Pleckstrin Phosphorylation Research Articles

Protein Kinase C-Dependent Phosphorylation of Human Pleckstrin Reduces its Capacity for Self-Association and Impairs its Ability to Bind Phosphoinositide

Pleckstrin is a major substrate of protein kinase C in lymphocytes, macrophages, monocytes, granulocytes and platelets. In these cells, pleckstrin is expressed at high levels and represents approximately 1 % of total cellular protein. Pleckstrin plays an important role in protein kinase C-dependent secretion, and abberant pleckstrin phosphorylation has been associated with disease. The mechanism, however, by which phosphorylation regulates pleckstrin function is unknown. Here, we show that native pleckstrin self-associates to form dimers that reduce its binding capacity to phosphoinositides. Phosphomimetic amino acid substitutions at residues normally phosphorylated by protein kinase C result in significantly reduced pleckstrin dimerization. Although phosphomimetic forms of pleckstrin exhibit subtle conformational changes they retain most of their phosphoinositide binding properties. These findings suggest that phosphorylation regulates pleckstrin interaction with membranes via a dimerization-dependent mechanism.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Inhibitory Effect of 20(S)-Ginsenoside Rg3 on Human Platelet Aggregation and Intracellular Ca2+ Levels via Cyclic Adenosine Monophosphate Dependent Manner.

Intracellular Ca2+ ([Ca2+]i) induces platelet aggregation, and influences the activation of aggregation associated-molecules. The increased [Ca2+]i activates both the Ca2+/calmodulin-dependent phosphorylation of the myosin light chain and the diacylglycerol-dependent phosphorylation of pleckstrin to trigger granule secretion (i.e., dense body and α-granule) and platelet aggregation. This study was carried out to elucidate the antagonistic effect of 20(S)-ginsenoside Rg3 (G-Rg3) present in Panax ginseng Mayer on Ca2+. G-Rg3 inhibited thrombin-induced human platelet aggregation in a dose-dependent manner and suppressed thrombin-induced elevation of [Ca2+]i mobilization. G-Rg3 increased the levels of cAMP, and subsequently, elevated the phosphorylation of inositol 1,4,5-triphosphate receptor I (Ser1756) during thrombin-induced human platelet aggregation. Moreover, G-Rg3 inhibited thapsigargin-induced Ca2+ influx and the thrombin-induced elevation of extracellular signal-regulated kinase 2 phosphorylation. G-Rg3 exhibited an inhibitory effect on [Ca2+]i levels leading to granule release and thus a therapeutic potential against platelet-mediated thrombotic disease is suggested.

Transcription Factor RUNX1 Regulates Pctp (Phosphatidylcholine Transfer Protein) in Platelets: A Potential Role in Regulating Platelet Function

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a critical hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, numerous platelet function defects, and increased leukemia risk. We have previously described a patient with a heterozygous RUNX1 nonsense mutation in the conserved runt domain necessary for DNA binding (Sun et al Blood 2004;103;948-54). The patient's platelet abnormalities included impaired aggregation and secretion in response to multiple agonists, granule deficiency, and decreased phosphorylation of myosin light chain and pleckstrin, activation of GPIIb-IIIa, 12-HETE production, and platelet protein kinase C-θ. Transcript expression profiling of patient platelets (Sun et al, J Thromb Haemost 2007;5:146-54) showed numerous genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP is a member of the START (Steroidogenic Acute Regulatory Protein-Related Transfer) domain superfamily and is responsible for the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Several findings indicate that PCTP is important for platelet function. PC is the main fraction of platelet phospholipids and source of arachidonic acid (AA) upon activation. Release of free AA from PC is the rate-limiting step in thromboxane production. PC is also a substrate for phospholipase D, which yields phosphatidic acid that can generate the second messenger diacylglycerol. Importantly, platelet PCTP has recently been demonstrated to have a major role in the racial differences in platelet responses - increased PCTP expression has been linked to the higher platelet aggregation and calcium mobilization induced by thrombin receptor protease-activated receptor 4 (PAR4) in blacks as compared to whites (Edelstein et al Nat Med 2013;19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to 995 bp of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding sites 1 (-232/-227) and 2 (-345/-340), at site 3 (-519/-514), and encompassing sites 4 and 5 (-861/-856, -884/-879). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-232/-227) and both sites 4 and 5 (-861/-856, -884/-879). To assess the effect of modulating RUNX1 expression on PCTP expression, PMA-treated HEL cells were transfected with RUNX1 overexpression plasmid or siRNA. PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown, suggesting that PCTP is regulated by RUNX1.Conclusions: Our results provide evidence that PCTP is a direct transcriptional target of and regulated by RUNX1, and a cogent molecular mechanism for downregulation of platelet PCTP in our patient with RUNX1 haplodeficiency. Regulation of PCTP by RUNX1 may be relevant to the platelet dysfunction in RUNX1 haplodeficiency as well as to racial differences in platelet responses linked to the differential platelet expression of PCTP. DisclosuresNo relevant conflicts of interest to declare.

Activation of phosphatidylinositol 3-kinase β by the platelet collagen receptors integrin α2β1 and GPVI: The role of Pyk2 and c-Cbl

Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kβ upon recruitment of the two main platelet collagen receptors, integrin α2β1 and GPVI. PI3Kβ-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2β1, but occurred normally upon GPVI ligation. Integrin α2β1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kβ activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KβKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kβ activity also hampered GPVI-induced tyrosine–phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kβ inhibitor TGX-221. These results outline important differences in the regulation of PI3Kβ by GPVI and integrin α2β1 and suggest that inhibition of Pyk2 may target PI3Kβ activation in a selective context of platelet stimulation.

Characterization of UBO-QIC as a Gαq inhibitor in platelets

Gαq plays an important role in platelet activation by agonists such as thrombin, adenosine diphosphate (ADP) and thromboxane. The significance of Gαq signaling in platelets was established using YM254890, a Gαq/11-specific inhibitor and Gαq knockout murine platelets. However, YM-254890 is no longer available for investigators and there is a need to characterize other Gαq inhibitors. The aim of this study is to characterize the specificity of a compound, {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)} (UBO-QIC), as a Gαq inhibitor in platelets. Human platelets treated with UBO-QIC showed a concentration-dependent inhibition of platelet aggregation and secretion by protease-activated receptors (PAR) agonists, U46619 and ADP. UBO-QIC also abolished Gαq pathway signaling events such as calcium mobilization and pleckstrin phosphorylation. UBO-QIC had no nonspecific effects on the Gα12/13 pathway since platelet shape change was intact in Gαq knockout murine platelets stimulated with PAR agonists in the presence of the inhibitor. In addition, UBO-QIC-treated platelets did not affect collagen-related peptide-induced platelet activation suggesting that this inhibitor had no non-specific effects on the GPVI pathway. Furthermore, Akt phosphorylation downstream of the Gαi and Gαz pathways, and vasodilator-stimulated phosphoprotein phosphorylation downstream of the Gαs pathway were not inhibited in UBO-QIC-treated platelets. UBO-QIC is a specific inhibitor for Gαq, which can be a useful tool for investigating Gαq-coupled receptor signaling pathways in platelets.

RUNX1 Regulates PLDN (Pallidin) Expression In Platelets: A Potential Mechanism For Dense Granule Deficiency In RUNX1 Haplodeficiency

Transcription factor RUNX1 plays a major role in hematopoiesis. Haplodeficiency of RUNX1 is associated with familial thrombocytopenia, impaired platelet function and megakaryopoiesis, and predisposition to acute myeloid leukemia. Platelet abnormalities include impaired aggregation, secretion, protein phosphorylation, and activation of GPIIb-IIIa on platelet activation. Dense granule deficiency, either alone or in combination with alpha granule deficiency, has been reported in patients with RUNX1 haplodeficiency, but the mechanisms involved are unknown. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood 87:1368-76, 1996) associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood 103: 948-54, 2004). The platelets showed impaired aggregation, dense granule secretion, phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-θ. Platelet mRNA expression profiling showed downregulation of several genes, including myosin light chain 9 (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRKB), and 12-lipoxygenase (ALOX12) in the patient compared to 6 normal subjects (Sun et al, J. Thromb Haemost. 5: 146-54, 2007). (We have shown that these genes are direct transcriptional targets of RUNX1.) In addition, PLDN, which encodes for protein pallidin, was four-fold down-regulated in platelets (fold change 0.239, p=0.029). In mouse model pallid, knock out of pldn leads to dense granule deficiency. Recent reports document mutations in PLDN in human subjects with platelet dense granule deficiency and the Hermansky-Pudlak syndrome. Pallidin is one of 8 subnunits that constitute the BLOC-1 (biogenesis of lysosome-related organelles complex-1), which plays a major role in granule/vesicle biogenesis. PLDN has two known human transcripts; PLDN1 is expressed ubiquitously, with exception of brain, while PLDN2 is expressed in brain, testes and leukocytes. We have addressed that PLDN is a direct transcriptional target of RUNX1 and hypothesize that its decreased expression constitutes a mechanism for the platelet dense granule deficiency in patients with RUNX1 haplodeficiency.We validated the decreased expression of PLDN on platelet profiling by quantitative real-time PCR - PLDN1 mRNA expression was decreased by 16-fold in the patient relative to normal subjects. We studied the regulation of PLDN1 by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. In silico analyses revealed the presence of 6 RUNX1 consensus binding sites in 2288bp of PLDN1 5' upstream region from ATG. To determine endogenous interaction of RUNX1 with PLDN promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. There was RUNX1 binding to PLDN chromatin at regions encompassing the putative RUNX1 binding site 1 (-184 to -179 bp) and site 3 (-1370/-1365 bp). We performed electrophoretic mobility shift assay (EMSA) using probes with RUNX1 motifs and PMA-treated HEL cell nuclear extracts. With 30–34 bp probes encompassing site 1 (-184 to -179 bp) and site 3 (-1370 to -1365 bp), protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. To study the functional effect of the binding sites, the wild type PLDN1 promoter construct –2288/-2 bp containing the putative RUNX1 sites or mutant constructs with each site individually mutated were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Mutation of RUNX1 sites 1 and 3 individually caused 60-70% reduction in promoter activity indicating that these sites were functional. Studies on the other RUNX1 consensus sites in PLDN promoter are underway. ConclusionsOur results provide the first evidence that PLDN gene is transcriptionally regulated by and is a direct target of RUNX1. These studies provide a cogent mechanism for the PLDN transcript downregulation observed in the patient platelets. More importantly, they provide a mechanism for the dense granule deficiency and impaired vesicle formation associated with RUNX1 haplodeficiency. Disclosures:No relevant conflicts of interest to declare.

Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

AbstractPatients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca2+ mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F-positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin αIIbβ3 activation.

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca2+ activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1−/− mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.

Abstract 40: Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation

Introduction: Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis which is a major cause of myocardial infarction and stroke. Following activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation as inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Objectives: Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. Methods: To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured in human platelets including platelet aggregation, calcium mobilization, integrin activation, and phosphorylation of PKC substrates. Results: Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ 3 activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Additionally, direct PKC activation rescued deficits in phosphorylation of pleckstrin and other PKC substrates mediated by inhibition of 12-LOX. Finally, inhibition of 12-LOX had no effect on PKC-mediated aggregation indicating that 12-LOX is upstream of PKC. Conclusions: These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for anti-platelet therapy.

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-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 overexpression. Lastly, PRKCQ promoter activity and PKC-θ 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-θ deficiency associated with RUNX1 haplodeficiency.

Epinephrine-mediated protein kinase C and Rap1b activation requires the co-stimulation of Gz-, Gq-, and Gi-coupled receptors

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.

Mechanism of Platelet Factor (PF4) Deficiency with RUNX1 Mutations: RUNX1 Is a Transcriptional Regulator of PF4.

Abstract 227RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesis. RUNX1 mutations are associated with mild thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. Several patients with mutations in RUNX1 have been shown to have alpha granule deficiency characterized by decreased platelet factor 4 (PF4) content. The mechanisms leading to PF4 deficiency remain unclear in most patients with α-granule abnormalities or the Gray platelet Syndrome (GPS). GPS is a heterogeneous disorder, and defective granule formation and targeting of proteins to the granule have been postulated. Previous studies from our group have documented a patient with mild thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, which was associated with a heterozygous mutation in transcription factor RUNX1. Platelet expression profiling of this patient showed decreased expression of several genes including chemokine PF4 and its non-allelic variant PF4V1. Platelet PF4 protein was also decreased. PF4 is mainly expressed in megakaryocytes and platelets, and it serves as a lineage-specific marker of megakaryocytic differentiation. Because PF4 is downregulated in platelets from our patient with a RUNX1 mutation, we addressed the hypothesis that PF4 and PF4V1 may be direct transcriptional targets of RUNX1. Computer based TFSEARCH analysis showed six RUNX1 consensus sites on PF4 upstream (2 kb) region and eleven RUNX1 sites on PF4V1 upstream sequence (2 kb). To assess in vivo binding of RUNX1 to PF4 upstream region chromatin immunoprecipitation (ChIP) assay was performed using HEL cell genomic DNA and RUNX1 antibody. These studies were done in HEL cells treated with phorbol 12-myristate 13-acetate (PMA) to induce megakaryocytic transformation. ChIP assay revealed in vivo RUNX1 binding in two regions encompassing RUNX1 sites at −1768 (TGTGGT) and −151 (ACCGCA) on PF4 promoter. These sites were pursued with electrophoretic mobility shift assay (EMSA) using PMA treated HEL cell nuclear extract. EMSA showed specific protein binding to DNA probes encompassing each of the above sites; this was abolished by RUNX1 antibody. The ChIP and EMSA suggest that RUNX1 binds to the PF4 promoter region. To test the functional relevance of the RUNX1 binding sites wild type PF4 upstream promoter region (−1936/−27) containing both RUNX1 sites (−1768, and −151) or containing one or both sites mutated were cloned into firefly luciferase reporter gene vector pGL4 and expressed in PMA-treated HEL cells. Mutation of the −1768 site caused ∼50% reduction in luciferase activity, mutation at −151 site caused 60% reduction in activity and mutation of both sites caused 75-80% reduction in activity. These studies suggest that each RUNX1 site contributes to the transcriptional activity of PF4 promoter. Moreover, the upstream region (−1837 to +25) of the non-allelic variant PF4V1 was cloned into pGL4 plasmid; it showed negligible luciferase activity as compared to the wild PF4 promoter containing plasmid. These studies suggest that the regulation of PF4 and its variant PF4V1 is distinctly different in HEL cells. Conclusions: Our results provide the first evidence that PF4 promoter is regulated by RUNX1, and the two RUNX1 sites at −1768 and −151 are involved in its regulation. These studies provide a cogent explanation for the α-granule PF4 deficiency in our patient and others with RUNX1/CBFA2 haplodeficiency. They extend our understanding of the potential mechanisms involved in the pathogenesis of the Gray platelet syndrome. Disclosures:No relevant conflicts of interest to declare.

Platelet/Megakaryocyte PKC-Θ Is a Transcriptional Target of RUNX1/ CBFA2: Studies in Human RUNX1 Haplodeficiency.

Protein kinase C Θ (PKC-Θ) is an important signaling molecule and regulates platelet responses to activation including aggregation and secretion. In a patient with lifelong thrombocytopenia, impaired platelet aggregation and secretion, we have shown (Gabbeta et al 1996, Blood 87:1368–1376) that phosphorylation of pleckstrin (a PKC substrate) and myosin light chain (MLC) is impaired along with diminished GPIIb-IIIa activation. Platelet protein and mRNA levels of PKC-Θ were decreased with normal levels of other PKC isozymes. These findings were associated with a heterozygous nonsense mutation in transcription factor RUNX1 (also known as CBFA2 or AML1) (Sun et al 2004, Blood 103:948–54). RUNX1 is transcription factor that plays a major role in megakaryopoiesis, megakaryocytic maturation, and platelet production. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. Because of the important role of PKC-Θ in platelet activation and of RUNX1 in hematopoiesis, we addressed the hypothesis that PKC-Θ is a direct transcriptional target of RUNX1. Studies were performed using human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. Chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody revealed RUNX1 binding to chromatin in the PKC-Θ 5' upstream region −1225/−1056 bp from ATG codon. This region includes a RUNX1 consensus binding site ACCGCA at −1081/−1076 bp identified by TFSEARCH. We performed electrophoretic mobility shift assay (EMSA) using 20-mer probe −1088/−1069 containing the RUNX1 site and nuclear extracts from PMA-treated HEL cells. Protein binding to the probe was observed, which was competed by excess unlabelled probe, and anti-RUNX1 antibody inhibited this binding, indicating that RUNX1 was involved in the DNA binding. Moreover, protein binding to the wild type probe was not competed by an oligo with 4 nucleotides deleted from the RUNX1 consensus site. To determine the functional relevance of RUNX1 binding to PKC-Θ, transient transfections were performed in HEL cells with luciferase reporter constructs. The full length construct −1085/−206 showed ~14-fold activity compared to empty vector. A mutant construct with deletion of the RUNX1 site resulted in a ~50% decrease in activity indicating that the site was functional. siRNA-mediated knockdown of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and PKC-Θ protein.Conclusion: These results and our findings in the patient provide the first evidence that PKC-Θ gene transcription in the megakaryocyte/platelet is regulated by RUNX1. They provide a cogent mechanism for the platelet PKC-Θ downregulation associated with RUNX1 haplodeficiency in our patient. RUNX1 dysregulation of PKC-Θ in megakaryocytic cells is an important aspect of the abnormal platelet function and production associated with human RUNX1 mutations.

RUNX1/CBFA2 Regulates Myosin Light Chain 9 (MYL9) in Megakaryocytic Cells: Decreased MYL9 Expression in Human RUNX1 Haplodeficiency.

RUNX1 (also known as CBFA2 or AML1) is a transcription factor that plays a major role in hematopoiesis. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood 87:1368–76, 1996). The patient platelets showed impaired phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-θ. This was associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood 103: 948–54, 2004). Platelet transcript profiling showed a striking downregulation of myosin light chain 9 (MYL9) by ~77-fold relative to normal platelets (Sun et al, J. Thromb Haemost. 5: 146–54, 2007). Myosin light chains (MLCs) play an important role in platelet responses to activation, in platelet biogenesis, and are involved in cellular processes such as cytokinesis, cell adhesion, cell contraction, cell migration. We have addressed the hypothesis that MYL9 is a direct transcriptional target of RUNX1. Studies were performed in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. To determine endogenous interaction of RUNX1 with MYL9 promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. These studies revealed RUNX1 binding to MYL9 chromatin at −742/−529 bp upstream of the ATG codon. TFSEARCH revealed four RUNX1 sites within this region. We performed electrophoretic mobility shift assay (EMSA) using probes containing each of the RUNX1 motifs and PMA-treated nuclear extracts from HEL cells. With each probe, protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. This protein binding was not competed by oligos containing mutations in the specific RUNX1 sites. No binding was noted directly to the mutant probes. To further corroborate our findings, we performed transient-ChIP analysis where wild type luciferase reporter construct −691/+4 and constructs with each of the RUNX1 sites individually mutated were transiently transfected into HEL cells. ChIP was performed using these cells and anti-RUNX1 antibody, and the expression analyzed by PCR amplification with a forward primer from MYL9 promoter sequence and reverse primer from luciferase vector sequence. Amplification was observed with immunoprecipitated wild type construct but not with any of the mutant constructs. Thus, RUNX1 interacts in vivo with MYL9 promoter, and the multiple RUNX1 sites interact with each other, as also shown for other genes. To test the functional relevance, the wild type construct −691/+4 containing all 4 RUNX1 sites or mutant constructs with each site individually deleted were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Deletion of RUNX1 site 1, 2, 3 or 4 caused ~60–90% reduction in the activity indicating that each site was functional. Lastly, siRNA mediated knock down of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and MYL9 protein.Conclusions: Our results provide the first evidence that MYL9 gene is transcriptionally regulated by RUNX1. They provide evidence for the presence of multiple RUNX1 sites in MYL9 promoter, as also observed in other genes. Moreover, these studies provide a cogent mechanism for the MYL9 transcript downregulation and the impaired MLC-phosphorylation we have previously described in association with RUNX1 haplodeficiency.

The Gi-coupled P2Y12 Receptor Regulates Diacylglycerol-mediated Signaling in Human Platelets

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.

The PKB inhibitor Akti-1/2 potentiates PAR-1-mediated platelet function independently of its ability to block PKB.

The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small-molecule inhibitors and limiting genetic models. Recently, a selective, non-ATP-competitive PKB inhibitor, Akti-1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. To determine the effect of the PKB inhibitor Akti-1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Akti-1/2 potently inhibited thrombin-mediated PKB phosphorylation on Thr(308) and Ser(473) and phosphorylation of its downstream substrate GSK3beta, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti-1/2 strongly potentiated PAR-1-mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti-1/2 was associated with increased [Ca(++)](i), PKC activation and degranulation and was ablated by agents that antagonized this pathway. The PKB inhibitor Akti-1/2 increases PAR-1-mediated platelet responses in a PKB-independent, Ca(++)/PKC-dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti-1/2 and structurally related compounds.

Phosphorylation of Pleckstrin Increases Proinflammatory Cytokine Secretion by Mononuclear Phagocytes in Diabetes Mellitus

The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-alpha and IL-1beta) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.

Inhibitory effect by new monocyclic 4-alkyliden-beta-lactam compounds on human platelet activation

In the present study some new beta-lactam compounds were screened for their ability to inhibit human platelet activation. In particular four compounds differing in the group on the nitrogen atom of the azetidinone ring were investigated. A beta-lactam having an ethyl 2-carboxyethanoate N-bound group was demonstrated to inhibit, in the micromolar range, both the Ca2+ release from endoplasmic reticulum, induced either by thrombin or by the ATPase inhibitor thapsigargin, and the Ca2+ entry in platelets driven by emptying the endoplasmic reticulum. The compound also inhibited the platelet aggregation induced by a variety of physiological agonists including ADP, collagen, thrombin and thrombin mimetic peptide TRAP. The beta-lactam reduced the phosphorylation of pleckstrin (apparent MW 47 kDa), elicited by thrombin but not by the protein kinase C activator phorbol ester. Accordingly it did not significantly affect the aggregation evoked by phorbol ester or Ca2+ ionophore. It was concluded that the beta-lactam likely exerts its anti-platelet-activating action by hampering the agonist induced cellular Ca2+ movements. The beta-lactam concentration, which significantly inhibited platelet activation, only negligibly affected the cellular viability. Even if it is still premature to draw definitive conclusions, the present results suggest that this new compound might constitute a tool of potential clinical interest and the starting-point for the synthesis of new more beneficial anti-thrombotic compounds.

Tyrosine Phosphorylation-Independent Activation of PLCγ2 Downstream Integrin α2β1 in Platelets: A Possible Role for the Small GTPase Rac.

We have recently documented the existence of a cross-talk between platelet integrins α2β1 and αIIbβ3 involving phospholipase C (PLC)-mediated activation of the small GTPase Rap1b (Bernardi B et al, Blood 2006; 107:2728–35). Here we report that integrin α2β1-mediated platelet adhesion to monomeric collagen and to other specific ligands, such as decorin and collagen-derived peptides, actually induced a robust activation of PLC, evaluated as phosphorylation of pleckstrin, the main platelet substrate for protein kinase C. This was paralleled by a robust tyrosine phosphorylation of the PLCγ2. We found that inhibition of Src kinases by PP2 completely abolished integrin α2β1-promoted PLCγ2 tyrosine phosphorylation, but did not affect either Rap1b stimulation or integrin αIIbβ3 activation in adherent platelets. Surprisingly, phosphorylation of pleckstrin occurred normally under conditions in which tyrosine phosphorylation of PLCγ2 was totally suppressed. The Src-kinase-independent PLC activity in integrin α2β1-adherent cells was not inhibited by pretreatment of platelets with aspirin and apyrase, excluding any possible contribution of Gq-regulated PLCβ isoforms stimulated by released thromboxane A2 or secreted ADP. In addition, PLC-dependent activation of Rap1b and integrin αIIbβ3 were not affected by aspirin and apyrase. Phosphorylation of pleckstrin induced by adhesion to monomeric collagen was not detected in platelets from PLCγ2 knockout mice, confirming that this was the only isoform activated downstream of integrin α2β1. In addition, mouse platelets lacking PLCγ2 showed impaired integrin α2β1-mediated outside-in signaling, and were unable to promote both GTP binding to Rap1b and integrin αIIbβ3 activation. These results indicate that although PLCγ2 is the only PLC isoform stimulated downstream integrin α2β1 and is responsible for the cross-talk to integrin αIIbβ3 through the small GTPase Rap1b, its activation in adherent platelets can occur independently of Src-mediated tyrosine phosphorylation. A recent work has reported that, in vitro, the activity of PLCγ2, but not that of PLCγ1, can be stimulated by the active, GTP-bound form of the small GTPase Rac, with a mechanism independent of phosphorylation on tyrosine residues (Piechlek T el al, J Biol Chem 2005; 208:38923–31). In platelets adherent through integrin α2β1, Rac was found to be activated upstream PLC. Activation of Rac was efficiently prevented by the inhibitor NSC23766, which is able to bind and block Rac-specific GEFs. In integrin α2β1-adherent platelets, inhibition of Rac by NSC23766 did not affect PLCγ2 tyrosine phosphorylation and activation of PLC and Rap1b. However, upon inhibition of Src kinases by PP2 and consequent prevention of PLCγ2 tyrosine phosphorylation, NSC23766 almost completely abolished PLC activation, GTP binding to Rap1b and integrin αIIbβ3 stimulation. These results demonstrate that PLCγ2 is the main PLC isoform involved in integrin α2β1-mediated activation of Rap1b and integrin αIIbβ3, and that its activation can occur through two alternative mechanisms involving either tyrosine phosphorylation or stimulation by Rac GTPase.