Guangfen Mao

Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region -729/-542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.

RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency.

Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonist-induced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at -1498, -1491, -708, -526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction.

Mechanism of Platelet Factor 4 (PF4) Deficiency with RUNX1 Haplodeficiency: RUNX1 is a Transcriptional Regulator of PF4

Background: Platelet factor 4 (PF4) is an abundant protein stored in platelet α‐granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of α‐granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non‐allelic variant PF4V1. Objectives: To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/results: Chromatin immunoprecipitation and gel‐shift assays with phorbol 12‐myristate 13‐acetate‐treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at −1774/−1769 and −157/−152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions: Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1‐regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in α‐granule deficiency.

Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation PRKCQ Is a Transcriptional Target of RUNX1

Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-&thgr; (PKC-&thgr;, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-&thgr; in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-&thgr; protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-&thgr; protein were decreased by short interfering RNA knockdown of RUNX1. Conclusion—Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-&thgr; deficiency associated with RUNX1 haplodeficiency.Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-θ in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-θ protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overex...

Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation

Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-&thgr; (PKC-&thgr;, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-&thgr; in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-&thgr; protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-&thgr; protein were decreased by short interfering RNA knockdown of RUNX1. Conclusion—Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-&thgr; deficiency associated with RUNX1 haplodeficiency.Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-θ in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-θ protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overex...

Alternative splice variants of phospholipase C-β2 are expressed in platelets: Effect on Gαq-dependent activation and localization

Phospholipase C (PLC) β2 plays a pivotal role in G-protein dependent signal transduction in platelets. We have previously demonstrated in platelets, leukocytes and human erythroleukemia cells the presence of transcripts of two forms of PLC-β2 generated by alternative splicing. They differ by 45 nucleotides in the carboxyl-terminal region and are designated as PLC-β2a and PLC-β2b, with and without by 15 amino acid residues (corresponding to 864–878). The presence of the two variants has not been shown at the protein level in cells. Moreover, the carboxy-terminal region of PLC-β has been implicated in Gαq activation, particulate association, and nuclear localization, suggesting that the PLC-β2 splice variants may be regulated differentially. We demonstrate for the first time that both PLC-β2 isoforms are expressed in platelets at the protein level. Studies in CV-1 cells transfected with PLC-β2a or β2b cDNAs, along with constitutively activated Gαq (Q209L), showed that inositolphosphate formation was comparable between the two variants. However, the nuclear localization of the two isoforms was different with a higher cytoplasmic to nuclear ratio for PLC-β2b compared to PLC-β2a, suggesting that a great proportion of the total PLC-β2a was in the nucleus relative to PLC-β2b. There was no difference in the relative distribution of the two variants between the cytosol and particulate fractions. Both PLC-β2 alternative splice variants are expressed at the protein level in platelets. In transfected CV-1 cells, PLC-β2a is relatively more enriched in the nuclei than PLC-β2b suggesting that the two variants may have different effects in cell proliferation and differentiation.

Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency.

Essentials Platelet dense granule (DG) deficiency is a major abnormality in RUNX1 haplodeficiency patients. The molecular mechanisms leading to the platelet DG deficiency are unknown. Platelet expression of PLDN (BLOC1S6, pallidin), involved in DG biogenesis, is regulated by RUNX1. Downregulation of PLDN is a mechanism for DG deficiency in RUNX1 haplodeficiency.

Alterations in insulin-signaling and coagulation pathways in platelets during hyperglycemia-hyperinsulinemia in healthy non-diabetic subject

INTRODUCTION Diabetes mellitus (DM) is a prothrombotic and proinflammatory state. Hyperglycemia (HG) is encountered even in patients without DM. We have shown that combined HG and hyperinsulinemia (HI) in healthy non-diabetic subjects increased circulating tissue factor (TF) and thrombin generation. To understand the changes in platelet and monocyte pathways induced by combined HG and HI in healthy non-diabetic state, we performed whole genome expression profiling of leukocyte-depleted platelets and monocytes before and after 24 hours of combined HG (glucose ~200mg/dL) and HI by glucose infusion clamp in a healthy non-diabetic subject. RESULTS We defined time-dependent differential mRNA expression (24 versus 0 hour fold change (FC) ≥ 2) common to platelets and monocytes. Ingenuity Pathways Analysis revealed alterations in canonical insulin receptor signaling and coagulation pathways. A preliminary group of 9 differentially expressed genes was selected for qRT-PCR confirmation. Platelet 24 hour sample was compared to the 0 hour sample plus 4 controls. Five transcripts in platelets and 6 in monocytes were confirmed. Platelet GSK3B and PTPN1 were upregulated, and STXBP4 was downregulated in insulin signaling, and F3 and TFPI were upregulated in coagulation pathways. Monocyte, PIK3C3, PTPN11 and TFPI were downregulated. Platelet GSKβ3 and PTPN11 protein and TF antigen in platelets and monocytes was increased. CONCLUSIONS Even in non-diabetic state, HG+HI for 24 hours induces changes in platelets and monocytes. They suggest downregulation of insulin signaling and upregulation of TF. Further studies are needed to elucidate cellular alterations leading to the prothrombotic and proinflammatory state in DM.

Platelet PKC-θ deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1

Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-&thgr; (PKC-&thgr;, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-&thgr; in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-&thgr; protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-&thgr; protein were decreased by short interfering RNA knockdown of RUNX1. Conclusion—Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-&thgr; deficiency associated with RUNX1 haplodeficiency.Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-θ in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-θ protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overex...

Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events Differential Effects of RUNX1 Variants

Background: PCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls. Methods: We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease. Results: Platelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6–2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression. Conclusions: PCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.