Decreased Platelet Expression of MYL9 (Myosin Regulatory Light Chain Polypeptide) and Other Genes with Platelets Dysfunction and CBFA2 Mutation: Insights from Platelet Expression Profiling.

We have previously reported (Blood, 87:1368, 1996) a patient with a mild thrombocytopenia, and markedly abnormal aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and activation of GPIIb-IIIa complex. We recently showed (Blood 2004, 103: 948–954) in this patient a heterozygous mutation in hematopoietic transcription factor CBFA2 (Core Binding factor A2, also called AML1) and decreased platelet PKC-theta. We postulated that platelet expression profiling would provide insights into the pathophysiological mechanisms and gene expression changes resulting from a CBFA2 mutation. We performed platelet expression profiling in 4 control subjects (6 profiles, 2 individuals profiled twice) and the patient (profiled twice, 10 months apart) using the Affymetrix microarray Human U133A and B GeneChips (total ~44000 probe sets). ~10,000 probe sets (genes and ESTs) are expressed in human platelets. Iterative comparisons of data from each individual platelet profiles had correlation coefficients (r) > 0.85. Using GeneSpring® analytical software we selected genes that showed at least 1 present call in the 8 profiles, and p 2 were considered as significant changes. In the patient, 298 probe sets were significantly down regulated and 29 significantly upregulated. We focused on genes showing a > 5-fold decrease (fold change, fc < = 0.20). There was a striking ~75 fold decrease (fc = 0.013; p=0.00022) in myosin regulatory light chain polypeptide (MYL9 gene). Other down-regulated genes included calcium binding protein 5 (CABP5, fc = 0.02; a protein with similarity to calmodulin), Na/K/Ca exchanger member 3 (SLC24A3, fc = 0.11), b3-endonexin (ITGB3BP, fc = 0.01), platelet factor 4 variant 1 (PF4V1, fc = 0.03), chemokine CXCL5 (ENA-78) (fc = 0.20), tropomyosin I (alpha) (TPM1, fc = 0.13) and arachidonate 12-lipoxygenase (ALOX-12, fc = 0.22). The expression array findings were validated by decreased platelet mRNA levels on real time PCR or by immunoblotting (platelet PF4). Several down-regulated genes are directly relevant to the patient’s platelet defect. Agonist-stimulated MLC phosphorylation is impaired in patient platelets. The strikingly decreased expression of MYL9 with normal expression of MLC kinase (MLCK, by array and real time PCR) suggests that the blunted myosin phosphorylation arises from decreased MYL9 than due to decreased MLCK. Myosin light chains play a role in diverse processes including in cell contractility, mobility and cytokinesis, and are relevant to impaired platelet function and production. The decreased expression of the Na/K/Ca exchanger and b3-endonexin 3 (both implicated in activation of GPIIb-IIIa) provide mechanisms for the diminished GPIIb-IIIa activation. Chemokine CXCL5 and PF4V1 are expressed in megakaryocytes, and the genes are colocalized to “chemokine cluster” on chromosome 4. 12-lipoxygenase, a major platelet enzyme, regulates arachidonic acid metabolism and production of the 12-hydroxy eicosanoids. This is the first proof of concept that platelet expression profiling can be applied to obtain new insights into the molecular basis of inherited platelet dysfunction. Further studies will provide new information on the genes regulated by CBFA2, and on mechanisms regulating platelet production and activation.