Platelet hyperreactivity is a risk factor for thrombosis in elderly patients with cardiovascular diseases. However, the mechanism of platelet hyperactivation has not been elucidated. This study aims to investigate alterations in the proteomes of platelets and their correlation with platelet hyperreactivity among elderly individuals.

This study included 10 young (28.1 ± 1.9 years), 10 middle-aged (60.4 ± 2.2 years), and 10 old (74.2 ± 3.0 years) subjects. Washed platelets were used in the present study. Platelet samples were analysed by using data-independent acquisition (DIA) quantitative mass spectrometry (MS).

The results showed that the platelet proteomic profile exhibited high similarity between the young and middle-aged groups. However, there were significant differences in protein expression profiles between the old group and the young group. By exploring the dynamic changes in the platelet proteome with ageing, clusters of proteins that changed significantly with ageing were selected for further investigation. These clusters were related to the initial triggering of complement, phagosome and haemostasis based on enrichment analysis. We found that platelet degranulation was the major characteristic of the differentially expressed proteins between the old and young populations. Moreover, complement activation, the calcium signalling pathway and the nuclear factor-κB (NF-κB) signalling pathway were enriched in differentially expressed proteins.

The present study showed that there are obvious differences in the protein profiles of the elderly compared with young and middle-aged populations. The results provide novel evidence showing changes in platelet hyperactivity and susceptibility to thrombosis in the elderly population.

Endometriosis is estrogen-dependent disorder. Two theories provide the explanations for the increased estrogen production. One is the feed-forward loop model linking inflammation and estrogen production. The more recent model evokes the tissue hypoxia resulting from endometrial debris detached and then regurgitated to the peritoneal cavity. Both models tacitly assume that everything occurs within the endometriotic stromal cells, seemingly without the need for exogenous factors. This study was undertaken to investigate as whether platelets may be responsible for local estrogen overproduction. We employed in vitro experimentation that evaluated the 17β-estradiol (E₂) levels in endometriotic stromal cells treated with activated platelets, and the genes and protein expression levels of StAR, HSD3B2, aromatase, and HSD17B1, as well as their upstream genes/proteins such as NF-κB, TGF-β1, HIF-1α, SF-1 and phosphorylated CREB. In addition, we conducted 2 animal experimentations using platelet depletion/infusion and also neutralization of NF-κB and TGF-β1, followed by immunohistochemistry analysis of involved in StAR, HSD3B2, aromatase, and HSD17B1, as well as SF-1 and p-CREB. We found that treatment of endometriotic stromal cells by activated platelets increase the E₂ production by 4.5 fold, and concomitant with increased gene and protein expression of StAR, HSD3B2, aromatase, and HSD17B1, the four genes/enzymes important to estrogen synthesis, along with their upstream genes HIF-1α, SF-1 and phosphorylated CREB. Moreover, platelets activate these genes through the activation of NF-κB and/or TGF-β1, and antagonism of either signaling pathway can abolish the induction of the 4 genes and thus increased estrogen production. The two animal experimentations confirmed these changes. Thus, platelets increase the E₂ production in endometriotic stromal cells through upregulation of StAR, HSD3B2, aromatase, and HSD17B1 via the activation of NF-κB and/or TGF-β1. These findings provide a yet another compelling piece of evidence that endometriotic lesions are indeed wounds undergoing repeated tissue injury and repair. They strongly indicate that non-hormonal therapeutics for endometriosis is theoretically viable, with anti-platelet therapy being one promising avenue.

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.

Leukotactin-1 (Lkn-1)/CCL15 is a recently cloned CC-chemokine that binds to the CCR1 and CCR3. Although Lkn-1 has been known to function as a chemoattractant for neutrophils, monocytes and lymphocytes, its cellular mechanism remains unclear. To understand the mechanism of Lkn-1-induced chemotaxis signaling, we examined the chemotactic activities of human osteogenic sarcoma cells expressing CCR1 in response to Lkn-1 using inhibitors of signaling molecules. Inhibitors of G(i)/G(o) protein, phospholipase C (PLC) and protein kinase Cdelta (PKCdelta) inhibited the chemotactic activity of Lkn-1 indicating that Lkn-1-induced chemotaxis signal is transduced through G(i)/G(o) protein, PLC and PKCdelta. The activities of PLC and PKCdelta were also enhanced by Lkn-1 stimulation. Chemotactic activity of Lkn-1 was inhibited by the treatment of cycloheximide and actinomycin D suggesting that newly synthesized proteins are needed for chemotaxis. Nuclear factor-kappaB (NF-kappaB) inhibitor reduced chemotactic activity of Lkn-1. DNA binding activity of NF-kappaB was also enhanced by Lkn-1 stimulation. These results suggest that Lkn-1 transduces the signal through G(i)/G(o) protein, PLC, PKCdelta, NF-kappaB and newly synthesized proteins for chemotaxis.

The goal of the present study was to assess whether cardiac myocytes exposed to anoxia-reoxygenation (A/R) could generate a chemotactic gradient for polymorphonuclear neutrophil (PMN) transendothelial migration. Exposure of neonatal mouse cardiac myocytes to A/R induced an oxidant stress in the myocytes. Supernatants obtained from A/R-conditioned myocytes promoted mouse PMN migration across mouse myocardial endothelial cell monolayers. This increase in PMN transendothelial migration could be prevented if catalase or a platelet-activating factor (PAF) antagonist was added to the supernatants before assay. Supernatants from A/R-conditioned myocytes activated endothelial cells by inducing an intracellular oxidant stress. The oxidant stress and PMN transendothelial migration induced by supernatants from A/R-conditioned myocytes were substantially reduced when endothelial cells derived from manganese superoxide dismutase overexpressing mice were used in the assays. Supernatants from A/R-conditioned myocytes also increased endothelial cell surface levels of E-selectin and intercellular adhesion molecule-1. Our results indicate that cardiac myocytes exposed to A/R can generate a chemotactic gradient, presumably due to production and release of stable oxidants and PAF. The ability of supernatants from A/R-conditioned myocytes to promote PMN transendothelial migration was largely dependent on induction of an oxidant stress in endothelial cells. In addition, these supernatants also induced a proadhesive phenotype in the endothelial cells.

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.

Platelet-activating factor (PAF) is a phospholipid mediator that belongs to a family of biologically active, structurally related alkyl phosphoglycerides. PAF acts via a specific receptor that is coupled with a G protein, which activates a phosphatidylinositol-specific phospholipase C. In this review we focus on the aspects that are more relevant for the cell biology of the cardiovascular system. The in vitro studies provided evidence for a role of PAF both as intercellular and intracellular messenger involved in cell-to-cell communication. In the cardiovascular system, PAF may have a role in embryogenesis because it stimulates endothelial cell migration and angiogenesis and may affect cardiac function because it exhibits mechanical and electrophysiological actions on cardiomyocytes. Moreover, PAF may contribute to modulation of blood pressure mainly by affecting the renal vascular circulation. In pathological conditions, PAF has been involved in the hypotension and cardiac dysfunctions occurring in various cardiovascular stress situations such as cardiac anaphylaxis and hemorrhagic, traumatic, and septic shock syndromes. In addition, experimental studies indicate that PAF has a critical role in the development of myocardial ischemia-reperfusion injury. Indeed, PAF cooperates in the recruitment of leukocytes in inflamed tissue by promoting adhesion to the endothelium and extravascular transmigration of leukocytes. The finding that human heart can produce PAF, expresses PAF receptor, and is sensitive to the negative inotropic action of PAF suggests that this mediator may have a role also in human cardiovascular pathophysiology.

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.

In recent studies, using a swine model of single lung transplantation, we demonstrated that IRI alone increased MHC II expression in the host's peripheral T lymphocytes. The inhibition of increased MHC II expression with TCV-309, a specific platelet-activating factor (PAF) antagonist suggested that PAF might play a role in the mechanism of increased MHC II expression. The purpose of the current study was two fold: 1) to investigate the mechanism of PAF-induced increased expression of MHC II in T lymphocytes, 2) to determine whether a specific PAF-antagonist, TCV-309, is capable of inhibiting the increased expression in an in vitro system. This study was subdivided, using four in vitro conditions: 1) purified resting T cells, 2) purified proliferating T cells, 3) PBL treated with PAF, and 4) PBL preincubated with TCV-309 and treated with PAF. The level of MHC II on T cells were measured by two color flow cytometry analysis (swine anti-CD3, MHC II-DR-(beta)antibodies). Both MHC II intensity and the number of CD3+MHC+ T cells did not change in resting purified T cells once treated with PAF, Furthermore, MHC II intensity did not change in purified proliferating T cells treated with PAF. The number of CD3+MHC+ T cells, however, increased significantly (p<0.05) from day 1 to day 4 as compared with pre-treatment value (day 0) for purified proliferating T cells. Treatment of PBL with PAF (10(-7)M) resulted in a significant (p<0.05) increase in MHC II expression from day 2 to day 4 post-treatment. The number of CD3+MHC+ T cells in PBL, however, did not change significantly upon treatment with PAF. The results of this study indicated that PAF did not have a direct effect on increased MHC II expression in resting or proliferating purified T lymphocytes. However, the mechanism of PAF-induced increased expression of MHC II in T cells may be via an indirect pathway involving accessory cells. TCV-309, a specific PAF receptor antagonist, is capable of inhibiting this PAF-induced increased expression of MHC II in T cells.