Triggering of β1-Integrin Chain Induces Platelet Adhesion to Cultured Endothelium
Abstract We report here that platelets adhere to cultured endothelial cells (EC) on exposure to the integrin β1 activating monoclonal antibody (mAb) BV7. The effect of BV7 is exerted mostly on platelets rather than EC. BV7 does not induce platelet aggregation or 5-hydroxytyptamine (5-HT) release and does not increase platelet adhesion to matrix proteins. Another activating β1 mAb, Lia1/2, triggers an effect similar to BV7. Blocking antibodies to α2 and β1, but not to other integrin chains, are able to inhibit BV7-mediated adhesion. Moreover, the effect of BV7 requires active cellular metabolism and is not inhibited by platelet treatment with aspirin, by the PAF receptor antagonist BN50730, the phosphokinase C inhibitor staurosporin, or by the cAMP or cGMP enhancers prostaglandin E1 and sodium nitroprusside, respectively. Finally, BV7-mediated adhesion was enhanced by the endoperoxide analogue U46619. These data describe a novel mechanism of platelet adhesion to endothelial cells. This adhesion pathway appears to be mediated by α2β1-integrin on platelets and a still-undefined endothelial counter receptor.
- Received February 2, 1997.
- Accepted April 8, 1997.
In normal conditions, platelets do not adhere to resting endothelial cells (EC) with antithrombotic properties, attributed in part to their capacity to synthesize and release anti-adhesive molecules, including prostacyclin, nitric oxide, and 13-hydroxyoctadecadienoic acid.1,2 However, platelets may adhere to apparently intact virally transformed3 or bacteria-infected4 EC, as well as to thrombin-stimulated EC2 5 6 7 or following their pretreatment with thrombin.8,9 Moreover, platelet-activating factor (PAF) induces platelet adhesion to EC in presence of polymorphonuclear leukocytes10 and platelets from diabetic patients show a significant extent of adhesion to resting EC.11,12 Finally, PECAM-1–mediated platelet adhesion and aggregation was observed in arterioles in absence of endothelial denudation and exposure of the subendothelial matrix in vivo.13–15
Platelets adherent to the vascular endothelium could contribute to thrombotic and inflammatory reactions at least by two different ways: (1) a direct vascular injury induced by platelet activation, which causes subendothelial matrix exposure16,17 and (2) P-selectin–mediated promotion of polymorphonuclear leukocyte accumulation18–20 and fibrin deposition.19
The molecular mechanisms responsible for platelet adhesion to EC are still in large part unknown. A recent report showed that platelet “rolling” on intact endothelium is mediated by P-selectin.21 However, the adhesive molecules implicated in a more firm adhesion remain undefined.
In this study we found that an integrin-β1–activating mAb, BV7, was able to markedly increase platelet adhesion to EC. This effect was mostly directed to platelets and was substratum specific since BV7 did not increase platelet adhesion to matrix proteins. In addition, only α2 and β1 blocking antibodies were able to inhibit BV7 effect. These data describe a novel mechanism of platelet adhesion to endothelial cells. This new adhesion pathway appears to be mediated by α2β1 integrin on platelets and a still undefined endothelial counter receptor.
The suppliers for chemicals were Sigma Chemical Co (St Louis, Mo) for 2-deoxy-d-glucose, Dulbecco’s phosphate buffered saline (PBS; with and without 1 mmol/L Ca2+/Mg2+), EGTA, fibronectin, fluorescein-labeled phalloidin, phenyl-methyl sulfonylfluoride (PMSF), prostaglandin E1 (PGE1), sodium deoxycholate, sodium dodecyl sulfate (SDS), sodium nitroprusside (SNP), staurosporin, Tris-(hydroxymethyl)amino methane (Tris); Telios Pharmaceuticals Inc (San Diego, Calif) for vitronectin; Genzyme (Boston, Mass) for human recombinant tumor necrosis factor-α (TNF); Upjohn Co. (Kalamazoo, Mich) for 15(S)hydroxy-11(epoxymethano)prosta-(5Z,13E)-dienoic acid (U46619); Kabi Vitrum (Stockholm, Sweden) for human fibrinogen; Maggioni (Milano, Italy) for acetylsalicylic acid (ASA) in the form of its soluble lysine salt; Gibco (Paisley, UK) for Medium 199 (M199) and all other reagents for endothelial cell culture; Merck Inc (Darmstadt, Germany) for paraformaldehyde and sodium azide; Becton Dickinson Labware (Lincoln Park, NJ) for collagen (type I) and tissue culture plates and flasks; Technogenetics (Boston, Mass) for fluorescein isothiocyanate–conjugated goat F(ab′)2 anti-mouse IgG (FITC anti-mouse IgG); Serotec (Oxford, UK) for phycoerythrin-conjugated anti-human P-selectin; Amersham (Buckinghamshire, UK) for Na251CrO4; du Pont de Nemours (Firenze, Italy) for 5-[1,2–3H] hydroxytryptamine binoxalate (3H-5-HT; specific activity 15 to 30 Ci/mmol). Mouse laminin purified from the murine tumor EHS was kindly supplied by Dr G. Taraboletti from Istituto Mario Negri (Bergamo, Italy). PAF antagonist BN50730 was kindly supplied by Dr E. Pirotzky from Institut Henri Beaufour (Les Ulis, France).
BV7 mAb (IgG1 isotype) was generated from BALB/c mice immunized with EC as previously described.22 F(ab′)2 and Fab’ fragments of BV7 were subsequently obtained by standard procedures as described.22,23 The mAbs to integrin β1 subunit were kindly donated by the following investigators: Lia 1/2 by Dr F. Sanchez-Madrid from Hospital de la Princesa (Madrid, Spain) and P4C10 by Dr M.J. Elices from Cytel Co (San Diego, Calif); mAb K20 was from Immunotech (Marseilles, France). The following mAbs against integrin-α chains were used: 5E8 (anti-α2) was kindly supplied by Dr R.B. Bankert from T and B Bioclone Inc (Buffalo, NY); P1D6 (anti-α5) from Telios Pharmaceuticals Inc. (San Diego, Calif); GoH3 (anti-α6) was kindly supplied by Dr A. Sonnenberg from the Netherlands Cancer Institute (Amsterdam, The Netherlands). The anti-β2 mAb TS1/18 was from ATCC (Rockville, Md). The mAb against the glycoprotein IIb/IIIa complex AP2 was kindly supplied by Dr T.J. Kunicki from The Blood Center of Southeastern Wisconsin (Milwaukee). A rabbit antiserum (poly-β1) raised by injecting placenta-purified α5β1 was prepared in our laboratory.
Venous blood from healthy donors who had not received any medication for at least 2 weeks was anticoagulated with 3.8% trisodium citrate. Blood was centrifuged at 150g for 10 minutes to obtain platelet-rich plasma, which in turn was centrifuged at 500g for 10 minutes in presence of 1 (mol/L PGE1. Platelets were resuspended in PBS (with 1 (mol/L PGE1 and 10 mmol/L EGTA), centrifuged (12 000g for 15 s), and resuspended in PBS (containing 1 (mol/L PGE1). Finally, platelets were centrifuged (12 000g for 15 s) and resuspended in PBS containing 1 mmol/L Ca2+ as described.24
To prepare 3H-5-HT-labeled platelets, platelet-rich plasma was incubated with 0.1 μCi/mL of 5-[1,2–3(H)]hydroxytryptamine binoxalate in 2% ethanol at room temperature for 30 minutes. Platelets were then washed following standard procedure and resuspended in PBS (with 1 mmol/L Ca2+), as described.25 5-HT uptake was about 80% of total radioactivity.
EC were isolated from human umbilical vein, cultured in M199 supplemented with NCS (20%), endothelial cell growth supplement (50 μg/mL), and heparin (100 μg/mL) and kept in a 37°C, 5% CO2 humidified atmosphere. For the adhesion assay, EC were detached by brief exposure to trypsin (0.25%)/EDTA (0.022%), plated, and grown to confluence in 96-well plates, as previously described.26
51Cr-labeled platelets (5×108/mL) were preincubated with BV7 mAb (at the concentrations specified in the text) for 15 minutes at 37°C. Platelets (100 μL) were then layered on 96-well cultured EC and incubated for 45 minutes at 37°C unless otherwise specified. Wells were washed three times to remove nonadherent cells, the remaining bound cells were lysed with 0.1% SDS, and individual lysates were counted by an Beckman gamma 5500 counter (Fullerton, Calif). Data are expressed as the number of adherent platelets/well (×103).
In a series of experiments, we evaluated platelet adhesion to subendothelial matrix (ECM). EC monolayers were removed by two different methods, which have been shown to leave an intact extracellular matrix27: the first one consisted of three washings of EC monolayers with PBS (without Ca2+/Mg2+) and a subsequent incubation with 5 mmol/L EGTA in PBS for 10 to 15 minutes; the second of three washings with PBS and a subsequent incubation (10 minutes) with 0.5% of sodium deoxycholate and 1 mmol/L PMSF in 10 mmol/L Tris-HCl buffered saline. EC detachment was morphologically assessed by light microscopy. The exposed matrix was then washed twice with PBS before platelet adhesion assay performed under the experimental conditions indicated above.
For the experiments of platelet adhesion to protein-coated surfaces, 96-well plates were coated overnight at 4°C with collagen, laminin, vitronectin, and fibronectin (5 to 10 μg/ml, in PBS without Ca2+). Plates were washed with PBS (+ 1 mmol/L Ca2+/Mg2+) and then coated with PBS containing 2% BSA, for 1 hour at 37°C. Plates were washed twice and 51Cr-labeled platelets (5×108/mL) were subsequently incubated for 45 minutes at 37°C. The number of adherent platelets was calculated as described above.
Fluorescence Flow Cytometric Analysis
Phenotyping of platelets was performed by indirect immunofluorescence. Platelet suspensions (5×107 cells/sample) were preincubated with the following antibodies: BV7, AP2, MAR1, P4C10, TS1/18 (at the saturating concentrations specified in the text) for 30 minutes at 4°C. Platelets were then washed in presence of PGE1 (1 μmol/L) and incubated for 30 minutes at 4°C with FITC anti-mouse IgG (1:10 final dilution). Cells were washed, fixed with PBS containing 1% formalin, and fluorescence measured by an FACStar Plus apparatus (Becton Dickinson, Mountainview, Calif).
Cytofluorimetric analysis was also utilized to determine platelet adhesion to EC in suspension. Platelets (5×108/mL) were first incubated with BV7 (5 and 10 μg/mL), P4C10 (1:10 final dilution), and TS1/18 (1:10 final dilution) for 15 minutes at 37°C and then washed (in presence of 1 μmol/L PGE1) to eliminate unbound mAb. Antibody-treated platelets were subsequently resuspended in 1 mL of trypsin (EDTA)-detached EC (3×105/mL) in presence of FITC anti-mouse IgG (1:10 final dilution), for 45 minutes at 37°C. Cell suspensions were washed, fixed with 3% paraformaldehyde, and fluorescence, corresponding to EC bearing–bound mAb-treated platelets, was measured as described above.
To investigate the surface expression of P-selectin, platelets (5×106/mL), resting and treated with 5 μg/mL BV7 (15 minutes) and 0.1 U/mL thrombin (3 minutes), were exposed to the phycoerythrin-conjugated anti P-selectin mAb (1:10 final dilution) for 30 minutes at 4°C. Platelets were washed, fixed with PBS containing 1% formalin, and analyzed by flow cytometry.
Monolayer integrity was assessed by phase contrast microscopy and F-actin staining. EC were exposed to BV7 mAb, platelets and BV7-treated platelets for 45 minutes at 37°C. At the end of the incubation time, cells were washed and fixed with 0.2% fast green (3 minutes at 22°C) and stained with cresyl violet 0.5% (in 20% methanol) for 5 minutes at 22°C. Endothelial integrity was subsequently observed in a Nikon TMS microscope and images recorded on Kodak T-max p400 film.
For F-actin staining, cells to be examined were grown on glass coverslips coated with 10 μg/mL of human fibronectin (2 hours at 37°C). Coverslip-attached EC were exposed to BV7 mAb, platelets and BV7-treated platelets for 45 minutes at 37°C. Cells were subsequently washed, exposed for 3 minutes to 3% paraformaldehyde (containing 0.5% Triton X-100), and subsequently exposed for 15 minutes to 3% paraformaldehyde (without Triton X-100); washed and exposed to fluorescein-labeled phalloidin; and then processed for immunofluorescence microscopy as previously described.28 Observations were carried out in a Zeiss Axiophot photomicroscope equipped for epifluorescence and fluorescence images were recorded on Kodak T-max p3200 film.
Platelet Aggregation and 5-HT Release
5-HT-labeled platelets (108/mL) were preincubated at 37°C (under constant stirring) in a Platelet Ionized Calcium Aggregometer (PICA; Chrono-Log, Hevertown, Pa) for 3 minutes before the addition of BV7 (5 μg/mL) or U46619 (300 nmol/L). Aliquots (10 μL) of the samples were taken after 5, 30, and 60 minutes of BV7 addition and platelet aggregation quantified as the fall in singlet-platelet count measured with a platelet analyzer from (Baker Instruments; Allentown, Pa), as previously described.29 At the end of the incubation time, samples were immediately centrifuged in presence of 5 mmol/L EGTA and 1% paraformaldehyde (14 000g; 2 minutes) and release of radioactive 5-HT measured in the supernatants.
BV7 mAb Induced Platelet Adhesion to Resting EC
Preincubation of platelets with BV7 dramatically increased their adhesion to resting EC (Fig 1⇓, panel A). Phase contrast microscopy observation and F-actin staining, indicated that endothelial integrity was not affected by treatment with BV7, or by resting and BV7-treated platelets (Fig 1⇓, panel A).
A quantitative evaluation of the effect of BV7 on platelet adhesion to EC is reported in Fig 1⇑ (panel B). Purified BV7 mAb (0.2 to 5 μg/mL) induced a dose-dependent increase of platelet adhesion that was four-fold with the higher concentration of BV7 utilized. This effect was comparable when the mAb was used as F(ab′)2 or F(ab′) fragments. As shown in Fig 2⇓, the extent of platelet adhesion induced by purified BV7 (5 μg/mL) was related to the number of platelets seeded on EC (panel A) and the time of cellular contact (panel B). In all the conditions used the control irrelevant mAb TS1/18 was ineffective.
BV7-treated platelets adhered to TNF (100 U/mL, for 6 hours)-activated EC to a similar extent than to resting EC (not shown). This indicates that endothelial adhesion molecules induced by TNF do not contribute to the BV7 effect.
BV7 Activity Was Mainly Directed to Platelets
As assessed by flow cytometry, 1 μg/mL of F(ab′)2 fragment of BV7 could bind to resting platelets, similarly to 1 μg/mL of the anti-GpIIb/IIIa complex mAb AP2 (88% and 89% of positive cells, respectively). BV7 could also bind to EC (96% of positive cells) (not shown).
We therefore investigated whether the effect of BV7 was directed to platelets, EC or both. Platelets and EC were treated separately with F(ab′)2 fragment of BV7 (5 μg/mL) for 15 minutes. At the end of the incubation, cells were washed and tested for adhesion as described above. When only EC were treated, platelet adhesion was increased twofold. In contrast when only platelet were treated adhesion was five times higher than basal adhesion, an effect comparable to that obtained when BV7 was incubated with both cell types (Fig 3⇓). These results indicate that, although EC pretreatment with the antibody induces a slight increase of platelet adhesion, BV7 activity is essentially directed to platelets.
BV7 Activity on Platelet Adhesion to Extracellular Matrix Proteins
The effect of BV7 mAb could be due to an increased adhesion of platelets to extracellular matrix proteins incidentally exposed at interendothelial gaps. We therefore tested the effect of BV7 on platelet adhesion to EC exposed extracellular matrix.
The matrix was exposed after EC treatment either with EGTA or with deoxycholate and PMSF, as previously described.27 Table 1⇓ shows that, as expected, the extent of adhesion of untreated platelets to EC matrix was considerably higher than to EC. However, BV7 significantly increased platelet adhesion to EC but not to matrix proteins. We next tested platelet adhesion to protein-coated surfaces. Platelets, untreated or treated with 5 μg/mL of BV7, adhered to collagen, laminin, vitronectin, and fibronectin to a similar extent, as showed in Fig 4⇓. Platelet treatment with BV7 induced a slight, however not significant, decrease only when adhesion was tested on laminin.
This observation was further supported by experiments performed with EC in suspension. Platelets were treated with BV7 (5 and 10 μg/mL) or with another anti-β1 mAb P4C10 (1:10 final dilution) and with the irrelevant control mAb TS1/18 (1:10 final dilution), for 15 minutes at 37°C. At the concentrations used, the binding of BV7 and P4C10 to platelets alone was comparable: 90% and 92%, respectively. Antibody-treated platelets were then washed and exposed to EC in suspension (at the usual platelet/EC ratio) in presence of FITC anti-mouse IgG for 45 minutes at 37°C. EC associated fluorescence was subsequently analyzed by flow cytometry, as described in “Materials and Methods.” The assumption was that the fluorescence associated to EC was related to the amount of adherent platelets since the FITC anti-mouse IgG could only bind to those that were precoated with anti-β1 mAbs. As shown in Fig 5⇓, fluorescence corresponding to EC incubated with TS1/18-treated platelets resulted negative (5.5% positive cells) and that corresponding to EC incubated with P4C10-treated platelets was slightly increased (18.1% positive cells). However, 44.9% and 73.9% of EC were positive after their exposure to platelets treated with 5 μg/mL and 10 μg/mL of BV7 mAb, respectively.
Overall these data indicate that BV7 is able to induce platelet binding to EC in suspension further excluding the role of matrix proteins on this process.
Platelet Adhesion Was Inhibited by Anti-α2β1 mAbs
Platelets express different integrin-α chains linked to β1 such as α2, α3, α5, and α6. We therefore tried to define which integrin was responsible for BV7-induced adhesion. Platelets were preincubated with BV7 (5 μg/mL for 15 minutes at 37°C) and then exposed (20 minutes at 37°C) to the following anti-α chain mAbs: 5E8 (anti-α2; 5 μg/mL), P1D6 (anti-α5; 1:10 final dilution), and GoH3 (anti-α6; 1:100 final dilution), or to the anti-β1 antiserum (poly-β1; 1:100 final dilution) and the anti-β1 mAb K20 (1:50 final dilution). As shown in Fig 6⇓, basal platelet adhesion was unaffected by the mAbs tested. On the contrary, BV7-induced adhesion was significantly reduced after platelet incubation with 5E8 and poly-β1 antibodies. The combination of 5E8 with poly-β1 was not more effective than the two antibodies tested separately (Fig 6⇓). Moreover, BV7-induced platelet adhesion was unchanged by the anti-GpIIbIIIa mAb AP2 (5 μg/mL) or a nonimmune serum (1:100 final dilution), as shown in Fig 6⇓. These results strongly suggest that BV7-induced platelet adhesion is mediated by α2β1.
Comparative Study With Other Anti-β1 mAbs
The activity of BV7 mAb in promoting platelet adhesion to EC was compared with that of other anti-β1 mAbs known to activate β1 integrins. Platelets were pretreated (15 minutes at 37°C) with the following anti-β1 mAbs: BV7 (5 μg/mL), Lia1/2 (1:10 final dilution), and P4C10 (1:10 final dilution) and then layered on resting EC for 45 minutes. Lia1/2 was able to significantly increase platelet adhesion to resting EC in a way comparable to BV7, whereas P4C10 was ineffective (Fig 7⇓).
These results suggest that BV7 and Lia1/2 binds to a close related epitope on platelet β1-integrin and indicate that not all β1-activating mAbs can efficiently induce platelet adhesion to EC.
Modulation of BV7-Induced Platelet Adhesion
To better define the mechanism in which BV7 upregulates platelet adhesion, we tested the effect of different inhibitors. Platelet pretreatment with the anti-cyclooxygenase aspirin (0.3 mmol/L), the adenylate cyclase enhancer PGE1 (10 and 25 μmol/L), the guanylate cyclase enhancer SNP (0.3 mmol/L), the PAF receptor antagonist BN 50730 (10 μmol/L), and the protein kinase C inhibitor staurosporin (0.2 μmol/L) did not significantly affect BV7 (5 μg/mL)-induced adhesion (Fig 8⇓). Aspirin and PGE1 treatment exerted a slight enhancement of platelet adhesion induced by BV7 mAb.
In contrast, the adhesion was inhibited by platelet pretreatment with a combination of 2-deoxyglucose (50 mmol/L) and NaN3 (0.3%), which inhibits glucose metabolism, by 20 minutes of treatment with 1% paraformaldehyde and by cell chilling at 4°C (Fig 8⇑).
Finally, EC pretreatment with aspirin (0.5 mmol/L; 1 hour at 37°C) or heparin (250 μg/mL; 15 minutes at 37°C) did not change the effect of BV7 (not shown).
BV7 Activity on Platelet Aggregation, 5-HT Release, and P-Selectin Expression
As shown in Fig 9⇓, BV7 (5 μg/mL) was unable to directly induce platelet aggregation (panel A) or 5-HT release (panel B). As a control, platelet suspensions were exposed to the endoperoxide analog U46619 (300 nmol/L), that largely induced both aggregation and 5-HT release (Fig 9⇓). Similar results were obtained when platelet activation was tested on platelet suspensions (3×108/mL) exposed to F(ab′)2 fragment of BV7 (5 μg/mL) for up to 30 minutes (not shown).
Results in Fig 9⇑ shows that 5-HT was not released from dense bodies on platelet treatment with BV7. We next investigate if the antibody was able to induce the surface expression of P-selectin that is normally stored in α-granules. Therefore, resting, BV7 (5 μg/mL)-activated platelets and thrombin (0.1 U/mL)-activated platelets were exposed to a phycoerythrin-conjugated anti-P–selectin mAb and fluorescence analyzed by flow cytometry, as described in “Methods.” Resting, BV7, and thrombin-activated platelets resulted 8.2%, 10.2%, and 79.8% positive, respectively, for P-selectin, suggesting that the antibody was unable to induce an increase of P-selectin exposure on the platelet membrane.
Effect of Platelet Activation on BV7-Induced Adhesion
We next investigate whether the adhesion induced by BV7 may be modified by platelet pretreatment with a specific agonist. Platelets were pretreated (15 minutes at 37°C) with BV7 (5 μg/mL) in the presence or absence of different concentrations of U46619 (180 to 1500 nmol/L). As shown in Fig 10⇓ (panel A), platelet adhesion induced by BV7 was dose-dependently enhanced by U46619 pretreatment. At the higher concentration of U46619, the adhesion induced by BV7 was about 2.5-fold increased respect to the absence of the agonist. As reported in Fig 10⇓ (panel B), the extent of the enhancement induced by U46619 (360 nmol/L) increased with the time of cellular contact.
In this article we describe a novel mechanism of platelet adhesion to EC induced by the anti-β1 integrin mAb BV7.
Phase contrast microscopy indicated that after BV7 treatment, platelets adhered to the endothelial surface mainly as single cells and seldom in the form of small aggregates (Fig 1⇑). The effect of BV7 seems to be specific for EC and was not mediated by an increased adhesion of platelet to the subendothelium since the antibody was poorly effective on platelet adhesion to endothelial matrix proteins and could promote platelet adhesion to endothelial cells in suspension.
The different effect of BV7 on platelet adhesion to the endothelium or to matrix proteins suggests that these activities are mediated by two different epitopes on the β1-chain.
Another anti-β1 mAb Lia1/2 was able to increase platelet adhesion to endothelium. Interestingly, Lia1/2 inhibits cell adhesion to collagen and laminin and can compete with BV7 for binding to β1-chain,22 indicating that these two antibodies recognize closely related epitopes. In contrast, the anti-β1 P4C10 did not increase platelet adhesion to EC and was unable to compete for BV7 binding to β1-integrin chain.22 Overall these data suggest that BV7 and Lia1/2 binding to β1-chain causes a change in conformation/availability for the endothelial ligand without major modifications of the integrin chain avidity for endothelial matrix.
Platelets express different α chains linked to the β1-chain, however only an anti-α2 mAb was able to inhibit BV7-induced platelet adhesion to EC (Fig 6⇑), indicating that α2β1 integrin plays a major role in this effect. It is noteworthy that α2β1 integrin binding to collagen seems not to be involved, since adhesion assay was done at Ca2+ concentration that can efficiently inhibit this linkage30 and EC monolayer integrity was unaffected (Fig 1⇑).
Other studies showed that α2β1 integrin promotes cell-to-cell binding in EC, keratinocytes, and α2-transfected cells.31–35 In these studies it was shown that conditions that inhibit cell-to-cell adhesion did not affect cell binding and spreading on extracellular matrix. This supports the idea that different mechanisms and/or epitopes on α2β1 are responsible for cell-to-cell or cell-to-matrix binding.
An important question is related to the endothelial counter-receptor for α2β1 integrin. We can exclude that it may be the Fc receptor because F(ab′)2 fragments of the antibody were equally effective. The possibility that BV7 could act through a bridging effect by binding to platelet and endothelial α2β1 can also be discarded since F(ab′) fragment of BV7 was as active as the native antibody. Finally, it is unlikely that BV7 promotes homophilic interaction between α2β1 molecules present in the two types of cells since the effect of BV7 seems to be essentially directed to platelets and only to a minor extent to the endothelium.
It is noteworthy that EC activation with inflammatory cytokines, such as TNF, did not change the effect of BV7 on platelet adhesion, suggesting that cytokine-induced endothelial adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1, do not play a significant role in the effect exerted by BV7. Moreover, ICAM-2, although expressed on platelets and EC, do not play a role in this process since both cells lack of counter receptors for this molecule.
Another aspect that, despite our efforts, remains in large part unsolved is the mechanism of BV7 activation of platelet adhesion. BV7 treatment was unable to induce platelet aggregation and 5-HT release, even in the presence of exogenous fibrinogen (Fig 9⇑). Thus, while BV7 upregulated an adhesive mechanism it was unable to determine a more general platelet activation. In addition, the effect of BV7 was not related to either cyclooxygenase metabolites or to the active phospholipid PAF, since adhesion was unchanged after platelet treatment with aspirin or the PAF receptor antagonist BN 50730 (Fig 8⇑). Moreover, the effect was not prevented by enhanced cytoplasmic levels of cAMP or cGMP, obtained by platelet treatment with PGE1 and SNP (Fig 8⇑), or by staurosporin suggesting that BV7 acts on platelets by a mechanism unrelated to the stimulus response coupling normally evoked by classic agonists.36 However, the effect of the antibody requires active platelet metabolism. As reported in Fig 8⇑, the adhesion was prevented by platelet chilling at 4°C, by fixation with paraformaldehyde and by a combination of 2-deoxyglucose and sodium azide, which blocks glucose metabolism and electron transport.37 This indicates that the effect of BV7 was not mediated by unspecific cell agglutination and that it requires active cellular metabolism.
Results reported in Fig 10⇑ show that the endoperoxide analogue U46619, while inactive per se on platelet adhesion to EC, can increase the effect of BV7. These results support the hypothesis that, whereas BV7 binding to α2β1 causes a change in conformation/availability for the endothelial ligand, U46619 may amplify the process by enhancing the availability of α2β1-integrin.
Indeed, immunolocalization studies show that, in resting platelets, β1-integrins are present on the plasma membrane as well as in an intracellular pool from where they translocate toward the external membrane on platelet activation.38 This suggests that U46619 may enhance the number of α2β1-integrin molecules on the platelet surface that become available for BV7 binding.
The evidence reported in this work suggests that BV7 binding does not change expression and/or activity of platelet integrins, since platelet adhesion to collagen, laminin, fibronectin, and vitronectin is unaffected by the antibody (Fig 4⇑). Moreover, BV7 did not directly rearrange αIIbβ3-integrin since it did not induce platelet aggregation in presence of fibrinogen (Fig 9⇑). This effect was also unrelated to an enhancement of P-selectin expression, as assessed by flow cytometry. Finally, BV7 did not change GpIb activity since platelet adhesion to von Willebrand-rich EC matrix did not increase (Table⇑ I).
The EC monolayer normally constitutes an anti-adhesive surface for circulating platelets39; however, it has been reported that in some circumstances these cells may adhere to an apparently intact EC monolayer2-9. This work presents evidence for a novel platelet adhesive mechanism to resting endothelium. This pathway includes activation of α2β1 integrin on the platelet surface and its binding to a still undefined EC ligand. Other cells, such the colon carcinoma cell lines were found to respond in a similar way.22
The biological significance of platelet adhesion to EC is far from being fully understood. In this context, platelets may be relevant in promoting thrombotic events by injuring the vasculature.17 Moreover, the evidence that platelets roll along the vessel wall21 and migrate when stimulated by allergens40 supports the idea of a direct proinflammatory role for these cells. This effect can be exerted by virtue of their ability to accumulate with neutrophils,41 generate inflammatory cytokines that regulate endothelial expression of adhesion molecules,42,43 and promote leukocyte infiltration by a P-selectin−dependent mechanism.19,20
This work was supported by the Associazione Italiana per la Ricerca sul Cancro and by the European Community (BIOMED projects BMH4CT360669 and CT950875, and BIOTECH project CT960036) and Istituto Superiore di Sanitá (Sottoprogetto1; Tema 1.3).
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