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Keywords:

  • platelet dysfunction;
  • Ca2+ ionophore;
  • impaired utilization of intracellular Ca2+;
  • pathogenetic analysis

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.

Ca2+ ionophores such as A23187 and ionomycin, which transport divalent cations across membranes, were reported to induce platelet activation. A number of reports suggested that secretion and phospholipase C activation induced by Ca2+ ionophores are totally dependent upon intact thromboxane formation (Holmsen & Dangelmaier, 1981; Rittenhouse, 1984). This suggests that platelets with impaired thromboxane formation or an impaired response to thromboxane show defective Ca2+ ionophore-induced platelet aggregation. In fact, we previously reported that Ca2+ ionophore A23187-induced platelet aggregation was markedly reduced in patients with congenital platelet cyclo-oxygenase deficiency (Fuse, 1996) or with defective thromboxane A2 (TXA2)-induced platelet aggregation (Fuse et al, 1993, 1996). Regarding the latter disorder, we clarified that an Arg60 to Leu mutation in the first cytoplasmic loop of the TXA2 receptor (TXR) causes impaired coupling between TXR and phospholipase C activation (Hirata et al, 1994; Higuchi et al, 1999; Fuse et al, 2000).

Alternatively, several cases with bleeding tendencies whose platelets respond defectively to Ca2+ ionophores without those causes have been reported (Hattori et al, 1981; Hardisty et al, 1983; Machin et al, 1983; Rao et al, 1989). However, as these cases are rare, the defective site has not been fully elucidated.

Here, we report three patients with this type of platelet dysfunction and discuss the pathogenesis.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Patients Patient 1 was a 55-year-old woman, who has been reported previously (Hattori et al, 1981). She had a life-long history of mucosal bleeding and easy bruising, and had menorrhagia for over 30 years. The patient had a delivery when she was 24 years old and required a blood transfusion owing to excessive bleeding at that time. The family history revealed a possible tendency to bruise easily in her father and elder brother, but they could not be investigated because they had already died.

Patient 2 was an 18-year-old man who was evaluated because of excessive post-operative bleeding following appendectomy, which required a blood transfusion. He had a life-long history of easy bruising and bleeding from the gums when brushing his teeth. His elder brother may also have a history of easy brusing, but has not been evaluated.

Patient 3 was an 18-year-old woman with a life-long history of easy bruising, frequent epistaxis and bleeding from the gums when brushing her teeth. She has had menorrhagia for 6 years and had iron deficiency anaemia at the time of our evaluation. Her parents were unrelated and were both asymptomatic, with normal bleeding times and platelet aggregation and secretion studies.

None of these patients had episodes of major bleeding such as haematuria, gastrointestinal bleeding or haemarthrosis. The bleeding times, using a modified template method with Simplate 1, were 14·5 min, 12·5 min and > 15 min in patients 1,2, and 3 respectively (normal range: 3·5–9·5 min). The coagulation and fibrinolysis tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and fibrin degradation products (FDP) were all within normal limits.

Platelet aggregation study and adenosine triphosphate release Venous blood was collected into polyethylene tubes containing a 1:10 volume of 3·8% sodium citrate from the patients and healthy volunteers after at least 12 h of fasting. Platelet rich plasma (PRP) was obtained by centrifugation at 150 g for 10 min, and the platelet count in the PRP was adjusted to 300 × 109/l by dilution with autologous platelet-poor plasma (PPP). Aggregation induced using various agonists was measured in 200 μl of PRP by the turbidometric method described by Born (1962) in an NKK HEMA-tracer 1 aggregometer. Measurement of washed platelet aggregation induced by thrombin suspended in HEPES-Tyrode's buffer containing CaCl2 (1 mmol/l) was carried out as described previously (Fuse et al, 1986).

To measure the release of platelet adenosine triphosphate (ATP), 450 μl of citrated PRP and 50 μl of firefly lantern extract were pipetted into the cuvette of a Lumi-aggregometer Model 400 (Chrono-Log.). After 1 min, 50 μl of various agonists was added to induce platelet aggregation and the release of ATP. At the peak of ATP secretion of luminescent intensity, 1 μmol/l ATP was added to calibrate the amount of released ATP (Charo et al, 1977).

The stimulating agents used were adenosine diphosphate (ADP; Sigma), collagen (Horm), arachidonic acid (Sigma), STA2 (9,11-epithio-11, 12-methano-TXA2, a stable TXA2 mimetic; Ono Pharmaceutical; Katsura et al, 1983), A23187 (Calbiochem), ristocetin (Lundbeck) and thrombin (Yoshitomi).

Platelet adenine nucleotide contents The ATP and ADP content of platelets was determined by the method of Holmsen et al (1972), using the luciferin-luciferase technique.

Platelet membrane glycoprotein analysis Washed platelets in phophate-buffered saline (PBS) from the patients and normal controls were incubated with 10 μg/ml of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody for 1 h at 4°C, followed by washing with the suspension buffer three times and analysis on a Beckton-Dickinson FACScan cytometer. The monoclonal antibodies used were SP.2/0 (anti-GPIb monoclonal antibody), SZ.22 (anti-GPIIb monoclonal antibody) and SZ.21 (anti-GPIIIa monoclonal antibody). These monoclonal antibodies were purchased from Immunotech SA.

Platelet thromboxane B2 (TXB2) formation Washed platelet suspensions (5 × 108 platelets/ml) in Ca2+, Mg2+-free Tyrode's buffer were stimulated with thrombin (0·5 IU/ml), arachidonic acid (0·2 mmol/l) or STA2 (0·4 μmol/l) at 37°C for 3 min. The reaction was terminated with 0·03 ml of 1 mol/l HCl and then the platelets were pelleted by centrifugation at 1900 g for 10 min. The supernatant was neutralized with 1 mol/l Tris-base and TXB2 was assayed using a TXB2 radioimmunoassay (RIA) kit (Amersham).

Measurement of platelet cytosolic calcium The agonist-stimulated increase of platelet cytosolic-free calcium ([Ca2+]i) was determined in fura-2-loaded platelets. PRP was incubated with 3·3 μmol/l fura-2 AM (Molecular Probes) at 37°C for 30 min. The fura-2-loaded platelets were washed twice and suspended in modified HEPES-Tyrode's buffer (1 × 108 platelets/ml) (Poll & Westwick, 1986). Changes in fluorescence of the fura-2-loaded platelets at excitation wavelengths of 340 and 380 nm after addition of various agonists were recorded using a Kowa F-100 spectrofluorophotometer. The ratio of fluorescence at an excitation wavelengh of 340 nm to fluorescence at an excitation wavelength of 380 nm (relative fluorescence intensity) was a measure of the [Ca2+]i concentration (Grynkiewicz et al, 1985).

Measurement of inositol triphosphate production The synthesis of IP3 was assayed according to a previously published modified method (Palmer et al, 1989; Nakashima et al, 1991). PRP was prepared as described from blood collected in a 1:9 volume of 3·8% sodium citrate. Platelets were centrifuged (at 500 g for 10 min) and resuspended in Ca2+-free Tyrode's buffer (1 × 109 platelets/ml). The washed platelets (120 μl) were equilibrated to 37°C and stimulated with 30 μl of agonist (STA2, A23187 or thrombin) at 37°C for 10 s. The final concentrations (f.c.) of STA2, A23187 and thrombin were 0·4 μmol/l, 0·5 μmol/l and 0·2 IU/ml respectively. The reactions were terminated by the addition of 50 μl of ice-cold 10% HClO4. The reaction tubes were placed on ice for 30 min and centrifuged. The supernatants were neutralized and the amount of IP3 in the supernatant was determined according to the manufacturer's instructions (Amersham).

Phosphorylation of 20 kDa and 47 kDa protein Phosphorylation of platelet 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) was assessed as described previously (Fuse et al, 1986). Briefly, PRP from the patients and normal individuals were incubated with [32P]-PO4 (3·7 MBq/ml, New England Nuclear) for 90 min at 37°C. After the platelets were centrifuged and washed, the platelet pellet was resuspended in Tyrode's buffer. Aliquots of the platelet suspension (190 μl) were activated with 10 μl of thrombin (0·05 IU/ml, f.c.), A23187 (0·5 μmol/l, f.c.) or STA2 (0·4 μmol/l, f.c.). The reaction was terminated at 30 s and 60 s by the addition of 100 μl of sodium dodecyl sulphate (SDS) sampling buffer. After the samples were boiled at 100°C for 3 min, the labelled platelet proteins were subjected to 13·5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were dried and subjected to autoradiography using Kodak X-AR X-ray film. The bands corresponding to pleckstrin and MLC were excised, placed in liquid scintillation fluid and counted for radioactivity. The results were expressed as the fold of the basal radioactivity in pleckstrin and MLC.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Platelet aggregation, secretion studies, platelet adenine nucleotide contents and the expression of platelet membrane glycoproteins

The platelets of the patients showed abnormal aggregation and release responses to various stimuli, including ADP, collagen, arachidonic acid, STA2 and A23187. These responses were studied several times and the results were reproducible. Figure 1 shows typical aggregation tracings for the patients. The aggregation response to ADP was markedly reduced. Even 10 μmol/l ADP induced only the primary phase of aggregation and was completely reversible. The aggregation responses to collagen, arachidonic acid and STA2 were also much reduced. A23187-induced platelet aggregation was defective or dramatically reduced even at 20 μmol/l. However, thrombin- and ristocetin-induced platelet aggregation were always normal.

image

Figure 1. Typical platelet aggregation tracings of the patients' platelets. C, normal control; 1, patient 1; 2, patient 2; 3, patient 3.

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ATP was not released from the patients' platelets stimulated with arachidonic acid, STA2 or A23187. However, the amounts of ATP and ADP in the patient's platelets were all within normal ranges and the surface expression of glycoprotein Ib and IIb/IIIa was normal (data not shown).

Platelet TXB2 formation

TXB2 formation by the washed patients' platelets stimulated with arachidonic acid (0·2 mmol/l), thrombin (0·5 IU/ml) or STA2 (0·4 μmol/l) were all within normal ranges (data not shown).

Platelet cytoplasmic Ca2+ mobilization

The elevation of [Ca2+]i in response to thrombin, STA2 or A23187 was normal both in the presence and absence of extracellular Ca2+ in all patients' platelets (Table I).

Table I.  [Ca2+]i of fura-2-loaded platelets after stimulation with thrombin, STA2 and A23187 in the presence (1 mmol/l) or absence of extracellular Ca2+ (EGTA 2 mmol/l).
 ExtracellularPeak [Ca2+]i (μmol/l)  
AgonistCa2+Patient 1Patient 2Patient 3Normal controls
  1. The patient's values represent the means of two separate experiments. Normal controls represent mean Å ± SD, n = 16.

Thrombin (0·25 IU/ml)1 mmol/l 5·90 6·80 6·13 6·42 ± 2·35
 EGTA 5·32 4·56 4·02 4·17 ± 1·32
Thrombin (0·5 IU/ml)1 mmol/l 7·02 9·01 8·77 7·53 ± 3·05
 EGTA 5·51 5·59 5·14 4·86 ± 1·32
STA2 (0·2 μmol/l)1 mmol/l 6·78 6·56 7·78 6·57 ± 1·31
 EGTA 6·57 6·54 7·73 6·38 ± 1·57
STA2 (0·4 μmol/l)1 mmol/l 8·57 8·58 8·93 7·83 ± 2·54
 EGTA 7·04 7·15 7·57 7·08 ± 1·37
A23187 (0·25 μmol/l)1 mmol/l 8·74 8·05 9·17 8·57 ± 3·62
 EGTA 2·76 3·00 3·46 2·78 ± 1·36
A23187 (0·5 μmol/l)1 mmol/l10·1711·0511·3710·52 ± 3·34
 EGTA 4·78 4·96 6·15 5·04 ± 2·62

Formation of inositol triphosphate

In all patients' platelets, the formation of IP3 in response to thrombin, STA2 or A23187 were within normal ranges (Fig 2).

image

Figure 2. Agonist-induced inositol 1,4,5-triphosphate (IP3) formation in platelets. Washed platelet suspension from the patients or normal volunteers were stimulated with thrombin (0·2 IU/ml), STA2 (0·4 μmol/l) or A23187 (0·5 μmol/l) for 10 s. IP3 was extracted with trichloroacetic acid and measured using a radioreceptor assay (Amersham). The vertical bars represent mean ± SD (n = 4). Normal control, white bars; patient 1, black bars; patient 2, forward hatched bars; patient 3, back hatched bars.

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Phosphorylation of MLC and pleckstrin

The phosphorylation of MLC and pleckstrin in response to A23187 was normal in the patients' platelets (Figs 3 and 4). Furthermore, those induced by thrombin or STA2 were normal (data not shown).

image

Figure 3. Time course of A23187 (0·5 μmol/l)-induced pleckstrin (P-47) and MLC (P-20) phosphorylation in the patients' platelets. The results are expressed as the percentage increase of the basal (unstimulated) radioactivity. The shaded area represents mean ± 2SD in normal controls (n = 7). The mean basal radioactivity (in dpm) were as follows; normal subjects, 284 (range, 107–532); patient 1, 262 and 370; patient 2, 288 and 343; patient 3; 236 and 307. Patient 1, ▪––▪; patient 2, ●––●; patient 3, ○––○.

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image

Figure 4. Autoradiograph of [32P]-labelled platelets induced by A23187 (0·5 μmol/l) shows phosphorylation of P-20 (MLC) and P-47 (pleckstrin) in patient 1.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

In the present study, we have reported three patients with bleeding tendencies characterized by impaired aggregation to Ca2+ ionophore A23187. As platelet adenine nucleotide contents and arachidonic acid metabolism were normal in the patients' platelets, the defective platelet function was not caused by either a storage pool deficiency or by defective synthesis of TXA2/prostaglandin H2. Furthermore, the platelet aggregation responses and platelet membrane glycoprotein analysis ruled out Bernard–Soulier syndrome and Glanzmann's thrombasthenia.

The analysis of second messenger formation showed that IP3 formation induced by thrombin, STA2 or A23187 was normal, and that Ca2+ mobilizations induced by those agonists were also normal, both in the presence and absence of extracellular Ca2+. This suggests that the defective site in these patients' platelets lies beyond phospholipase C activation and Ca2+ mobilization. However, the phosphorylation of pleckstrin and MLC induced by the agonists was also normal in the patients' platelets. This suggests that the defective site lies in the process distal to or independent of these biochemical events.

Pathogenetically, two possible causes have been described in the platelet disorder characterized by defective Ca2+ ionophore-induced aggregation with normal TXA2 production. One is impaired intracellular Ca2+ mobilization and the other is impaired utilization of Ca2+. The patients whose platelet dysfunction was caused by the former aetiology were reported by Rao et al (1989). They described that, in two patients, mother and son, the peak [Ca2+]i following activation with ADP, platelet-activating factor (PAF), collagen, U46619 and thrombin were diminished in quin-2-loaded platelets and that the peak [Ca2+]i following thrombin stimulation was also diminished in aequorin-loaded platelets. Furthermore, in a patient reported by Machin et al (1983), washed platelet aggregation induced by A23187 was corrected by the addition of exogenous Ca2+, which also suggests the presence of an intracellular Ca2+ mobilization defect in the patient's platelets. However, as agonist-induced cytoplasmic Ca2+ mobilizations were normal in our patients' platelets, this pathogenesis can be excluded.

However, a patient whose platelet dysfunction was caused by the latter aetiology has been reported by Hardisty et al (1983). They reported a 16-year-old boy with Silver–Russel syndrome whose platelets showed defective Ca2+ ionophore-induced platelet aggregation despite a normal increase in cytoplasmic Ca2+. The causes of impaired utilization of intracellular Ca2+ mobilization can be further divided into two major categories. One is the defect of Ca2+-calmodulin-dependent MLC phosphorylation and the other is either distal to or independent of the above phenomenon. Although Hardisty et al (1983) did not show the Ca2+ ionophore-induced MLC phosphorylation in their study, they showed that the calmodulin content was normal in the patient's platelets. However, in the present patients' platelets, MLC phosphorylation induced by several agonists, including A23187, occurred normally. This suggests that the former aetiology can also be excluded in these patients' platelets.

Considering the defective site in the present patients' platelets, one candidate is an abnormal cytoskeleton assembly. Indeed, in animal platelets, Searcy et al (1994) investigated the defective site in Simmental cattle with a congenital, inherited bleeding disorder, whose platelets showed impaired ADP and Ca2+ ionophore-induced platelet aggregation, and showed that the platelet dysfunction was caused by an abnormal cytoskeleton assembly following calcium mobilization and MLC phosphorylation. Although this point should be investigated further in the present patients' platelets, it must be emphasized that thrombin-induced platelet aggregation was normal in our three patients. As similar findings were reported in the platelets from the patient with Silver–Russel syndrome described by Hardisty et al (1983) and those from bleeding Simmental cattle, it seems to be a common feature in this platelet disorder. Although it is not clear why this type of defect did not affect thrombin-induced platelet aggregation, it is probable that thrombin-induced post-Ca2+ mobilization events are different from other agonist-induced ones or the defect in this platelet disorder can be overcome or by-passed by other intracellular process when the platelets are challenged by thrombin. This point should be also investigated in the future.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References
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