Calcium Mobilization In Platelets Research Articles

Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood. 2006; 107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

Calcium Mobilization in Human Platelets is Differentially Modulated by PAR‐1 and PAR‐4 through Gi/o and PI3K

Protease activated receptors (PARs) on the platelet surface play an essential role in mediating hemostasis. Human platelets express PAR-1 and PAR-4, and these receptor subtypes are activated by thrombin. Knockout mice for the various G-alpha subunits of the heterotrimeric G-protein family have provided good tools for the study of PAR-4 signaling in platelets. However, differences in G-protein coupling between human and mouse PAR-4 could exist, and mouse platelets do not express PAR-1. Therefore, determining which G-alpha subunits PAR-1 and PAR-4 activate in human platelets is paramount in understanding the physiological effects of thrombin and its inhibitors. Both receptors have been shown to couple to Gq and G12/13 pathways. However, neither receptor has been shown to have a Gi/o signaling component. The main objective of our research was to determine the role of Gi/o in human platelet PAR-mediated calcium mobilization and integrin activation. We have found that PAR-1, but not PAR-4, directly couples to Gi/o family members to activate PI3K in human platelets using flow cytometry. We have also shown that this PI3K potentiates intraplatelet calcium mobilization in a conditional manner using Fura-2 loaded platelets, and also causes integrin glycoprotein IIbIIIa activation using flow cytometry. PAR-4 seems to lack this Gi/o pathway, and we are currently developing a cell permeable peptide based strategy to explore this possibility. Demonstration of a Gi/o signaling component downstream of PAR-1 in human platelets is a novel finding with potential therapeutic implications. This work was supported by the Training Grant in the Pharmacological Sciences (Vanderbilt-NIH).

Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized ‘secondary’ receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Gαq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors.

Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse.

Platelet accumulation at sites of vascular injury arrests bleeding but also plays a critical role in the pathogenesis of thrombosis, leading to ischemia in myocardial infarction or stroke. Intracellular calcium mobilization in platelets is a critical step in the activation of platelets and formation of the platelet thrombus. Here we show the relationship of the dynamics of intracellular calcium mobilization with platelet accumulation into the developing thrombus in a living mouse. Following injection of 100 x 106 fura-2 loaded platelets into a living mouse we used high speed intravital multi-channel digital fluorescence microscopy to monitor calcium status in circulating and thrombus-bound platelets during thrombus development. One population of platelets binds transiently to the developing thrombus but does not mobilize calcium. The mean duration of platelet-thrombus interaction for these platelets is 11 sec. Another population of platelets undergoes calcium mobilization after binding to the developing thrombus. The time interval from attachment to calcium mobilization for individual platelets varied from 1.0 to 12 sec, with a median of 3.5 sec. More than 90% of platelets that undergo calcium mobilization do so with in 5 sec of attachment. The calcium mobilization in the thrombus bound platelets is reversible. About two thirds of the platelets return rapidly to the basal Ca2+ state while the remaining thrombus bound platelets maintain an elevated Ca2+ level for an extended period. The mean duration of platelet-thrombus interaction is 35 sec with a range of 1.5 sec to 284 sec (median duration 39.5 sec) as calculated from multiple independent observations of single platelets. In each platelet studied, only one calcium peak is detected per platelet. There is a close correlation between the duration of calcium mobilization in an individual platelet and the time that the platelet remains attached to the developing thrombus, suggesting a relationship of calcium-dependent events and platelet-thrombus affinity. A population of platelets binds to the thrombus, mobilizes calcium and remains associated with the thrombus. Using widefield deconvolution techniques to obtain planar images and increased numbers of dye-loaded platelets, individual platelets could be observed undergoing sustained calcium elevation within the thrombus. As the platelet thrombus reaches maximal size at about 120 sec, calcium mobilization continues in the stable core of the thrombus for several minutes, then decreases. These studies describe thrombus formation in a living animal under conditions in which the endothelium and vessel wall, blood cells and plasma components, and flowing blood are preserved in the absence of anticoagulants. Our results indicate that stable platelet thrombus formation is dependent upon durable calcium mobilization, and that intracellular calcium regulates thrombus development and maturation in vivo.

Role of Lipid Rafts in GPVI Agonist-Induced Platelet Signaling.

The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.

Interference of neutrophil–platelet interaction by YC-1: A cGMP-dependent manner on heterotypic cell–cell interaction

N-Formyl-Met-Leu-Phe (fMLP) activated neutrophils and then induced neutrophil–platelet complex formation in co-incubation condition. In addition, fMLP induce intracellular calcium mobilization in platelets, only when it is incubated along with neutrophils. This data established that fMLP-stimulated neutrophils activate platelets. 9E1, a monoclonal antibody of P-selectin, significantly blocks the formation of neutrophil–platelet complex induced by fMLP, indicating the involvement of P-selectin in the neutrophil–platelet complex formation. 3-(5′-hydroxymethyl-2′-furyl-1-benzylindazole (YC-1), an unique nitric oxide-independent activator of soluble guanylate cyclase, was evaluated for its effect on neutrophil–platelet complex. YC-1 inhibits fMLP-induced neutrophil–platelet complex formation in a concentration-dependent manner with an IC50 value of 15.3±3.5 μM. However, this effect of YC-1 is partially reversed by pre-treatment of 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ; 10 μM), which is a soluble guanylate cyclase inhibitor. Pre-treatment of either neutrophils or platelets with YC-1 (50 μM) prevent the fMLP-induced neutrophil–platelet complex formation, indicating that YC-1 could potentially exert its effects individually on either neutrophils or platelets alone. Cathepsin G released from fMLP-stimulated neutrophil activates the nearby platelets. YC-1 was also shown to inhibit this release of cathepsin G in a concentration-dependent manner. The IC50 value was 6.2±0.2 μM. This inhibitory effect of YC-1 on cathepsin G release is reversed by ODQ (10 μM) and a protein kinase G inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carbooxylic acid methyl ester (KT5835); 1 μM]. YC-1 inhibits cathepsin G-induced P-selectin expression on human platelet at the IC50 value of 32.5±2.6 μM. A further study showed that YC-1 inhibits fMLP-induced neutrophil–platelet complex formation in whole blood at the IC50 value of 35.8±8.1 μM in a concentration-dependent manner. According to these data, it was hypothesized that fMLP stimulates neutrophils to release cathepsin G, which subsequently activates the nearby platelets, creating neutrophil–platelet complexes. YC-1 inhibits fMLP-induced neutrophil from releasing cathepsin G via a cGMP-dependent pathway. This inhibitory effect of YC-1 on cathepsin G release is a major mechanism for affecting fMLP-induced neutrophil–platelet complex. YC-1's inhibition P-selectin expression on platelet may potentiate its effects. These inhibitory effects may contribute to the inhibition of neutrophil–platelet complex formation in whole blood.

Cytoplasmic free calcium mobilization in platelets, expression of P-selectin, phosphatidylserine, and microparticle formation, measured by whole blood flow cytometry, in hypertensive patients. Effect of doxazosin GITS

The effects of doxazosin on expression of CD62 (P-selectin) and phosphatidylserine on platelet membrane and platelet calcium flux were studied in 50 uncomplicated essential hypertensive patients (World Health Organization stages 1–2) and 80 normotensive control subjects, matched for age, sex, and cardiovascular risk factors. Hypertensive patients showed greater in vivo platelet activation at baseline than control patients (percentage of CD62-positive platelets, 4.1 ± 2.2% versus 2.4 ± 1.5%, p < 0.001; percentage of phosphatidylserine-positive platelets, 0.8 ± 0.5% versus 0.5 ± 0.3%, p < 0.001). Increased platelet activation was associated with significant changes in the mobilization of free intraplatelet calcium, evaluated by a whole blood flow cytometric kinetic method. With this method, an arbitrary Ca 2+ mobilization index was defined as the ratio of cytoplasmic free calcium before activation with thrombin to the slope of the calcium removal rate following the action of the agonist. This index was significantly higher in untreated hypertensive patients than in normotensive controls (0.12 ± 0.06 versus 0.05 ± 0.08, p < 0.001). Treatment of hypertensive patients with doxazosin gastrointestinal therapeutic system (4 mg/day as a single dose) for 2 months normalized both platelet activation and Ca 2+ mobilization. Changes in the expression of CD62 and phosphatidylserine in the platelet membrane after treatment with doxazosin gastrointestinal therapeutic system may be related to normalization of the kinetics of cytoplasmic free Ca 2+. Normalization of platelet activation may represent an additional beneficial effect to the known antihypertensive action of doxazosin gastrointestinal therapeutic system.

Human platelets exhibit chemotaxis using functional N-formyl peptide receptors

Activated platelets participate in inflammatory and microbicidal processes by upregulation of surface selectins, shedding of CD40 ligand, and release of platelet microbicidal proteins and microparticles. Given their myeloid lineage, we hypothesized that platelets express functional N-formyl peptide receptors and respond to the bacterially derived chemotactic peptide N-formyl peptide with gradient-driven chemotaxis. Here we show specific binding of N-formyl peptides to the surface of activated platelets. Platelet expression and function of the formyl peptide receptor, FPR, was verified by RT-PCR of the differentiated megakaryocyte MEG-01 cell line, immunoblotting of platelet proteins, and calcium mobilization in platelets with formyl peptide binding. Furthermore, we demonstrate gradient-driven chemotaxis of platelets by video microscopy and transwell migration toward formyl peptides. We also show that endogenous formyl peptides, released by eukaryotic mitochondria from necrotic cells, induce chemotaxis using formyl peptide receptors expressed by thrombin-activated platelets. Conversely, supernatants from cells undergoing apoptotic cell death do not induce platelet chemotaxis. Platelet chemotaxis to formyl peptides was blocked with FPR-specific antibody as well as by pertussis toxin inhibition of the formyl peptide G-coupled receptor. These data establish a new role for platelets in host defense and suggest reexamination of their active function in microbicidal and other host defense activities.

Treahlose Loaded, Freeze-Dried Rehydrated Human Platelets Maintain and Regulate Their Intracellular pH.

Maintenance of intracellular pH, a critical cellular function, is required for generation of proton gradients and platelet response to agonist. Previously, we have demonstrated that freeze-dried rehydrated platelets are able to respond to agonists generate a rise in intracellular calcium (Auh et al. 2004 Calcium mobilization in freeze-dried platelets. Cell Preservation Technology in press) and maintain normal membrane and protein secondary structure (Wolkers et al. 2002 Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175–188). As part of our ongoing studies of trehalose loaded freeze-dried rehydrated human platelets we examined their ability to maintain their intracellular pH. Platelet proton regulation is achieved through the Na-H exchanger (NHE1) which is dependent on the concentration of extracellular sodium. Freeze-dried and fresh control human platelets were loaded with the pH ratio dye bis-carboxyfluorescein acetomethyl ester (BCECF-AM), washed over a Sepharose 2B column and examined by fluorescence spectroscopy. Fresh and freeze-dried rehydrated human platelets maintained virtually identical resting intracellular pH, 7.273 +/− 0.015, and 7.270 +/− 0.034 respectively. Both cell types responded to increased extracellular sodium by increasing their pH in a virtually identical manner. The addition of 0.5U/ml thrombin (in the presence of 135 mM NaCl) resulted in an initial acidification and subsequent alkalinzation of both fresh and freeze-dried rehydrated cells. Prior to the addition of thrombin both cell populations had an [pH]i of 6.9, while after thrombin stimulation the pH rose to 7.012 for fresh cells and 7.001 for freeze-dried rehydrated cells. Thrombin stimulation in the absence of extracellular sodium resulted in a significant acidification of both cell populations to a final pH of 6.6 for fresh cells and 6.7 for freeze-dried rehydrated cells. Specific inhibition of the NHE1 transporter by 5-(N-methyl-N-isobutyl) amiloride (MIA) completely abolished the response of all cells to increasing concentrations of sodium. In the parallel control experiment both freeze-dried and fresh cells acidified to pH 6.2 and incubated with 135mM NaCl responded by generating a rise in intracellular pH to 7.1. These results demonstrate that freeze-dried rehydrated platelets are able to maintain normal pH homeostasis and respond to agonist in a specific manner. Studies funded by DARPA.

Direct Real Time Visualization of Platelet Calclium Signaling In Vivo: Role of Platelet Activation and Thrombus Formation in a Living Mouse.

Intracellular calcium mobilization plays a critical role in platelet signaling. Upon platelet activation, an intracellular calcium mobilization leads to the activation of various intracellular and membrane proteins, including integrins involved in both platelet shape change and aggregation. The goal of the present study was to monitor platelet calcium mobilization in vivo in an intact animal and to determine which intracellular pathways are dominant in platelet accumulation into the developing thrombus. Platelets were isolated from mice, washed, loaded with a calcium-sensitive fluorochrome, Fura2-AM and then infused into a recipient mouse. We studied Fura2-AM loaded platelet incorporation during arterial thrombus development following laser injury of the vessel wall in the cremaster microcirculation of living mice using high speed intravital widefield digital microscopy. Fura-2 loaded platelets were monitored by excitation at 380 nm; this fluorescence reports the basal calcium levels in platelets. Calcium mobilization was monitored by excitation at 340 nm where the fluorescence intensity reflects Fura2-calcium complex formation. We observed that platelets bind to the growing thrombus independent of calcium mobilization. However, the stable incorporation of platelets into the thrombus correlated with a significant intracellular calcium increase. Once the thrombus reached maximal size at about 100 seconds, the calcium mobilization also reached maximal intensity. Subsequently, platelets that did not mobilize calcium dissociated from the thrombus. We confirmed these observations by using platelets treated with the calcium chelators, BAPTA-AM or EGTA-AM. We observed a significant inhibition of platelet accumulation into the thrombus, indicating that the intracellular calcium increase is necessary in vivo for the stable accumulation of the platelets into the thrombus. We also evaluated the involvement in vivo of two platelet agonists, ADP and thromboxane A2 (TxA2), on calcium mobilization and platelet incorporation into thrombi. When platelets were treated with aspirin or with the P2Y1 antagonist A3P5P (adenosine 3′-phosphate-5′-phosphate), we observed a partial decrease in both calcium mobilization and platelet accumulation into the thrombus. These results indicate that TxA2 and ADP via the P2Y1 receptor are involved in vivo in platelet activation upon vessel wall injury in this thrombosis model. When platelets were treated with both compounds, we completely inhibited the calcium increase and the incorporation of platelets into the thrombus. Altogether, our results directly show, for the first time in vivo, the importance of the calcium mobilization on platelet accumulation into the developing thrombus. The platelet agonists TxA2 and ADP both play an important and complementary role on platelet activation by acting on the mobilization of the intracellular calcium.

Effect of eprosartan on cytoplasmic free calcium mobilization, platelet activation, and microparticle formation in hypertension

Hypertensive patients show greater platelet activation than do normotensive individuals. Platelet activation is characterized by increased phosphatidylserine (PS) exposure in the external hemilayer of the membrane, a larger number of platelet microparticles (PMP), and changes in intraplatelet-free calcium kinetics. This study evaluated whether eprosartan can protect against undesirable platelet activation. A total of 30 hypertensive patients (systolic blood pressure [SBP] 140 to 189 mm Hg; diastolic blood pressure [DBP] 90 to 109 mm Hg) without renal, liver, or cardiac organic lesions and with a mean age of 47.6 +/- 9.4 years and mean body mass index (BMI) of 27.9 +/- 3.9 kg/m2 received eprosartan (600 mg/day). They were compared with 31 normotensive individuals with a mean age of 43.3 +/- 6.7 years and a mean BMI of 26.8 +/- 3.9 kg/m2. Blood pressure measurements and platelet function changes were assessed at baseline (control and hypertensive patients) and after 1 and 2 months of eprosartan monotherapy (hypertensive patients only). Significant baseline to endpoint (month 2) changes in SBP and DBP were noted in the eprosartan group (SBP: baseline 152.2 +/- 16.8 mm Hg, endpoint 142.2 +/- 16.9 mm Hg, P <.01; DBP: baseline 93.5 +/- 9.9 mm Hg, endpoint 85.8 +/- 11.9 mm Hg, P <.001). Native circulating activated platelets increased in both groups after shear stress or Ca2+ ionophore activation, and were reduced by eprosartan (after shear exposure from 104% at month 1 to 76% after 2 months of therapy). Eprosartan therapy normalized the number of microparticles after blood shear exposure (P <.01) and after exposure to Ca2+ ionophore activation (P <.05) and significantly reduced the trend for platelets to be more readily activated in hypertensive compared with normotensive subjects (baseline to endpoint change P <.001; increase/shear versus baseline P <.001). Eprosartan partially normalizes cytoplasmic-free calcium mobilization in platelets. Eprosartan significantly reduces blood pressure and normalizes undesirable changes in platelet function.

Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats

BackgroundIn the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy.ResultsBasal calcium levels were always elevated in the platelets of the L-NAME-treated rats, both in the presence and in the absence of extracellular calcium. The administration of thrombin in the absence and in the presence of extracellular calcium induced important elevations in calcium levels that were always of greater magnitude in the platelets of the L-NAME-treated rats than in those of the controls. The addition of calcium to thapsigargin-treated platelets produced a massive elevation in calcium levels in both groups that was significantly greater in the platelets obtained from the hypertensive rats than in those of the controls.ConclusionsIt is concluded that the arterial hypertension induced by the reduction of nitric oxide alters the regulation of platelet calcium levels so that elevated baseline levels and calcium entry and mobilization are enhanced. This could be the result of direct or indirect effects of the lack of nitric oxide synthesis in platelets or in other tissues.

Cardiology patient page. Platelets and cardiovascular disease.

Platelets are specialized disk-shaped cells in the blood stream that are involved in the formation of blood clots that play an important role in heart attacks, strokes, and peripheral vascular disease. In most people, the more than 200 million platelets in a milliliter of blood act as tiny building blocks to form the basis of a clot to stop bleeding from cuts or injuries. Platelets can detect a disruption in the lining of a blood vessel and react to build a wall to stop bleeding (Figure 1). Figure 1. Platelets form a platelet plug to stop bleeding from an injured blood vessel. In cardiovascular disease, abnormal clotting occurs that can result in heart attacks or stroke. Blood vessels injured by smoking, cholesterol, or high blood pressure develop cholesterol-rich build-ups (plaques) that line the blood vessel; these plaques can rupture and cause the platelets to form a clot. Even though no bleeding is occurring, platelets sense the plaque rupture and are confused, thinking that an injury has taken place that will cause bleeding. Instead of sealing the vessel to prevent bleeding as would occur with a cut, a clot forms in an intact blood vessel, causing a blockage of blood flow (Figure 2). Without …

Increased coronary events in depressed cardiovascular patients: 5-HT2A receptor as missing link?

Major depressive disorder and depressive symptoms have been identified as independent risk factors for cardiac morbidity and mortality in patients with ischemic heart disease. Increased susceptibility to platelet activation has been proposed as one of the mechanisms by which depression acts as a significant risk factor for thrombotic events. In this review, data on platelet activation and platelet aggregation measures in depressed patients with or without concomitant cardiovascular disease are given. Data on the influence of antidepressants on parameters of platelet activation are summarized. A literature search was done by checking MEDLINE Advanced and PsycInfo from 1990 to 2003 and through checking the bibliographies of these sources. The following key words were used for this search: platelet activation, platelet aggregation, depression, depressive disorder, ischemic heart disease, calcium, and serotonin. There is an indication of enhanced platelet activation and aggregation in depressed patients. Next, patients with a depressive disorder show signs of a hyperactive platelet 5-HT2A receptor signal transduction system as measured by increased platelet calcium mobilization after stimulation of platelets with serotonin. Depression appears to be associated with an increased susceptibility for serotonin-mediated platelet activation. Upregulation and/or increased sensitivity of 5-HT2A/1B receptors and downregulated 5-HT transporter receptors in the periphery may contribute to increased risk of thromboembolic events in patients with depression and cardiovascular disease. Increased platelet reactivity based on a hyperreactive 5-HT2A receptor signaling system might be influenced by antidepressive medication that antagonizes platelet 5-HT2A receptors.

Lack of association between suicidal ideation and enhanced platelet 5-HT2A receptor-mediated calcium mobilization in cancer patients with depression

Background Increased density of 5-HT2A receptors was observed in the platelets of depressive patients with suicidal ideation. Enhanced 5-HT2A receptor-mediated platelet calcium mobilization has been proposed as a biological marker for the pathophysiology of major depression in cancer patients as well as in physically healthy patients. To examine whether depressive cancer patients with suicidal ideation have enhanced 5-HT2A receptor-mediated platelet response compared with those without suicidal ideation, we compared 5-HT-induced platelet calcium mobilization in depressive cancer patients with and without suicidal ideation. Methods 5-HT-induced platelet calcium mobilization was examined in 24 cancer patients diagnosed as having major depression according to the DSM-IV criteria. Suicidal ideation was evaluated by the Hamilton Depression Rating Scale and Zung’s Self Depression Scale, as well as by the DSM-IV criteria. Results There was no significant differences in 5-HT-induced platelet calcium response between the depressive cancer patients with ( n = 8) and without suicidal ideation ( n = 16). 5-HT-induced platelet calcium response was also not significantly associated with the severity of suicidal ideation or with the severity of depression assessed by Hamilton Depression Rating Scale and Zung’s Self Depression Scale. Conclusions These findings suggest that enhanced 5-HT2A receptor-mediated response was not associated with suicidal ideation in cancer patients with depression.

The CC Chemokines MDC and TARC Induce Platelet Activation Via CCR4

While chemokines have received considerable attention for their role in leukocyte chemotaxis, their effects on platelets have not been well described. We found that two CC chemokine receptor 4 (CCR4) ligands, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) induce concentration-dependent platelet aggregation and calcium flux. Flow cytometric analysis revealed the expression of CCR4 on platelets and a monoclonal antibody (mAb) to CCR4 inhibited MDC- and TARC-induced platelet aggregation, confirming that this effect is mediated through their common receptor CCR4. MDC fully desensitized TARC-induced calcium mobilization in platelets, while TARC was unable to completely desensitize a subsequent MDC response, which is similar to observations made in Th2 CD4 + lymphocytes and CCR4-transfected cells. Aspirin (ASA) treatment of platelets allowed reversible primary aggregation but inhibited irreversible complete aggregation, suggesting that MDC- and TARC-induced full platelet aggregation is dependent on cyclooxygenase metabolites of arachidonic acid. MDC and TARC were unable to induce platelet aggregation and platelet secretion in washed human platelets, even though they induced a calcium flux, suggesting that plasma components are required for MDC- and TARC-induced platelet aggregation. Since Th2-type cytokines induce the release of MDC and TARC from cells and the expression of these chemokines is increased in Th2-type inflammation, we hypothesize that MDC and TARC may play a role in platelet activation seen in Th2 diseases, such as asthma and atopic dermatitis.

Three sets of diagnostic criteria for major depression and correlations with serotonin-induced platelet calcium mobilization in cancer patients

Enhanced serotonin-induced platelet calcium mobilization has been proposed to be a biological marker for the pathophysiology of major depression in physically healthy patients. To determine the most appropriate method of diagnosing major depression in cancer patients, we compared serotonin-induced platelet calcium mobilization between patients with and without major depression diagnosed according to three different sets of diagnostic criteria (inclusive, substitutive and exclusive). Among the cancer patients referred to our institution between June 1997 and March 1998, 24 patients diagnosed as having major depression according to the inclusive approach (in which the nine traditional symptoms of major depression contribute towards the diagnosis of depression regardless of its presumed etiology) participated in the study. Serotonininduced platelet calcium mobilization was examined in these patients and in the same number of non-depressed controls matched for age, sex, cancer stage and cancer site. The depressed patients were then re-evaluated using substitutive and exclusive criteria, and calcium mobilization comparisons with the relevant controls were repeated. Compared with the controls, an enhanced serotonin-induced platelet calcium response was only observed in the patients with major depression according to the exclusive criteria. No significant enhancement was observed when the inclusive or substitutive approaches were used. These findings, based on the use of enhanced serotonin-induced platelet calcium mobilization as a biological marker, suggest that the exclusive approach might be the most valid and appropriate method of diagnosing major depression in cancer patients, while the inclusive and substitutive approaches might overestimate the occurrence of major depression in these patients.

Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41–Ser42. In addition, a rapid cleavage of the peptide bond at Arg46–Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41–42 and 46–47 peptide bonds, and fragment 42–103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.

Serotonin stimulation of calcium mobilisation in human platelets: choice of units of measurement, effects of age and tobacco use, and correlation with serotonin 2A receptor density

The calcium responses to serotonin and thrombin, as assessed using the fluorescent indicator Fura-2, have been investigated in platelets taken from 59 non-smokers and 17 smokers. The peak responses above baseline, calculated either as fluorescence ratios or calibrated calcium concentrations, were in the order of magnitude thrombin 520 mU/ml>thrombin 52 mU/ml≫serotonin 1 μmol/l≈serotonin 100 μmol/l. Multiple regression analyses indicated that the responses to 1 μmol/l serotonin, but not the responses to thrombin, were significantly correlated with the serotonin 2A receptor density measured using [ 3H]LSD as radioligand. No effects of age, gender, smoking habit or the time of year of sampling were seen on the calcium responses to serotonin and thrombin. It is concluded that cellular processes distal to the serotonin 2A receptor recognition site may compensate to some extent for the large differences in recognition site expression, thus underlining the importance of providing a functional correlate in addition to [ 3H]LSD binding site densities when studying platelet serotonin 2A receptors in neuropsychiatric disorders. The measurement of Ca 2+ responses to serotonin provides a useful such functional correlate.

Decreased intraplatelet Ca2+ release and ATP secretion in pediatric nephrotic syndrome.

Platelets play an important role in the natural history of idiopathic nephrotic syndrome (NS). Although thromboembolic events are rare, the activation of circulating platelets is generally considered an important factor in the prethrombotic state in children with NS. Platelet-activating factor (PAF), a potent endogenous phospholipid mediator of inflammation, stimulates intracellular free calcium (Ca2+) mobilization, aggregation, and release reactions in platelets obtained from normal donors. Platelet-related effects of PAF in children with NS are unknown. We studied PAF-induced intracellular Ca2+ mobilization in washed platelets and ATP secretion in platelet-rich plasma in 34 children with idiopathic NS and in 7 healthy children. There was a significant decrease in ATP secretion: 0.021+/-0.011 microg/10(7) cells with 20 nM PAF and 0.089+/-0.017 microg/10(7) platelets with 200 nM PAF versus control values (0.195+/-0.004 microg/10(7) and 0.813+/-0.09 microg/10(7), respectively). Moreover, PAF-evoked increase in intracellular free Ca2+ concentration was twofold lower in NS patients than in control subjects (230.1+/-22.4 nM versus 455.6+/-15.3 nM). Also, thrombin-induced intracellular free Ca2+ mobilization was diminished in children with NS compared with the control group. Thus, contrary to expectations, a decrease of platelet reactivity in response to PAF in vitro was observed in children with idiopathic NS. We suggest that this decreased platelet reactivity may reflect a period refractory to PAF and may be considered as platelet desensitization to PAF released in vivo in children with NS.