the vessel wall. Together with the study by Lindemann et al, our data suggest that GP IIb/IIIa engagement that occurs during homotypic or heterotypic platelet interactions may induce a cascade of activation pathways in the respective coadhesive cell types, which might be triggered by inflammatory platelet compounds such as CD40L, CD62P, or IL-1β. We have described one representative activation pathway, triggered by platelet-CD40L in endothelial cells.
The present data may have important pathophysiological and therapeutic implications for atherosclerotic diseases. CD40L-CD40 signaling substantially contributes to atherogenesis, because inhibition of CD40L retarded the progression of atherosclerosis in mice22 and led to a collagen-rich stable plaque phenotype very likely attributable to decreased MMP-1 and MMP-2 activity.23 Consistent with our data, CD40L-expressing T-cell membranes have been shown to enhance endothelial secretion of both pro-MMP-9 and activated MMP-2 in vitro.16
Recently, it was shown that platelets enhance the release of soluble uPAR by endothelial cells; however, the underlying mechanism remained unclear.24 Our data present evidence that platelets can induce matrix-degrading activity in HUVEC via platelet-associated CD40L and that this mechanism is regulated by GP IIb/IIIa, a receptor involved substantially in platelet-endothelial interaction.13 The present data show that irrespective of the nature of GP IIb/IIIa antagonists, platelet-induced endothelial matrix degradation is substantially inhibited by all tested inhibitors, although maximal effects were found for inhibitors that block both GP IIb/IIIa and αvβ3. Whether these differential effects might be of clinical significance has to be answered in future studies.
In conclusion, our data indicate that platelets can focus matrix-degrading activity to the particular sites of platelet adhesion at the vessel wall. GP IIb/IIIa blockade may not only inhibit platelet aggregation at the vulnerable plaque and thereby prevent physical vessel occlusion but also may prevent platelet-CD40L–mediated inflammatory cascades, leading to matrix degradation and plaque rupture. This mechanism may contribute to the understanding of beneficial effects of GP IIb/IIIa antagonists on ischemic complications in patients with acute coronary syndromes.
The study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ga 381/4-1) and Wilhelm Sander-Stiftung.
↵*These authors contributed equally to this study.
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