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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Mario Negri Institute for Pharmacological
Research, Bergamo, Italy, and the Division of Nephrology and Dialysis,
Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy.
Verotoxin-1 (VT-1)-producing Escherichia coli is
the causative agent of postdiarrheal hemolytic uremic
syndrome (D+HUS) of children, which leads to renal and other organ
microvascular thrombosis. Why thrombi form only on arterioles and
capillaries is not known. This study investigated whether VT-1
directly affected endothelial antithrombogenic properties promoting
platelet deposition and thrombus formation on human microvascular
endothelial cell line (HMEC-1) under high shear stress. Human umbilical
vein endothelial cells (HUVECs) were used for comparison as a
large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused
at 60 dynes/cm2 with heparinized human blood prelabeled
with mepacrine. Results showed that VT-1 significantly increased
platelet adhesion and thrombus formation on HMEC-1 in comparison with
unstimulated control cells. An increase in thrombus formation was also
observed on HUVECs exposed to VT-1, but to a remarkably lower extent.
The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor
(20-fold more) as documented by FACS analysis. The HMEC-1 line had a
comparable susceptibility to the thrombogenic effect of VT-1 as primary
human microvascular cells of the same dermal origin (HDMECs). The
adhesive molecules involved in VT-induced thrombus formation were also
studied. Blocking the binding of von Willebrand factor to platelet
glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of
platelet Verotoxin (VT)-producing Escherichia
coli infection has been strongly implicated as the causative agent
for most cases of postdiarrheal hemolytic uremic syndrome (D+HUS), a
disorder of microangiopathic hemolytic anemia, thrombocytopenia, and
acute renal failure that mainly affects infants and small
children.1-3 The characteristic lesion, thrombotic
microangiopathy, consists of swelling and detachment of endothelial
cells from the basement membrane and deposition of platelet thrombi
that occlude the microcirculation of the kidneys and other
organs.4 Why thrombi form only in arterioles and
capillaries is not known.
It is now clear that endothelial dysfunction plays a crucial role in
the sequence of events leading to the microangiopathic processes, and
evidence points to VT-1 and VT-2 as critical determinants for the
development of vascular lesions. Verotoxins (also called Shiga toxins)
are formed by a biologically active A subunit and a number of B
subunits by which the toxin binds to a specific glycosphingolipid
globotriaosyl ceramide (Gb3) receptor on the endothelial
surface.2,5 After binding, the toxin penetrates the
cytosol by endocytosis and exerts its cytotoxic effect by inhibiting
protein synthesis and causing cell death.6-8
Monocytes/macrophages in response to VT release cytokines such as
interleukin-1 (IL-1) and tumor necrosis factor (TNF) that remarkably
potentiate sensitivity of vascular endothelial cells to VT by
up-regulating endothelial Gb3 receptor.9 Evidence also
shows that renal microvascular endothelial cells have a higher
sensitivity to the cytotoxic effect of VT as compared to endothelial
cells derived from large vessels.7,8
The interaction between leukocytes and endothelial cells is
instrumental in the development of microvascular injury in
VT-associated HUS. Thus, evidence suggests that neutrophils isolated
from children in the acute phase of D+HUS adhered to endothelial cells
in culture more than normal neutrophils and induced endothelial injury
by local release of proteases.10 We have demonstrated in
vitro that VT-1 directly induced a massive leukocyte adhesion to
cultured endothelial cells under flow conditions, by up-regulating the adhesive proteins E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1
(VCAM-1).11 Furthermore, a preliminary report has shown
that glomerular endothelial cells exposed to VT became more susceptible
to neutrophil-mediated oxidant injury.12 Taken together
these studies indicate that VT causes cell injury by altering cell
adhesive properties and by increasing endothelial susceptibility to
leukocyte-mediated injury. The resulting injured endothelium changes
its normal thromboresistant phenotype and becomes thrombogenic,
initiating microvascular thrombus formation.
In HUS, structural damage of microvessels associated with narrowing of
the lumina determines major changes in fluid shear stress, which would
favor persistent endothelial damage, platelet activation, and
progression of microvascular thrombosis.13 Changes in
shear stress, the tractive force produced by blood flowing over the
endothelial surface, have a profound influence on von Willebrand factor
(vWF) handling by enhancing its susceptibility to proteolytic
cleavage.14 Under conditions of high shear stress, vWF
undergoes conformational changes and serves to bridge the subendothelial matrix to glycoprotein (GP) Ib expressed on platelet membranes.15 The engagement of this receptor
promotes activation of the platelet Several distinct endothelial cell molecules have been reported to be
involved in the binding of platelets to endothelial cells. P-selectin,
which is stored in intracellular granules of platelets and endothelial
cells together with vWF and which rapidly mobilizes to the cell surface
on stimulation,20 is required for platelet rolling and
adhesion on activated endothelium.21 Increased plasma levels of P-selectin have been measured in patients with HUS, possibly
reflecting activation/damage of platelets and endothelial cells.22 Evidence also indicates that platelet-endothelial
cell adhesion molecule-1 (PECAM-1) expressed on endothelial
cells23 contributes to platelet adhesion and aggregation at
sites of injured endothelium, as documented by the finding that
anti-PECAM-1 antibody injection delayed thrombus formation in
laser-induced microvessel injury of mouse brain.24
In the present study, we sought to (1) assess whether VT-1 directly
affected the antithrombogenic properties of the endothelium under high
shear stress; (2) evaluate whether microvascular endothelium had a
higher sensitivity to VT-1-induced thrombus formation, as compared to
endothelium derived from large vessels; and (3) identify platelet and
endothelial cell adhesive proteins involved in the thrombotic process
promoted by VT-1.
Endothelial cell culture and incubation
Human umbilical vein endothelial cells (HUVECs) were obtained by
collagenase digestion according to the method of Jaffé and coworkers.27 The cells were grown in Medium 199 (Gibco)
supplemented with 10% newborn calf serum (Gibco) and 10% human serum,
5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic
acid (HEPES; Sigma Chemical, St Louis, MO), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL fungizone, 2 mM glutamine (Gibco), 15 U/mL heparin (Parke-Davis, Milan, Italy), and 50 µg/mL endothelial
cell growth factor. Cultured cells were identified as endothelial by
their cobblestone morphology and the presence of vWF, using indirect
immunofluorescence microscopy. Confluent HUVECs were used for
experiments between the first and fifth passage.
Primary human microvascular endothelial cells of dermal origin (HDMECs)
(Promocell, Heidelberg, Germany) were cultured in endothelial cell
growth medium MV plus SupplementMix (Promocell). HDMECs were used
between the second and sixth passage.
For the experiments, endothelial cells were plated on 60 × 20-mm
plastic coverslips (Thermanox; Nunc, Naperville, IL) and used 1 day
after reaching confluence.
To study the effect of VT-1 in inducing platelet adhesion and thrombus
formation, HMEC-1 and HUVECs were pre-exposed for 24 hours in static
condition to 2, 10, and 50 pM VT-1 (kindly provided by Dr M. A. Karmali, Hospital for Sick Children, Toronto, ON, Canada; endotoxin
content <0.05 EU/mL using Limulus amoebocyte lysate assay) in medium
plus 2% fetal calf serum (Hyclone Laboratories, Logan, UT); then cells
were perfused at 60 dynes/cm2 in a parallel plate flow
chamber with human blood. Blood was drawn from an antecubital vein
through a 19-gauge needle (infusion set) directly into a polypropylene
tube and prelabeled for 5 minutes with fluorescent dye mepacrine (10 µM, quinacrine dihydrochloride BP; Sigma Chemical). Blood was
then transferred (5-mL aliquots) to test tubes and not disturbed until
assay. The percentage of the surface occupied by thrombi was calculated
by analysis of fluorescent thrombus images acquired by confocal microscopy.
The concentrations of VT-1 used for the adhesion experiments did not
affect cell count after 24 hours of incubation either in HMEC-1 (10 pM:
75 ± 0.5 × 104, 50 pM: 75 ± 5 × 104
versus control: 70 ± 5 × 104 cells) or in HUVECs (10 pM: 38.2 ± 1.1 × 104, 50 pM:
37.4 ± 0.7 × 104 versus control:
36.5 ± 0.5 × 104 cells).
To evaluate endothelial integrity, HMEC-1 pre-exposed for 24 hours to
VT-1 (10 pM) were perfused with blood without mepacrine and then fixed
with 0.5% glutaraldehyde (Fluka, Milan, Italy), dehydrated with methyl
alcohol, and stained with May-Grunwald Giemsa technique (Carlo Erba
Reagents, Milan, Italy).
By selected experiments we verified whether the HMEC-1 cell line
exhibited a similar sensitivity to VT-1 To compare the effect of VT-1 with respect to other thrombogenic
stimuli, HMEC-1 and HUVECs were exposed to thrombin (2 U/mL, 10 minutes; Biosciences, La Jolla, CA), TNF- To identify platelet receptors involved in VT-induced thrombus
formation, HMEC-1 treated for 24 hours with VT-1 (10 pM) were perfused
with human blood preincubated with inhibitors of GPIb and
To determine the endothelial adhesive proteins involved in VT-induced
thrombus formation, HMEC-1 pretreated with VT-1 (10 pM) for 24 hours
were incubated with chimeric 7E3 Fab (20 µg/mL) for 20 minutes, mouse
monoclonal antibody (mAb) antihuman vitronectin receptor LM609 (10 µg/mL; Chemicon International, Temecula, CA) for 10 minutes, mouse
mAb anti-GPIb The involvement of P-selectin and PECAM-1 in VT-1-induced thrombus
formation on HDMECs was also assessed, using the same experimental condition described above for HMEC-1.
Adhesion assay under flow conditions and fluorescence
confocal microscopy
Images of platelet thrombi on endothelial cell surface were acquired by a confocal inverted laser microscope (InSight plus; Meridian Instruments, Okemos, MI). An argon laser emission filter at 488 nm was used to excite specimens. Fifteen fields, systematically digitized along the adhesion surface, were acquired using a computer-based image analysis system. The area occupied by thrombi was evaluated by automatic edge detection using built-in specific functions of the software Image 1.61 (National Institutes of Health, Bethesda, MD), and expressed as µm2/field analyzed (total area: 474 473 µm2/field). Scanning electron microscopy For scanning electron microscopy analysis HMEC-1 grown on coverslips were treated with VT-1 (10 pM, 24 hours), perfused with blood, and then fixed overnight at 4°C with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. The slides were rinsed in 0.1 M sodium cacodylate buffer, osmicated for 1 hour, and then dehydrated in an ascending series of ethanol. The dehydration series concluded with 2 × 15-minute exchanges in 100% ethanol. After drying, the slides were coated with gold and examined in a scanning electron microscope (Stereoscan 200, Cambridge Instruments, Cambridge, MA).Flow cytometry analysis The surface expression of VT-1 receptor on HMEC-1 and HUVECs was evaluated by flow cytometry analysis (FACS; FACSort, Becton Dickinson). Endothelial cells in suspension were incubated for 45 minutes at room temperature with 1 µg/100 µL fluorescein isothiocyanate (FITC)-labeled albumin (Sigma Chemical) as control or 1 µg/100 µL FITC-labeled VT-1B subunit (a kind gift from Dr C. A. Lingwood, The Hospital for Sick Children, Toronto, ONT),33 washed 3 times, fixed with 2% paraformaldehyde, and assayed within 1 hour.Fluorescence confocal microscopy The HMEC-1 line and HDMECs grown on coverslips were incubated with medium alone or VT-1 (10 pM) for 24 hours and then fixed in 3% paraformaldehyde plus 2% sucrose in PBS, pH 7.4, for 15 minutes at room temperature. After 3 washings (5 minutes) with PBS plus 3% bovine serum to prevent nonspecific antibody binding, cells were treated with anti-P-selectin antibody (50 µg/mL) or antivitronectin receptor antibody LM609 (10 µg/mL). For PECAM-1 assessment, cells were permeabilized with Triton X-100 (0.1% in PBS; Sigma Chemical) for 4 minutes before incubation with anti-PECAM-1 antibody (10 µg/mL). Then cells were incubated with FITC-conjugated F(ab')2 goat antimouse IgG + IgM (Jackson Immunoresearch Laboratories, West Grove, PA).Negative control experiments with FITC-conjugated antibody alone were performed. Coverslips were washed, mounted in 1% N-propyl-gallate in 50% glycerol, 0.1 M Tris-HCl, pH 8, and examined under confocal inverted laser microscopy. Representative fields were digitized with millions of colors and printed. Statistical analysis Results are expressed as mean ± SE. Statistical analysis was performed using ANOVA followed by the Tukey test for multiple comparisons, as appropriate.34 Statistical significance was defined as P less than .05.
VT-1 promotes platelet adhesion and thrombus formation on endothelial cells We studied the effect of VT-1 on platelet adhesion and thrombus formation under laminar flow at high shear rate on HMEC-1. HUVECs were used for comparison as large-vessel endothelium. Heparinized blood was prelabeled with mepacrine and perfused at 60 dynes/cm2 over resting or VT-1-treated endothelial cells; then thrombus formation was quantified by analyzing images acquired by confocal microscopy. On resting HMEC-1 only limited platelet deposits were observed, usually less than 0.3% of the total perfused area, corresponding to about 1200 µm2/field analyzed. Exposure of HMEC-1 to VT-1 for 24 hours led to a significant (P < .01) increase in platelet adhesion and thrombus formation in comparison to control cells, with a maximum effect at 10 pM (2 pM: 7754 ± 1592; 10 pM: 10 090 ± 2246; 50 pM: 8244 ± 2874 versus control: 1077 ± 140 µm2 area covered by thrombi) (Figure 1).
In these experimental conditions the endothelial cell integrity was preserved as indicated by staining with May-Grunwald Giemsa of HMEC-1 treated with VT-1 (10 pM) and then perfused with blood. As shown in Figure 1, VT-1 also promoted platelet adhesion and thrombus formation on HUVECs but to a remarkably lower extent than on HMEC-1 (2 pM: 3406 ± 297; 10 pM: 3849 ± 540; 50 pM: 2497 ± 99 versus control: 1311 ± 263 µm2). Figure 2 depicts digitized images from a
representative experiment acquired by confocal fluorescent microscopy
showing an increase in the number of mepacrine-labeled thrombi on
HMEC-1 exposed to VT-1 in comparison to HUVECs.
Scanning electron microscopy evaluation of VT-treated HMEC-1
illustrates the attachment of platelets to the endothelial cell monolayer to form organized thrombi in which leukocytes at different stages of activation are entrapped (Figure
3).
In selected experiments, the behavior of the HMEC-1 cell line in response to the thrombogenic effect of VT-1 was compared with that of primary microvascular endothelial cells of similar dermal origin. VT-1 (10 pM) induced thrombus formation on the HMEC-1 line and primary HDMEC to a similar extent (HMEC-1: 11 831 ± 1303; HDMEC: 9226 ± 1979 µm2 area covered by thrombi). HUVECs used in these settings for comparison were significantly less susceptible to the effects of VT-1 (4061 ± 553 µm2, P < .01 versus HMEC-1 and P < .05 versus HDMECs). VT-1 is more thrombogenic than thrombin and cytokines We compared the capability of VT-1 to induce thrombus formation on HMEC-1 and HUVECs with other thrombogenic agonists like thrombin, TNF- , and IL-1 . As shown in Figure
4, thrombin and cytokines were less
effective in promoting platelet deposition than VT-1 in both
endothelial cell types. The superior thrombogenic effect of VT-1
translated in larger thrombus size with respect to thrombin, TNF-,
and IL-1, indicating a selective pattern of endothelial activation
implemented by VT-1 (mean area of thrombi, HMEC-1 + VT-1:
2584 ± 511 µm2; HMEC-1 + thrombin: 1140 ± 132 µm2; HMEC-1 + TNF-: 1204 ± 588
µm2; HMEC-1 + IL-1: 950 ± 362
µm2; and HUVEC + VT-1: 1748 ± 399
µm2; HUVEC + thrombin: 637 ± 128
µm2; HUVEC + TNF-: 427 ± 149
µm2; HUVEC + IL-1: 1406 ± 618
µm2).
VT-1 receptor expression on endothelial cells To investigate whether the higher sensitivity of HMEC-1 to VT-1 with respect to HUVECs was due to a different expression of VT-1 receptors, FACS studies were performed using fluorescent VT-1B subunit. Surface expression of VT-1 receptor, as a percentage of fluorescent cells, is depicted in Figure 5. The percentage of resting HMEC-1 stained with fluorescent VT-1B subunit was approximately 20-fold higher than that observed for HUVECs. FITC-albumin, used as control, bound to HMEC-1 and HUVECs at comparable extents (1% ± 0.08% and 0.9% ± 0.1% of fluorescent cells, respectively).
Effect of functional blockade of platelet and endothelial adhesive molecules on VT-1-induced thrombus formation in HMEC-1 To identify adhesive proteins involved in thrombus formation induced by VT-1 at high shear rate, we first evaluated the effect of blocking the interaction between vWF and platelet receptors GPIb andIIb3. As shown in Figure
6, ATA, which inhibits vWF-GPIb interaction, completely prevented platelet deposition and thrombus formation induced by VT-1 (10 pM) on HMEC-1 surface (VT-1 + ATA: 919 ± 256 µm2 versus VT-1: 9502 ± 1475
µm,2 P < .01). A similar significant (P < .01) reduction in the area occupied by thrombi was
observed by blocking the platelet receptor
IIb3 with the chimeric 7E3 Fab (VT-1 + 7E3: 952 ± 278 µm2).
Considering that GPIb is also expressed on endothelial cells, we investigated the role of this receptor on VT-1-induced thrombus formation in HMEC-1. Functional blocking of GPIb with LJ-Ib1 mAb did not affect thrombus formation in response to VT-1 (VT-1 + anti-GPIb: 12 096 ± 2716 µm2 versus VT-1: 12 696 ± 1677 µm2), suggesting that in this experimental setting endothelial GPIb was not involved in vWF-induced thrombi at high shear stress. Because it is known that vWF, besides binding extracellular matrix
proteins, can interact with endothelial
To investigate endothelial adhesive proteins that can directly interact with platelet receptors, HMEC-1 preincubated for 24 hours with VT-1 were exposed to anti-P-selectin and anti-PECAM-1 antibodies. Anti-P-selectin antibody almost completely (P < .01) prevented platelet adhesion (VT-1 + anti-P selectin: 2386 ± 826 µm2). Blocking of PECAM-1 had a less pronounced but still significant (P < .05) inhibitory effect on VT-1-induced thrombus formation with the area covered by thrombi averaging 4553 ± 532 µm2 (Figure 7). Irrelevant antibody did not significantly modify VT-1-induced platelet deposition (percent of reduction in area covered by thrombi compared with VT-1 alone: 10 µg/mL, 2%; 25 µg/mL, 9%; and 50 µg/mL, 15%). The involvement of P-selectin and PECAM-1 in platelet deposition elicited by VT-1 was also confirmed on primary microvascular endothelial cells by using functional blocking antibodies that reduced by 79% ± 7% and 86% ± 4%, respectively, the area covered by thrombi (P < .01 versus VT-1). Endothelial adhesive proteins involved in VT-1-induced thrombus formation We characterized by confocal fluorescence microscopy the distribution on the endothelial surface of the adhesive molecules found in the above experiments to be implicated in the thrombotic process induced by VT-1. As shown in Figure 8A,B, HMEC-1 treated with VT-1 (10 pM, 24 hours) exhibited an increased expression of vitronectin receptor, as small diffuse granules on the luminal surface, in comparison to unstimulated cells.
The HMEC-1 cell line in a resting condition did not stain for P-selectin on the apical surface (Figure 8C). In contrast, on VT-1 challenge a strong fluorescence was observed with the P-selectin staining pattern of granules distributed on the apical side (Figure 8D). PECAM-1 localized to the cell-cell border of adjacent unstimulated HMEC-1 as a linear staining (Figure 8E). After treatment with VT-1 PECAM-1 redistributed away from intercellular junctions and formed irregular patches of staining along the periphery of the cell or diffuse granules on the luminal surface and/or at the intracellular level (Figure 8F). By studying the effect of VT-1 on the expression of these adhesive
proteins in primary HDMECs we observed a distribution similar to that
of the HMEC-1 line (Figure 9A-F).
Verotoxin-producing E coli, the causative agent of D+HUS, activates endothelial cells to acquire a prothrombotic phenotype with corresponding lesions confined to microvessels mostly of renal glomeruli.1-4 In this report we show for the first time that VT-1 directly induces platelet adhesion and thrombus formation on cultured endothelial cells perfused with whole blood in a flow chamber system under shear stress levels high enough to mimic the ones encountered in the microcirculation. The effect of VT-1 was superior to that of other known thrombogenic agonists such as thrombin and cytokines. The area occupied by thrombi was more pronounced on VT-1-treated endothelial cells of microvascular (HMEC-1) in comparison with large-vessel (HUVEC) origin. The HMEC-1 line had a similar sensitivity to the thrombogenic effect of VT-1 as HDMECs. That microvascular endothelium is indeed more susceptible to the prothrombotic activity of VT-1 is consistent with previous findings. Thus, renal microvascular endothelial cell viability, as well as their protein synthesis capacity, were reduced by VT concentrations that were instead not cytotoxic for HUVECs.7 Basal Gb3 levels in renal microvascular endothelial cells were 50-fold higher than in HUVECs, which suggested a relationship between the degree of VT sensitivity and the amount of Gb3 receptor expressed by these cells.7 Similar to renal microvascular endothelial cells, we found that HMEC-1 expressed about 20-fold more VT-1 receptors than HUVECs, which might account for the different sensitivity of different vascular beds to VT-mediated disease. In the attempt to identify the adhesive proteins involved in
platelet-endothelial cell interactions elicited by VT-1, which eventually resulted in thrombus formation on HMEC-1, we first focused
on vWF, which is the indispensable adhesive substrate to promote
platelet thrombus formation in high shear stress
environments.15 We found that ATA, an inhibitor of
vWF-platelet GPIb interaction, completely prevented the deposition of
thrombi. Furthermore, blockade of It has been widely described that interaction of vWF with platelet
GPIb/ Our finding that treatment of endothelial cells with anti-GPIb antibody did not inhibit thrombus formation induced by VT-1 suggested that GPIb expressed on endothelium is not engaged in the interaction with soluble vWF in a high shear stress environment. The endothelial adhesive molecules P-selectin20 and PECAM-123 have been involved in the process of platelet deposition on activated or damaged endothelium by their direct binding to platelets.21,24,37 Thus, it has been shown by intravital fluorescence microscopy that ischemia/reperfusion injury caused overexpression of P-selectin on intestinal microvascular endothelial cells, in association with platelet rolling and adhesion.37 Antibodies against P-selectin significantly reduced microvascular thrombosis, which implied a direct role of this endothelial adhesive molecule in platelet deposition under flow conditions.37 We have found that inhibition of P-selectin with a specific antibody caused a significant decrease in VT-induced thrombus formation on HMEC-1. These data, along with our observation of a strong expression of P-selectin on the apical surface of HMEC-1 after VT-1 challenge, provide evidence for the involvement of P-selectin in the thrombotic process elicited by VT-1 at high shear stress. As for endothelial PECAM-1, its contribution to platelet deposition was proved in a model of laser-induced endothelial injury in mouse brain arterioles by the observation that anti-PECAM-1 antibody reduced microvascular thrombosis over damaged but not denuded endothelium.24 Our present study showed that functional blocking of PECAM-1 resulted in a significant reduction of the area covered by thrombi in HMEC-1 exposed to VT-1. In addition, confocal microscopy experiments revealed that VT-1 induced a redistribution of this protein away from cell junctions, a pattern similar to that described in human endothelial cells after cytokine stimulation.38 We speculate that PECAM-1 once redistributed on the endothelial surface may undergo phosphorylation,23 which would render this adhesive receptor available for platelet interaction. In primary microvascular endothelial cells treated with VT-1, expression of vitronectin receptor, P-selectin, and PECAM-1 was similar to that observed in the HMEC-1 line. Moreover, as in HMEC-1, blockade of P-selectin and PECAM-1 by specific antibodies markedly limited VT-1-induced thrombus formation, thus suggesting that thrombotic response elicited by VT-1 involved activation of the same endothelial adhesive proteins in both line and primary microvascular endothelial cells. In conclusion, our results indicate for the first time that (1) VT-1 is
a potent promoter of platelet adhesion and thrombus formation on
endothelial cells under high shear stress; (2) microvascular endothelial cells demonstrate a remarkably greater sensitivity to the
thrombogenic effect of VT-1 than endothelium derived from large
vessels, possibly due to the higher expression of VT-1 receptor; (3) at
high shear stress interaction of vWF with platelet
GPIb/ These findings might help to clarify why thrombi in HUS preferentially localize in microvessels and provide insights on the determinants possibly involved in the process of microvascular thrombosis associated with D+HUS.
We are indebted to Prof Giuseppe Silva and Piero Pellini for helpful cooperation during scanning electron microscopy evaluations (Politecnico di Milano, Italy). We thank Dr Anna Falanga (Unit of Hematology, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Italy) for kind cooperation, and Stefania Angioletti, Chiara Rossi, and Federica Casiraghi for technical assistance.
Submitted May 2, 2000; accepted May 15, 2001.
E.B. is a recipient of a fellowship from "Foppolo aiuta i bambini."
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marina Morigi, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy; e-mail: morigi{at}marionegri.it.
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Role of integrin |
Top
Abstract
Introduction
as for the VT effect to induce
thrombus formation
