© 1996 American Heart Association, Inc.
Articles |
Defective Thrombin-Induced Calcium Changes and Aggregation of Bernard-Soulier Platelets Are Not Associated With Deficient Moderate-Affinity Receptors
From the Departments of Oral Biology and Pharmacology, University of Manitoba, Winnipeg, Manitoba (A.M.), and the Departments of Laboratory Medicine (M.S.) and Medicine (R.Z., J.D.), University of Ottawa, Ottawa, Ontario, Canada.
Correspondence to Dr A. McNicol, Department of Oral Biology, University of Manitoba, 780 Bannatyne Ave, Winnipeg, Manitoba, R3E 0W2, Canada. E-mail mcnic@bldghsc.lan1.umanitoba.ca.
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Abstract |
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Abstract Cloning of the moderate-affinity, serpentine thrombin receptor has helped clarify the mechanism of thrombin-induced platelet activation. Proteolytic cleavage by thrombin generates a new amino terminal that autostimulates the receptor, leading to activation of multiple signaling pathways and the platelet response. The function of other thrombin receptors, such as high-affinity glycoprotein Ib (GPIb), on platelets and their relationships to the moderate-affinity receptor remain unclear. The present study examined the role of the moderate-affinity thrombin receptor in Bernard-Soulier syndrome (BSS) platelets, which contain low amounts of GPIb. Platelets from four BSS subjects displayed normal aggregation profiles and cytosolic calcium changes in response to moderate or high concentrations of thrombin. In contrast, the BSS platelet aggregation response was delayed and calcium changes were absent in response to low thrombin concentrations. Platelets from an asymptomatic BSS heterozygote displayed an activation profile similar to those of control individuals. Specific activation of the moderate-affinity receptor by a synthetic peptide caused similar aggregation in platelets from all individuals. The synthetic peptide also elicited calcium responses in BSS platelets. Platelets from the BSS subjects and from an individual with the May-Hegglin anomaly showed increased expression of the moderate-affinity thrombin receptor by flow-cytometric analyses. These results suggest that BSS platelets possess high levels of a functional moderate-affinity thrombin receptor, probably due to large platelet size, and provide indirect evidence that a high-affinity thrombin receptor is associated with GPIb.
Key Words: platelets • thrombin • TRAP • calcium • Bernard-Soulier syndrome
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Introduction |
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The serine protease thrombin is by far the most potent known platelet agonist, stimulating a wide range of biochemical and physical responses.1 The mechanism whereby a protease can specifically stimulate a cell has been the subject of debate for many years. However, the cloning of the moderate-affinity serpentine receptor has allowed partial characterization of the action of thrombin on platelets.2 3 Thrombin proteolytically cleaves the external region of the receptor to generate a new amino terminal called the tethered ligand. The tethered ligand in turn interacts with and autostimulates the receptor in the conventional manner.2 3 4 5 Synthetic peptides of amino acid sequence corresponding to the tethered ligand, termed TRAPs, mimic many of the effects of thrombin on platelets, including aggregation, secretion, calcium changes, phosphoinositide hydrolysis, phosphatidylinositol 3-kinase activity, and protein phosphorylation.2 5 6 7 8 9 10
Although most of the effects of thrombin on platelets are attributable to stimulation of the moderate-affinity receptor, a role for a high-affinity, nonproteolytic binding site has also been postulated. Several high-affinity thrombin-binding sites on platelets have been identified, including GPIb.11 12 The specific role of GPIb in thrombin-induced platelet activation is unclear, although a GPIb-dependent component of the process has been demonstrated.13 14
BSS, an inherited bleeding disorder,15 is characterized by thrombocytopenia, giant platelets, and mucocutaneous hemorrhages. The platelets of BSS individuals either lack, or have an abnormality in, the complex of GPs Ib, V, and IX.16 17 18 19 20 21 These platelets demonstrate delayed aggregation, secretion, phospholipase C activity, and cytosolic calcium changes in response to thrombin,22 23 24 25 consistent with a role for one or more of these GPs as a thrombin receptor.
Little is known about the role of the moderate-affinity thrombin receptor in BSS platelets, although a preliminary study has determined its presence on the surface of BSS platelets.26 Therefore, in the present study the presence of a moderate-affinity receptor on BSS platelets and the responses of these platelets to TRAP were examined.
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Methods |
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For the aggregation and cytosolic calcium studies, blood was collected by sterile venipuncture into ACD solution (3.8 mmol/L citric acid, 7.5 mmol/L trisodium citrate, and 125 mmol/L dextrose, with 1.9 mL anticoagulant per 8.1 mL blood) from BSS subjects, an M-HA subject, or healthy volunteers (control subjects). All subjects denied taking any medication known to interfere with platelet function within 2 weeks before blood collection.
PRP was obtained by centrifugation of blood from control subjects at 800g for 5 minutes27 or of BSS and M-HA blood at 150g for 10 minutes.20 Plasma-free platelet suspensions were obtained by centrifugation of PRP at 800g for 15 minutes, and the resultant pellet was resuspended in 1 plasma volume of HEPES-buffered Tyrode's solution (in millimoles per liter, 134 NaCl, 12 NaHCO3, 2.9 KCl, 0.34 Na2HPO4, 1 MgCl2, 10 HEPES, and 5 dextrose, together with 0.3% BSA, pH 7.4). Platelet aggregation in response to the agonists was monitored photometrically as described.27 In each case baseline values were set by comparison with a buffer blank, representing complete aggregation.
Cytosolic calcium changes were monitored by the method of Pollock and colleagues28 as reported.27 In brief, PRP was incubated with 3 µmol/L fura 2-AM for 45 minutes at 37°C. The platelets were then washed by centrifugation and resuspended at a concentration of 7.5x1010/L in physiological saline.28 Fluorescence levels were determined at 339 nm excitation and 500 nm emission and converted to cytosolic calcium levels.28
Flow-cytometric analysis of the platelet thrombin receptor was performed in an Epics Profile II Analyzer (Coulter Electronics) with an anti–thrombin receptor monoclonal antibody that recognizes the cleavage site of intact receptors.4 29 PRP was obtained from fresh blood collected in EDTA by gravity sedimentation for 20 minutes to obviate the loss of large platelets. A 15-µL aliquot of patient or control subject PRP was incubated for 30 minutes at room temperature with diluted anti–thrombin receptor monoclonal antibody (final concentration, 1:1000) in Tyrode's buffer. The platelets were pelleted by centrifugation at 500g for 10 minutes and washed once in Tyrode's buffer. Samples were centrifuged again and the supernatant was discarded. The pellet was suspended in 117 µL Tyrode's buffer, and 3 µL FITC-conjugated goat anti-mouse antibody was added. After a 20-minute incubation at room temperature the samples were run in the flow cytometer. Control samples that had been incubated with the secondary antibody only were included. The bitmap was drawn to include the platelet population. Fluorescence intensity was plotted on the x axis and full-scale counts on the y axis. The flow rate was 70 cells/s and 15 000 cells were analyzed for light-scatter and green fluorescence signals.
Materials
The anti–thrombin receptor antibody was a kind gift from Dr
L.F. Brass (University of Pennsylvania). The TRAP (single-letter
amino acid code, SFLLRN) was synthesized by Dr D. Litchfield
(University of Manitoba). Thrombin was obtained from Sigma Chemical Co
and fura 2-AM from Molecular Probes. All other reagents were of the
highest purity available.
Patients
The BSS subjects have been described in detail. M.R.H.
(proposita), M.R., and Mad.H. are symptomatic sisters in a
large French-Canadian family. I.R. is the asymptomatic
daughter of M.R.19 20 N.O. is an unrelated BSS
subject.25
E.L. is an unrelated individual with M-HA anomaly. She has experienced lifelong bruising and epistaxis and has suffered from menorrhagia. Laboratory studies have shown thrombocytopenia (3.0 to 6.0x1010 platelets/L), giant platelets, and neutrophils with Döhle bodies.
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Results |
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Aggregation studies with all BSS platelets (M.R.H., M.R., Mad.H., and N.O.) displayed slightly delayed aggregation profiles at thrombin concentrations down to 0.01 U/mL. At 0.005 U/mL thrombin, aggregation of BSS platelets was dramatically reduced at 2 minutes after addition of the agonist (Fig 1
), although normal
aggregation was observed at 7 to 10 minutes. Platelets from I.R.
showed an aggregation profile similar to that of control platelets
at all thrombin concentrations tested (0.005 to 0.1 U/mL; Fig 1).
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) to washed platelet suspensions (containing 1 mmol/L
CaCl2) and aggregation monitored continuously as an
increase in light transmission. M.R., M.R.H., Mad.H., and N.O. are BSS
individuals; I.R. is the asymptomatic daughter of M.R.
Results are a typical tracing of one to three similar experiments with
BSS and matched control platelets.
The TRAP (2 to 5 µmol/L) caused similar aggregation responses in all
BSS patients and control individuals (Fig 2). Rapid
maximal aggregation was normally obtained at 5 µmol/L TRAP, although
some day-to-day and interdonor variations were observed.
Concentrations of TRAP <2 µmol/L failed to cause aggregation in
control, I.R., or BSS platelets.
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Cytosolic calcium changes were monitored with fura 2. Resting levels of
cytosolic calcium were 90±4 nmol/L, consistent with previous
reports for this method,27 28 and both thrombin and TRAP
caused concentration-dependent increases in cytosolic calcium in
control platelets (data not shown). TRAP (5 µmol/L) and higher
thrombin concentrations (0.03 U/mL) caused elevations in cytosolic
calcium levels in BSS, I.R., and control platelets (Table 1). With TRAP, the BSS platelet response was lower
than that in control platelets. The lower thrombin concentration
(0.01 U/mL) raised cytosolic calcium levels in control and I.R.
platelets, but there was no detectable increase in BSS
platelets from M.R., M.R.H., Mad.H., or N.O. (Table 1).
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The presence of the moderate-affinity thrombin receptor was
examined by flow cytometry. In each case nonspecific
fluorescence was assessed by using nonimmune serum.
Representative histograms illustrating the
fluorescence intensity of platelets are shown in Fig 3
and the mean channel numbers and nonspecific
fluorescence in Table 2. The mean channel number
for control platelets was similar to that for platelets from
I.R. whereas the values for platelets from the BSS patients were
higher (Table 2). Therefore, there was an apparent rightward shift of
the fluorescence pattern for BSS compared with control
platelets, which may be consistent with increased
expression of the thrombin receptor on platelets from BSS
individuals.
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To investigate any effect of platelet size on the
fluorescence profile, one M-HA patient was also studied. M-HA
is associated with large platelets although there is no abnormality
in membrane GPIb.30 The M-HA platelets had a
pronounced rightward shift in their fluorescence pattern (Table 2
) but showed normal aggregation responses to thrombin and TRAP (data
not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The identity and function of thrombin receptors on human platelets have been the subject of interest for many years. Cloning of the moderate-affinity thrombin receptor has helped elucidate a proteolytic mechanism for platelet activation.1 2 3 4 5 6 7 8 9 10 However, there is also substantial evidence for a nonproteolytic mechanism of thrombin-induced activation, potentially through a high-affinity receptor.1 7 13 14 31 32 Although direct evidence from receptor-binding studies is lacking,33 indirect evidence from functional studies implicates GPIb as a prime candidate for this role.11 12 32 34 35 36 37 38 Platelets from BSS individuals have either aberrant levels or an aberrant structure of GPIb15 16 17 19 20 21 and have been used to examine the potential role of GPIb in thrombin-induced platelet activation.
In contrast, a potential defect in the moderate-affinity thrombin receptor in BSS platelets has not been thoroughly addressed. In the present study platelets from 3 related BSS subjects, 1 related BSS heterozygote, and 1 unrelated BSS individual were examined. The BSS platelets did not show decreased expression of the moderate-affinity thrombin receptor as assessed by flow cytometry. Indeed, platelets from the BSS individuals may have increased levels of the moderate-affinity receptor, although the significance of this is unknown. Comparison with platelets from an M-HA patient (in whom there are no apparent GP abnormalities) shows a similar apparent increase in receptor levels compared with control platelets. Therefore, it is probable that in both conditions platelets express more moderate-affinity receptors owing to their large size.
The specific moderate-affinity thrombin receptor agonist TRAP increased cytosolic calcium levels in, and aggregation of, platelets from BSS individuals. Together with the flow-cytometry results, the data are consistent with the notion that BSS platelets contain a functional moderate-affinity thrombin receptor, consistent with findings in a preliminary report.26
Platelets from each BSS subject demonstrated an abnormal aggregation response to low concentrations of thrombin, as had been reported.22 23 24 Similarly, there were no detectable changes in cytosolic calcium levels in BSS platelets that had been stimulated with low thrombin concentrations, although changes in calcium levels were observed in response to higher concentrations of thrombin. These observations are consistent with the notion that BSS platelets lack a high-affinity thrombin receptor and provide indirect evidence that the receptor is GPIb.
The function of the high-affinity thrombin receptor is unclear. Some studies have suggested that GPIb is not directly linked to signal transduction mechanisms24 and that the high-affinity receptor merely accelerates the action of moderate-affinity receptors.39 However, these observations contradict several other studies in which biochemical processes have been found to be associated with the high-affinity rather than the moderate-affinity autoproteolytic thrombin receptors.7 25 31 32 40 41 It is interesting to note that recent studies have indicated subtle differences in platelet responses to thrombin and TRAP at the biochemical level.10 42 These data may suggest a role for the high-affinity receptor in thrombin-induced activation, which is not mimicked by TRAP. In addition, Sindt and colleagues,32 using pertussis toxin, have provided evidence that GPIb is linked to platelet calcium changes, secretion, and aggregation. These authors did not address the specific second-messenger system activated. However, phospholipase C–mediated phosphoinositide metabolism is a possibility. Phospholipase C activity in response to thrombin was significantly reduced in platelets from several of the BSS individuals reported in the present study, although this was at least partially due to a signaling defect common to several agonists.25
In addition, Kramer and colleagues42 have recently demonstrated that the phosphorylation and activation of the cytosolic form of phospholipase A2 differs in platelets stimulated with thrombin from those stimulated with TRAP. One potential explanation for this observation is that thrombin and TRAP act via different receptors to stimulate phospholipase A2, and indeed platelet activation by TRAP appears to be more susceptible to inhibition by cyclooxygenase inhibitors than is thrombin (A. McNicol and C.A. Robson, unpublished observations, 1995).
In summary, the present study demonstrates the presence of the moderate-affinity thrombin receptor on the surface of BSS platelets. Activation of this receptor results in full platelet activation. In contrast, these data are consistent with a deficiency of the high-affinity thrombin receptor on BSS platelets, which fulfills an important function in platelet second-messenger generation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was funded by grants from the Heart and Stroke Foundation of Manitoba and the Children's Hospital of Winnipeg Research Foundation (to A.M.) and the Heart and Stroke Foundation of Ontario (to J.D.). The anti–thrombin receptor antibody was a kind gift from Dr L.F. Brass (University of Pennsylvania).
Received April 27, 1995; accepted January 23, 1996.
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