By
From the Division of Hematology, University of Washington, Seattle, Washington 98195-7710
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Abstract |
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Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC)
monolayers were incubated with washed, stained, and thrombin-activated human platelets. To
exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was
measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with
different receptor antagonists. Whereas blockade of platelet 1 integrins, GPIb
, GPIV, P-selectin,
and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to
HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to
GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin,
or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC
receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and
v
3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIb
. Blockade of
1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet
adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is
mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1,
v
3 integrin, and, to a lesser extent, GPIb
.
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Introduction |
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Although the pathophysiologic consequences of activated platelets in circulation are not yet fully understood, it is well established that increased platelet activation is associated with an enhanced risk of thrombotic complications in different clinical disorders, such as diabetes, preeclampsia, unstable angina, peripheral vascular disease, and stroke and after angioplastic and fibrinolytic therapy (1). Because activated, but not resting, platelets have been shown to adhere to intact endothelium, it has been suggested that platelet thrombi may also occur in the absence of endothelial cell denudation, particularly in the microvasculature (2). However, while the platelet receptors involved in aggregate formation and matrix adhesion have been studied extensively, the pathways responsible for the interaction of platelets and the endothelium are not well characterized.
So far, three different platelet receptors have been reported to be involved in the binding to endothelium. Rolling of activated platelets on high endothelial venules was
found to depend primarily on platelet P-selectin (IIb
3;
CD62P; 6), whereas firm adhesion to human saphenous
vein endothelial cells was inhibited by anti-GPIIbIIIa (CD41a/
CD61) antibodies and RGD peptides (7). Furthermore, it has been shown that platelet-sialylated glycoproteins
may, at least in part, be responsible for the increased adhesion
of platelets from diabetics to bovine valvular endothelial
cells (8).
Likewise, several distinct endothelial cell molecules have
been reported to be involved in the binding of resting and
activated platelets. Both endothelial-sialylated glycoproteins
(6), as well as P-selectin on activated endothelium (9), have
been proposed to mediate platelet rolling. With human
umbilical vein endothelial cells (HUVEC)1 infected with
herpes virus or stimulated with IL-1 or plasma containing
chemotherapeutic drugs, platelet adhesion was effectively inhibited by antibodies to endothelial von Willebrand factor (vWF) and v
3 integrin (CD51/CD61), respectively
(10). Moreover, a recent in vivo study has presented
evidence that platelet-endothelial cell adhesion molecule-1
(PECAM-1; CD31) on endothelial cells may contribute to
platelet adhesion and aggregation at a site of injured but not
denuded endothelium (13).
Thus, this study was designed to further clarify the role
of the different receptors that have been implicated in the
adherence interaction of platelets with endothelial cells. Because both resting and activated platelets adhere primarily
to matrix proteins, rather than to endothelial cells, many investigators have used fixed endothelial cells in the adhesion
assay in an attempt to maintain complete confluence. However, fixation can alter the receptor function and does not
exclude the involvement of matrix proteins exposed by small
intercellular gaps or expressed on the endothelial cells themselves. Hence, to avoid this problem, platelet binding to
HUVEC was determined in suspension using flow cytometry. Our results show that thrombin-activated platelets bind
to HUVEC by a GPIIbIIIa-dependent bridging mechanism
involving platelet-bound adhesive proteins, including fibrinogen, fibronectin, and vWF. Importantly, activated platelet binding did not involve endothelial cell-associated adhesive proteins such as collagen IV, fibronectin, and vWF,
but instead used intercellular adhesion molecule-1 (ICAM-1; CD54) and v
3 integrin. In addition, we also found evidence for the involvement of endothelial GPIb
(CD42b).
Thus, these endothelial adhesion molecules may contribute
to the recruitment of activated platelets to intact endothelium
and, consequently, to the formation of intravascular platelet
aggregates, thereby promoting thrombotic processes.
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Materials and Methods |
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Endothelial Cell Culture.
HUVEC were obtained by collagenase treatment of umbilical cord veins as previously described (14). Cells were cultured on gelatin-coated dishes and propagated in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 20% bovine calf serum (Hyclone Laboratories Inc., Logan, UT), 90 µg/ml heparin (Sigma Chemical Co., St. Louis, MO), and 50 µg/ml endothelial cell growth factor prepared from bovine hypothalamus (15). For flow cytometry assays, HUVEC derived from passages two or three were allowed to grow to confluence in 12-well dishes. For assays with HUVEC matrix, cells were cultivated in 48-well plates and harvested with trypsin after at least 4 d. Before the adhesion assay, HUVEC were washed twice with RPMI medium and incubated with receptor antagonists in phenol red-free RPMI medium for 30 min at 37°C.Platelet Preparation.
Blood was obtained by venipuncture from healthy adult volunteers according to a protocol approved by the Human Subjects Division of the University of Washington (Seattle, WA). The volunteers did not take any drugs for the previous 10 d. Isolation of platelets was performed as described by Baenziger and Majerus (16). In brief, blood was drawn into polypropylene syringes containing one-tenth volume of 0.11 M sodium citrate and centrifuged at 1,000 g for 4 min to obtain platelet-rich plasma. Platelets were sedimented by centrifugation at 2,000 g for 10 min and washed twice with 10 ml of Hepes buffer (10 mM Hepes, 0.5 mM MgCl2, 130 mM NaCl, 4 mM KCl, 1 mM CaCl2, and 5 mM glucose, pH 7.4). To pellet erythrocytes selectively, platelets were resuspended in the same buffer and centrifuged twice at 120 g for 3 min. Subsequently, the content of erythrocytes was <1% as calculated using a hemocytometer. Platelets were stained with 2.5 µM calcein-acetoxymethyl ester (Molecular Probes Inc., Eugene, OR) in the dark for 15 min. To avoid platelet activation, all centrifugations were done at room temperature and in the presence of 1 µM prostaglandin E1 (Alprostadil, Prostin VR Pediatric®; The Upjohn Co., Kalamazoo, MI). After washing, platelets were adjusted to a final concentration of 2 × 109/ml and activated with 0.5 U/ml human thrombin (Sigma Chemical Co.) for 10 min. Thrombin was inactivated with 2 U/ml hirudin (Sigma Chemical Co.) for 10 min. Platelets were incubated with receptor antagonists for 30 min at room temperature.Antibodies.
To blockOther Receptor Antagonists.
Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES) peptides (Peninsula Laboratories, Belmont, CA) and recombinant annexin V (provided by Dr. J.F. Tait, University of Washington, Seattle, WA) were used at final concentrations of 50 µg/ml and 5 µM, respectively. Treatment with neuraminidase from Vibrio cholerae (Calbiochem-Novabiochem Corp.) was performed at a final concentration of 0.5 U/ ml for 4 h (platelets 60 min). For divalent cation depletion assays, cells were incubated with 1 mM EDTA (Sigma Chemical Co.) for 30 min. Adhesive proteins were added to HUVEC immediately before addition of platelets. Purified human vWF (American Diagnostica Inc., Greenwich, CT), fibrinogen (Sigma Chemical Co.), fibronectin (Calbiochem-Novabiochem Corp.), and bovine albumin (Sigma Chemical Co.) were used at concentrations as indicated in the figure legends. Ristocetin (Sigma Chemical Co.) was added together with vWF at concentrations as indicated in the figure legends.Determination of HUVEC-associated Adhesive Proteins.
Washed , suspended HUVEC were incubated with antibodies to either collagen IV, fibronectin, or vWF (same antibodies as described above) for 30 min. After washing twice, the secondary FITC-conjugated antibody, either goat anti-mouse IgG (Caltag Lab., San Francisco, CA) or mouse anti-rabbit IgG (Sigma Chemical Co.), was incubated for 30 min (final dilution of 1:200). At least 10,000 cells were then analyzed by a FACScan® (Becton Dickinson). Cells treated with FITC-conjugated isotype-specific IgG were used as negative controls.Adherence Assay (Flow Cytometry).
HUVEC were washed twice with RPMI medium and incubated with 600 µl of Hepes buffer (same buffer as described above, but with 2 mM CaCl2 added) and 40 µl of the suspension of activated and stained platelets for 30 min at 37°C. The final platelet concentration was 1.25 × 108/ ml. After removing unbound platelets followed by two washes, HUVEC were harvested mechanically by vigorous pipetting, washed once, and then immediately fixed with 80% ethanol on ice for 30 min. To differentiate HUVEC from residual unbound platelets, HUVEC were stained with the DNA-binding dye propidium iodide. The cells were resuspended in 200 µl PBS buffer, pH 7.4, and 0.1% Triton X-100 containing 5 µg/ml propidium iodide and 50 µg/ml ribonuclease A (R-6513; all Sigma Chemical Co.). Subsequently, specimens were analyzed by a FACScan®. At least 10,000 cells that stained positive for propidium iodide (FL-2-H) were evaluated. Platelet adhesion to HUVEC was expressed by the median fluorescence (FL-1-H) of the entire HUVEC population. Based on the distribution of the DNA content (FL-2-H), HUVEC were routinely tested for homotypic aggregate formation. On average, ~5% of the cells exhibited more than tretraploid DNA content indicating that they were clumped. Statistical significance was determined using Student's t test.Adherence Assay (Fluorescence Plate Reader).
Assays of platelet adhesion to HUVEC matrix were performed in 48-well dishes. Platelets were isolated, stained, and activated as described above. After harvesting the HUVEC with EDTA, 2.5 × 107 platelets per well were allowed to adhere to the matrix for 30 min at 37°C. Adherence was then assessed in a Cytofluor Series 4000 fluorescence plate reader (PerSeptive Biosystems, Framingham, MA). Plates were scanned before and after washing for total and adherent cells, respectively. Adherence was expressed as a percentage of the total fluorescence.Transmission Electron Microscopy.
Adhesion assays were performed as described above. HUVEC were then harvested, washed, and fixed with 3% glutaraldehyde in PBS buffer. After postfixation with 2% osmium tetroxide for 60 min, cells were washed and dehydrated with increasing concentrations of ethanol and finally propylene oxide. Fragments of the cell pellet were infiltrated with 100% Medcast in vacuum for 4 h and subsequently embedded in 100% fresh (Ted Pella, Reading, CA) Medcast. After polymerization for 48 h at 60°C, specimens were visualized by a JEOL 1200-EX11 (JEOL USA Inc., Peabody, MA). ![]() |
Results |
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Over a time period of 30 min, the number of HUVEC with bound resting platelets was quite low (Fig. 1). However, with thrombin-activated platelets HUVEC exhibited a significant increase in fluorescence after only 5 min. Maximal binding of activated platelets was found after 20-30 min. As determined by an arbitrary gate, ~10 and 65% of HUVEC bound resting and activated platelets, respectively. Using flow cytometry, it was not possible to determine whether the increased fluorescence of HUVEC was due to multiple single platelets bound to HUVEC or to a few large platelet aggregates attached to HUVEC. However, large platelet aggregate formation on HUVEC was found to be a rare event (see below), suggesting that the adhesive interaction was platelet- HUVEC rather than platelet-platelet.
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The striking difference in the fluorescence intensity between HUVEC incubated with either resting or activated platelets was also evident by transmission electron microscopy (TEM). As shown in Fig. 2 A, resting platelets bound minimally to HUVEC. However, activated platelets bound avidly to HUVEC with single or clumped platelets coating the endothelial cells (Fig. 2 B). Occasionally, large platelet aggregates adherent to HUVEC could also be found (Fig. 2 C). However, on average, we observed only one large aggregate in every 10-12 samples.
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Treatment of HUVEC with annexin V, neuraminidase, and the blocking anti-1 mAb P4C10 did not affect
platelet adhesion (Fig. 3 A). When HUVEC were treated
with the anti-PECAM-1 mAb M89D3, platelet adhesion
decreased by ~10%. However, this decrease was not significant. Although HUVEC were not stimulated by any exogenous agents before the assay, we also tested stimulation-dependent HUVEC receptors, assuming that platelets
might be able to activate HUVEC. Blocking mAbs to
VCAM-1, E-selectin, or P-selectin did not inhibit platelet
binding. However, a significant decrease in platelet binding
was observed when HUVEC were treated with mAbs to
ICAM-1,
v
3 integrin, or GPIb
. All four mAbs to
ICAM-1 exhibited a blocking effect. HUVEC treated with
anti-ICAM-1 mAb R6.5 or 6E6, which both block the
2
integrin-binding site of ICAM-1, decreased platelet binding by ~26%. Similarly, the mAb VF27, which has been
found to block adhesion of rhinoviruses to endothelial
ICAM-1, also reduced platelet binding by ~32%. However, the greatest inhibition (50%) was found when HUVEC
were treated with the mAb 2D5, which blocks the fibrinogen-binding site of ICAM-1 (22). Involvement of
v
3
integrin was evident when platelet adhesion decreased
by ~38% after treating HUVEC with the anti-
v
3 integrin mAb LM609, whereas the nonblocking anti-
v
3 integrin mAb LM142 had no effect. Interestingly, the anti-GPIb
mAb SZ2 also exhibited an inhibitory effect, reducing
platelet binding significantly by ~22%. A second blocking
anti-GPIb
mAb 2E4 inhibited platelet adhesion by ~12%,
which, however, was not statistically significant. Incubation
with RGD peptides and EDTA decreased platelet binding
by 60 and 71%, respectively. A combined treatment with mAbs to ICAM-1 (2D5), GPIb
(SZ2), and
v
3 integrin
(LM609) decreased platelet adhesion by ~59%, but still did
not reduce binding to the level of resting platelets.
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Platelets are well known to bind rapidly to the adhesive proteins that are exposed by an injured vessel wall. Thus, HUVEC were tested for the exposure of matrix proteins known to be important for platelet adhesion, such as collagen IV, fibronectin, and vWF. When analyzed by flow cytometry, all three of these proteins were found to be exposed on detached HUVEC with vWF staining greater than fibronectin and collagen IV. Incubation of HUVEC monolayers with the same antibodies to collagen IV, fibronectin, and vWF did not affect platelet adhesion (Fig. 3 B). However, each of the three antibodies was found to inhibit platelet adhesion to a matrix of the corresponding purified protein such as collagen IV, vWF, and fibronectin (data not shown). This suggests that the HUVEC monolayers do not express these adhesive proteins on the luminal surface to a degree sufficient to contribute to platelet binding. Indeed, when platelets were allowed to adhere to HUVEC in suspension, treatment of HUVEC with the antifibronectin and anti-vWF mAb markedly decreased platelet binding (data not shown). Thus, matrix proteins relevant for platelet binding, such as fibronectin and vWF, seem to be expressed primarily on the abluminal side of endothelial cells.
Activated Platelets Bind to HUVEC Matrix viaTo clarify further the role of matrix proteins in the
adhesion of activated platelets to HUVEC, we tested whether
the platelet receptors involved in the binding to HUVEC
were the same as those involved in the binding to the
HUVEC matrix. Measured by a fluorescence plate reader,
activated platelets bound exclusively by 1 integrins to the
HUVEC matrix (Fig. 4). Blockade of other receptors including GPIb
, GPIV, and GPIIbIIIa did not significantly
affect platelet adhesion. Similarly, platelet
1 integrins were
the primary receptors mediating the binding of activated
platelets to HUVEC in suspension (data not shown). However, platelet adhesion to adherent HUVEC monolayers
did not involve platelet
1 integrins (see below), which further indicates that exposed matrix proteins on the HUVEC monolayer did not serve as ligands for activated platelet receptors.
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As shown
in Fig. 5 A, thrombin-activated platelet adhesion to HUVEC was found to be mediated by the platelet receptor
GPIIbIIIa. Blockade of phosphatidlyserine by annexin V and
enzymatic degradation of sialylated glycoproteins by neuraminidase did not affect adhesion. As well, incubation of
platelets with mAbs to 1 integrins (clone P4C10), GPIb
(clones SZ2 and 2E4), GPIX (clone BL-H6), GPIV (clone FA6.152), and P-selectin (clones G1 and WAPS12.2) did
not reduce platelet binding to HUVEC. Similar to HUVEC, treatment of platelets with the anti-PECAM-1 mAb
M89D3 led to a slight decrease of platelet binding, which,
however, was not significant. Because in separate experiments we found that platelets did not bind to recombinant PECAM-1 (our unpublished observation), involvement of
PECAM-1 in the interaction with HUVEC seemed very
unlikely. On the other hand, when platelets were treated
with either RGD peptides or the anti-GPIIbIIIa mAb P2,
platelet binding was reduced by >50%. Furthermore, EDTA
(which dissociates GPIIbIIIa) decreased platelet binding almost to the level of resting platelets. Because it was not
possible to remove the mAbs from the platelet suspension
after activation with thrombin (centrifugation would cause
an irreversible clot), it was necessary to consider the possibility that unbound mAbs could also block HUVEC receptors.
However, when using mAbs and peptides at concentrations
corresponding to the concentration in the platelet suspension after dilution in the adhesion buffer, platelet binding
was not affected (data not shown).
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Upon activation, platelets express different adhesive proteins, such as fibrinogen, fibronectin, and vWF (28, 29). Because all three of these proteins are known to mediate platelet aggregation, their involvement in the binding to HUVEC seemed very likely. Indeed, as shown in Fig. 5 B, blockade of vWF, fibrinogen, and fibronectin on platelets decreased adhesion to HUVEC monolayers by 56, 46, and 39%, respectively. This result indicates that, similar to platelet aggregation, platelet-exposed adhesive proteins participate in activated platelet binding to HUVEC monolayers.
Exogenous Adhesive Proteins Enhance Platelet Binding to HUVEC.To investigate further the role of adhesive proteins in the binding mechanism, platelet adhesion was examined in the presence of purified fibrinogen, fibronectin,
vWF, and albumin (Fig. 6). All proteins but albumin were
found to induce a significant increase in platelet binding.
The minimal concentrations required for a maximal increase were 75 µg/ml for fibrinogen, 15 µg/ml for fibronectin, and 0.25 µg/ml for vWF, which correspond to
plasma concentrations of 2.5, 5, and 2.5%, respectively.
Addition of vWF increased platelet adhesion only in the
presence of ristocetin, but had no effect when added alone.
This indicates an involvement of endothelial and/or platelet GPIb. Participation of endothelial GPIb
seemed more likely, because blockade of GPIb
on HUVEC, but
not platelets, was found to affect platelet adhesion (Figs. 4
A and 5 A). Albumin added at concentrations up to 4 mg/ml
did not alter platelet binding, confirming that the effect of
adhesive proteins was specific.
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Results to this point suggested that adhesive proteins may play an important role in mediating the binding of activated platelets to HUVEC monolayers. This hypothesis was further supported by experiments using zinc or manganese, rather than thrombin, to activate platelets (Fig. 7). Both cations are known to activate GPIIbIIIa without inducing secretion and aggregation (30, 31). In the presence of 0.5 mM zinc or 2 mM manganese only, platelets did not adhere to HUVEC. However, when exogenous fibrinogen (375 µg/ml) was added, both zinc- and manganese-activated platelets bound to HUVEC to the same degree as thrombin-activated platelets. Again, this binding could be partly inhibited by the anti-GPIIbIIIa mAb P2 and the antifibrinogen mAb D1G10VL2. Hence, the ability of platelets to bind to HUVEC monolayers depended not only on an activated GPIIbIIIa receptor, but also on the presence of a bridging adhesive protein such as fibrinogen.
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Fig. 8 further demonstrates a GPIb-dependent
component of platelet adhesion to HUVEC. In the presence of 1 µg/ml vWF, ristocetin dose dependently increased binding of resting and activated platelets. However,
when HUVEC were treated with the anti-GPIb
mAb
SZ2, binding of resting and activated platelets decreased by
~30 and 23%, respectively. Treatment of resting platelets
with the mAb SZ2 reduced binding to HUVEC by ~80%,
whereas treatment of activated platelets did not affect adhesion significantly. This result indicates that the vWF/ristocetin-induced increase in the binding of resting platelets to
HUVEC depended on both platelet and endothelial GPIb
, whereas increased binding of activated platelets required only endothelial GPIb
.
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Discussion |
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In this report we show that adherence of thrombin-activated platelets to HUVEC monolayers is mediated by platelet GPIIbIIIa and distinct endothelial cell adhesive molecules (Fig. 9). As has previously been demonstrated (32), flow cytometry is a very sensitive and accurate method to measure heterotypic adhesion of platelets and its use allowed us to exclude the involvement of matrix proteins in the adhesive interaction.
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Besides other mechanisms, activated platelet adhesion to
leukocytes and tumor cells has been found, similar to platelet aggregation, to involve fibrinogen bound to GPIIbIIIa
(33). Thus, it seemed very likely that the same mechanism mediated platelet binding to endothelial cells. Indeed,
blockade of GPIIbIIIa by the mAb P2 or RGD peptides
decreased platelet binding to HUVEC monolayers by ~50%,
which is comparable to a recent report (7). Although both
RGD peptides and the mAb P2 have been shown to block
completely platelet aggregation (19), platelet binding to
HUVEC was not inhibited 100% by these antagonists. Because platelets were activated before treatment with antagonists, adhesion of small preformed platelet aggregates to
HUVEC had to be considered. However, when platelets
were treated with the mAb P2 before thrombin activation to prevent aggregate formation, platelet binding to HUVEC did not further decrease (data not shown). Thus, activated platelets that are unable to aggregate may still adhere
to HUVEC, perhaps by an additional binding mechanism.
However, blockade of 1 integrins, GPIb
, GPIX, GPIV,
P-selectin, or PECAM-1 did not reveal an inhibitory effect.
We found that adhesive proteins play an important role in activated platelet adhesion to HUVEC. Blockade of platelet-exposed fibrinogen resulted in a significant decrease of bound platelets. Moreover, addition of fibrinogen further increased platelet adhesion, indicating that platelets may be able to use both endogenous and exogenous fibrinogen to bind to endothelial cells. Importantly, blockade of fibronectin and vWF, which are both also exposed on GPIIbIIIa on activated platelets (29), decreased adhesion as well. This is in accordance with previous studies showing that platelet-released fibronectin and vWF are capable of cross-linking platelets or platelets and tumor cells (35, 36). It is conceivable that thrombospondin (TSP)-1, another platelet-released adhesive protein, may also participate in the binding to HUVEC. Although blockade of the major TSP-1 receptor on platelets, GPIV (CD36), did not affect platelet adhesion to HUVEC, treatment of platelets with the anti-TSP-1 mAb 5G11 revealed a minimal inhibitory effect (data not shown). This indicates that TSP-1 may be bound by a different receptor, most likely GPIIbIIIa. However, because this inhibition was very modest and this mAb does not block platelet aggregation (37), involvement of TSP-1 remains uncertain.
The significant role of adhesive proteins in platelet adhesion to HUVEC monolayers is further demonstrated by the fact that binding was not observed when platelets were activated without secretion by using zinc or manganese. Although these cations activate the GPIIbIIIa receptor, only the addition of exogenous fibrinogen allowed platelets to bind to HUVEC or to form aggregates (30, 31), indicating that platelet-platelet and platelet-HUVEC interactions are mediated by the same bridging mechanism. Thus, it is possible that the reduced fluorescence of HUVEC after treatment of platelets with anti-GPIIbIIIa antagonists may be due to the reduction of platelet aggregates rather than of platelet-HUVEC interactions. However, extensive platelet aggregate formation was not observed on TEM, suggesting that the inhibition was primarily due to blockade of platelet adhesion to HUVEC.
Interestingly, evaluation of the HUVEC receptors involved in activated platelet adhesion revealed participation
of three different receptors. Treatment of HUVEC with
RGD peptides resulted in the greatest inhibition of platelet
adhesion, suggesting that RGD-dependent adhesive protein receptors were predominantly involved. However, blockade of 1 integrins on HUVEC did not affect platelet
adhesion. This is in accordance with a previous study
showing that
5
1 integrins serve as RGD-dependent fibrinogen receptors only when HUVEC are suspended, but
not attached (38). Under our experimental conditions,
v
3 integrin was the only RGD-binding receptor found to be involved in platelet binding, although its blockade by
the mAb LM609 did not reduce platelet adhesion to the
level observed with RGD peptides. Similarly, two previous
reports have shown that platelet adhesion to activated endothelial cells could be partly inhibited by the same anti-
v
3 mAb (11, 12). Because
v
3 integrin binds different
adhesive proteins (39), it is likely that platelets bind to endothelial
v
3 integrin by forming bridges with fibrinogen,
fibronectin, and vWF.
Besides the binding site for 2 integrins, endothelial
ICAM-1 has recently been shown to contain an epitope for
the binding of fibrinogen that mediates the binding of leukocytes (22, 40). We now present evidence that a component of platelet binding to HUVEC uses this mechanism.
Treatment of HUVEC with the mAb 2D5, which blocks
the fibrinogen-binding site of ICAM-1, decreased binding
of activated platelets by ~50%. The fact that the other anti-ICAM-1 mAbs, that recognize either the epitope for
2 integrins (R6.5 and 6E6) or rhinoviruses (VF27) also decreased platelet binding suggests some cross-reactivity with
the fibrinogen-binding region. This region has recently
been localized within the first Ig domain of ICAM-1 (41),
which is also involved in the
L
2-dependent binding of
leukocytes (42) and rhinoviruses (23).
Besides v
3 integrin and ICAM-1, we additionally found
evidence for involvement of endothelial GPIb
in the
binding of activated platelets to HUVEC monolayers.
Platelet adhesion could be blocked significantly by treating
HUVEC with the anti-GPIb
mAb SZ2. Furthermore, addition of exogenous vWF increased platelet binding only
in the presence of ristocetin, which is known to allow soluble vWF to interact with GPIb
. Also, this vWF/ristocetin-dependent binding of activated platelets to HUVEC
could be partially inhibited with the mAb SZ2. However,
the mAb 2E4, another blocking anti-GPIb
mAb, did not
exhibit a significant inhibitory effect, raising some question
as to the significance of endothelial GPIb
in platelet binding. Indeed, it has been debated whether HUVEC express
GPIb
. Whereas one study has recently questioned the
synthesis of GPIb
in HUVEC (43), other investigators
have found that GPIb
on HUVEC participated in different functions, including binding to vWF, binding of sickle
erythrocytes, and homotypic binding of HUVEC in the
presence of vWF and ristocetin (44). We found that unstimulated HUVEC expressed GPIb
at a modest level
when measured by flow cytometry with the mAb SZ2 and
2E4 (data not shown). Thus, based on our data, we conclude that platelet-exposed vWF may bind not only to
v
3 integrin but, in part, also to GPIb
. The potential relevance of this observation is enhanced by a recent study
showing that human endothelial cells also express GPIb
in
vivo (48).
Although activated platelet adhesion to endothelial cells
was demonstrated some 20 years ago (2), the involvement
of either surface-bound or exposed intercellular matrix proteins has not been conclusively evaluated. We believe that
our studies measuring platelet binding to HUVEC monolayers by flow cytometry convincingly demonstrate that activated platelets can bind directly to endothelial cells rather
than to exposed intercellular matrix proteins. Moreover, we present evidence that endothelial cell surface-associated
matrix proteins are likewise not involved in the binding of
activated platelets. Although suspended HUVEC were found
to express vWF, collagen IV, and fibronectin, platelet adhesion to HUVEC monolayers was not inhibited by blocking
mAbs to these proteins. HUVEC-associated vWF and fibronectin participated in platelet binding only when HUVEC
were detached and suspended before the addition of platelets. Furthermore, we found that activated platelet adhesion
to HUVEC matrix (or to HUVEC in suspension) was mediated by platelet 1 integrins, whereas binding to confluent HUVEC monolayers was dependent upon GPIIbIIIa.
This participation of different platelet receptors in binding
to matrix versus cells further confirms that HUVEC-associated matrix proteins were not significantly involved in activated platelet binding to HUVEC monolayers.
It is notable that platelet adhesion was not affected by blockade of platelet P-selectin, a major pathway for platelet binding to leukocytes and tumor cells (49, 50). Both anti- P-selectin mAbs G1 and WAPS12.2 have previously been shown to block platelet adhesion and rolling, respectively (6, 24). Similarly, neuraminidase-induced degradation of endothelial sialylated glycoproteins that may be potential endothelial ligands for platelet P-selectin also did not alter platelet binding. Although in our assays HUVEC were not stimulated by any exogenous agonists, platelet-induced stimulation of HUVEC could not be excluded. Thus, we additionally tested the contribution of activation-dependent adhesion molecules, including P-selectin, E-selectin, and VCAM-1. However, none of these proteins participated in the binding mechanism. This may be due, in part, to the brief incubation with platelets (30 min), which may have been too short to induce significant upregulation of E-selectin and VCAM-1. Finally, PECAM-1 has recently been proposed to participate in platelet binding to endothelium (13). However, treatment of platelets and HUVEC with two different anti-PECAM-1 mAbs produced only minimal inhibition that was not significant.
As summarized in Fig. 9, our data provide evidence that,
similar to platelet aggregation, activated platelet adhesion to
HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism, involving platelet-bound fibrinogen, fibronectin, and vWF. Consequently, administration
of GPIIbIIIa-blocking drugs may inhibit not only the formation of platelet aggregates, but also their adhesion to the
intact endothelium. This effect might be important, particularly in the microvasculature, as it has recently been shown
in an in vivo thrombosis model using the anti-GPIIbIIIa
mAb 7E3 (51). Furthermore, the involvement of the endothelial cell receptors ICAM-1, v
3 integrin, and GPIb
in the binding of activated platelets suggests that blockade
of these receptors may have antithrombotic effects. Finally,
because these endothelial adhesion molecules are known to
become upregulated by various exogenous stimuli, endothelial cell activation may further increase binding of activated platelets and thus contribute to a prothrombotic state.
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Footnotes |
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Address correspondence to John M. Harlan, MD., Division of Hematology, Box 357710, 1959 Pacific Street NE, University of Washington, Seattle, WA, 98195-7710. Phone: 206-685-7866; Fax: 206-685-3062; E-mail: jharlan{at}u.washington.edu
Received for publication 3 June 1997 and in revised form 22 October 1997.
1 Abbreviations used in this paper: HUVEC, human umbilical vein endothelial cells; ICAM, intercellular adhesion molecule; PECAM, platelet endothelial cell adhesion molecule; RGD, Arg-Gly-Asp; TEM, transmission electron microscopy; TSP, thrombospondin; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.This work was supported by the United States Public Health Service Grants HL 18645 and 30541. T. Bombeli was supported by the Swiss Foundation for Medical and Biological Grants and the Anniversary Foundation of Swiss Life for Public Health and Medicinal Research, Switzerland.
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References |
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---|
1. | Abrams, C., and S.J. Shattil. 1991. Immunological detection of activated platelets in clinical disorders. Thromb. Haemostasis 65: 467-473 [Medline]. |
2. | Czervionke, R.L., J.C. Hoak, and G.L. Fry. 1978. Effect of aspirin on thrombin-induced adherence of platelets to cultured cells from the blood vessel wall. J. Clin. Invest 62: 847-856 [Medline]. |
3. | Fry, G.L., R.L. Czervionke, J.C. Hoak, J.B. Smith, and D.L. Haycraft. 1980. Platelet adherence to cultured vascular cells: influence of prostacyclin (PGI2). Blood. 55: 271-275 [Medline]. |
4. |
Kaplan, J.E.,
D.G. Moon,
L.K. Weston,
F.L. Minnear,
P.J. Del Vecchio,
J.M. Shepard, and
J.W. Fenton.
1989.
Platelets adhere to thrombin-treated endothelial cells in vitro.
Am. J. Physiol.
257:
H423-H433
|
5. | Tloti, M.A., D.G. Moon, L.K. Weston, and J.E. Kaplan. 1991. Effect of 13-hydroxy octadeca-9,11-dienoic acid (13-HODE) on thrombin induced platelet adherence to endothelial cells in vitro. Thromb. Res 62: 305-317 [Medline]. |
6. | Diacovo, T.G., K.D. Puri, R.A. Warnock, T.A. Springer, and U.H. von Andrian. 1996. Platelet-mediated lymphocyte delivery to high endothelial venules. Science. 273: 252-255 [Abstract]. |
7. | Li, J.M., R.S. Podolsky, M.J. Rohrer, B.S. Cutler, M.T. Massie, M.R. Barnard, and A.D. Michelson. 1996. Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa. J. Surg. Res 61: 543-548 [Medline]. |
8. | Manduteanu, I., M. Calb, C. Lupu, N. Simionescu, and M. Simionescu. 1992. Increased adhesion of human diabetic platelets to cultured valvular endothelial cells. J. Submicrosc. Cytol. Pathol 24: 539-547 [Medline]. |
9. | Frenette, P.S., R.C. Johnson, R.O. Hynes, and D.D. Wagner. 1995. Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. Proc. Natl. Acad. Sci. USA. 92: 7450-7454 [Abstract]. |
10. | Etingin, O.R., R.L. Silverstein, and D.P. Hajjar. 1993. Von Willebrand factor mediates platelet adhesion to virally infected endothelial cells. Proc. Natl. Acad. Sci. USA. 90: 5153-5156 [Abstract]. |
11. | Buchanan, M.R., M.C. Bertomeu, T.A. Haas, F.W. Orr, and L.L. Eltringham-Smith. 1993. Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro. Blood. 81: 3303-3312 [Abstract]. |
12. | Bertomeu, M.C., S. Gallo, D. Lauri, M.N. Levine, F.W. Orr, and M.R. Buchanan. 1990. Chemotherapy enhances endothelial cell reactivity to platelets. Clin. Expl. Metastasis. 8: 511-518 [Medline]. |
13. |
Rosenblum, W.I.,
G.H. Nelson,
B. Wormley,
P. Werner,
J. Wang, and
C.C.Y. Shih.
1996.
Role of platelet-endothelial
cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo
and ex vivo.
Stroke.
27:
709-711
|
14. | Jaffe, E.A., R.L. Nachman, C.G. Becker, and C.R. Minick. 1973. Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest 52: 2745-2756 [Medline]. |
15. | Maciag, T., J. Cerundolo, S. Ilsley, P.R. Kelley, and R. Forand. 1979. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc. Natl. Acad. Sci. USA 76: 5674-5678 [Abstract]. |
16. | Baenziger, N.L., and P.W. Majerus. 1974. Isolation of human platelets and platelet surface membranes. Methods Enzymol 31: 149-155 [Medline]. |
17. |
Carter, W.C.,
E.A. Wayner,
T.S. Bouchard, and
P. Kaur.
1990.
The role of integrins ![]() ![]() ![]() ![]() |
18. | Cheresh, D.A.. 1987. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc. Natl. Acad. Sci. USA. 84: 6471-6475 [Abstract]. |
19. | McGregor, J.L., J. Brochier, F. Wild, G. Follea, M.C. Trzeciak, E. James, M. Dechavanne, L. McGregor, and K. Clemetson. 1983. Monoclonal antibodies against platelet membrane glycoproteins. Eur. J. Biochem 131: 427-436 [Abstract]. |
20. | Ruan, C., X. Du, X. Xi, P.A. Castaldi, and M.C. Berndt. 1987. A murine antiglycoprotein Ib complex monoclonal antibody SZ 2 inhibits platelet aggregation induced by both ristocetin and collagen. Blood. 74: 570-577 . |
21. | Smith, C.W., R. Rothlein, B.J. Hughes, M.M. Mariscalco, H.E. Rudloff, F.C. Schmalstieg, and D.C. Anderson. 1988. Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration. J. Clin. Invest 82: 1746-1756 [Medline]. |
22. | Languino, L.R., A. Duperray, K.J. Joganic, M. Fornaro, G.B. Thornton, and D.C. Altieri. 1995. Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc. Natl. Acad. Sci. USA. 92: 1505-1509 [Abstract]. |
23. | Staunton, D.E., V.J. Merluzzi, R. Rothlein, R. Barton, S.D. Marlin, and T.A. Springer. 1989. A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell. 56: 849-853 [Medline]. |
24. | Hamburger, S.A., and R.P. McEver. 1990. GMP-140 mediates adhesion of stimulated platelets to neutrophils. Blood. 75: 550-554 [Abstract]. |
25. |
Kyan-Aung, U.,
D.O. Haskard,
R.N. Poston,
M.H. Thornhill, and
T.H. Lee.
1991.
Endothelial leukocyte adhesion
molecule-1 and intercellular adhesion molecule-1 mediate
the adhesion of eosinophils to endothelial cells in vitro and
are expressed by endothelium in allergic cutaneous inflammation in vivo.
J. Immunol.
146:
521-528
|
26. | Bevilacqua, M.P., S. Stengelin, M.A. Gimbrone Jr., and B. Seed. 1989. Endothelial leukocyte adhesion molecule-1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science. 243: 1160-1165 [Medline]. |
27. |
Thornhill, M.H.,
S.M. Wellicome,
D.L. Mahiouz,
J.S. Lanchbury,
U. Kyan-Aung, and
D.O. Haskard.
1991.
Tumor necrosis factor combines with IL-4 or IFN-![]() |
28. | Thurlow, P.J., D.A. Kenneally, and J.M. Connellan. 1990. The role of fibronectin in platelet aggregation. Br. J. Haematol 75: 549-556 [Medline]. |
29. | Peerschke, E.I.. 1992. Adhesive protein expression on thrombin-stimulated platelets: time-dependent modulation of anti-fibrinogen, -fibronectin, and -von Willebrand factor antibody binding. Blood. 79: 948-953 [Abstract]. |
30. | Heyns, A.P., A. Eldor, R. Yarom, and G. Marx. 1985. Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis. Blood. 66: 213-219 [Abstract]. |
31. | Azuma, H., T. Shigekiyo, S. Miura, Y. Uno, and S. Saito. 1989. Mechanism of potentiation by manganese ion of aggregation of porcine pancreatic elastase-treated human platelets. Thromb. Haemostasis. 62: 984-988 [Medline]. |
32. | Rinder, H.M., J.L. Bonan, C.S. Rinder, K.A. Ault, and B.R. Smith. 1991. Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood. 78: 1760-1769 [Abstract]. |
33. | Spangenberg, P., H. Redlich, I. Bergmann, W. Losche, M. Gotzrath, and B. Kehrel. 1993. The platelet glycoprotein IIb/IIIa complex is involved in the adhesion of activated platelets to leukocytes. Thromb. Haemostasis. 70: 514-521 [Medline]. |
34. | Spangenberg, P.. 1994. Adhesion of activated platelets to polymorphonuclear leukocytes. Thromb. Res 74: S35-S44 [Medline]. |
35. | Nierodzik, M.L., A. Plotkin, F. Kajumo, and S. Karpatkin. 1991. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J. Clin. Invest 87: 229-236 [Medline]. |
36. | DeMarco, L., A. Girolami, T.S. Zimmerman, and Z.M. Ruggeri. 1986. von Willebrand factor interaction with the glycoprotein IIb/IIIa complex: its role in platelet function as demonstrated in patients with congenital afibrinogenemia. J. Clin. Invest 77: 1272-1277 [Medline]. |
37. | Kieffer, N., A.T. Nurden, M. Hasitz, M. Titeux, and J. Breton-Gorius. 1988. Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody. Biochim. Biophys. Acta. 967: 408-415 [Medline]. |
38. |
Suehiro, K.,
J. Gailit, and
E.F. Plow.
1997.
Fibrinogen is a
ligand for integrin ![]() ![]() |
39. | Cox, D., T. Aoki, J. Seki, Y. Motoyama, and K. Yoshida. 1994. The pharmacology of the integrins. Med. Res. Reviews. 14: 195-228 . |
40. | Languino, L.R., J. Plescia, A. Duperray, A.A. Brian, E.F. Plow, J.E. Geltosky, and D.C. Altieri. 1993. Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway. Cell. 73: 1423-1434 [Medline]. |
41. |
D'Souza, S.E.,
V.J. Byers-Ward,
E.E. Gardiner,
H. Wang, and
S.S. Sung.
1996.
Identification of an active sequence
within the first immunoglobulin domain of intercellular cell
adhesion molecule-1 (ICAM-1) that interacts with fibrinogen.
J. Biol. Chem
271:
24270-24277
|
42. | Staunton, D.E., M.L. Dustin, H.P. Erickson, and T.A. Springer. 1990. The arrangement of immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinoviruses. Cell. 61: 243-254 [Medline]. |
43. |
Perrault, C.,
H. Lankhof,
D. Pidard,
D. Kerbiriou-Nabias,
J.J. Sixma,
D. Meyer, and
D. Baruch.
1997.
Relative importance of the glycoprotein Ib-binding domain and the RGD
sequence of von Willebrand factor for its interaction with endothelial cells.
Blood.
90:
2335-2344
|
44. | Beacham, D.A., M.A. Cruz, and R.I. Handin. 1995. Glycoprotein Ib can mediate endothelial cell attachment to a von Willebrand factor substratum. Thromb. Haemostasis 73: 309-317 [Medline]. |
45. |
Beacham, D.A.,
L.P. Tran, and
S.S. Shapiro.
1997.
Cytokine treatment of endothelial cells increases glycoprotein Ib![]() |
46. | Wick, T.M., J.L. Moake, M.M. Udden, and L.V. McIntire. 1993. Unusually large von Willebrand factor multimers preferentially promote young sickle and nonsickle erythrocyte adhesion to endothelial cells. Am. J. Hematol 42: 284-292 [Medline]. |
47. | Asch, A.S., B. Adelman, M. Fujimoto, and R.L. Nachman. 1988. Identification and isolation of a platelet GPIb-like protein in human umbilical vein endothelial cells and bovine aortic smooth muscle cells. J. Clin. Invest 81: 1600-1607 [Medline]. |
48. |
Wu, G.,
D.W. Essex,
F.J. Meloni,
T. Takafuta,
K. Fujimura,
B.A. Konkle, and
S.S. Shapiro.
1997.
Human endothelial
cells in culture and in vivo express on their surface all four
components of the glycoprotein Ib/IX/V complex.
Blood.
90:
2660-2669
|
49. | Larsen, E., A. Celi, G.E. Gilbert, B.C. Furie, J.K. Erban, R. Bonfanti, D.D. Wagner, and B. Furie. 1989. PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell. 59: 305-312 [Medline]. |
50. | Stone, J.P., and D.D. Wagner. 1993. P-selectin mediates adhesion of platelets to neuroblastoma and small cell lung cancer. J. Clin. Invest 92: 804-813 [Medline]. |
51. |
Taylor, F.B.,
B.S. Coller,
A.C. Chang,
G. Peer,
R. Jordan,
W. Engellener, and
C.T. Esmon.
1997.
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