© 2000 American Heart Association, Inc.
Thrombosis |
Platelets From a Patient Heterozygous for the Defect of P2CYC Receptors for ADP Have a Secretion Defect Despite Normal Thromboxane A2 Production and Normal Granule Stores
Further Evidence That Some Cases of Platelet ‘Primary Secretion Defect’ Are Heterozygous for a Defect of P2CYC Receptors
From the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center (M.C., A.L., R.L., M.L.Z.), Department of Internal Medicine. IRCCS Ospedale Maggiore. University of Milan, Milan, Italy and INSERM U.311 (C.G.), Biologie et Pharmacologie de l’Hémostase et de la Thrombose, Etablissement Français du Sang-Alsace, Strasbourg, France.
Correspondence to M. Cattaneo, MD, Hemophilia and Thrombosis Center, Via Pace 9, 20122 Milano, Italy. E-mail marco.cattaneo{at}unimi.it
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Abstract |
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Abstract—Two unrelated patients with a congenital bleeding diathesis associated with a severe defect of the platelet ADP receptor coupled to adenylate cyclase (P2CYC) have been described so far. In one of them, platelet secretion was shown to be abnormal. We recently showed that platelets with the primary secretion defect (PSD; characterized by abnormal secretion but normal granule stores, thromboxane A2 production, and ADP-induced primary wave of aggregation) have a moderate defect of P2CYC. Therefore, the interaction of ADP with the full complement of its receptors seems to be essential for normal platelet secretion, and PSD patients may be heterozygotes for the congenital severe defect of P2CYC. In this study, we describe 2 new related patients with a severe defect of P2CYC and the son of one of them, who is to be considered an obligate heterozygote for the defect. The 2 patients with the severe defect had lifelong histories of abnormal bleeding, prolonged bleeding times, abnormalities of platelet aggregation and secretion, lack of inhibition of adenylate cyclase by ADP, and a deficiency of platelet-binding sites for [33P]2 MeS-ADP (240 and 225 sites per platelet; normal range, 530 to 1102). The son of one of them had a mildly prolonged bleeding time and abnormalities of platelet aggregation and secretion similar to those found in patients with PSD. In addition, his platelets showed a moderate defect of binding sites for [33P]2 MeS-ADP (430 sites per platelet) and of adenylate cyclase inhibition by ADP. This study of a family with the platelet disorder characterized by a defect of the platelet P2CYC receptor supports our hypothesis that the full complement of the platelet ADP receptors is essential for normal platelet secretion and that some patients with the common, ill-defined diagnosis of PSD are actually heterozygous for the defect.
Key Words: ADP • purinoceptors • platelet secretion • congenital disorders of platelet function • thromboxane A2
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Introduction |
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Platelet secretion defects are the most common congenital abnormalities of platelet function.1 2 They include storage pool deficiency (characterized by a diminished content of platelet granule constituents), defective arachidonate metabolism (aspirin-like defects), and an ill-defined and heterogeneous group of abnormalities characterized by abnormal secretion but a normal content of platelet granule constituents and arachidonate metabolism (sometimes referred to as primary secretion defects, or PSD).1 2 3
We recently showed that released ADP potentiates platelet secretion induced by the thromboxane mimetic U46619 independently of the formation of large aggregates and thromboxane A2 production, and that at least some of the PSD patients have a partial reduction of platelet-binding sites for [33P]2 MeS-ADP.3 This finding suggested to us that some PSD patients have a partial defect of platelet ADP receptors, which is responsible for their secretion defect. Because the inhibition of adenylate cyclase in PSD platelets was partially impaired, we hypothesized that these individuals may be heterozygotes for the severe defect of platelet ADP receptors coupled to adenylate cyclase and termed P2TAC or P2CYC.4 5 6 7 8 9 10 11 Two unrelated patients with a congenital, severe defect of P2CYC have been described so far.12 13 Their platelets, when exposed to ADP, change shape normally but undergo no or only very slight and rapidly reversible aggregation and do not exhibit the normal inhibition of prostaglandin (PG) E1–stimulated adenylyl cyclase. In addition, they have a severely reduced number of binding sites for the ADP analogue 2 MeS-ADP.13 14 Platelet secretion induced by several agonists was studied in 1 of the 2 patients and found to be severely impaired.12
In the present study, we describe 2 sisters with a severe defect of platelet-binding sites for 2 MeS-ADP and abnormalities of platelet function identical to those of the 2 previously described patients, suggesting that they also have a severe defect of P2CYC. The son of one of them had a moderate deficiency of platelet-binding sites for 2 MeS-ADP, and his platelets had a secretion defect and partial impairment of inhibition of adenylate cyclase by ADP, similar to that found in platelets with PSD. The results of this study, therefore, support our initial hypothesis that some patients with PSD are heterozygous for the congenital, severe defect of the platelet P2CYC receptors for ADP and confirm our previous demonstration that released ADP, by interacting with its platelet P2CYC receptor, potentiates platelet secretion.
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Methods |
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Patients
The proband (II-4 in Figure 1
), a
57-year-old woman born from first cousins and living in southern Italy,
was referred to our center for a lifelong history of easy bruising,
menorrhagia, and severe bleeding complications after dental extractions
or major surgery. Of her family members, only her 48-year-old sister
(II-6), who was living in Milan, could be studied. She had a lifelong
history of easy bruising, epistaxis, menorrhagia, and moderate bleeding
complications after dental extractions. Her 13-year-old son (III-1) and
45-year-old husband (II-7), who had never suffered abnormal bleeding
episodes, also accepted to be investigated. All studied subjects had
normal coagulation tests (prothrombin time and activated
partial thromboplastin time) and normal von Willebrand factor
plasma levels. None of the other family members could be investigated
or directly interviewed. Patients II-4 and II-6 reported that their
mother (I-1) and 1 of their brothers (II-1), who died of Parkinson’s
disease at 60 years of age had a mild bleeding tendency. The nature of
the study was explained to the proband and her family members and to 55
healthy controls (male/female 25:30; median age, 38 years; range, 25 to
52), who gave their informed consent.
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Materials
2 MeS-ADP was from Boehringer.
[33P]2 MeS-ADP was from New England Nuclear.
ADP, adenosine 2'-phosphate 5'-phosphate (A2P5P),
epinephrine, collagen, the
thromboxane/prostaglandin
endoperoxide analogue
9,11-dideoxy-11,9-epoxymethano-prostaglandin
F2 (U46619), platelet-activating factor
(PAF-acether), o-phthaldialdehyde, prostaglandin
I2 (PGI2),
prostaglandin E1
(PGE1), and acetylsalicylic
acid (ASA) were from Sigma. AR-C69931 MX, a P2CYC
antagonist, was a kind gift of AstraZeneca R&D (Charnwood,
UK). Fura 2 AM was from Calbiochem. Apyrase was a kind gift of Dr R.L.
Kinlough-Rathbone (McMaster University, Hamilton, Ontario, Canada). All
other products were of reagent grade or better. Commercial
preparations of luciferin/luciferase reagent were used to measure the
platelet ATP and ADP contents (ATP assay kit, BioOrbit Oy) and
platelet secretion concurrently with platelet aggregation
(Chronolume). The radioimmunoassay kit for measurement of
TxB2 was from New England Nuclear. The bleeding
time was measured with the Symplate II disposable device (Organon
Teknika Corp).
Preparation of Platelet-Rich Plasma (PRP) and Washed
Platelet Suspensions
Nine volumes of blood was drawn into 1 volume of 129 mmol/L
trisodium citrate for preparation of PRP, and 6 volumes was drawn into
1 volume of acid-citrate-dextrose anticoagulant for preparation of
washed platelet suspensions. Twice, washed platelet suspensions
were prepared according to the method described by Mustard et
al,15 with the exception that 500 nmol/L
PGI2 was added during the first and second
wash.14 Platelet counts were adjusted to
300x109/L in PRP and to
400x109/L in washed platelet suspensions.
For measurement of cytoplasmic concentrations of ionized calcium
([Ca2+]i), platelets
were loaded with 2 µmol/L fura 2 AM for 45 minutes at 37°C as
previously described.12
Platelet Aggregation and Secretion Studies
Samples of PRP (0.45 mL) were incubated with 50 µL of
luciferin/luciferase reagent at 37°C for 30 seconds and stirred at
1000 rpm in a lumiaggregometer (Lumi-aggrometer, Chrono-log Corp).
After incubation, 10 µL of an aggregating agent was added, and the
aggregation and ATP secretion tracings were recorded for 3 minutes.
Measurement of Platelet Shape Change
Platelet shape change induced by 0.1 or 1 µmol/L ADP
was measured in the aggregometer by using citrated PRP to which 2
mmol/L EDTA had been added to prevent platelet aggregation.
Decreases in oscillations of the basal tracings and
increases in optical density were interpreted as being caused by
platelet shape change.
Measurement of [Ca2+]i
Aliquots of fura 2 AM–loaded platelets were transferred to
quartz cuvettes maintained at 37°C. Fluorescence was
monitored continuously before and after stimulation with ADP by using a
spectrofluorometer (LS50B, Perkin-Elmer Co). The excitation wavelength
was alternatively fixed at 340 and 380 nm, and fluorescence
emission was determined at 510 nm.
Measurement of Platelet Granule Content
Total platelet ADP and ATP content was measured by the
firefly luciferin/luciferase method16 in a luminometer
(luminometer 1250, LKB, BioOrbit Oy). Platelet
serotonin (5-HT) content was measured with the
o-phthaldialdehyde method.17 Fibrinogen
was measured in washed platelet lysates by an ELISA with a
polyclonal anti-fibrinogen antibody (Atlantic Antibodies) conjugated
with peroxidase (type I, Boehringer Mannheim).18
The sensitivity of the method was 0.02 µg/mL.18
Measurement of Platelet Thromboxane B2
Production
Thromboxane B2
(TxB2) was measured in supernatant serum of blood
samples collected in glass tubes containing no anticoagulant and
clotted at 37°C for 2 hours.
Binding of [33P]2 MeS-ADP to Washed
Platelets
Binding of [33P]2 MeS-ADP (872 to 1044
Ci/mmol) to washed platelets was measured as
described.14 [33P]2 MeS-ADP (0.1
nmol/L) with increasing concentrations of unlabeled ligand (0 to 49.9
nmol/L) was incubated with washed platelet suspensions (1 mL final
volume) at 37°C for 5 minutes. The ligand bound to platelets was
separated from free ligand by filtration through Whatman GF/C glass
filters under vacuum. Radioactivity bound to the platelets on the
filters was measured by scintillation counting (Minibeta 1211, LKB).
Nonspecific binding, determined by incubation in the presence of 1
µmol/L 2 MeS-ADP, amounted to 1% to 3% of total binding. Data were
analyzed by a computer program
(LIGAND).19
Measurement of Platelet cAMP
Platelet cAMP was measured by a radioisotopic assay with a
commercially available kit (Amersham International). Duplicate samples
of 1 mL of citrated PRP containing 1 mmol/L theophylline were
incubated with Tyrode’s solution, PGE1 (1
µmol/L) plus Tyrode’s solution, or PGE1 plus
ADP (0.1, 1, and 10 µmol/L). After incubation at 37°C for 2
minutes, 1 mL of 5% trichloroacetic acid was added, and the samples
were snap-frozen in dry ice and methanol, thawed at room temperature,
and then shaken at 4°C for 45 minutes. After
centrifugation at 4°C for 30 minutes, the supernatant
was extracted 3 times with 5 mL of water-saturated ether, dried under a
stream of N2 at 60°C, and stored at -20°C.
Before assay, the samples were reconstituted with 0.05 mol/L Tris
buffer containing 4 mmol/L EDTA.
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Results |
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The bleeding time was prolonged in the proposita (II-4), her sister (II-6), and her nephew (III-1) and normal in II-7, her brother-in-law (II-6 in Table 1
). The
platelet count and contents of ADP, ATP, 5-HT, and fibrinogen were
normal in all subjects except in II-7, whose platelet content of
5-HT and ADP was slightly lower than normal (Table 1),
suggesting that he had a mild 
-storage-pool deficiency.
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Platelet Aggregation and ATP Secretion
ADP at concentrations ranging from 2 to 20 µmol/L induced a
very small and rapidly reversible wave of platelet aggregation in
PRP of II-4 and II-6 (Figure 2).
Platelet aggregation induced by 2 or 4 µmol/L ADP was
reversible in III-1 (son of II-6 and II-7) and comparable to that of
normal platelets that had been incubated in vitro with 0.5
mmol/L ASA for 30 minutes (Figure 2). Higher concentrations of
ADP (up to 20 µmol/L) induced normal platelet aggregation in
PRP of III-1 (Figure 2). The P2Y1
antagonist A2P5P (1 mmol/L), which was tested in II-6
(Figure 3) and III-1 (not shown),
completely abolished platelet aggregation induced by 2
µmol/L ADP. Platelet aggregation induced by 0.2 µmol/L
PAF-acether or 0.5 µmol/L U46619 was reversible in II-4, II-6,
and III-1, whereas it was irreversible in normal controls (not shown).
The extent of platelet aggregation induced by collagen (2 µg/mL)
was normal in PRP of III-1 and 
50% of normal in that of II-4 and
II-6 (not shown). Platelet aggregation induced by any agonist
tested was normal in II-7 (husband of II-6), despite his mild
-storage-pool deficiency, confirming that platelet aggregation
may be normal in patients with this abnormality.20
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The platelet ATP secretion induced by any of the tested agonists
was absent or severely impaired in the proposita (II-4) and her sister
(II-6) (Table 2). In III-1 (son of II-6
and II-7), it was absent or severely reduced when induced by ADP or
U46619 and borderline-low when induced by PAF-acether or collagen
(Table 2). It was borderline-low with any tested agonist in
II-7 (Table 2), the husband of II-6 and father of III-1, whose
platelet concentration of ADP and 5-HT was slightly reduced.
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Platelet Shape Change
ADP (0.1 and 1 µmol/L) caused normal shape change of
platelets from II-6 (Figure 3
) and III-1 (not shown),
which was completely inhibited by 1 mmol/L A2P5P. Platelet
shape change was not studied in II-4 and II-7.
ADP-Induced Increase in [Ca2+]i
In the presence of 2 mmol/L external
CaCl2, stimulation with 5 µmol/L ADP
caused a rapid increase in
[Ca2+]i which was similar
in platelets from II-6 and a normal control; in the absence of
external Ca2+ (ie, with 0.2 mmol/L EGTA),
the mobilization of
[Ca2+]i was lower, but
again it was similar in II-6 and a normal control; the ADP-induced
increases in [Ca2+]i were
completely abolished by 0.5 mmol/L A2P5P in both II-6 and
normal platelets (data not shown). The mobilization of
[Ca2+]i was not studied
in the other patients.
Inhibition of PGE1-Induced Increase in Platelet
cAMP
The basal levels of platelet cAMP in the 4 patients studied
were normal (5.9 pmol/109 in II-4, 12.5 in II-6,
9.1 in III-1, and 12.5 in II-7; normal range, 6 to 14.2
pmol/109, n=18) and increased normally after
stimulation with PGE1 (32.3 in II-4, 54.8 in
II-6, 35.6 in III-1, and 44.3 in II-7; normal range, 22.5 to 68.6). ADP
(0.1 to 10 µmol/L) did not affect the
PGE1-induced increase in cAMP levels in
platelets from patients II-4 and II-6, whereas it inhibited the
cAMP increase in a concentration-dependent manner in platelets from
healthy subjects and patients II-7 and III-1 (Figure 4). However, although the degree of
inhibition of the PGE1-induced increase in
platelet cAMP was very similar to that seen in healthy subjects for
patient II-7, it was much less for patient III-1 at all ADP
concentrations tested (Figure 3). AR-C69931 MX (0.1
mmol/L), a P2CYC antagonist,
completely abolished the residual inhibitory effect of ADP
on the PGE1-induced increase in the platelet
cAMP of patient III-1, whereas A2P5P (1 mmol/L), a
P2Y1 antagonist, had no effects (not
shown).
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[33P]2 MeS-ADP Binding to Washed Platelets
Binding experiments were performed in 29 healthy subjects and in
at least 2 different sessions in the 4 patients studied. Specific
binding of [33P]2 MeS-ADP to washed normal and
patient platelets was saturable and the Scatchard
plot21 was linear. For all patients, the
Kd values were comparable to those
calculated for normal volunteers (Table 3). In contrast, the number of binding
sites was severely decreased in the proposita II-4 and her sister II-6,
moderately decreased in III-1, and normal in II-7 (Table 3).
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Discussion |
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Three purinergic receptors are currently believed to contribute separately to the complex platelet responses to ADP: the P2X1 ionotropic receptor, responsible for rapid influx of ionized calcium into the cytosol; the P2Y1 metabotropic receptor, responsible for mobilization of ionized calcium from internal stores, platelet shape change, and initial aggregation; and an as-yet-unidentified receptor coupled to G
i2,
which is essential for the full aggregation response to ADP and is
termed P2CYC, or
P2TAC.4 5 6 7 8 9 10 11 The elusive
P2CYC receptor is probably the molecular target
of thienopyridine compounds and other selective inhibitors
of ADP-induced platelet aggregation.10 22 23 24 25 In
addition, it is probably defective in patients with a congenital
bleeding diathesis characterized by a severe defect of platelet
responses to ADP. Two such patients have been described so far by our
group12 and by Nurden et al13 : the many
similarities between the 2 patients suggest that they are affected by
the same type of abnormality of platelet ADP
receptors.26
In this study, we describe 2 sisters with a congenital bleeding
tendency characterized by a platelet function defect that is
similar to that of the 2previously described patients: (1) severe
impairment of aggregation but normal shape change induced by ADP; (2)
lack of adenylate cyclase inhibition by ADP; (3) normal
mobilization of Ca2+ from internal stores induced
by ADP, which was completely inhibited by the
P2Y1 receptor antagonist A2P5P; and
(4) decreased number of binding sites for
[33P]2 MeS-ADP. In addition, similar to those
of the first patient described by our group,12 the
patients’ platelet secretion of -granule constituents induced
by several agonists was severely impaired, confirming our previous
observation that ADP plays an important role in the potentiation of
platelet secretion stimulated by release-inducing
agonists.3
Another similarity between the 2 patients described in this study and those previously described12 13 is that all were born from consanguineous parents, indicating that their platelet defect is most likely inherited as a recessive trait. As expected, the platelets of the son of 1 of them, who is an obligate heterozygote, had a number of binding sites for 2 MeS-ADP that was intermediate between normal and that of his mother and aunt. ADP induced a normal primary wave of aggregation of his platelets and partially inhibited adenylate cyclase. In addition, a variety of agonists, including the TxA2 mimetic U46619, PAF-acether, and collagen, induced impaired or borderline-low secretion of ATP from his platelets. This secretion defect was not caused by impaired production of TxA2 or low concentrations of platelet granule contents and is therefore, very similar to that described in patients with an ill-defined and probably heterogeneous group of congenital defects of platelet secretion, sometimes referred to by the general term "primary secretion defect" (PSD).3 This defect, which is the most common congenital abnormality of platelet secretion, is characterized by abnormal/borderline-low platelet secretion induced by different agonists, a normal primary wave of aggregation induced by ADP, normal granule stores, and normal arachidonate metabolism.1 The results of this study therefore confirm our previous hypothesis that some patients with PSD are heterozygous for the severe defect of P2CYC, the platelet ADP receptor that is coupled to adenylate cyclase.3 The important role of ADP interaction with this receptor in primary hemostasis is emphasized by the finding that patient III-1, like others with PSD, has, despite the mild defect of P2CYC, a mild prolongation of the bleeding time. The bleeding history, which is usually mild in PSD, was negative in patient III-1, probably because he had never been exposed to situations at risk for excessive bleeding owing to his young age.
ADP is a weak agonist because by itself it does not trigger
platelet secretion directly.27 When exogenous
ADP is added to a normal platelet suspension, it causes shape
change and aggregation. It is the close platelet-to-platelet
contact brought about by the aggregation process that, in a minority of
individuals, triggers the formation of trace amounts of
TxA2, which stimulates the secretion of small
amounts of -granule constituents and reinforces
aggregation.10 28 29 30 This effect is greatly enhanced and
can be observed in most individuals when the concentration of ionized
calcium in the extracellular medium is artificially decreased to the
micromolar level, such as in citrated PRP.28 30 On the
other hand, the mechanism that is responsible for the potentiation of
platelet secretion by ADP released from the platelet
-granules, which was demonstrated for the first time by our group in
1997,3 is quite different from that triggered by exogenous
ADP because it is independent of TxA2 synthesis
and the formation of large platelet aggregates.3 It is
likely that although ADP by itself cannot directly stimulate the
secretion of -granule constituents, it does potentiate platelet
secretion induced by other agonists. The results of the present
study and of our previous one3 indicate that the full
complement of platelet ADP receptors is necessary for the
potentiation of platelet secretion by ADP, whereas it is not
essential for normal aggregation.
Based on current knowledge, it is unknown whether the potentiation of platelet secretion by ADP is mediated solely by its interaction with P2CYC or also by the other platelet purinoceptors. In 2 preliminary reports of congenital defects of platelet P2Y131 and P2X132 receptors, both associated with abnormal platelet aggregation and a mild bleeding diathesis, platelet secretion was not studied. However, recent unpublished data in P2Y1 knockout mice34 (Gachet et al, 2000) and in human platelets incubated with selective P2Y1 and P2CYC antagonists (Cattaneo et al, 2000) indicate that the P2CYC receptor is the main receptor responsible for the potentiation of platelet secretion, suggesting that the Gi pathway cross-talks with other pathways of platelet activation. The molecular mechanisms for this are still unknown but could involve phosphoinositide 3-kinase activation,35 tyrosine kinases specifically linked to this receptor and/or the vasodilator-stimulated phosphoprotein.36 37 Thus, the patients described here should help in better defining the selective role of the P2CYC receptor in primary hemostasis.
In conclusion, our study describes 2 new patients with a congenital bleeding diathesis associated with a severe defect of platelet P2CYC receptors. In addition, the possibility of studying the son of 1 of the 2 patients, who is an obligate heterozygote for the disease, allowed us to confirm our previous hypothesis that some patients with the common PSD are heterozygotes for the disease and that the interaction of released ADP with the full complement of its platelet receptors is essential for normal platelet secretion.
Received March 14, 2000; accepted June 14, 2000.
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