Intracellular Calcium Mobilization In Platelets Research Articles

Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation

The healthy vascular endothelium constantly releases autacoids which cause an increase of intracellular cyclic nucleotides to tame platelets from inappropriate activation. Elevating cGMP and cAMP, in line with previous reports, cooperated in the inhibition of isolated human platelet intracellular calcium-mobilization, dense granules secretion, and aggregation provoked by thrombin. Further, platelet alpha granules secretion and, most relevant, integrin αIIaβ3 activation in response to thrombin are shown to be prominently affected by the combined elevation of cGMP and cAMP. Since stress-related sympathetic nervous activity is associated with an increase in thrombotic events, we investigated the impact of epinephrine in this setting. We found that the assessed signalling events and functional consequences were to various extents restored by epinephrine, resulting in full and sustained aggregation of isolated platelets. The restoring effects of epinephrine were abolished by either interfering with intracellular calcium-elevation or with PI3-K signalling. Finally, we show that in our experimental setting epinephrine likewise reconstitutes platelet aggregation in heparinized whole blood, which may indicate that this mechanism could also apply in vivo.

A human platelet calcium calculator trained by pairwise agonist scanning.

Since platelet intracellular calcium mobilization [Ca(t)]i controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Pairwise Agonist Scanning (PAS) deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50; 154 conditions including a null condition) to stimulate platelet P2Y1/P2Y12 GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t)]i data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t)]i beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.

The PAK Signaling System Links Rho Gtpase Activation to Platelet Lamellopodia Formation, Aggregation and Aggregate Stability Under Shear

Abstract 1060Platelets serve as the primary mediators of hemostasis and thrombosis, circulating as surveyors for gaps in vascular integrity. As platelets encounter exposed extracellular matrix proteins, receptors on the platelet surface trigger intracellular signaling events that result in rapid platelet activation and a complex rearrangement of platelet morphology to form filopodia and lamellipodia. Rac1, a member of the Rho GTPase family, has emerged as a key regulator in platelet actin dynamics. However, the specific downstream events following Rac1 activation that mediate platelet actin cytoskeleton reorganization remain ill-defined.The Rho GTPase, Rac, supports the autocatalytic activation of the p21 activated kinases, or PAKs, to mediate actin reorganization processes in focal adhesion formation and cell migration. Upon activation by GTP-bound Rac, the PAKs phosphorylate a number of substrates to coordinate actin dynamics. Platelets express a number of PAK isoforms, and like Rac, PAK has been shown to be activated as platelets spread on collagen in a Src and PI3K dependent manner. Furthermore, the adaptor protein SLP-76 has been proposed to potentiate PAK activity downstream of Rac activation to mediate platelet lamellipodia formation. However, the specific roles of PAK in platelet function have yet to be characterized. Thus we set out to elucidate the role of PAK in platelet function and to define the connection between Rac activation, PAK, and platelet cytoskeletal reorganization.Our initial experiments with mass spectrometry revealed that following platelet activation, Rac1 associates with a set of PAK effectors, GIT1, GEFH1, LIMK1, and Merlin. We next demonstrated a co-localization of Rac1 and PAK with actin at the leading edge of spread platelets on fibrinogen. In addition, inhibition of PAK signaling by two different pharmacologic inhibitors blocked platelet focal adhesion and lamellopodia formation on both fibrinogen and collagen. Inhibition of PAK signaling abrogated intracellular calcium mobilization in platelets, prevented platelet aggregation to the GPVI-agonist, CRP, and destabilized platelet lamellipodia, resulting in the retraction of lamellipodia in spread platelets. Finally, inhibition of PAK resulted in the disaggregation of platelet aggregates formed under shear flow conditions.Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation to PAK effector function and platelet lamellopodia formation, thus filling an important gap in the understanding of platelet actin cytoskeletal organization. In addition, these data characterize the integral role of PAK in platelet spreading, aggregation, and aggregate stability. Elucidating the mechanisms that mediate platelet spreading and aggregate formation may highlight important steps in the platelet activation cascade at which to pharmacologically intervene in order to inhibit or treat pathologic thrombi formation. Disclosures:No relevant conflicts of interest to declare.

Blood Clots Are Rapidly Assembled Hemodynamic Sensors

Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction. Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Characterization of human platelet binding of recombinant T cell receptor ligand

BackgroundRecombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.MethodsWe have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function.ResultsOur data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.ConclusionsPlatelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase

Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.

Modulation of platelet and leucocyte function by a Chinese herbal formulation as compared with conventional antiplatelet agents

Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic® Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y12 receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 µmol/L adenosine diphosphate (ADP), 1.0 µmol/L platelet activating factor (PAF), 5.0 µmol/L adrenaline and 0.5 µg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 µmol/L ADP, 10 µmol/L PAF, 2 µg/mL collagen and 15 µmol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 µmol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.

Low Concentrations of Resveratrol Potentiate the Antiplatelet Effect of Prostaglandins

Resveratrol, a polyphenolic compound found in grapes and other fruits, is thought to contribute to the cardioprotective effect of red wine. While resveratrol exhibits some antiplatelet effect in vitro, the concentrations needed are much higher than those in plasma after consumption of red wine. In the present study, we investigate if resveratrol is able to potentiate the effect of endogenous antiplatelet substances--prostaglandin (PG) I2 and PGE1. In human platelet suspension resveratrol at relatively low concentrations (2 or 5 microM), which did not affect platelet function, significantly enhanced the inhibitory activity of PGs on platelet aggregation caused by collagen. The mechanisms underlying this effect may be associated with the inhibition of protein kinase C activation and protein tyrosine phosphorylation, but not with cyclic nucleotide levels and intracellular calcium mobilization in platelets. Our results might provide a possible explanation for the in vivo antiplatelet effect of resveratrol despite the poor bioavailability and the weak in vitro activity.

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.

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.

Effects of lithium and valproate on agonist-induced platelet intracellular calcium mobilization: relevance to myosin light chain kinase

Serotonin (5-HT)- or thrombin-stimulated platelet intracellular calcium (Ca) mobilization has been reported to be enhanced in patients with bipolar disorders. However, the mechanism of this enhancement is unknown. As a preliminary study, the authors examined the effects of a myosin light chain kinase (MLCK) inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazepine hydrochloride (ML-9), and two drugs that are mainstays of treatment for bipolar disorder, lithium and valproate, on 5-HT- or thrombin-induced Ca increase in the platelets of normal subjects. When preincubated with 30 μM ML-9, Ca responses to both agonists were enhanced. Valproate showed a dose-dependent attenuation of agonist-induced intracellular Ca rise, both in the absence and presence of ML-9. Although lithium alone had no significant effect on the Ca increase, a high concentration of lithium significantly decreased Ca mobilization only in the presence of ML-9. These results suggest that the enhanced Ca response observed in bipolar disorder might be relevant to decreased function of MLCK and that the mechanism of action of lithium may include a compensatory effect on MLCK modulation.

Serotonin-induced platelet intracellular calcium mobilization in various psychiatric disorders: is it specific to bipolar disorder?

Background: Serotonin (5-HT)-stimulated platelet intracellular calcium (Ca) mobilization has been reported to be enhanced in unmedicated depressive patients compared to those of normal healthy subjects, which suggests increased 5-HT2A receptor function in these patients. It has not been ascertained whether this enhanced response is specific to some type of affective disorders among various mental disorders. Methods: We examined 5-HT-induced platelet intracellular Ca response in 152 unmedicated outpatients with various psychiatric disorders including bipolar disorder (BD), major depressive disorder with melancholia (DM), major depressive disorder without melancholia (DN), schizophrenia (SCH), panic disorder (PD), obsessive–compulsive disorder (OCD), social phobia (SP) and bulimia nervosa (BN), and 30 normal controls. Results: We observed no significant differences in basal intracellular Ca concentration among all patient subgroups and normal controls. While the 5-HT-induced Ca response was significantly and specifically higher in patients with BD than in normal controls, no significant differences were found in the Ca response to 5-HT between patients with DM, DN, SCH, PD, OCD, SP and BN, and normal controls. Limitations: The sample sizes of each group are still small. Therefore, they have to be enlarged in the continuation of the study so as to increase the power of the statistical tests. Conclusion: These results indicate the possibility that enhanced signal transduction, mediated by the 5-HT2A receptor, may be specific to bipolar disorder.

Evidence for Two Distinct G-protein-coupled ADP Receptors Mediating Platelet Activation

The identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1 receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTPgammaS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2YI receptor. By contrast, the P2Y1 receptor antagonist adenosine 3',5'-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1 receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1 receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Gi proteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1 and coupled to Gq.

Genetic variation in human 5-HT receptors: potential pathogenetic and pharmacological role.

Mutation screening identified variants of h5-HT1A (Gly-22-Ser, Ile-28-Val, Arg-219-Leu), h5-HT1B (Phe-124-Cys), h5-HT2A (Thr-25-Asn, His-452-Tyr), h5-HT2C (Cys-23-Ser) and h5-HT7 (Thr-92-Lys, Pro-279-Leu) receptors. Screening of h5-HT1D, h5-ht1e, h5-ht1f and h5-ht5 receptor genes failed to detect any significant mutations. No differences in radioligand binding properties were observed between the h5-HT1A Ile-28-Val variant receptor (VR) and the wildtype receptor (WTR). Binding profiles of the h5-HT1A Gly-22-Val variant and the WTR were also very similar, but the 8-OH-DPAT-induced down-regulation and desensitization of the VR was attenuated. The h5-HT1B Phe-124-Cys variant leads to considerable changes in [3H]5-carboxamidotryptamine binding: Bmax was decreased and the affinity of various h5-HT1B ligands was modified (usually increased; e.g., in the case of sumatriptan). The h5-HT2A His-452-Tyr variant causes an alteration of the amplitude and timing of intracellular calcium mobilization in platelets from 452-His/452-Tyr heterozygous compared to 452-His/452-His homozygous individuals. Most, but not all, of the VRs listed above were examined for association with, e.g., bipolar depression and schizophrenia, yet no relation was observed. The most consistent finding was an association between a silent mutation (102T/C) in the h5-HT2A receptor gene and schizophrenia; this association may be explained by linkage disequilibrium with a functional variant in the regulatory region of the gene. Studies of the therapeutic response to clozapine produced no homogeneous results with respect to the pharmacogenetic significance of the various mutations in the h5-HT2A and h5-HT2C receptor genes.

Serotonin-stimulated intracellular calcium mobilization in platelets from alcoholic men

Numerous lines of evidence have suggested a key role for serotonin (5-hydroxytryptamine, 5HT) pathways in the regulation of alcohol consumption. To explore the functioning of the 5HT 2 receptor in alcoholism, 5HT-stimulated intracellular calcium response was measured in platelets from abstinent alcoholic patients and normal comparison subjects. No difference in resting or stimulated calcium levels was observed. This finding suggests that 5HT 2 receptor function is unaffected in non-drinking alcoholic subjects. The previously reported 5HT inhibition in actively drinking alcoholic subjects is likely to be a state rather than trait marker of alcoholism.

Magnolol inhibits collagen-induced platelet serotonin release

Magnolol is one of the major phenolic constituents of Magnolia bark (1,2). The bark of Magnolia has been used as a folk medicine by Chinese for the treatment of anxiety, neural and cardiovascular disorders (3). Nowadays medicinal herbs have been gaining renewed popularity in the world as an alternative choice in health care very much reminiscent of the pre-pharmacology days. Recent studies indicate that magnolol inhibits intracellular calcium mobilization in platelets (4), relaxes vascular smooth muscle (5), and has antihemostatic and antithrombotic effects (6). For the 5-HT determination, the more commonly used chemical method is spectrophotofluorometric method. However, its detection limit is low (7). Radioimmunoassay and enzymatic assay have higher sensitivity (8,9). The newly developed method of labelling the granule-bound pool by incorporation of radioactive 5-HT then measuring the appearance of radioactivity extracellularly for the quantitation of 5-HT secretion was reported (10). However, they are all complicated and time-consuming. The purpose of this study is to establish a reliable and sensitive microbore HPLC ECD in order to measure platelet 5-HT and evaluate antiplatelet effect of magnolol on collagen-induced 5-HT release.

Increased intracellular calcium mobilization in platelets from patients with Type 2 (non-insulin-dependent) diabetes mellitus

Enhanced platelet functions have been reported in patients with diabetes mellitus. Our recent study demonstrated that phosphoinositide turnover is increased in platelets from diabetic patients. In the present study, we evaluated the abnormality in platelet intracellular calcium mobilization in patients with Type 2 (non-insulin-dependent) diabetes mellitus using fura-2, a fluorescent calcium indicator. Washed platelets were prepared from six diabetic patients with increased platelet aggregation rates (DM-A group), seven diabetic patients with normal platelet aggregation rates (DM-B group), and eight age-matched healthy control subjects. The basal intracellular free calcium concentrations in platelets were similar among the three groups. Thrombin (0.025-0.1 U/ml) induced a dose-dependent increase in intracellular calcium in both the presence and the absence of extracellular calcium. This increase in the presence of extracellular calcium, which depends on calcium influx and release, was significantly higher in the DM-A group than in the DM-B and control groups. However, there was no significant difference between the control group and the DM-B group. In the absence of extracellular calcium, thrombin-induced calcium increase, which depends only on calcium release, was also significantly enhanced in the DM-A group. Furthermore, the calcium increase stimulated by platelet-activating factor (10 nmol/l) with and without extracellular calcium was significantly higher in the DM-A group than in the other groups. Additionally, calcium ionophore A23187 (100 nmol/l) caused a significantly higher calcium increase in the DM-A group with extracellular calcium, while the calcium increase without extracellular calcium showed no significant difference among the three groups. These observations suggest that enhanced intracellular calcium mobilization due to increased calcium influx and release may be closely related to platelet hyperfunctions in diabetes mellitus.