Association of CBFA2 mutation with decreased platelet PKC-θ and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation

Abstract

The mechanisms by which agonists activate glycoprotein (GP) IIb-IIIa function remain unclear. We have reported data on a patient with thrombocytopenia and impaired receptor-mediated aggregation, phosphorylation of pleckstrin (a protein kinase C [PKC] substrate), and activation of the GPIIb-IIIa complex. Abnormalities in hematopoietic transcription factors have been associated with thrombocytopenia and platelet dysfunction. To define the molecular mechanisms, we amplified from patient platelet RNA exons 3 to 6 of core-binding factor A2 (CBFA2) cDNA, which encompasses the DNA-binding Runt domain; a 13-nucleotide (nt) deletion was found (796-808 nt). The gDNA revealed a heterozygous mutation (G>T) in intron 3 at the splice acceptor site for exon 4, leading to a frameshift with premature termination in the Runt domain. On immunoblotting, platelet CBFA2, PKC-, albumin, and IgG were decreased, but pleckstrin, PKC-␣, -␤I, -␤II, -, -⑀, -␦, and -, and fibrinogen were normal. Our conclusions are that (1) CBFA2 mutation is associated with not only thrombocytopenia, but also impaired platelet protein phosphorylation and GPIIb-IIIa activation; (2) proteins regulated by CBFA2 are required for inside-out signal transduction-dependent activation of GPIIbIIIa; and (3) we have documented the first deficiency of a human PKC isozyme (PKC-), suggesting a major role of this isozyme in platelet production and function. IntroductionSignal transduction-dependent activation of integrin glycoprotein (GP) IIb-IIIa complex and subsequent fibrinogen binding is a prerequisite for platelet aggregation on exposure to receptormediated agonists. However, the molecular mechanisms by which GPIIb-IIIa function is regulated on cell activation are not fully understood. We have previously reported 1 detailed studies in a patient with lifelong mucocutaneous bleeding manifestations, mild thrombocytopenia, and markedly abnormal platelet aggregation (including in primary wave) and dense granule secretion in response to multiple agonists. The patient's father and grandfather also had a history of easy bruising, and the father (who had thrombocytopenia) had died of acute leukemia. Phosphorylation of pleckstrin (a protein kinase C [PKC] substrate) and myosin light chain (MLC) was diminished in the patient's platelets on activation with platelet-activating factor (PAF) and thrombin. PKC activation has been linked to insideout signal transduction-dependent activation of GPIIb-IIIa on platelets. 2,3 In detailed studies in this patient, we demonstrated by flow cytometry 1 that the platelets had a full complement of GPIIb-IIIa complexes but receptor-mediated signal transductiondependent activation of GPIIb-IIIa was impaired on stimulation with PAF, adenosine diphosphate (ADP), and protease-activated receptor 1 (PAR-1) agonist SFLLRN. The expression of ligandinduced binding sites (LIBSs) induced by a number of agonists was normal. We concluded 1 that the patient's platelets had a defect in GPIIb-IIIa activation due to an upstream defect in the signaling mechanisms...

Familial myelodysplastic syndromes: a review of the literature

“…Both platelet storage pool deficiency 14 and impaired GPIIbIIIa activation 12 have been described. Importantly, the presence of thrombocytopenia is not mandatory for a diagnosis of FPD/AML, as some affected individuals have displayed normal platelet counts, and could, therefore, escape detection within an affected family.…”

Toward individualized dosing in pediatric ALL

“…The St. Jude's group has already shown that individualized dosing of methotrexate improves outcome in pediatric ALL. 5 Decreased platelet Mpl receptor in AML1 haplodeficiency: another piece of the puzzle…”

“…4 PKC-⑀ cooperates with the transcription factor GATA-1 in the activation of the glycoprotein IIb (GPIIb) promoter. 3 Of particular relevance, platelet PKC-expression (protein and mRNA) has been reported to be decreased in AML1 haplodeficiency, 5 suggesting a potential mechanism for the impaired megakaryopoiesis and platelet production. This PKC deficiency has also been associated with impaired platelet responses to activation, including in protein phosphorylation and activation of GPIIb-IIIa.…”

A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies

“…Genetic and molecular studies reveal that all reported pedigrees of FPD/MM display a mutation in the RUNX1 gene or LOH at the locus. [5][6][7][8][9][10] In this report, we present a Japanese family with FPD/MM and a novel RUNX1 mutation.…”

“…So far, 13 pedigrees have been reported, mainly in European countries. [5][6][7][8][9][10] Clinically, the disorder is characterized by moderate thrombocytopenia from birth, impaired platelet function and a propensity to develop myeloid malignancies. Genetic and molecular studies reveal that all reported pedigrees of FPD/MM display a mutation in the RUNX1 gene or LOH at the locus.…”