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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-07-2149.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Service d'Hématologie Biologique and
INSERM Unité 428, Hôpital Européen Georges Pompidou,
and Laboratoire de Génétique Moléculaire, UPRES-JE
2195, Faculté des Sciences Pharmaceutiques et Biologiques,
Université Paris V, Paris, France.
Protease-activated receptor 1 (PAR-1), the main thrombin receptor
on vascular cells, plays a key role in platelet activation. We examined
the range of PAR-1 expression on platelets, obtained twice, 1 week
apart, from 100 healthy subjects and found a 2-fold interindividual
variation in receptor numbers (95% CI = 858-1700). Because PAR-1
density was stable with time (r2 = 76%,
P < .001), we sought a genetic explanation for the
observed variability. To validate this approach, we also analyzed the
Platelets can be activated by a variety of
physiologic agonists, such as collagen, adenosine diphosphate (ADP),
and thrombin through interactions with specific membrane
receptors.1 Activation results in platelet adhesion to
extracellular matrix components and subsequent platelet aggregation.
Thrombin, a key enzyme in blood coagulation, activates human platelets
via two 7-transmembrane G-protein-coupled protease-activated receptors
(PAR-1 and PAR-4) and activation of either is sufficient to trigger
platelet secretion and aggregation.2-4 Thrombin at
picomolar concentrations activates platelet PAR-1 by cleaving its
amino-terminal end.5,6 The unmasked amino-terminus
sequence acts as a tethered ligand, resulting in a rapid response that
can be reproduced by a hexapeptide corresponding to the amino terminus
(SFLLRN).5,7 PAR-4 can mediate platelet activation but
only in the presence of nanomolar thrombin
concentrations.3 PAR-1 density on resting platelets has
been evaluated in a small number of subjects, by using radioiodinated
monoclonal antibodies (mAbs).8,9 According to Brass et
al,8 the PAR-1 number is approximately 1500/platelet. We
have previously reported variable platelet responsiveness to SFLLRN in
a study of 100 healthy volunteers.10 The response was
stable over time in a given individual, pointing to genetic control.
The PAR-1 gene is about 27-kilobase (kb) long and
comprises 2 exons separated by a large intron (~22
kb).11,12 Two polymorphisms have been identified in the 5'
regulatory region: a C>T transition 1426 base pair (bp) upstream of
the transcription start site ( Most of the genes encoding the platelet receptors have been sequenced,
and polymorphisms have been described in coding and regulating regions.
The consequences of these polymorphisms for platelet functions and
their involvement in predispositions to excessive bleeding or thrombus
formation begin to be determined.14-17 For instance,
marked differences in platelet surface
The purpose of this study was to determine the impact of
PAR-1 gene polymorphisms on the expression and function of
PAR-1 receptors on platelets from healthy individuals. We used
quantitative flow cytometry to evaluate receptor density and real-time
reverse transcription-polymerase chain reaction (RT-PCR) to quantify
mRNA. To validate this approach, we also analyzed the
Subjects
Sample preparation
Platelet aggregation studies Aggregation studies were performed within 2 hours after blood collection. A 280-µL aliquot of PRP was incubated for 3 minutes at 37°C and was then stirred at 1100 rpm for 2 minutes before adding 20 µL of one of the following agonists: collagen Horm (a 95% type I/5% type III mixture, Nycomed, Munich, Germany; 1 µg/mL), SFLLRN peptide (Serbio, Gennevilliers, France), or sodium chloride (spontaneous aggregation test). Platelet aggregation was recorded for 5 minutes by using a photometric method derived from the Born technique, on a 4-channel aggregometer (Regulest, Amneville, France). Results were expressed as the maximum percent increase in light transmission over that of the platelet suspension, relative to that of autologous PPP (arbitrarily 100%). The collagen lag time, that is, the interval between collagen addition and the onset of aggregation, was also recorded. SFLLRN aggregation was performed in the presence of 100 µM amastatin (Sigma-Aldrich, Saint-Quentin Fallavier, France), an aminopeptidase M inhibitor, to ensure the stability of the synthetic peptide in plasma.10,26 At visit 1, the tests were performed with 7, 10, and 15 µM SFLLRN (final concentration) to determine the range of SFLLRN concentrations causing irreversible aggregation. At the second visit, the precise SFLLRN concentration inducing biphasic aggregation was determined by varying by 1-µM intervals. In all samples the rate of spontaneous aggregation was lower than 5%.Quantitative flow cytometry Platelet receptors on resting and activated platelets were quantified in PRP by means of quantitative flow cytometry with a calibrator kit (Platelet Calibrator, Biocytex, Marseille, France), according to the manufacturer's instructions. The kit includes a mixture of 4 calibration beads coated with increasing concentrations of mouse IgGs (360, 8600, 29 000, and 90 000 molecules for the batch used throughout the study). Platelets were stained, in a no-wash indirect immunofluorescence technique, with the following mouse IgG1 mAbs (Immunotech, Marseille, France): anti-P-selectin (CD62p, clone CLB-Thromb/6), anti- 2 1 (CD49b, clone
Gi9), and anti-PAR-1 WEDE15 and SPAN12. WEDE15 recognizes amino acids
51-64 of the PAR-1 N-terminus and is directed against both cleaved and uncleaved receptors. Anti-PAR-1 SPAN12 is directed against uncleaved receptors because it recognizes amino acids 35-46 (NATLDPR/SFLLR), where the virgule indicates the putative thrombin cleavage site. Combined use of the 2 mAbs theoretically yields the uncleaved receptor
level. All mAbs were used at saturating conditions (10 µg/mL final
concentration), as determined in preliminary experiments with
concentrations ranging from 1 to 20 µg/mL. A negative isotypic control IgG1 was included in each series. The staining reagent was a
polyclonal antimouse IgG-fluorescein isothiocyanate (FITC) antibody.
To assess the platelet response to agonists, PRP samples were incubated
(15 minutes, 37°C) in static conditions with Horm collagen
at 100 µg/mL (final concentration), 100 µM thrombin
receptor-activating peptide (TRAP), or 10 µM U46619
(Calbiochem, VWR International, Meudon, France). To avoid platelet
aggregation,27,28 PRP was first incubated for 3 minutes at
37°C with 4 µg/mL eptifibatide (Schering-Plough,
Levallois-Perret, France), a cyclic heptapeptide inhibitor of the
platelet fibrinogen receptor
A calibration curve was constructed for each sample series, and a
negative isotype control was run with each PRP sample. Five thousand
events were acquired on a FACScan flow cytometer (Becton Dickinson),
and data were analyzed using CellQuest software (Becton Dickinson).
Receptor numbers were derived from the calibration curve, after
subtracting the negative isotype control value (Figure 1). The coefficient of variation of the
method was below 10%. Experiments were performed within 3 hours after
blood collection.
Platelet RNA extraction and reverse transcription Four milliliters PRP (1 × 109 platelets) was pelleted by centrifugation for 15 minutes at 2300g and immediately stored at 80°C. Platelet was extracted with the RNeasy
kit (Qiagen, Courtab uf, France) according to the manufacturer's
instructions, and eluted in 30 µL Rnase-free water. Eight microliters
RNA solution was immediately used for cDNA synthesis. The remaining RNA
was stored at 80°C.
Reverse transcription was performed in a final volume of 20 µL
containing 1 × RT-PCR buffer (3 mM MgCl2, 75 mM KCl, 50 mM Tris [tris(hydroxymethyl)aminomethane]-HCl, pH 8.3;
Invitrogen, Cergy-Pontoise, France), 500 µM each deoxyribonucleoside
triphosphate (dNTP; Amersham Pharmacia Biotech, Orsay, France),
10 U RNasin ribonuclease inhibitor (Promega, Charbonnières,
France), 10 mM dithiothreitol (Invitrogen), 100 U Superscript II Rnase
H Real-time RT-PCR Theoretical basis.
Quantitative values are obtained from the threshold cycle number (Ct
value) at which the increase in fluorescent signal associated with
exponential growth of PCR products begins to be detected by the laser
detector of the ABI Prism 7700 Sequence Detection System (Applera,
Courtab Ct target gene
sample) (Ct RPLP0 reference sample R Ct target gene
reference sample R))
Each data point was determined in duplicate, and data with a Ct
coefficient of variation of more than 2% were determined again.
Primers and PCR consumables.
Primers for PAR-1,
PCR amplification. All PCRs were performed using an ABI Prism 7700 Sequence Detection System (Applera), according to the manufacturer's instructions, and the SYBR Green PCR Core Reagents kit (Applera). For each PCR run, a master mixture was prepared on ice with 1 × Sybr Green buffer, 200 µM deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxyguanosine trhiphosphate (dGTP), 400 µM deoxyuridine triphosphate (dUTP), 1.25 U AmpliTaq Gold DNA polymerase, 5 mM MgCl2, and 200 nM each primer (Invitrogen). Ten microliters of each appropriately diluted reverse transcription sample was added to 40 µL of the PCR master mix. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 minutes, and 50 cycles at 95°C for 15 seconds and 65°C for 1 minute. Analysis of the exon 1/exon 2 junction sequences in platelet PAR-1 cDNA We amplified the platelet cDNA fragment flanking the region at the junction of the 2 PAR-1 exons by using a reverse primer 5'-ACA ATG GGG CCG CGG CGG-3' and a forward primer 5'-CCG GAG GCA TCT TCT GAG ATG-3' corresponding to the exon 1 and exon 2 sequences, respectively. PCR products were then purified using the Pre-Sequencing Kit (Amersham Pharmacia Biotech) and directly sequenced with an ABI Prism 3700 (Applera) and DNA Sequencing Analysis 3.6 NT software (Applera).Genotyping of polymorphisms gDNA was isolated from peripheral blood leukocytes by using the Qiamp Maxi Kit (Qiagen) according to the manufacturer's instructions and was stored at 4°C until analysis. Polymorphisms were determined by means of genomic PCR. All primers were from Genset (Proligo, Paris, France).
Statistical analysis Data are shown as means ± SEM, except for skewed variables, which are expressed as medians. The distribution of continuous variables was estimated by using the Schapiro-Wilk test. Skewed variables were log-transformed before analysis. Individual subjects' values obtained at visits 1 and 2 (1 week apart) were compared using a concordance test.30 When values of a given parameter obtained at the 2 visits were concordant (defined as r2 > 50%), the mean of the 2 measurements was used for subsequent analyses. The 2 test was used to compare the
observed allele and genotype frequencies with the Hardy-Weinberg
equilibrium prediction. A paired t test was used to estimate
changes in receptor numbers before and after platelet activation. An
univariate linear regression was used to compare phenotypic results.
The association between genotype and phenotype was tested using
analysis of variance (ANOVA). Genotype-phenotype or phenotype-phenotype
associations were further tested after adjustment for other parameters
(white blood cell count, fibrinogen plasma level, mean platelet volume,
CRP, von Willebrand factor plasma level,
PlA1/PlA2 polymorphism), by using multiple
linear regression model.
Statistical tests were performed using the STATA 7.0 software package (Stata, College Station, TX) and differences with P < .05 were considered statistically significant.
Platelet PAR-1 quantification The number of PAR-1 receptors on platelets was determined from a calibration curve, after subtracting nonspecific binding. A typical example is given in Figure 1. As shown in Figure 2, PAR-1 expression, as determined by mAb WEDE15 binding, ranged from 853 to 1713 copies/platelet (mean, 1262 ± 198 copies/platelet) among the 100 volunteers. Values obtained in each subject at the 2 visits (1 week apart) showed a high degree of concordance (r2 = 76%, P < .001; Figure 2). We also tested a subset of 63 donors with mAb SPAN12, which is specific for uncleaved PAR-1 receptors. The mean number of uncleaved PAR-1 receptors was 687 ± 154/platelet (range, 407-1114/platelet) and, again, the 2 measurements performed 1 week apart showed good concordance (r2 = 61%, P < .001). Thus, 45% of PAR-1 receptors on resting platelets were not recognized by SPAN12, that is, were cleaved. Such an observation has already been reported by others.31 To rule out the possibility of platelet activation during PRP preparation, we quantified the expression of P-selectin, a marker of-granule exposure.32,33 P-selectin expression on resting platelets
was below 1000 sites in most cases (mean, 502 ± 335 sites) and
"cleaved" PAR-1 expression did not correlate with the P-selectin
level. One possible explanation for artefactual PAR-1 cleavage is the generation of trace amounts of thrombin in citrated PRP. We thus randomly selected 20 of the 100 subjects and quantified total and
uncleaved PAR-1 on resting platelets isolated from a third blood sample
collected on hirudin or citrate as anticoagulant. The ratio of
uncleaved PAR-1 to total PAR-1 was stable whatever the anticoagulant
(data not shown), implying that PAR-1 was not cleaved by thrombin
during PRP preparation.
PAR-1 density was also determined after ex vivo platelet stimulation
with collagen, SFLLRN, and the thromboxane A2 receptor agonist U46619. U46619 did not modify PAR-1 expression, whereas SFLLRN
and collagen induced a small but statistically significant decrease in
total PAR-1 receptor expression (mean decrease of 27 and 133 sites,
respectively, P < .001; Figure
3). The number of PAR-1 copies after
platelet activation was also measured on the second blood sample. As
with resting platelets, the 2 values showed good concordance
(r2 > 68% whatever the agonist,
P < .001).
Platelet aggregation and secretion responses to SFLLRN SFLLRN is specific for PAR-1 and does not therefore bind other thrombin receptors on platelets. The median SFLLRN concentration inducing a double-wave aggregation profile (Figure 4A inset) was 9.75 µM (range, 5-19 µM; Figure 4A). These results confirmed the interindividual variability of platelet sensibility to SFLLRN.10 The SFLLRN concentration causing double-wave aggregation on sample 2 was always close to the SFLLRN concentration determined on sample 1, suggesting that the platelet aggregation response to SFLLRN is stable with time in a given subject. We also observed interindividual differences in the platelet secretory response to SFLLRN. Indeed, P-selectin density on platelets activated by 100 µM SFLLRN ranged from 6613 to 15 933 sites (11 223 ± 1934 sites; Figure 4B) and this response was stable at a 1-week interval (r2 = 50%, P < .001). We therefore used the mean value of the 2 visits in each subject for subsequent analyses.
Although the transduction mechanisms underlying platelet activation are
complex and multiple, the platelet response to SFLLRN correlated with
PAR-1 expression; the SFLLRN concentration causing double-wave
aggregation correlated negatively with the number of PAR-1 sites on the
platelet surface (r2 = 23%, P < .001;
Figure 5A). This correlation remained
significant after adjustment for the PlA1/PlA2
genotype, the white blood cell count, the fibrinogen level, platelet mean volume, and CRP and von Willebrand factor levels
(P < .001). Platelet secretion (P-selectin expression)
also correlated with PAR-1 density (r2 = 30%,
P < .001; Figure 5B). Adjustment for other blood
parameters did not influence the correlation. Importantly, P-selectin
expression did not correlate with PAR-1 density when platelets were
activated with collagen (P = .28; Figure 5C). Thus,
response capacity to SFLLRN activation of platelets from healthy
volunteers was associated with platelet surface PAR-1 density.
Genotype-phenotype relationships To determine whether the platelet PAR-1 phenotype was associated with genetic variations, we genotyped the volunteers for 3 previously described PAR-1 polymorphisms, namely1426 C/T, 506 I/D, and
IVSn14 A/T. The respective allelic frequencies of 1426T,
506I, and IVSn14T were 0.12, 0.27, and 0.14, and the distribution
of heterozygotes was close to that predicted by Hardy-Weinberg
equilibrium. These allelic frequencies were similar to those previously
obtained in 1214 healthy subjects.13 The 506 I/D and
1426 C/T polymorphisms were not associated with the platelet PAR-1
phenotype, whereas the intronic polymorphism IVSn14 A/T was
significantly associated with the platelet PAR-1 expression level
(P = .003; Figure 6A).
Homozygous carriers of the IVSn14A allele (n = 74) had a
significantly higher PAR-1 level (1297 ± 186) than heterozygous
carriers (1182 ± 199, n = 24, P = .013). The 2 subjects homozygous for the IVSn14T allele had 857 and 1022 PAR-1
sites. The presence of an IVSn14T allele was also associated with
decreased platelet sensitivity to SFLLRN (Figure 6B). Indeed, biphasic
aggregation occurred with 9.1 ± 2.1 µM SFLLRN in AA subjects and
10.6 ± 2.5 µM in AT subjects (P = .005). The highest
SFLLRN concentration required for biphasic aggregation (19 µM) was
found in 1 of the 2 TT homozygotes (subject H1), who had only 857 PAR-1
sites/platelet. The IVSn14 A/T polymorphism was also associated with
the maximal platelet secretory response to 100 µM SFLLRN (Figure 6C).
Indeed, higher P-selectin expression was observed in carriers of 2 A
alleles (11 548 ± 1735 sites/platelet) than in carriers of a single
T allele (10 530 ± 2078 sites/platelet, P = .011). The
TT homozygous subjects H1 and H2 had, respectively, 6778 and 10 211
P-selectin sites per platelet after SFLLRN stimulation.
The relationship between the IVSn PAR-1 expression might also be regulated at the transcriptional level.
We used real-time RT-PCR to quantify gene expression at the mRNA level.
The NPAR-1 value, calculated as described in "Patients,
materials, and methods," was determined in a subset of 75 subjects in
whom platelet mRNA isolation and reverse transcription yielded enough
material for a reliable quantification (Ct values below 30).
NPAR-1 values ranged from 0.03 to 3.61 (1.02 ± 0.74) for
PAR-1, but did not correlate either with the PAR-1 receptor number or
with the intronic polymorphism IVSn Platelet 2-integrin,
the density of which on the platelet surface is known to be associated with the 807 C/T polymorphism. We confirmed the strong variability of
2-integrin expression on resting platelets from healthy
subjects. Density ranged from 1607 to 5631 receptors/platelet (mean,
3587 ± 833 receptors/platelet) and remained stable in a given
individual at a 1-week interval (r2 = 77%,
P < .001). No correlation was found between
2-receptor expression and either maximal platelet
aggregation or P-selectin expression induced by collagen. However, the
aggregation lag time (mean, 62 ± 16 seconds; range, 35-120 seconds),
which did not vary between the 2 samples 1 week apart
(r2 = 55%, P < .001),
correlated with 21-receptor density
(r2 = 8%, P = .004).
We also genotyped the volunteers for the 807 C/T polymorphism in the
PAR-1, the main thrombin receptor on vascular cells, plays a key role in platelet activation. Here, we studied the variability of the platelet PAR-1 phenotype in 100 healthy male volunteers, to avoid fluctuating hormonal influences on platelet functions. The number of PAR-1 molecules on the platelet surface was measured by using a new quantitative flow cytometry method with the WEDE15 antibody, which recognizes both intact and cleaved PAR-1. The mean receptor number per platelet (1200 copies) was similar to the reported number of moderate-affinity 125I-thrombin-binding sites on platelets, and to the number obtained by radioimmunoassay in a small number of subjects.8,9,34 We observed 2-fold variability of PAR-1 levels, in keeping with the expression range (1000-1800 sites) reported in the literature. PAR-1 levels were stable on 2 occasions 1 week apart (ie, when most platelets had been renewed) and, in a subset of 20 subjects, over a period of 6 months (data not shown). This strongly suggests that genetic factors are involved in the control of platelet PAR-1 expression. We also quantified PAR-1 expression after platelet activation because
previous data suggest that activation of U46619 induces expression of
surface-connecting system PAR-1 receptors, whereas SFLLRN induces a
strong decrease of PAR-1.31 We did not find such a wide
change in PAR-1 expression on platelet activation; the number of
receptors fell only slightly on SFLLRN and collagen activation and did
not change significantly after U46619 activation. This implies that
only small numbers of PAR-1 receptors are internalized in platelets, in
contrast to nucleated cells such as endothelial cells and
megakaryocytes.35-37 Noteworthy,
The in vivo PAR-1 cleavage by thrombin is complex because it is
modulated by other thrombin-binding sites on platelets (PAR-4 and GP
Ib).3,4,39,40 Thus, to determine whether platelet activation via PAR-1 is dependent on the number of receptors, we used
the specific PAR-1-activating peptide SFLLRN, which allows platelets
to be studied in plasma, their natural environment. SFLLRN is widely
used to monitor antithrombotic therapy with
Analysis of the platelet PAR-1 genotype according to the PAR-1
phenotype showed that the intronic polymorphism IVSn In conclusion, we report a relationship between the platelet PAR-1
phenotype and the IVSn
We thank Alvine Bissery for her help with the statistical analysis, and the nursing staff of the Clinical Investigation Center 9201-INSERM AP-HP of Hôpital Européen Georges Pompidou. We thank Véronique Remones for excellent technical assistance. We are grateful to Biocytex for generously providing the calibrator kits for flow cytometry.
Submitted July 18, 2002; accepted October 4, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-07-2149.
Supported in part by a grant from Programme Hospitalier de Recherche Clinique (Ministère chargé de la Santé, PHRC AOR01023, sponsor: INSERM) and the Claude Bernard Association. A.D. was supported by a grant from Assistance Publique-Hôpitaux de Paris.
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: Pascale Gaussem, INSERM U 428, Service d'Hématologie Biologique A, Hôpital Européen Georges Pompidou, 20 rue Leblanc, F-75908 Paris Cedex 15, France; e-mail: pascale.gaussem{at}egp.ap-hop-paris.fr.
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S. L. Hetherington, R. K. Singh, D. Lodwick, J. R. Thompson, A. H. Goodall, and N. J. Samani Dimorphism in the P2Y1 ADP Receptor Gene Is Associated With Increased Platelet Activation Response to ADP Arterioscler. Thromb. Vasc. Biol., January 1, 2005; 25(1): 252 - 257. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
reverse transcriptase (Invitrogen), 1.5 mM
random hexanucleotides (Amersham Pharmacia Biotech), and 8 µL mRNA.
The samples were incubated at 20°C for 10 minutes then at 42°C for
30 minutes; reverse transcriptase was inactivated by heating for 5 minutes at 99°C, then cooling for 5 minutes at 5°C. The cDNA was
stored at 
2 = 2.12 ± 2.71; range, 0.02-13)
did not differ according to the polymorphism nor to the platelet
effect in thromboembolic diseases.
Thromb Haemost.
1999;81:951-956