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