Sodium–Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation

2020 ◽  
Author(s):  
Alessandro Aliotta ◽  
Debora Bertaggia Calderara ◽  
Maxime G. Zermatten ◽  
Lorenzo Alberio
Keyword(s):  
Platelet Activation ◽  
Intracellular Signaling ◽  
Critical Role ◽  
Ion Fluxes ◽  
Sodium Calcium Exchanger ◽  
Potassium Efflux ◽  
Sodium Calcium ◽  
Activated Platelets ◽  
Thrombin Activation

AbstractProcoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium–calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

scholarly journals The Inflammatory Role of Pro-Resolving Mediators in Endometriosis: An Integrative Review

2021 ◽  
Vol 22 (9) ◽  
pp. 4370
Author(s):  
Cássia de Fáveri ◽  
Paula M. Poeta Fermino ◽  
Anna P. Piovezan ◽  
Lia K. Volpato
Keyword(s):  
Intracellular Signaling ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Integrative Review ◽  
Inflammatory Factors ◽  
Resolvin D1 ◽  
Endogenous Factors

The pathogenesis of endometriosis is still controversial, although it is known that the inflammatory immune response plays a critical role in this process. The resolution of inflammation is an active process where the activation of endogenous factors allows the host tissue to maintain homeostasis. The mechanisms by which pro-resolving mediators (PRM) act in endometriosis are still little explored. Thus, this integrative review aims to synthesize the available content regarding the role of PRM in endometriosis. Experimental and in vitro studies with Lipoxin A4 demonstrate a potential inhibitory effect on endometrial lesions’ progression, attenuating pro-inflammatory and angiogenic signals, inhibiting proliferative and invasive action suppressing intracellular signaling induced by cytokines and estradiol, mainly through the FPR2/ALX. Investigations with Resolvin D1 demonstrated the inhibition of endometrial lesions and decreased pro-inflammatory factors. Annexin A1 is expressed in the endometrium and is specifically present in women with endometriosis, although the available studies are still inconsistent. Thus, we believe there is a gap in knowledge regarding the PRM pathways in patients with endometriosis. It is important to note that these substances’ therapeutic potential is evident since the immune and abnormal inflammatory responses play an essential role in endometriosis development and progression.

scholarly journals Role of Endothelial Cells in Acute and Chronic Thrombosis

2019 ◽  
Vol 39 (02) ◽  
pp. 128-139 ◽  
Author(s):  
Magdalena L. Bochenek ◽  
Katrin Schäfer
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Blood Clot ◽  
Vascular Occlusion ◽  
Coagulation Factors ◽  
Activated Platelets ◽  
Endothelial Cell Response ◽  
Von Willebrand

AbstractHaemostasis encompasses a set of strictly regulated actions, such as vasoconstriction, platelet activation and blood coagulation. Endothelial cells play a crucial role in all of these processes and are an integral part of the vascular response to injury resulting in thrombus formation. Healthy endothelium expresses mediators to prevent platelet activation, including prostacyclin and nitric oxide, and to inhibit coagulation, such as thrombomodulin or RNase1. Upon activation, endothelial cells expose von Willebrand factor, integrins and other receptors to interact with activated platelets, erythrocytes and coagulation factors, respectively, resulting in blood clot formation. The endothelial cell response to cytokines and growth factors released from activated platelets and immune cells abundantly present in arterial and venous thrombi also plays an important role for thrombus resolution, whereas failure to completely resolve thrombi may initiate fibrotic remodelling and chronic vascular occlusion both in the arterial and venous tree. Therefore, endothelial cells are increasingly recognized as potential target to prevent thrombotic events and to accelerate thrombus resolution. Here, we discuss recent publications from our group in the context of other studies on the role of the endothelium during acute and chronic thrombotic events.

The Abcc4 Knockout Reveals An Important Role for Abcc4 in Platelet Aggregation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1141-1141
Author(s):  
Satish Babu Cheepala ◽  
Kazumasa Takenaka ◽  
Tamara I. Pestina ◽  
Carl W. Jackson ◽  
Schuetz John
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Critical Role ◽  
Antiplatelet Drugs ◽  
Null Mouse ◽  
Dense Granules

Abstract Abstract 1141 Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides. We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels. Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10. This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures: No relevant conflicts of interest to declare.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

2005 ◽  
Vol 98 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
Antonino Coppola ◽  
Ludovico Coppola ◽  
Liliana dalla Mora ◽  
Francesco M. Limongelli ◽  
Antonio Grassia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
B Lymphocytes ◽  
Platelet Activation ◽  
Critical Role ◽  
Strenuous Exercise ◽  
Platelet Glycoprotein ◽  
Vigorous Exercise ◽  
Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

2003 ◽  
Vol 65 (3) ◽  
pp. 283-288 ◽  
Author(s):  
Robert Moreau ◽  
Georges Daoud ◽  
Andr� Masse ◽  
Lucie Simoneau ◽  
Julie Lafond
Keyword(s):  
Calcium Atpase ◽  
Sodium Calcium Exchanger ◽  
Human Term Placenta ◽  
Term Placenta ◽  
Sodium Calcium

scholarly journals Calcium-dependent inactivation controls cardiac L-type Ca2+ currents under β-adrenergic stimulation

2019 ◽  
Vol 151 (6) ◽  
pp. 786-797 ◽  
Author(s):  
Danna Morales ◽  
Tamara Hermosilla ◽  
Diego Varela
Keyword(s):  
Calcium Current ◽  
Calcium Currents ◽  
Adrenergic Stimulation ◽  
Sodium Calcium Exchanger ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Calcium Dependent ◽  
Sodium Calcium ◽  
Dependent Inactivation

The activity of L-type calcium channels is associated with the duration of the plateau phase of the cardiac action potential (AP) and it is controlled by voltage- and calcium-dependent inactivation (VDI and CDI, respectively). During β-adrenergic stimulation, an increase in the L-type current and parallel changes in VDI and CDI are observed during square pulses stimulation; however, how these modifications impact calcium currents during an AP remains controversial. Here, we examined the role of both inactivation processes on the L-type calcium current activity in newborn rat cardiomyocytes in control conditions and after stimulation with the β-adrenergic agonist isoproterenol. Our approach combines a self-AP clamp (sAP-Clamp) with the independent inhibition of VDI or CDI (by overexpressing CaVβ2a or calmodulin mutants, respectively) to directly record the L-type calcium current during the cardiac AP. We find that at room temperature (20–23°C) and in the absence of β-adrenergic stimulation, the L-type current recapitulates the AP kinetics. Furthermore, under our experimental setting, the activity of the sodium–calcium exchanger (NCX) does not affect the shape of the AP. We find that hindering either VDI or CDI prolongs the L-type current and the AP in parallel, suggesting that both inactivation processes modulate the L-type current during the AP. In the presence of isoproterenol, wild-type and VDI-inhibited cardiomyocytes display mismatched L-type calcium current with respect to their AP. In contrast, CDI-impaired cells maintain L-type current with kinetics similar to its AP, demonstrating that calcium-dependent inactivation governs L-type current kinetics during β-adrenergic stimulation.

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