Keywords
junctional adhesion molecule-A (F11R/JAM-A) - platelets - adhesion - thrombosis
Introduction
F11Receptor/Junctional adhesion molecule-A (F11R/JAM-A) is a transmembrane protein
which belongs to the immunoglobulin superfamily (IgSF).[1]
[2] The protein was first discovered in blood platelets as a target of an activating
F11 antibody.[3] Shortly after, the same protein was identified as a functional component of tight
junctions in endothelium and in epithelium.[1] Later, its expression was also detected in immune cells[4] and in smooth muscle cells.[5]
The molecule of F11R/JAM-A is composed of an extracellular part, which contains two
Ig-like domains, a single-pass transmembrane domain and a short cytoplasmic tail with
a PDZ domain-binding motif.[6]
[7] The extracellular part contains two Ig-like domains, a membrane distal D1 domain
and membrane proximal D2 domain. F11R/JAM-A molecules are capable of forming homodimers:
in cis-configuration when the dimer is formed by the molecules located in the same
cell and in trans-configuration when the interacting molecules are located on membranes
of adjacent cells.[8] Both cis- and trans-homophilic interactions are mediated by D1 domain, but the sites
responsible for these two types of interactions are located in distinct parts of the
molecule, as it was described in detail by Monteiro et al.[8] The only known heterodimeric ligand of F11R/JAM-A is the lymphocyte function-associated
antigen (LFA-1) - a leukocyte integrin involved in adhesion and extravasation of lymphocytes.
The LFA-1 interacts with JAM-A/F11R via a site located in the proximal D2 domain.[9]
A majority of knowledge of the function of F11R/JAM-A comes from epithelial cells,
endothelial cells, and lymphocytes, while its role in regulation of blood platelets'
activity is still not fully explained, even though the protein was primarily discovered
in these elements of blood.[3]
[10] As a component of tight junctions in endothelial and epithelial cells, the molecules
of F11R/JAM-A are assembled as cis-homodimers and interact via trans-homodimerisation
with cis-homodimers on adjacent cells.[1] The intracellular part of the protein integrates tight junctions with the actin
filaments. Importantly, upon inflammatory activation of endothelial cell, the protein
translocates from tight junctions to the apical side of the cell and thus to the luminal
surface of the blood vessel facilitating the adhesion of monocytes and platelets to
the inflamed vascular wall.[11]
[12]
In blood platelets F11R/JAM-A is associated with αIIbβ3–platelet most dominant integrin.[13] This association, however, is limited to a resting platelet, as platelet activation
results in dissociation of the protein from the complex.[14] Murine platelets devoid of the F11R/JAM-A protein not only maintained their ability
to aggregate and to form thrombi but were more reactive than their wild-type counterparts.
The effect was dependent on Csk kinase.[14]
[15] This demonstrated that F11R/JAM-A, at least in murine platelets, is not essential
to the process of thrombus formation. The activity of platelet F11R/JAM-A, however,
is not limited to the regulatory role, as it was shown to form trans-homophilic interactions
with its counterpart immobilised on the surface of activated endothelial cells in
static conditions.[11]
[16] Clusters of F11R/JAM-A on contacts between thrombin-activated platelets were identified
by electron microscopy.[17] What is more, it was evidenced that a soluble form of F11R/JAM-A increased platelets'
ability to form aggregates, which implied that trans-homophilic interactions of this
protein play a role during thrombus formation.[18]
As can be inferred from the above-mentioned studies, one piece of evidence shows that
F11R/JAM-A is not indispensable to thrombus formation, whereas others suggest that
trans-homophilic interactions of this protein may occur between platelets. Therefore,
it can be speculated that trans-homophilic interactions of F11R/JAM-A play some as
of yet undefined role during thrombus formation.
Recent reports pointed to a potential role of platelet F11R/JAM-A in thromboinflammation[18] and drew attention to this new–old player in the process of thrombus formation;
hence, we decided to address two specific questions. One of the possible activities
of F11R/JAM-A addressed in presented study is the contribution of trans-homophilic
interactions of F11R/JAM-A to immobilisation of flowing platelets. Our previous study
showed that functional blockade of F11R/JAM-A limited the interaction of flowing platelets
with the inflamed vascular wall in vivo.[19] However, the exact contribution of F11R/JAM-A to this process was not elucidated.
The second question we addressed was whether the functional blockade of the protein
could affect thrombus formation in vitro and in vivo.
Materials and Methods
Chemicals
The list of chemicals has been included in the [Supplementary Material S1] (available in the online version).
Experimental Animals
Mice were housed and bred at the animal facility of the Medical University of Lodz.
Food and water were provided ad libitum and mice were housed in groups of up to six
animals per cage, under a 12:12 light–dark cycle. Male C57BL/6JRj mice (Janvier Labs,
France) were used. Animals included in the experiments were young adults of 8 to 12
weeks, 20 to 24 g. All mouse experimental procedures were approved by the Local Ethical
Committee on Animal Experiments, Medical University of Lodz, administrative decision
no 1/ŁB190/2021. Animals were randomly allocated to the treatment groups. The researcher
who performed the measurements was unaware of the type of treatment applied to an
animal.
Human Donors
Human blood was collected from healthy donors under the guidelines of the Helsinki
Declaration for human research and the study were approved by the Committee on the
Ethics of Research in Human Experimentation at Medical University of Lodz (RNN/323/20/KE).
Written informed consent, including detailed information regarding the study objectives,
study design, risks, and benefits, was obtained from each individual before blood
withdrawal. None of donors had taken aspirin or other drugs affecting platelet function
for at least 10 days prior to blood collection or had a history suggestive of underlying
haemostatic disorders. Blood was withdrawn to test tubes containing 0.105 mol/L sodium
citrate (citrate: blood volume ratio 1:9) or hirudin with a special caution to avoid
undesirable activation of circulating platelets. For the assessment of Src phosphorylation
in blood platelets apyrase was added at final concentration of 0.02 U/mL.
Flow Cytometry
Whole blood was incubated with activating factors for 5 minutes at room temperature
(RT) and fixed with CellFix (BD Bioscience, Franklin Lakes, New Jersey, United States)
at RT. The samples were then labelled with anti-CD41/PE antibodies, anti-JAM-A/FITC
(BioLegend, San Diego, California, United States) or J10.4/FITC (Santa Cruz Biotechnology,
Texas, United States) antibodies or IG1κ/FITC (BioLegend, San Diego, California, United States) as an isotype control in
final concentration of 15 µg/mL for 15 minutes at RT. Antibody titer was established
on the basis of separate experiments. The lowest saturating concentration was chosen,
i.e., when any higher concentrations did not increase the percentage of positive platelets
compared with isotype antibodies. Prior to the measurements, the samples were diluted
1:30 with phosphate-buffered saline (PBS), and the assay was performed by recording
10,000 CD41/PE-positive events using a FACS Canto II flow cytometer (BD Bioscience,
Franklin Lakes, New Jersey, United States). In the gated population of CD41/PE-positive
events, the percentage fractions of the platelets positive with regard to JAM-A (above
isotype cutoff) were measured.
Adhesion to Fibrinogen and F11R/JAM-A under Flow Conditions
Blood platelet adhesion was assessed with the use of VenaFlux platform (Celix, Dublin,
Ireland). Channels of Vena8 Endo+ biochip were coated with human fibrinogen (200 μg/mL)
overnight at 4°C and blocked with 0.1% bovine serum albumin (BSA) for 1 hour at 4°C.
Biochip was mounted on a thermo-controlled stage of an inverted AxioVert microscope
(Carl Zeiss, Oberkochen, Germany), heating plate maintained the constant temperature
of 37°C throughout the experiment. Prior to measurements, the channels were washed
with saline. In the experiments involving co-coating of the chips with F11R/JAM-A,
the chips were coated with 100 µg/mL Fbg and 100 µg/mL Fc-JAM-A. Blood samples were
recalcified with CaCl2 in a concentration of 1 mM shortly before measurement. Whole blood was perfused through
the channel at 40 dynes/cm2 (∼890 s−1) for 2 minutes. The channel was then perfused with the CellFix for 2 minutes at 5
dynes/cm2. Next, anti-CD41/PE antibodies were aspired to the channel, and incubated for 30 minutes.
The channel was then washed with 100 µL of saline to remove any unbound antibodies.
Images of labelled platelets were taken with AxioExaminer (Carl Zeiss, Oberkochen,
Germany) microscope using a 20× objective focusing on at least five different region
of interest (ROI). Area covered by platelets was quantified with the use of Fiji (ImageJ)
preceded by segmentation step performed with the use of Ilastik software.[20] Additional experiments were performed to quantify the number of platelets adhering
as a function of time. Perfusion conditions were as described above. Adhesion events
in real time were recorded. Movie sequences were analysed with the use of TrackMate
plugin for ImageJ.
Adhesion to Fibrin and F11R/JAM-A under Static Conditions
For the static adhesion study, the surface of Ibidi µ-Slide 15 wells (Ibidi, Martinsried,
Germany) was coated overnight with fibrinogen (37.5 μg/mL) at 4°C. After coating,
the excess of coating solution was removed, so the reaction with calcified (1 mM)
thrombin (1 U/mL over 15 minutes, 37°C) formed a flat net instead of a fully three-dimensional
mesh, which was critical to achieving a uniform plane of focus for platelet observation.
Surfaces were additionally treated with recombinant Fc-F11R /JAM-A (100 µg/mL) or
BSA (0.1%). Platelet-rich plasma was recalcified and incubated over the surfaces for
30 minutes. Next, the wells were washed with Tyrode solution and left to incubate
for an additional 30 minutes. Adhered platelets were fixed using CellFix solution,
permeabilized with Triton X-100 (0.1%), and stained with Phalloidin for 30 minutes
at RT, and excess staining was removed by washing with 100 µL of PBS.
Images were taken in five different fields of view in each well with the use of AxioExaminer
microscope (Carl Zeiss, Oberkochen, Germany). Ilastik software was used for machine
learning-based classification and quantification of distinct morphological types of
blood platelets. To this end a modified protocol described previously by Pike et al
was used.[21] Briefly, a model was trained to differentiate three morphological types of platelets:
(1) nonactivated, i.e., round-shaped, devoid of filopodia; (2) presenting at least
one filopodium but devoid of lamellipodia; and (3) presenting lamellipodium or fully
spread. Images were analysed with the trained model to obtain percentage of platelets
presenting each of the phenotypes. Trained Ilastik model is available in ZENODO repository
(https://doi.org/10.5281/zenodo.13378736).
Confocal Microscopy
Blood platelets incorporated into thrombi in VenaFlux chips channels were stained
with anti-CD41/PE and anti-JAM-A/AlexaFluor488 antibodies as described above. Blood
platelets adhered to fibrinogen and F11R/JAM-A in VenaFlux chips channels were stained
with anti-CD41/PE as described above. F11R/JAM-A staining pattern was visualised using
a confocal microscope (Nikon D-Eclipse C1) and analysed with EZ-C1 version 3.6 software
(Nikon, Japan).
Fab Enzymatic Preparation
For the preparation of Fabs we used the ficin enzymatic cleaving capacity provided
by Pierce murine IgG1 Fab Micro Preparation Kit (ThermoFisher Scientific), in accordance
with manufacturers Fab preparation protocol. Successful fragmentation of J10.4 to
Fabs of J10.4 was confirmed with the use of SDS-PAGE.
Whole-Blood Impedance Aggregometry
The measurements were performed using Multiplate analyzer (Hoffmann-La Roche, Basel,
Switzerland) according to the manufacturer's instructions. Briefly, whole blood was
preincubated with full J10.4 antibodies or their Fabs for 5 minutes at RT, then 300
µL of blood was transferred into the measurement cell and diluted with 300 μL 0.9%
NaCl and preheated to 37°C for another 3 minutes. In selected experiments 0.5 μM ADP
(final concentration) was added and platelet aggregation was recorded continuously
for 10 minutes. Area under the curve (AUC) was monitored. All measurements were completed
within 3 hours of blood collection.
Thrombus Formation Analysis
Total Thrombus formation Analysis System (Zacros, Fujimori Kogyo Co. Ltd., Tokyo,
Japan) was used to analyze in vitro thrombus formation.[22]
[23] In this method the blood is perfused through a capillary coated with a specific
substrate. The perfusion pressure that increases as the thrombi buildup is a measure
of the capillary occlusion. Reaching of a preset pressure value is considered as a
total occlusion of the capillary. The time to the total occlusion as well as AUC of
pressure changes over time are the quantitative parameters assessed in the method.
Depending on the substrate coating the capillary and blood preparation, the assay
measures primary or total haemostasis. In the first case, hirudin-anticoagulated blood
is perfused through collagen-coated capillary, whereas in the second case citrated
and recalcified blood is perfused through collagen and thromboplastin-coated capillary.
Blocking agents (IgG, Fabs, and soluble F11R/JAM-A His-tagged protein) were added
to the whole blood 5 minutes prior to perfusion.
Src Phosphorylation Analysis
Platelet-rich plasma was obtained from whole blood by centrifugation at 200 g for
12 minutes and carefully collected to avoid contamination with red blood cells. Platelets
were pelleted by centrifugation at 700 g for 15 minutes and suspended in Tyrode's
buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose, pH 7.4). Aliquots of platelet suspension (150 × 103/µL) were added to wells of a 6-well plate coated with 100 µg/mL fibrinogen. Then,
Fab fragments of J10.4 or isotype antibodies were added to final concentration of
50 µg/mL. After 1-hour incubation at 37°C nonadherent platelets were removed. Adherent
platelets were lysed by incubation with lysis buffer (1% NP40, 0.2% sodium deoxycholate,
150 mM NaCl, 50 mM Tris pH 7.5, 1 mM PMSF, protease and phosphatase inhibitors cocktail)
for 30 minutes at 4°C. Lysates were centrifuged at 16,000 g for 20 minutes at 4°C.
The supernatants were stored at −80°C. To evaluate the level of Src phosphorylation
samples with equal total protein content were loaded and separated on SDS–PAGE gels
(Mini-PROTEAN TGX Gels; Bio-Rad Laboratories, Hercules, California, United States)
and then transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories).
For the analysis of Src levels primary antibodies: goat anti-Src (R&D Systems) and
rabbit anti-phospho-Src (Y416) (R&D Systems) were used. GAPDH was used as a loading
control and detected using rabbit anti-GAPDH (Abcam, Cambridge, GB). Respective horseradish
peroxidase-conjugated anti-goat or anti-rabbit secondary antibodies (ThermoFisher
Scientific) were used. The signal was detected by measuring the chemiluminescence
with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Bands' intensity
was quantified with the use of ImageJ. The intensities of the phospho-Src bands were
normalized to the respective Src and GAPDH bands.
Ferric Chloride-Induced Injury
Total anaesthesia was induced using mix of ketamine (100 mg/kg) and xylazine (10 mg/kg)
protocol. The mixture was injected intraperitoneally. Intravenous administration of
staining antibodies and tested substances was performed by injection to the retro-orbital
plexus after confirming full anaesthesia. All surgical procedures were performed using
dissecting microscope to aid the operator. Carotid artery was exposed as described
in detail in [Supplementary Material S1] (available in the online version). A strip of a black polyethylene wrap and a strip
of filter paper (Whatman, Sanford, Maine, United States) were threaded under the blood
vessel to provide isolated field of view and a vehicle for FeCl3. The BV11 antibodies were administered intravenous into the retro-orbital plexus.
To saturate the filter paper, 3 µL of the 10% ferric chloride was applied. Immediately
after administration of FeCl3, the probe of laser Doppler flowmeter was placed directly over the cranial end of
the carotid at a height of approximately 1 mm and data collection ensued. Time of
the experiment was measured starting with the application of FeCl3 solution to the paper strip. The measurements were taken for 30 minutes or until
reduction of the blood flow to below 10% of the initial value expressed in arbitrary
units (LDU) for more than 2 minutes.
Data were collected with the use of a ML191 Blood Flowmeter (ADInstruments, Colorado
Springs, Colorado, United States). Doppler shift was analysed and recalculated to
LDF output signal by the flowmeter and transferred to ML870 PowerLab 8/30 data acquisition
system. All the LDF data were recorded as a function of time by Chart 5 software.
Statistical Analysis
Data were presented as mean ± standard deviation or median and interquartile range
(IQR), depending on the normality of data distribution. The Shapiro–Wilk test and
Levene's test were used to confirm that the data were normally distributed and homogenous.
For normally distributed and homoscedastic variables, the statistical significance
of differences between two groups was estimated using the paired or the unpaired Student's
t-test; for variables that departed from normality and/or variance homogeneity, Mann–Whitney
U test was applied instead. To compare differences between more than two groups with
a control group, analysis of variance for repeated measures and the Dunnett's post
hoc test for multiple comparisons were used. In time-dependent analyses, the Kaplan–Meier
model was adopted. Comparison of time to occlusion occurrence was performed with the
use of log-rank (Mantel–Cox) test. To compare the fractions of samples analysed with
the use of T-TAS in which embolisation occurs and to compare the fractions of animals,
which developed occlusion, the Fisher's exact test was used. Statistica v. 13.1 (Dell
Inc., Tulsa, Oklahoma, United States), GraphPad Prism v.9 (San Diego, California,
United States) were used for statistical calculations and to draw charts.
Results
Contribution of Homophilic F11R/JAM-A Interactions to Platelet Adhesion under Flow
Conditions
Surface coated with F11R/JAM-A alone had no capability to capture and immobilise blood
platelets under flow conditions ([Fig. 1A]). This lack of adhesion was also observed at lower, venous shear force conditions
([Supplementary Fig. S1], available in the online version). However, when the surface was coated with the
mix of both F11R/JAM-A and fibrinogen more platelets adhered to such combination of
proteins than to fibrinogen alone ([Fig. 1B]). Since the increased coverage observed in the presence of F11R/JAM-A could be assigned
either to an increased number of platelets adhering in a period of time, or to an
increased spreading of adhered platelets, in order to dispel these doubts the additional
experiments were performed where adhesion events were monitored in real time. Quantitative
analyses revealed increased frequency of adhesion events on surface covered with fibrinogen
in a combination with F11R/JAM-A when compared with adhesion on fibrinogen alone ([Fig. 1C]), confirming that the presence of F11R/JAM-A on the surface allowed more efficient
capture of flowing platelets.
Fig. 1 Capability of surface-bound F11R/JAM-A to capture blood platelets under flow conditions.
(A) Platelets firmly adhered to bovine serum albumin (BSA)-coated or Fc-F11R/JAM-A-coated
surface under flow conditions at 40 dyne/cm2 (∼890 s−1); (B) platelets firmly adhered to fibrinogen-coated surface or fibrinogen and Fc-F11R/JAM-A-coated
surface under flow conditions at 40 dyne/cm2 (∼890 s−1) P = 0.0017, paired two-tailed t-test, n = 12. (C) Number of firmly adhered platelets to a surface coated with fibrinogen and Fc-F11R/JAM-A
was monitored in real-time at 40 dyne/cm2 (∼890 s−1); P = 0.037, paired two-tailed Student's t-test, n = 7. Each pair of values represents a sample from individual donor. Right panel of
each chart presents differences calculated for each pair of values shown on the left
panel (coverage on Fc-F11R/JAM-A–coated surface upon subtraction of the fibrinogen
or BSA alone–coated surface) shown as mean ± standard deviation.
Compounds Blocking F11R/JAM-A-dependent Interactions
As a next step we aimed at verifying whether F11R/JAM-A-dependent enhancement of platelets
interactions with fibrinogen described above could contribute to thrombus formation.
To this end, we used two approaches to block F11R/JAM-A homophilic interactions. One
of them being J10.4 monoclonal antibodies with a defined ability to block homophilic
interactions of F11R/JAM-A[24]
[25] and the other was the use of soluble form of F11R/JAM-A.
Taking into consideration that monoclonal F11 antibodies against F11R/JAM-A were previously
reported to activate blood platelets in FcγRII-dependent manner, we first tested J10.4
clone to exclude its ability to exert similar effect. Interestingly, a whole-blood
aggregometry assay revealed that J10.4 antibodies induced platelet aggregation and
caused potentiation effect of subthreshold ADP concentration ([Supplementary Fig. S2], available in the online version). Therefore, in order to use these antibodies as
a blocking factor devoid of activating effect, their Fab fragments were prepared as
described in the methods section ([Supplementary Fig. S3], available in the online version). Prepared fragments were tested to assure the
lack of the stimulatory effect. As expected Fab fragments of J10.4 did not increase
ADP-induced platelet activation ([Supplementary Fig. S2], available in the online version). Murine isotype Fab fragments were prepared according
to the same protocol to be used as control.
Contribution of Homophilic F11R/JAM-A Interactions to Thrombus Formation Assessed
in Total Thrombus Formation Analysis System
In most of the blood samples treated with J10.4 antibodies the time to formation of
an occlusive thrombus was significantly increased when compared with isotype IgG treated
samples ([Fig. 2A]) indicating that antibodies impaired thrombus formation. In some donors however,
as depicted on the figure, the effect was opposite. Additionally, in 7 out of 13 samples
treated with J10.4 antibodies, the process of thrombus growth was interrupted with
drops of pressure reflecting thrombi embolization, which was not observed in the control
group (sample tracings shown under [Fig. 2A]). This fraction of embolisation occurrence was statistically significant (p = 0.0052, Fisher's exact test). In none of the donors, however, thrombus formation
was entirely abrogated. Importantly, no effect of J10.4 antibodies was observed when
primary haemostasis capacity was evaluated ([Fig. 2B]).
Fig. 2 Effects of J10.4 antibodies and soluble F11R/JAM-A on thrombus formation. Effects
of J10.4 antibodies on: (A) time of occlusive thrombus formation, P = 0.035 two-tailed paired Student's t-test (n = 13), panels below show representative registrations of occlusive thrombus formation
and the occurrence of embolisation in the presence of J10.4 antibodies in comparison
to unaffected thrombosis in the presence of isotype IgG; (B) primary haemostasis, not significant (n.s.) Effects of Fab fragments of J10.4 antibodies
on: (C) time of occlusive thrombus formation, P = 0.0068 Wilcoxon matched-pairs signed rank test (n = 11); (D) primary haemostasis, n.s.; (E) effects of soluble His-tagged F11R/JAM-A on time of occlusive thrombus formation,
P = 0.002 Wilcoxon matched-pairs signed rank test (n = 11); each pair of values represents a sample from individual donor. Right panels
of the charts present differences calculated for each pair of values shown on the
left panels (the aggregation in the presence of J10.4 or soluble F11R/JAM-A upon subtraction
of the aggregation in the presence of isotype or vehicle) shown as mean ± standard
deviation.
Similarly to the full J10.4 antibodies, Fab fragments of J10.4 significantly delayed
time to occlusion in the total haemostasis assay ([Fig. 2C]) and in contrast to the full antibodies in none of the donors the opposite effect
was observed. Interestingly, in one donor out of 11, the thrombus formation was completely
inhibited. Primary haemostasis was not affected by Fab fragments of J10.4 ([Fig. 2D]).
To further confirm the effect of the blockade of homophilic F11R/JAM-A interactions
on total thrombus formation, soluble recombinant F11R/JAM-A molecule with His tag
was used. Its presence significantly delayed thrombus formation ([Fig. 2E]).
Western Blot
To verify whether the inhibition of F11R/JAM-A cis-dimerisation using J10.4 Fab fragments
affects platelet intracellular signalling, we assessed Y416 phosphorylation of Src
in blood platelets adhering to fibrinogen in the presence of either J10.4 Fab or isotype
Fab. No significant effect of J10.4 Fab on the level of phospho-Src was observed ([Supplementary Fig. S4], available in the online version).
Flow Cytometry Analysis
Flow cytometry was used to evaluate the binding of monoclonal J10.4 antibodies to
resting and activated blood platelets. The level of platelet activation was assessed
with the monitoring of the P-selectin exposure. As shown on [Fig. 3A] only approx. 10% of resting platelets was positively stained with J10.4 antibodies.
This J10.4-positive fraction increased gradually along with platelets stimulation
by TRAP (1–20 µM), but it reached only 50% compared with nearly 100% of platelet P-selectin
exposure under the same conditions. Interestingly, platelets binding J10.4 antibodies
were located mainly in the fraction of larger platelets and platelet aggregates ([Fig 3B]).
Fig. 3 Flow cytometry analysis of F11R/JAM-A expression and platelet activation (P-selectin
exposure). Effects of TRAP on total or monomeric F11R/JAM-A expression: (A) percentage of blood platelets positive for monomeric F11R/JAM-A (J10.4 antibody
staining), total F11R/JAM-A (polyclonal antibody staining) in comparison with percentage
of platelet activation level revealed by P-selectin staining (n = 3–7); data presented as median with interquartile range [IQR] (B) representative dot-plots showing location of blood platelets positively stained
with polyclonal or J10.4 antibodies (green dots) in the population of CD41-gated blood
platelets (red dots) either resting or activated with 20 µM TRAP; (C) effects of soluble His-tagged F11R/JAM-A alone and in combination with ADP on platelet
activation, one-way analysis of variance (P < 0.0001) followed by Tukey's post hoc multiple comparisons test (P-value adjusted for multiple comparisons depicted on the plot), n = 6; data presented as median with IQR.
In turn, when polyclonal antibodies against F11R/JAM-A were used, approximately 60%
of resting platelets were positive and this number only slightly increased with platelet
activation ([Fig 3A]).
Flow cytometry was also used to verify the effects of soluble His-tagged F11R/JAM-A
on platelet reactivity. As shown on [Fig. 3C], His-tagged F11R/JAM-A enhanced platelet response to ADP (as revealed by an increased
P-selectin exposure) when used in the same concentration, which delayed thrombus formation
in T-TAS analyses. .
Expression of F11R/JAM-A on Activated and Adhered Blood Platelets
As flow cytometry analysis showed that the expression of F11R/JAM-A increases upon
platelet stimulation with the agonist, confocal imaging was performed to evaluate
if F11R/JAM-A-positive staining changes throughout thrombi. We revealed no specific
gradation of fluorescence intensity between the core and the outer layers of thrombus,
suggesting that platelets do not differ in terms of F11R/JAM-A expression throughout
the body of thrombus ([Fig. 4A, B]). Since staining was performed without permeabilisation it could be assumed that
only the pool of the protein presented on platelet surface is visible. Interestingly,
structures that may be identified as procoagulant platelets (the annexin V positive,
balloon-shaped objects located on the borders or outside the body of thrombus) were
characterised by relatively low expression of F11R/JAM-A when compared with platelets
located in the body of thrombus ([Fig. 4B]).
Fig. 4 Expression of F11R/JAM-A on blood platelets in thrombus formed on collagen. Images
show a confocal section in the medial position of the thrombus (100× oil objective).
Red staining represents (A) anti-CD-41 labeling or (B) annexin V labeling and green staining represents anti-F11R/JAM-A labeling. Arrows
point at ballooning platelets. Representative examples of images acquired on blood
samples from three different donors.
The Effects of F11R/JAM-A on Platelet Adhesion to Fibrin
Previous experiments showed that platelet F11R/JAM-A plays a role in thrombus formation,
but it is not related to platelet aggregation. Therefore, we tested how F11R/JAM-A
presence on the surface can affect platelet adhesion to fibrin-like structure. As
shown on [Fig. 5], platelets adhering to fibrin-like structure in the presence of F11R/JAM-A more
often acquired lamellipodial morphology than platelets adhering to fibrin-like mesh
alone. This was accompanied by a significant decrease of filopodial platelets.
Fig. 5 Effects of F11R/JAM-A on morphology of blood platelets adhered to fibrin (A) representative image of platelets adhered to fibrin (left panel) with platelets
assigned to one of three morphological classes by the trained classifier (right panel);
(B) representative image of platelets adhered to fibrin and Fc-F11R/JAM-A (left panel)
with platelets assigned to one of three morphological classes by the trained classifier
(right panel). (C) Comparison of fractions of platelets assigned to one of three morphological classes:
nonactivated P < 0.05, filopodia P < 0.05, lamellipodia P < 0.05 (paired t-test), n = 10. Each pair of values represents a sample from individual donor. Right panels
of the charts present differences calculated for each pair of values shown on the
left panels (adhesion to fibrin and Fc-F11R/JAM-A upon the subtraction of adhesion
to fibrin alone) shown as mean ± standard deviation.
Contribution of Homophilic F11R/JAM-A Interactions to Thrombus Formation In Vivo
The effects of F11R/JAM-A blockade were tested in an in vivo thrombosis models in
carotid artery in mouse. As the blocking agent, anti F11R/JAM-A antibodies (clone
BV11) were used that were previously shown to inhibit homophilic interactions of F11R/JAM-A[6] and to reduce migration of neutrophils through venules in inflammatory conditions
in mice.[26] Isotype IgGs were used as a control.
A rate of occlusion, measured as a decrease in blood flow, was significantly lower
in BV11-treated animals than in isotype-treated control. Also, a lower number of animals
developed full occlusion during observation period in BV11-treated animals than in
isotype-treated group ([Fig. 6]). To rule out that lower incidence of occlusion was caused by platelet depleting
effect of administering BV11 antibodies, in a separate group of animals an effect
of BV11 antibodies on platelet count was tested. In the animals treated with BV11
platelet count was decreased from 947 (831; 970) × 103 plt/µL (median with IQR) to 524 (492; 1,038) in a period of 30 minutes from the time
of antibody injection.
Fig. 6 Effects of BV11 antibodies on thrombus formation in a model of experimental thrombosis
in mouse carotid artery. Comparison of time to occlusion occurrence measured by means
of laser Doppler flowmetry; P = 0.0026, log-rank (Mantel–Cox) test, n = 11; the comparison of proportions of animals that developed occlusion; P = 0.03, Fisher's exact test, n = 11.
Discussion
Contribution of F11R/JAM-A to Platelet Immobilisation under Flow Conditions
Homophilic interactions between F11R/JAM-A expressed on blood platelets and that immobilised
on a surface were proven to support platelet adhesion and spreading under static conditions.[11]
[16] Our earlier research showed that in vivo under flow conditions F11R/JAM-A-dependent
interactions were involved in transient adhesion of platelets to inflamed endothelium.[19] In the present work we aimed at elucidating whether homophilic interactions of F11R/JAM-A,
which apparently facilitate platelet adhesion in static conditions, would provide
such support under flow conditions and how these interactions translate to thrombus
formation.
According to our present study, interactions of flowing platelets with F11R/JAM-A
immobilised on a surface as the sole adhesion protein were not sufficient to capture
and immobilise platelets, even under low-shear, venous flow conditions. On the other
hand, when F11R/JAM-A was co-coated with fibrinogen, it turned out to play a marked
supporting role in recruiting platelets to the surface under high shear arterial flow
conditions. Therefore, although F11R/JAM-A homophilic interactions per se are not
sufficient to immobilise a flowing platelet, they may increase the efficiency of adhesion
to surfaces where other proadhesive proteins are present. Such a milieu is present
in thrombus where activated platelets presenting a plethora of proadhesive molecules
are interweaved with fibrin. Whether this F11R/JAM-A-mediated facilitation of adhesion
is due to a physical tethering of flowing platelet by homophilic interaction between
surface-bound F11R/JAM-A and that present on platelet or this interaction only primes
platelet to develop physical interaction via another adhesive proteins remains to
be elucidated. However, taking into account that even at low shear forces F11R/JAM-A-mediated
interactions alone were not sufficient to arrest the flowing platelets suggests the
latter explanation more likely.
It is not clear why homophilic interactions of F11R/JAM-A alone can mediate platelet
adhesion and activation under static conditions, but they are insufficient on their
own under the flow conditions. It may be assumed that in flow these interactions are
too short lasting or that downstream signal is too weak to activate platelet to an
extent required for immobilisation. Under static conditions in turn, the period of
interactions could be sufficient to trigger the downstream signal resulting in platelet
adhesion.
Contribution of F11R/JAM-A to Thrombus Formation
To understand whether this F11R/JAM-A-mediated facilitation of adhesion translates
to thrombus formation we looked at the effects of compounds blocking F11R/JAM-A homophilic
interactions on formation of occlusive thrombus.
In the first place we used monoclonal J10.4 antibodies that are very well defined
in terms of binding site on F11R/JAM-A molecule, and which were shown to bind specifically
to monomeric F11R/JAM-A and to inhibit its homophilic cis-dimerisation.[25] The J10.4 antibodies themselves, however, activated blood platelets in Fc fragment-dependent
fashion that was similar to previously published studies, which had shown that stimulatory
monoclonal F11 antibodies alone activated platelets in an FcγRIIa-dependent manner.[10] Therefore, we performed these experiments in parallel with their Fab fragments devoid
of the activating effect. Application of either full antibodies or their Fab fragments
resulted in significant prolongation of time to formation of occlusive thrombus. The
effect of J10.4 full antibodies, however, was not unequivocal. In some of donors it
caused an acceleration of occlusive thrombosis rather than a delay. Moreover, in a
significant number of donors the antibodies application resulted in disturbed formation
of thrombus characterised by repetitive embolisation. This may be explained by the
fact that activating effect of full antibodies competed with their inhibitory effect.
The use of a soluble form of F11R/JAM-A was another mean of blocking F11R/JAM-A–dependent
interactions. His tag F11R/JAM-A delayed the formation of occlusive thrombus. Two
possible mechanisms can explain this effect. Soluble His tag F11R/JAM-A, as a monomeric
protein, can bind to monomeric F11R/JAM-A on blood platelet in the site responsible
for homodimeric cis-dimerisation, thus preventing cis-homodimerisation of platelet
portion of this protein. His tag F11R/JAM-A would therefore act in a way similar to
J10.4 antibodies. However, this hypothesis is not supported by the fact that under
physiological pH F11R/JAM-A tends to form cis-dimers;[6] therefore, we rather opt that the His tag F11R/JAM-A is more likely to be present
as a dimer in an experimental environment. As such it presumably binds to platelet
F11R/JAM-A in trans-configuration blocking the interplatelet interactions between
F11R/JAM-A molecules located on flowing platelets and platelets already incorporated
in thrombus.
Our finding that soluble F11R/JAM-A delayed formation of occlusive thrombus is in
contrast to the work of Rath et al who showed that soluble F11R/JAM-A enhanced thrombus
formation in flow chamber assay and increased platelet reactivity.[18] The soluble form used in the cited studies was, as the authors assumed, dimeric
under the experimental conditions. To evaluate whether soluble form used in our studies
exerts similar effects on platelet activation as that described by Rath et al we performed
similar assays and found that indeed it increased blood platelets response to ADP.
Why a molecule that induced activation of blood platelets could cause a delay in thrombus
formation in another experimental conditions? To our understanding, this apparent
disagreement may be due to the dual nature of the effects mediated by soluble F11R/JAM-A.
Binding of soluble form of the protein to platelet F11R/JAM-A has two consequences:
one of them being the sending of a downstream signal for platelet activation as shown
by Rath et al, and the other being the blocking the interaction of platelet F11R/JAM-A
molecule with its counterpart on another blood platelet. While the former effect is
obviously prothrombotic, the latter is antithrombotic, as shown by our experiments
with J10.4 antibodies that block cis-dimerisation.
Importantly, neither of the compounds caused a definitive abrogation of the formation
of occlusive thrombi. None of them also compromised primary haemostasis. Therefore,
we conclude that although homophilic interactions of F11R/JAM-A between platelets
facilitate formation of thrombus they are not a decisive factor.
In accordance with the in vitro results, the blockade of F11R/JAM-A interactions in
an animal model of thrombosis caused a significant decrease of thrombus formation.
However, in this case, antibodies were found to decrease the number of circulating
platelets. Although the final level of circulating platelets was in the range that,
according to literature,[27]
[28] still ensures haemostasis in mice, the observed outcome of reduced thrombosis could
be partially attributed to this effect.
The question arises as to which of the type of interactions of F11R/JAM-A, cis- or
trans- are affected by blocking antibodies and soluble F11R/JAM-A. Monoclonal J10.4
antibodies were shown to bind solely to F11R/JAM-A monomers and to inhibit cis-dimerisation.[25] According to some studies cis-dimerisation in turn is necessary for trans-dimerisation.[8] The antibodies would then inhibit trans-homodimerisation indirectly by inhibiting
cis-dimerisation. Soluble His tag F11R/JAM-A is expressed as a monomeric protein but
according to Bazzoni et al in physiological pH the protein forms cis-homodimers, which
renders cis-homodimerisation sites inaccessible.[6] Since cis- and trans-homodimerisation occurs at different sites[8] soluble His tag F11R/JAM-A still could bind to platelet F11R/JAM-A via trans-homodimerisation
site. At the same time cis-dimerisation of F11R/JAM-A per se does not enhance intracellular
signalling via Src kinase during platelet adhesion, as shown by the lack of the effect
on its phosphorylation in platelets adhering to fibrinogen in the presence of J10.4
Fabs. This way of reasoning therefore advocates the notion that the type of F11R/JAM-A
interactions involved in thrombus formation are the interactions between two molecules
of F11R/JAM-A located on opposing cells and that the dimerisation of monomeric F11R/JAM-A
facilitates this process.
This trans-homodimerisation of platelet F11R/JAM-A may occur not only with F11R/JAM-A
presented on another platelets in thrombus, but also with soluble F11R/JAM-A entrapped
in growing thrombus either as soluble F11R/JAM-A molecules or as F11R/JAM-A rich microvesicles.
As shown by Rath et al[18] soluble F11R/JAM-A increases platelet reactivity but, by their own admittance, humoral
levels of soluble JAM-A required to achieve this effect in vitro are several-fold
higher than those observed naturally in serum. As they suggested, it could be assumed
that concentration of soluble F11R/JAM-A is increased locally in thrombus milieu due
to accumulation of the protein. Considering the fact that activated platelets can
produce F11R/JAM-A-rich microvesicles,[29] this soluble fraction of the protein may actually become stationary from the functional
point of view by the way of incorporating to growing thrombus. Therefore, we suggest
that this localised high concentration of F11R/JAM-A may be present in stationary
phase either as accumulated soluble F11R/JAM-A or as F11R/JAM-A presented by platelets,
which were already incorporated in thrombus and thus provide target-rich environment
for local interactions.
In this context, the abundance of F11R/JAM-A accessible for trans-homophilic interactions
on the surface of resting and activated blood platelets is an important factor. As
shown by the staining with polyclonal antibodies, F11R/JAM-A is present on a majority
of both resting and activated platelets, which is consistent with previously published
reports.[13] However, only approximately 10% of resting platelets bound J10.4, the antibodies
that are known to bind specifically to monomeric F11R/JAM,[25] while according to previous reports, monomeric F11R/JAM-A dominates in resting platelets,
where it is associated with αIIbβ3.[14] It is therefore expected that majority of resting platelets should bind J10.4 antibodies.
The inconsistency could be possibly explained by low accessibility of the epitope
on F11R/JAM-A, which binds J10.4 antibodies under conditions when the protein is in
complex with αIIbβ3. When F11R/JAM-A dissociates from αIIbβ3 during platelet activation, the accessibility of F11R/JAM-A to J10.4 increases. Since monomeric F11R/JAM-A dissociated from αIIbβ3 plausibly tends to form dimers,[13] the capacity of J10.4 binding is limited. It would explain why only up to approximately
50% of activated platelets became positive against J10.4 in the same conditions when
more than 80% was stained with polyclonal antibodies.
It was reported earlier that the interaction of platelet F11R/JAM-A with JAM-A enriched
fibrinogen coating in static conditions changes platelet shape to more flattened when
compared with fibrinogen alone,[18] which shows that homophilic interactions of F11R/JAM-A have yet another intriguing
consequence, such as the modulation of platelet phenotype. Additionally, recent studies
showed platelets to acquire different phenotypes depending on whether they interact
with fibrin or with fibrinogen.[30] While platelets interacting with fibrin formed protrusions and remained stationary,
these adhered to fibrinogen were flattened and acquired migratory phenotype. Since
fibrin is a component of thrombus, we wondered whether homophilic interactions of
F11R/JAM-A could play a role in this process. Our experiments showed that a shift
in platelet shape occurred in platelets adhering to fibrin in the presence of F11R/JAM-A.
It suggests that F11R/JAM-A not only facilitates immobilisation of flowing platelets,
but also plays a role in the process of thrombus organisation. Interestingly, as shown
by confocal imaging of F11R/JAM-A staining in thrombus, the protein was present in
platelets across thrombus with relatively less to none staining on platelets, which
could be identified as procoagulant platelets, i.e., ballooned-shaped annexin V positive
ones.[31] It supports a notion that the regulatory role of F11R/JAM-A is important in the
inner part of thrombus rather than in its outer layers. Besides F11R/JAM-A contribution
to platelet–platelet interactions, the protein can also take part in platelet–endothelium
interactions where platelets take part in sealing gaps between adjacent endothelial
cells in inflamed vascular wall, a process that has been recently described.[30]
Conclusion
We show that interactions of F11R/JAM-A located on flowing platelets with its surface-bound
counterpart enhance platelets binding to fibrinogen under high shear stress conditions.
These homophilic interactions modulate thrombus formation but are not indispensable
for its completion. F11R/JAM-A plays a role in acquiring by platelets of adhesive
phenotype during interaction with fibrin mesh. While previously published studies
pointed at a significant role of soluble F11R/JAM-A in priming platelets during thrombus
formation, our results address the role of surface-bound F11R/JAM-A in this process.
What is known about this Topic?
What does this paper add?
-
F11R/JAM-A homophilic interactions facilitate blood platelets adhesion under flow
conditions.
-
Cis- and trans-homophilic interactions of platelet F11R/JAM-A take part in thrombus
formation, but they are not critical for this process.
