Platelet Protein Kinase Research Articles

Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin \u03b1IIb\u03b23 inside-out but not outside-in signals

BackgroundColumbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo.MethodsAggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study.ResultsCBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbβ3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbβ3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation.ConclusionThis study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.

Glutamate induces synthesis of thrombogenic peptides and extracellular vesicle release from human platelets

Platelets are highly sensitive blood cells, which play central role in hemostasis and thrombosis. Platelet dense granules carry considerable amount of neurotransmitter glutamate that is exocytosed upon cell activation. As platelets also express glutamate receptors on their surface, it is pertinent to ask whether exposure to glutamate would affect their signalling within a growing thrombus. In this study we demonstrate that, glutamate per se induced synthesis of thrombogenic peptides, plasminogen activator inhibitor-1 and hypoxia-inducible factor-2α, from pre-existing mRNAs in enucleate platelets, stimulated cytosolic calcium entry, upregulated RhoA-ROCK-myosin light chain/myosin light chain phosphatase axis, and elicited extensive shedding of extracellular vesicles from platelets. Glutamate, too, incited platelet spreading and adhesion on to immobilized matrix under arterial shear, raised mitochondrial transmembrane potential associated with generation of reactive oxygen species and induced activation of AMP-activated protein kinase in platelets. Taken together, glutamate switches human platelets to pro-activation phenotype mediated mostly through AMPA receptors and thus targeting glutamate receptors may be a promising anti-platelet strategy.

Antiplatelet Activity of a Newly Synthesized Novel Ruthenium (II): A Potential Role for Akt/JNK Signaling.

In oncotherapy, ruthenium complexes are considered as potential alternatives for platinum compounds, and have been proved as promising anticancer drugs with high efficacy and lesser side effects. Platelet activation plays a major role in cancer metastasis and progression. Hence, this study explored the effect of a newly synthesized ruthenium complex, [Ru(η6-cymene)(L)Cl]BF4(TQ5), where L = 4-phenyl-2-pyridin-2-yl-quinazoline), on human platelet activation. TQ5 (3–5 µM) inhibited concentration-dependent collagen-induced platelet aggregation in washed human platelets. However, this compound only inhibited platelet aggregation at a maximum concentration of 500 and 100 µM against thrombin and 9,11-dideoxy-11α, 9α-epoxymethanoprostaglandin (U46619)-induced stimulation, respectively. TQ5 inhibited collagen-induced ATP release and calcium mobilization ([Ca2+]i), without inducing cell cytotoxicity. In addition, neither SQ22536, an adenylate cyclase inhibitor, nor 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, significantly reversed the TQ5-mediated inhibition of platelet aggregation. TQ5 inhibited the collagen-induced phosphorylation of protein kinase B (Akt) and c-Jun N-terminal kinase (JNK), but did not effectively inhibit extracellular signal-regulated kinase 1/2 (ERK1/2) and p38-mitogen-activated protein kinase (p38-MAPK) in human platelets. Additionally, TQ5 significantly prolonged the closure time in whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. This study demonstrates, for the first time, that a newly synthesized ruthenium complex, TQ5, exhibits potent antiplatelet activity by hindering ATP release and [Ca2+]i, and by decreasing the activation of Akt/JNK signals. Together, these results suggest that TQ5 could be developed as a therapeutic agent that helps prevent or treat thromboembolic disorders, since it is found to be potently more effective than a well-established antithrombotic aspirin.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

SH2B3/LNK Loss of Function Promotes Atherosclerosis and Thrombosis

Human genome-wide association studies (GWAS) have revealed many novel genetic loci that are associated with coronary heart disease (CHD) but do not involve traditional risk factors. However, the relevant genes and mechanisms are largely unknown. One such locus resides in SH2B3/LNK, which is expressed in hematopoietic cells and suppresses thrombopoietin (TPO) signaling via its receptor (MPL). The common risk single nucleotide polymorphism (SNP) of SH2B3/LNK is associated with CHD, increased platelet and myeloid cell counts in peripheral blood and JAK2V617F positive myeloproliferative neoplasms.Analysis of human cord blood revealed that samples with TT risk SNP (R262W) in SH2B3/LNK was associated with expansion of hematopoietic stem cells (HSCs), increased MPL signaling in HSCs, increased megakaryopoiesis, and a paradoxical increased expression of LNK mRNA, indicating reduced LNK function but induction of LNK transcription downstream of increased MPL signaling. Since humans are hypercholesterolemic relative to mice, to assess the role of reduced LNK function in athero-thrombosis, we transplanted WT or Lnk-/- bone marrow (BM) cells into irradiated WT or low-density lipoprotein receptor knock-out (Ldlr-/-) recipient mice and fed the recipients with chow and western type diet (WTD), respectively. Although Lnk-/- BM recipients showed similarly increased platelet counts on both diets, we found that platelet activation and aggregation, formation of platelet/leukocyte aggregates, atherosclerosis and arterial thrombosis were markedly increased by the combination of hyperlipidemia and hematopoietic LNK deficiency. These diet-genotype interactions mirrored an increase in mean platelet volume, protease-activated receptor-4 (PAR4) agonist-induced P-selectin exposure on platelets and platelet protein kinase C (PKC) activity, suggesting increased platelet granule secretion. We also observed an increased number of bone marrow myeloid progenitor cells expressing elevated levels of the common-beta subunit (CBS) of the interleukin-3/granulocyte-macrophage colony-stimulating factor (IL-3/GM-CSF) receptor, which promotes leukocytosis and the formation of platelet/leukocyte aggregates in the bloodstream. The increased P-selectin exposure, platelet aggregation and PKC activity were largely reversed by high-density lipoprotein (HDL) or cyclodextrin, reagents that remove cholesterol from platelets. Consistently, cholesterol loading of platelets from the chow-fed mice led to increased P-selectin exposure and PKC activity upon PAR4 agonist stimulation but the increase was more pronounced in Lnk-/- platelets, indicating a cholesterol-genotype interaction. LNK deficiency also resulted in defective MPL-mediated TPO internalization in platelets, leading to increased plasma TPO levels in mice on both diets. Elevated TPO levels and enhanced MPL signaling likely acted together to increase the pool of HSCs and megakaryopoiesis, leading to marked thrombocytosis.These studies suggest that SH2B3/LNK loss of function variants lead to HSC expansion, overproduction of platelets, and in the setting of hypercholesterolemia, markedly increased myeloid cells, platelet activation and formation of platelet/leukocyte aggregates, leading to accelerated atherosclerosis and arterial thrombosis. Our studies point to the importance of treating dyslipidemia in the setting of platelet and myeloid cell overproduction, as observed in subjects with SH2B3/LNK risk alleles. DisclosuresLevine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Tall:Amgen: Consultancy; Arisaph: Consultancy; CSL: Consultancy.

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.

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.

Activation of platelet protein kinase C by ultraviolet light B mediates platelet transfusion–related acute lung injury in a two‐event animal model

We recently reported that infusion of ultraviolet light B (UVB)-exposed human platelets (HPs) can be the second event that mediates acute lung injury (ALI) in a two-event mouse model of transfusion-related acute lung injury (mTRALI). We have now identified changes in HPs induced by UVB light and responses of the recipient animal that mediate the mTRALI. Effects of UVB on HPs were monitored by flow cytometry and aggregation. HPs exposed to UVB, with or without inhibitors to specific biochemical pathways, were infused into lipopolysaccharide (LPS)-primed severe combined immunodeficient (SCID) mice. ALI was monitored by protein elevations in bronchoalveolar lavage fluid (BALF). UVB increased fibrinogen binding and potentiated HP aggregation. Infusion of UVB HPs into LPS-primed SCID mice led to macrophage inflammatory protein 2 (MIP-2) elevations in plasma and BALF and resulted in ALI. Protein kinase C (PKC) inhibitors prevented UVB-induced HP changes in vitro and reduced MIP-2 elevation and mTRALI in vivo. Blocking of fibrinogen binding to HP αIIbβ3 with c7E3 monoclonal antibody prevented mTRALI. MIP-2 elevation in vivo in response to UVB HPs was essential for ALI since blocking of MIP-2 receptor in vivo prevented mTRALI. PKC signaling mediates UVB-induced HP fibrinogen binding and aggregation in vitro. The host animal responds to an infusion of UVB HPs by MIP-2 elevation that mediates downstream mTRALI. Elucidation of molecular mechanisms in UVB HP-mediated mTRALI may provide insight into pulmonary adverse events reported with UV-irradiated pathogen-reduced platelets.

Platelet protein kinase C and brain-derived neurotrophic factor levels in borderline personality disorder patients

Borderline personality disorder (BPD) is a prevalent and difficult to treat psychiatric condition characterized by abrupt mood swings, intense anger and depression, unstable interpersonal relationships, impulsive self-destructive behavior and a suicide rate of approximately 10%. Possible underlying molecular dysregulations in BPD have not been well explored. Protein kinase C (PKC) and brain-derived neurotrophic factor (BDNF) have both been implicated in affective disorders, but their role in BPD has not been examined. Platelets were isolated from blood obtained from 24 medication-free BPD patients and 18 healthy control subjects. PKC-α, phosphorylated-PKC-α (p-PKCα), PKC-βII, and BDNF were measured in platelet homogenates by immunoblotting. In the males, platelet BDNF and PKC-α levels were lower in patients than controls. p-PKC-α and PKC-βII were lower at trend levels. In the entire sample, platelet p-PKCα and PKC-α activity were lower, at a trend level, in patients compared to controls. This is the first report to our knowledge of PKC and BDNF activity in BPD and calls for replication. These findings are consistent with altered PKC and BDNF activity in a range of neuropsychiatric conditions including bipolar disorder, depression and suicide.

Cisplatin induces platelet apoptosis through the ERK signaling pathway

Cisplatin (cis-diamminedichloroplatinum II) is one of the most widely used anti-tumor agents. However, cisplatin-based chemotherapy is usually accompanied by adverse side effects such as thrombocytopenia, and the mechanism remains unclear. Here we show that cisplatin induced several platelet apoptotic events including up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, mitochondrial translocation of Bax, mitochondrial inner transmembrane potential depolarization, caspase-3 activation and phosphatidylserine (PS) exposure. Cisplatin dose-dependently induced activation of extracellular signal-regulated protein kinase (ERK) in platelets. Caspase-3 inhibitor z-DEVD-fmk dramatically inhibited cisplatin-induced caspase-3 activation and PS exposure without affecting ERK activation. Blockade of the ERK pathway significantly prevented platelet apoptosis. Furthermore, levels of reactive oxygen species (ROS) and Ca2+ were significantly elevated by cisplatin, and scavenging of ROS and Ca2+ obviously inhibited platelet apoptosis induced by cisplatin. In addition, cisplatin did not induce platelet activation, whereas it obviously impaired platelet functions. These data indicate that cisplatin induces platelet apoptosis through the ERK signaling pathway, which might contribute to cisplatin-related haematological toxicity.

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.

Protein Kinase C Mediates UVB Light Induced Changes In Human Platelets that Lead to Inflammation In a Two-Event Animal Model of Transfusion Mediated Acute Lung Injury

AbstractAbstract 3358Pathophysiology of acute lung injury (ALI) includes an inflammatory component with recruitment and activation of neutrophils to the lungs. One proposed mechanism of transfusion related acute lung injury (TRALI) involves two events; the first is a generalized inflammatory response, as would occur in sepsis, which leads to activation of endothelial cells and sequestration of neutrophils to the lungs. The second is an infusion of a transfusion product that contains HLA or HNA antibodies or biologic modifiers such as lipids from stored cells. The second event activates the neutrophils sequestered in the lungs which lead to neutrophil degranulation, superoxide release and localized tissue damage. Growing evidence suggests that platelets exert proinflammatory actions which include supporting tissue infiltration of neutrophils in septic lung injury. In a separate 2010 ASH abstract we show that ultraviolet B light (UVB, 2.4 J/cm2) exposed human platelets (HPs) mediate lung injury in a two-event animal model of ALI. UVB exposure has been reported to activate platelet protein kinase C (PKC). We compared the effects of UVB exposure to PKC activation by a PKC agonist, PMA (30 nM), in aggregation, activation and potential to cause lung injury in the two-event animal model. HPs were collected by apheresis and stored overnight with experiments performed on day 1 post collection. Platelet aggregation induced by increasing concentrations of ADP (5-20 mM) was potentiated by pretreatment with UVB or PMA. TRAP (20 mM) induced aggregation was inhibited by UVB, but unchanged by PMA pretreatment. Both UVB and PMA increased platelet PAC-1 binding and p-selectin expression. Pretreating HPs with a PKC inhibitor prevented all of PMA induced PAC-1 binding and inhibited UVB induced PAC-1 binding by 40%. Furthermore, the PKC inhibitor partially reduced p-selectin expression on PMA and UVB treated HPs, whereas p-selectin expression on control HPs remained unchanged. The UVB HPs or PMA HPs were evaluated in the two-event animal model of ALI. Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). MIP-2 elevation in plasma is a marker of acute inflammation and was increased following LPS administration. Infusion of control HPs as the second event moderately increased MIP-2. When UVB HPs or PMA HPs were infused MIP-2 was significantly elevated compared to control HPs. Pretreatment of UVB HPs with the PKC inhibitor (RO31-8425) reduced MIP-2 elevation to the level of control platelets. In summary, UVB HPs can cause ALI in animals pretreated with LPS (separate 2010 ASH abstract as mentioned above). Changes to the platelets induced by UVB appear to be mediated by PKC since a PKC agonist (PMA) has similar effects on platelets in aggregation and activation as does UVB and PKC inhibitor partially inhibits UVB induced platelet activation. In vivo, both UVB and PMA treated HPs elevated MIP-2 plasma levels when injected after LPS and this response was prevented by treatment of platelets with a PKC inhibitor prior to UVB exposure. The UVB induced activation leads to a conformational change in GpIIb/IIIa which potentiates weak agonist induced aggregation and mediates an acute in vivo inflammatory response that may be responsible for the acute lung injury in the animal model. Understanding the underlying mechanisms of UVB exposure induced changes in platelets would be beneficial in designing methods to reduce the UVB associated ALI in an animal model and potentially in patients susceptible to TRALI by a primary sensitizing event and infused with high dose UVB exposed platelets. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

AbstractMitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway.

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.

Phosphorylation of cAMP hydrolyzing PDE (PDE3A) by cGMP-dependent protein kinase (PKG) in human platelets

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Inhibition of platelet adherence to mononuclear cells by alpha-tocopherol: role of P-selectin.

Platelet-leukocyte interaction is an early important event for thrombogenesis, and this process is mainly mediated by P-selectin on platelets. Although alpha-tocopherol has been shown to inhibit thrombotic disorders, the effect of alpha-tocopherol on platelet P-selectin expression and platelet-leukocyte interaction is little known. We examined whether alpha-tocopherol inhibited human platelet P-selectin expression and platelet-leukocyte interaction. Alpha-tocopherol (50 to 500 microg/mL) inhibited thrombin-induced or phorbol 12-myristate 13-acetate (PMA)-induced P-selectin expression on platelets. alpha-Tocopherol suppressed platelet-mononuclear cell (MNC) interaction, platelet aggregation, and platelet protein kinase C (PKC) activity stimulated with either PMA (100 nmol/L) or thrombin. Inhibitory actions of alpha-tocopherol against the platelet functions were mimicked by staurosporine, a selective PKC inhibitor. After oral supplementation of alpha-tocopherol (300 mg/d for 3 weeks) in healthy subjects, thrombin-mediated or PMA-mediated P-selectin expression, platelet-MNC interaction, and platelet aggregation ex vivo were suppressed. alpha-Tocopherol inhibited P-selectin expression on human platelets and thereby attenuated platelet-MNC interactions, which were mediated at least in part by the inhibition of intraplatelet PKC activity. These actions of alpha-tocopherol on platelet functions provide new insights into the antithromboatherogenic properties of alpha-tocopherol.

The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents

Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3′,5′-cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, β-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser 1177 and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of l-[ 3H]-arginine to l-[ 3H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 μmol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser 157 and Ser 239 and of ecNOS at Ser 1177 was evaluated by Western blot. NOS activity (pmol l-citrulline/10 8 platelets) increased from 0.090±0.002 to 0.148±0.013 with 500 μmol/l 8-Br-cAMP ( p<0.0001), to 0.140±0.008 with 30 μmol/l adenosine ( p<0.0001) and to 0.140±0.009 with 10 μmol/l forskolin ( p<0.0001). Rp-cAMPS decreased baseline NOS activity from 0.093±0.001 to 0.075±0.006 ( p<0.02) and prevented the stimulation by 8-Br-cAMP, adenosine and forskolin. Platelet exposure to 8-Br-cAMP and forskolin, beside the phosphorylation of the specific PKA substrate VASP, markedly increased the expression of ecNOS protein phosphorylated at Ser 1177. The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.