Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Rosado, Juan A.; Meijer, Else M. Y.; Hamulyák, Karly; Nováková, I.R.O.; Heemskerk, Johan W. M.; Sage, Stewart O.

doi:10.1182/blood.v97.9.2648

Cited by 34 publications

(29 citation statements)

References 48 publications

(50 reference statements)

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“…RGDS peptide was included in all experiments to prevent aggregation and therefore artefactual decreases in Fluo-5N fluorescence. RGDS at this concentration has previously been demonstrated not to affect [Ca 2+ ] cyt signals in human platelets [22]. It was not possible to calibrate Fluo-5N measurements made in the presence of PKC inhibitors, as there were differential fluorescence effects of these drugs at the maximal and minimal fluorescences of Fluo-5N.…”

Section: Measurement Of Intracellular Store Ca 2+ Concentration ([Ca mentioning

confidence: 98%

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

et al. 2015

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Rises in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca 2+ ] cyt , but these only monitor the net effect of manipulations on the processes controlling [Ca 2+ ] cyt (Ca 2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca 2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca 2+ signalling, we here monitor Ca 2+ flux around the platelet by measuring net Ca 2+ fluxes to or from the extracellular space and the intracellular Ca 2+ stores, which act as the major sources and sinks for Ca 2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na + concentration ([Na + ] cyt ), which influences platelet Ca 2+ fluxes via Na + /Ca 2+ exchange. The intracellular store Ca 2+ concentration ([Ca 2+ ] st ) was monitored using Fluo-5N, the extracellular Ca 2+ concentration ([Ca 2+ ] ext ) was monitored using Fluo-4 whilst [Ca 2+ ] cyt and [Na + ] cyt were monitored using Fura-2 and SFBI respectively. PKC inhibition using Ro-31-8220 or bisindolaemide I potentiated ADP-and thrombin-evoked rises in [Ca 2+ ] cyt in the absence of extracellular Ca 2+ . PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca 2+ release and Ca 2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca 2+ ] cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na + ] cyt which would be expected to reduce Ca 2+ removal via the Na + /Ca 2+ exchanger (NCX). Thrombinevoked rises in [Na + ] cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn 2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca 2+ ] cyt following SERCA inhibition and either removal of extracellular Na + or inhibition of Na + /K + -ATPase activity by removal of extracellular K + or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca 2+ ] cyt by acceleration of SERCA activity, whilst rises in [Ca 2+ ] cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na + /K + -ATPase activity, with the latter limiting the effect of thrombin on rises in [Na + ] cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca 2+ signalling.

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Section: Measurement Of Intracellular Store Ca 2+ Concentration ([Ca mentioning

confidence: 98%

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

et al. 2015

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“…Blood was taken from healthy volunteers and from a patient with Glanzmann thrombasthenia, with established deficiency in integrin α IIb b 3 , 16 after informed consent and in accordance with the Declaration of Helsinki. Platelet-rich plasma (PRP), defibrinated platelet-free plasma (PFP) and washed platelets were prepared from whole blood as described in the Online Supplementary Appendix.…”

Section: Blood Collection and Platelet Preparationmentioning

confidence: 99%

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

et al. 2015

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C oated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α IIb b 3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α IIb b 3 . Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α IIb b 3 . Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α IIb b 3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α IIb b 3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α IIb b 3 .

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“…As shown in Fig. 1, A (20). Cells heavily loaded with the Ca 2ϩ chelator dimethyl BAPTA were used for this study, so as to eliminate Ca 2ϩ -dependent, but not store depletion-dependent, surface expression of Orai1.…”

Section: Camentioning

confidence: 99%

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca²⁺ store depletion

Woodard¹,

Salido²,

Rosado³

2008

American Journal of Physiology-Cell Physiology

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Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Abstract: Effects of the occupation of integrin ␣

Cited by 34 publications

References 48 publications

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca²⁺ store depletion

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Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Abstract: Effects of the occupation of integrin ␣

Cited by 34 publications

References 48 publications

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Coated platelets function in platelet-dependent fibrin formation via integrin α IIb β 3 and transglutaminase factor XIII

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion

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Product

Resources

About

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca²⁺ store depletion