TMEM97 facilitates the activation of SOCE by downregulating the association of cholesterol to Orai1 in MDA-MB-231 cells.

The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.

Population genetic screening efficiently identifies carriers of autosomal dominant diseases.

Three inherited autosomal dominant conditions-BRCA-related hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS) and familial hypercholesterolemia (FH)-have been termed the Centers for Disease Control and Prevention Tier 1 (CDCT1) genetic conditions, for which early identification and intervention have a meaningful potential for clinical actionability and a positive impact on public health1. In typical medical practice, genetic testing for these conditions is based on personal or family history, ethnic background or other demographic characteristics2. In this study of a cohort of 26,906 participants in the Healthy Nevada Project (HNP), we first evaluated whether population screening could efficiently identify carriers of these genetic conditions and, second, we evaluated the impact of genetic risk on health outcomes for these participants. We found a 1.33% combined carrier rate for pathogenic and likely pathogenic (P/LP) genetic variants for HBOC, LS and FH. Of these carriers, 21.9% of participants had clinically relevant disease, among whom 70% had been diagnosed with relevant disease before age 65. Moreover, 90% of the risk carriers had not been previously identified, and less than 19.8% of these had documentation in their medical records of inherited genetic disease risk, including family history. In a direct follow-up survey with all carriers, only 25.2% of individuals reported a family history of relevant disease. Our experience with the HNP suggests that genetic screening in patients could identify at-risk carriers, who would not be otherwise identified in routine care.

Store-independent Orai1-mediated Ca2+ entry and cancer.

Ca2+ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca2+ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca2+ channels. Orai1 mediates the highly Ca2+-selective Ca2+ release-activated current (ICRAC) and participates in the less Ca2+-selective store-operated current (ISOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca2+ influx mechanisms, including the arachidonate-regulated Ca2+ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca2+ influx processes activated by the secretory pathway Ca2+-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca2+-activated K+ channel 3 (SK3) or the voltage-dependent Kv10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca2+ influx activation through Orai1 channels and their role in the development of different cancer features.

(-)­Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells.

Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (-)­oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that OLCT is unable to induce Ca2+ mobilization in non-tumoral cells. By contrast, OLCT induces Ca2+ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24-72 h downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT impairs the ability of breast cancer cells to proliferate and migrate via downregulation of TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.

Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation.

Store-operated Ca2+ entry (SOCE) is a major mechanism for the regulation of intracellular Ca2+ homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2+ imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Relationship between calcium mobilization and platelet α- and δ-granule secretion. A role for TRPC6 in thrombin-evoked δ-granule exocytosis.

Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca(2+) mobilization.

Immunophilins are involved in the altered platelet aggregation observed in patients with type 2 diabetes mellitus.

UNLABELLED: Platelet hyperaggregability might contribute to vascular complications associated with type 2 diabetes mellitus (DM2).Experimental evidence supports a direct link between altered Ca(2+) entry and hyperaggregability in DM2 patients. OBJECTIVES: We aimed to investigate whether altered immunophilin expression and function are involved in the abnormal Ca(2+) entry observed in platelets from DM2 patients. RESULTS: Inhibition of immunophilins by tacrolimus (FK506) and sirolimus (rapamycin) reduced Ca(2+) entry in platelets from healthy donors and DM2 patients. Similarly, immunophilin inhibitors reduced platelet degranulation in both healthy and DM2 subjects. Nevertheless, α-granule secretion reduction was greater than that observed for dense granules in platelets from DM2 patients. However, no difference was observed in the inhibition of secretion in platelets from healthy subjects. Additionally, altered expression of FK506 binding protein-52 (FKBP52) and coupling to Ca(2+) channels were found in platelets from DM2 patients compared to healthy subjects. Finally, reduction in platelet function from healthy subjects and DM2 patients in the presence of immunophilin antagonists was observed, being this dysfunction more evident in platelets from DM2 patients. CONCLUSIONS: We suggest that, among others, FKBP52 expression and function are altered in platelets from DM2 patients, contributing to the altered Ca(2+) entry and hyperaggregability in these cells.

Transient receptor potential channels in human platelets: expression and functional role.

Recent studies have demonstrated that mammalian homologues of Drosophila transient receptor potential (TRP) channels are widely expressed in human platelets. Occupation of G protein-coupled receptors by agonists results in activation of these channels, which results in Na+ and Ca2+ entry. Canonical or classic TRP (TRPC) family members have been reported to associate with different Ca2+-handling proteins, including the type II inositol 1,4,5-trisphosphate receptor, the endoplasmic reticulum Ca2+ sensor STIM1 (STromal Interaction Molecule-1) or the Ca2+ permeable channel Orai1. The dynamic interaction of TRPC channels with the above mentioned proteins has been found to be important for both store-operated and capacitative Ca2+ entry, as well as for non-capacitative Ca2+ influx. The former is a major mechanism for Ca2+ entry in human platelets. This mechanism, activated by a reduction in the concentration of free Ca2+ in the intracellular stores, results in the formation of signaling complexes involving STIM proteins, Orai1, Orai2, TRPC1 and TRPC6. There is a growing body of evidence supporting that Ca2+ signaling dysfunction plays an important role in the pathogenesis of several platelet-linked disorders, including those associated to type 2 diabetes mellitus. Abnormal Ca2+ signals in response to physiological agonists have been associated to platelet hyperactivity. The expression of several TRPCs, STIM1 and Orai1, as well as their interaction, has been reported to be altered in platelets from type 2 diabetic patients, which results in attenuated capacitative Ca2+ entry but enhanced non-capacitative Ca2+ influx; thus suggesting a role for Ca2+ handling proteins, including TRPs, in the pathomechanism of diabetic complications.

Orais and STIMs: physiological mechanisms and disease.

The stromal interaction molecules STIM1 and STIM2 are Ca(2+) sensors, mostly located in the endoplasmic reticulum, that detect changes in the intraluminal Ca(2+) concentration and communicate this information to plasma membrane store-operated channels, including members of the Orai family, thus mediating store-operated Ca(2+) entry (SOCE). Orai and STIM proteins are almost ubiquitously expressed in human cells, where SOCE has been reported to play a relevant functional role. The phenotype of patients bearing mutations in STIM and Orai proteins, together with models of STIM or Orai deficiency in mice, as well as other organisms such as Drosophila melanogaster, have provided compelling evidence on the relevant role of these proteins in cellular physiology and pathology. Orai1-deficient patients suffer from severe immunodeficiency, congenital myopathy, chronic pulmonary disease, anhydrotic ectodermal dysplasia and defective dental enamel calcification. STIM1-deficient patients showed similar abnormalities, as well as autoimmune disorders. This review summarizes the current evidence that identifies and explains diseases induced by disturbances in SOCE due to deficiencies or mutations in Orai and STIM proteins.

Immunophilin dysfunction and neuropathology.

In case of nervous damages, like nervous system trauma or various neurodegenerative diseases such as dementia or Parkinson, several treatments are available to restore neurological function. In spite of these treatments, results are often insufficient or not satisfactory in many neurologic diseases, especially for central nervous system (CNS) lesions. To minimize neurological dysfunction, it is critical to reduce neuronal death, avoiding loss of the synaptic connections, and securing viable neurons to extend axons. Unfortunately, there are no effective strategies to fulfill these basic needs except for some cases of peripheral neural damage up to now. Rescue of damaged neurons, stimulation of neurogenesis and transplantation of nervous tissue are strategies proposed to prevent neurodegenerative disorders. A number of studies have recently reported successful axon regeneration and neurological recovery by using immunosuppressants, such as FK506. Immunosuppressants act as excellent agents for enhancing the rate and extent of axon regeneration and neurological recovery. FK506 and other neuroimmunophilin ligands (NILs) might reverse neuronal degeneration. In several animal models mimicking Parkinson's disease, dementia and surgical damage, NILs induces resprouting, by acting as neurotrophic agents and preventing nerve damage, although more studies are necessary to identify new NILs with neuroprotective action, but lacking the side immunological effects observed in the ligands analyzed to date. This review explores the new clinical role of immunosuppressants in the treatment of nerve surgery of autologous, allografts or xenografts. Results of studies regarding immunosuppressant treatment of nervous system trauma and neurodegenerative diseases, like neurogenic erectile dysfunction, will be here considered.

Immunophilins and thrombotic disorders.

The immunophilin family includes a large group of proteins with peptidyl prolyl-isomerase activity (PPI-ase). Immunophilins chaperone activity has been documented to be crucial for the correct folding and activation of many proteins. Thus, they have been subjected of intense investigation since they were firstly described in the last decades of the past century. Many of these studies have been focused on leukocyte constitutively expressed immunophilins, due to their relevance in the correct folding, and subsequently, sensitization and activation of the glycoprotein receptor (RGBs) of lymphocyte T CD4+ and Treg, hence regulating immunological responses against pathogen insults. Several clinical trials have been completed in the last decade reporting that administration of immunophilin-binding drugs, derived from macrolide lactones, like cyclosporine A (CsA) and tacrolimus (FK506), induced successful results in preventing organ rejection. By contrast, the expression of immunophilins and their physiological function remain poorly investigated in others cell types, such as platelets, where a reduced number of studies presenting evidences of immunophilins expression and their physiological contribution have been published, despite a number of clinical trials have noticed side effects of these drugs in thrombosis and platelet count, thus suggesting a possible regulatory function of immunophilins in human platelets, which is reviewed here.

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation.

BACKGROUND: All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP(3)Rs)-binding (CIRB) site involved in the regulation of TRPC channel function. OBJECTIVES: To assess the basis of CaM/IP(3)Rs binding to the CIRB site of TRPC6 and its role in platelet physiology. METHODS: Protein association was detected by co-immunoprecipitation and Western blotting, Ca(2+) mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry. RESULTS: Co-immunoprecipitation of TRPC6 with CaM or the IP(3)Rs at different cytosolic free Ca(2+) concentrations ([Ca(2+)](c)) indicates that the association between these proteins is finely regulated by cytosolic Ca(2+) via association of CaM and displacement of the IP(3)Rs at high [Ca(2+)](c). Thrombin-stimulated association of TRPC6 with CaM or the IP(3)Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca(2+)-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP(3)Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca(2+) entry in the presence of low or high, respectively, extracellular Ca(2+) concentrations, as well as altered thrombin-evoked platelet aggregation. CONCLUSIONS: Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca(2+) entry and aggregation through its interaction with CaM and IP(3)Rs.

Modulation of platelet function and signaling by flavonoids.

Blood platelets play a crucial role in the primary hemostasis and vessel wall repair. However; platelet hyperactivation is implicated in the pathogenesis of cardiovascular diseases such as thrombosis, atherosclerosis and stroke. Epidemiological data have suggested that regular consumption of fruits and vegetables, which are rich in flavonoids, is associated to a reduction in cardiovascular events. The cardioprotective effect of flavonoids is partly due to the inhibition of platelet function. However; the mechanisms underlying the anti-platelet effect of these compounds remain unclear. The aim of this review is to discuss the role of platelets in cardiovascular disease and to provide an overview of the potential anti-platelet effect of flavonoids, focusing on the various platelet signaling pathways modulated by flavonoids, including oxidative stress, protein tyrosine phosphorylation, calcium mobilization and nitric oxide pathway. The understanding of these mechanisms will be helpful in the development of new anti-platelet agents based on flavonoids with fewer or no adverse effects.

Acidic Ca(2+) stores in platelets.

Changes in cytosolic free Ca(2+) concentration play a pivotal role in the regulation of platelet functions, from secretion of autocrine and procoagulant factors to reversible or irreversible aggregation. It has long been recognized that platelet agonists release Ca(2+) accumulated into the dense tubular system, the analogue of the endoplasmic reticulum. However, current evidence indicates that Ca(2+) can also be stored and released from a number of acidic organelles, including lysosomes and lysosome-related organelles. Ca(2+) release from the dense tubular system is mediated through phospholipase C-dependent synthesis of inositol 1,4,5-trisphosphate, whereas Ca(2+) efflux from the acidic stores seems to be associated to the second messenger nicotinic acid adenine dinucleotide phosphate. The biochemical and biophysical properties of both Ca(2+) stores in platelets have been reported to show significant differences. Selective discharge of one or both stores depends on the platelet agonist and the concentration used, which further supports the complexity of the Ca(2+) signals that regulate platelet function. In this paper, we summarize the current knowledge on the role of acidic organelles in agonist-evoked Ca(2+) mobilization and highlight recent progress in understanding the functional aspects of the acidic Ca(2+) stores in Ca(2+) signalling and platelet physiology.

Homocysteine, intracellular signaling and thrombotic disorders.

Homocysteine, a sulphur-containing amino acid derived from methionine, has been presented as an independent risk factor for cardiovascular disorders, including atherosclerosis and thrombogenesis. The mechanisms underlying homocysteine-induced effects have been intensively investigated over the last two decades. Homocysteine can induce oxidative stress promoting oxidant injury to vascular and blood cells. Hyperhomocysteinemia often results in intracellular Ca2+ mobilization, endoplasmic reticulum (ER) stress, with the subsequent development of apoptotic events, chronic inflammation leading to endothelial dysfunction and remodeling of the extracellular matrix. Homocysteine has also been reported to induce modulation of gene expression through alteration of the methylation status. The effects of elevated concentrations of circulating homocysteine on the vascular wall, platelet function and coagulation factors promote the development of a pro-coagulant state. The pathophysiological significance of homocysteine in the development of vascular disorders through the induction of endothelial dysfunction and abnormal platelet activity and blood coagulation is discussed in this review.

STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets.

Ca(2+) entry, particularly store-operated Ca(2+) entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca(2+) content of the stores, where the intraluminal Ca(2+) sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca(2+) stores to the store-operated Ca(2+)-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25-139) antibody, directed towards the Ca(2+)-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288-301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557-571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca(2+) entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and -8 in human platelets. A role for endoplasmic reticulum stress.

BACKGROUND: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca(2+) concentration ([Ca(2+)](ER)). OBJECTIVES: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an ER Ca(2+) chelator, in human platelets. METHODS: Changes in cytosolic free Ca(2+) concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. RESULTS: Our results indicate that TPEN reduces the amount of free Ca(2+) releasable by the Ca(2+)-mobilizing agonist thrombin. TPEN induced activation of caspase-3, -8 and -9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2 alpha). TPEN-mediated caspase-3 activation requires functional caspase-8, but is independent of H(2)O(2) generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis. CONCLUSION: These findings provide experimental evidence for the existence of ER stress-mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.

Effect of homocysteine on calcium mobilization and platelet function in type 2 diabetes mellitus.

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidative stress and abnormal intracellular Ca(2+) homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show that homocysteine induce a concentration-dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with homocysteine resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilization-induced by homocysteine consisted in two components, an initial slow increase in intracellular free Ca (+) concentration ([Ca2+]i) and a rapid and marked increase in [Ca2+]i, th second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to homocysteine. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that homocysteine might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.

Thrombin induces activation and translocation of Bid, Bax and Bak to the mitochondria in human platelets.

BACKGROUND: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. OBJECTIVES: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. METHODS: Changes in the mitochondrial membrane potential were registered using the dye JC-1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. RESULTS: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H(2)O(2) (10 microm) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. CONCLUSION: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.

Effect of homocysteine on calcium mobilization and platelet function in type 2 diabetes mellitus.

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidative stress and abnormal intracellular Ca2+ homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show that homocysteine (Hcy) induce a concentration-dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with Hcy resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilisation-induced by Hcy consisted in two components, an initial slow increase in intracellular free Ca2+ concentration ([Ca2+]i) and a rapid and marked increase in [Ca2+]i, the second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to Hcy. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that Hcy might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.