Calcium Mobilization In Human Platelets Research Articles

Platelet-derived microvesicles induce intracellular calcium mobilization in human platelets.

Platelet-derived microvesicles (PMVs) represent a significant proportion of microvesicles in circulation and have been linked to various pathophysiological complications. Recent research suggests that PMVs carry significant amounts of cargo that can affect cellular functions by influencing calcium oscillations in target cells. As calcium is involved in multiple cellular processes, including hemostasis and thrombosis, this study aimed to investigate the impact of PMVs on platelet calcium mobilization. The study found that PMVs increase platelet intracellular calcium levels via both intracellular storage and extracellular space in a dose-dependent manner. The study highlighted the critical role of the dense tubular system, acidic vacuoles, mitochondrial stores, and store-operated calcium entry (SOCE) in PMV-mediated calcium release in human platelets. Moreover, the study revealed that PMV-induced calcium rise in platelets does not occur via sarcoendoplasmic reticulum calcium ATPase, and extracellular calcium addition further increases the calcium level in platelets, demonstrating the involvement of SOCE. These findings provide insights into the platelet stimulation signaling mechanisms and contributes to our understanding of platelet and cell behavior when exposed to PMV-rich environments.

The PI3Kδ Inhibitor Idelalisib Diminishes Platelet Function and Shows Antithrombotic Potential.

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

Mechanism underlying N-(3-oxo-dodecanoyl)-L-homoserine lactone mediated intracellular calcium mobilization in human platelets

Acyl-homoserine lactones (AHLs), are the key autoinducer molecules that mediate Pseudomonas aeruginosa associated quorum sensing. P. aeruginosa produces two types of AHLs; N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 HSL) and N-butyryl-L-homoserine lactone (C4 HSL). AHLs are not only regulating the virulence gene of bacteria but also influence the host cell functions by interkingdom signaling. In this study, we explored the mechanism of AHLs induced calcium mobilization in human platelets. We found that 3-oxo-C12 HSL but not C4 HSL induces intracellular calcium release. 3-oxo-C12 HSL induced calcium mobilization was majorly contributed from the dense tubular system (DTS). Furthermore, 3-oxo-C12 HSL also stimulates the store-operated Ca2+ entry (SOCE) in platelet. Intracellular calcium rise was significantly lowered in rotenone, and bafilomycin pre-treated platelets suggesting partial involvement of mitochondria and acidic vacuoles. The significant effect of 3-oxo-C12 HSL on calcium mobilization can alter the platelet functions that might results in thrombotic disorders in individuals infected with P. aeruginosa.

Synthesis and Structure–Activity Relationship Studies of 4-((2-Hydroxy-3-methoxybenzyl)amino)benzenesulfonamide Derivatives as Potent and Selective Inhibitors of 12-Lipoxygenase

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in β-cells.

Signaling through Gα13 Switch Region I Is Essential for Protease-activated Receptor 1-mediated Human Platelet Shape Change, Aggregation, and Secretion

This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.

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).

Effect of picotamide on the calcium mobilization and phospholipase C activation in human platelets

The effect of picotamide (G137 or N,N′-bis-3-picolyl-4-methoxy-isophthalamide), a dual thromboxane A 2 (TxA 2) synthetase inhibitor/TxA 2 endoperoxide receptor antagonist, on the phospholipase C (PLC) activation and calcium mobilization in human platelets stimulated by arachidonic acid (AA) and TxA 2 receptor synthetic agonist U46619, has been studied. Preincubation with picotamide (10 −4 M) for 1 and 3 min significantly reduced (p<0.03 and p<0.005 respectively) the calcium concentration changes induced in gel-filtered platelets (GFP s) by U46619 125 nM and 250 nM. Picotamide also reduced the calcium concentration changes induced in GFP s by AA 75 and 150 μM and by ADP 5 and 10 μM. In thrombin degranulated platelets picotamide inhibited the effect of U46619 up to 500 nM. The PLC activation, as indicated by inositol-1,3,4 P 3 (Ins 1,3,4 P 3) formation in response to U46619 250 nM and AA 150 μM was also inhibited by picotamide. These results may suggest a dual effect of picotamide on the receptor/effector systems through which TxA 2 mediates platelet activation.

Wheat Germ Agglutinin-Induced Intracellular Calcium Mobilization in Human Platelets: Suppression by Staurosporine and Resistance to Cyclic AMP Inhibition

The lectin wheat germ agglutinin (WGA) elicited a prompt and sharp increase in intracellular Ca 2+ concentration in human platelets. The WGA- induced Ca 2+ mobilization was markedly inhibited by a protein kinase inhibitor staurosporine, whereas Ca 2+ mobilization by receptor-mediated agonists, including thrombin, platelet-activating factor, and arginine-vasopressin, was not. In contrast, the lectin-induced Ca 2+ mobilization was resistant to cyclic AMP inhibition, compared with that induced by receptor-mediated agonists. These findings indicate that the mechanism of intracellular Ca 2+ mobilization, or possibly phospholipase C activation, induced by WGA is different from that induced by receptor-mediated agonists in human platelets.

Heterologous supersensitization between serotonin2 and alpha 2-adrenergic receptor-mediated intracellular calcium mobilization in human platelets.

Recent reports suggest that serotonin (5-HT)2 receptor-mediated second messenger systems are enhanced in platelets of affective disorders. To make the mechanism of the enhanced response clear, we investigated 5-HT2 and alpha (alpha) 2-adrenergic receptor-induced intracellular calcium (Ca2+) mobilization in platelets of healthy volunteers, using fura-2. 5-HT2 and alpha 2-adrenergic receptor-mediated Ca2+ mobilization was enhanced by prior exposure to the other type of agonist, so called "heterologous supersensitization." The supersensitization was due to the enhancement of maximal response without change in agonist affinity. Chelating extracellular Ca2+ did not diminish the supersensitization. This enhancement of Ca2+ mobilization was not inhibited by H-7, an inhibitor of protein kinase C. However, this supersensitization was inhibited by pretreatment with sodium fluoride which directly activates guanine nucleotide binding regulatory proteins (G proteins). These results suggest that the supersensitization was caused from intracellular Ca2+ storage sites through a G protein-coupled pathway.

Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors

Inhibitors of the endoplasmic reticulum Ca 2+-ATPase like thapsigargin (TG) and 2,5-di ( tert-butyl)-1,4-benzohydroquinone ( tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 μM) mobilized 100% of sequestered Ca 2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 μM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway ( K i ≈ 7 μM). Thererore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A 2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca 2+-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A 2 to cause a similar response. We conclude that the calcium store sensitive to Ca 2+-ATPase inhibitors is a subset of the receptor sensitive calcium pool.

Calcium Mobilization in Human Platelets Using Indo-1 and Flow Cytometry

Abstract Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha- thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Calcium mobilization in human platelets using indo-1 and flow cytometry

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Altered hemodynamic response to dobutamine in relation to the degree of preoperative β-adrenoceptor blockade: Tarnow J, Komar K. Anesthesiology 68:912, 1988

The effect of picotamide (G137 or N,N′-bis-3-picolyl-4-methoxy-isophthalamide), a dual thromboxane A2 (TxA2) synthetase inhibitor/TxA2 endoperoxide receptor antagonist, on the phospholipase C (PLC) activation and calcium mobilization in human platelets stimulated by arachidonic acid (AA) and TxA2 receptor synthetic agonist U46619, has been studied. Preincubation with picotamide (10−4 M) for 1 and 3 min significantly reduced (p<0.03 and p<0.005 respectively) the calcium concentration changes induced in gel-filtered platelets (GFPs) by U46619 125 nM and 250 nM. Picotamide also reduced the calcium concentration changes induced in GFPs by AA 75 and 150 μM and by ADP 5 and 10 μM. In thrombin degranulated platelets picotamide inhibited the effect of U46619 up to 500 nM. The PLC activation, as indicated by inositol-1,3,4 P3 (Ins 1,3,4 P3) formation in response to U46619 250 nM and AA 150 μM was also inhibited by picotamide. These results may suggest a dual effect of picotamide on the receptor/effector systems through which TxA2 mediates platelet activation.

P-254 - Synergistic effect of epinephrine on ADP-induced cytoplasmic free calcium mobilization in human platelets

P-254 - Synergistic effect of epinephrine on ADP-induced cytoplasmic free calcium mobilization in human platelets

Synergistic potentiation by epinephrine of collagen or thrombin-induced calcium mobilization in human platelets

Changes in cytoplasmic free calcium in response to epinephrine in combination with other agonists were studied in human platelets loaded with the fluorescent calcium indicator, quin 2. In the presence of 1mM external calcium, epinephrine augmented the increase in cytoplasmic free calcium in response to collagen or thrombin, and this was inhibited by yohimbine. In the absence of extracellular calcium, thrombin and collagen only caused a small increase in cytoplasmic free calcium, and the response was not enhanced by epinephrine. High concentrations of epinephrine alone caused a small increase in cytoplasmic free calcium in platelets when calcium was present externally. Inhibition of cyclooxygenase by aspirin markedly reduced the calcium response to collagen and almost completely inhibited potentiation by epinephrine. The calcium response to thrombin was inhibited to a lesser extent by aspirin, and aspirin did not prevent potentiation by epinephrine. These findings suggest that augmentation of the cytoplasmic free calcium level may be involved in the potentiation of platelet responses to agonists by epinephrine, and that metabolites of arachidonic acid make an important contribution to this action of epinephrine.

Inhibitory action of guanosine 3′,5′-monophosphate on thrombin-induced calcium mobilization in human platelets

During thrombin-induced serotonin release from platelets, phosphorylation of myosin light chain was increased. This protein phosphorylation, which is initiated by Ca 2+ mobilization, and serotonin release were inhibited progressively by increasing concentrations of sodium nitroprusside, a potent stimulator of platelet guanylate cyclase, or 8-bromo-cyclic GMP. Thrombin-induced increase in cytoplasmic free Ca 2+, which was measured with the fluorescent Ca 2+ indicator quin2, was also inhibited by these drugs in a dose-dependent manner. These results provide evidence that an increase in platelet cyclic GMP inhibits thrombin-induced Ca 2+ mobilization and prevents platelet activation.