Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets

Hallam, Trevor J.; Daniel, James L.; Kendrick‐Jones, John; Rink, T J

doi:10.1042/bj2320373

Cited by 58 publications

(13 citation statements)

References 26 publications

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“…13,14 Accumulation of platelets on purified collagen under flow conditions is also inhibited by elevated cAMP. 35 Several other PKA substrates such as G ␣13 , 15 Rap1B, 39 myosin light chain kinase, 40 and actin-binding protein 41 may contribute to the effects of cAMP on platelet accumulation at injury sites following initial attachment. The inhibition of platelet accumulation in the presence of PDE3A observed in this study could be due to the phosphorylation of any of the aforementioned intracellular proteins.…”

Section: Discussionmentioning

confidence: 99%

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels

Sim

Merrill-Skoloff

Furie

et al. 2004

Blood

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Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. Initial platelet accrual into thrombi is mediated by interactions of platelet adhesion receptors with ligands on the injured endothelium or in the subendothelial matrix. The role of intracellular signals in initial platelet accumulation at sites of endothelial injury, however, is the subject of debate. We have used a newly discovered inhibitor of phosphodiesterase 3A (PDE3A) and the well-characterized PDE3A inhibitor, cilostazol, to modulate 3,5-cyclic adenosine monophosphate (cAMP) levels in an in vivo model that enables the kinetic analysis of platelet accumulation. These studies demonstrate that elevation of basal cAMP levels results in an overall decline in platelet accumulation at the site of vascular injury. In particular, the initial rate of accumulation of platelets is inhibited by elevation of cAMP. Analysis of the kinetics of individual platelets at injury sites using intravital microscopy demonstrates that cAMP directs the rate at which platelets attach to and detach from thrombi. These studies demonstrate that cAMP in circulating platelets controls attachment to and detachment from sites of arteriolar injury. Thus, the status of the intracellular signaling machinery prior to engagement of platelet receptors influences the rate of platelet accumulation during thrombus formation. ( IntroductionWhile it is well established that adhesion molecules affect signaling events that lead to platelet activation, it is also possible that intracellular signals present in circulating platelets prior to interactions with thrombi affect the ability of platelets to incorporate into thrombi. [1][2][3][4][5] The role of intracellular signaling in controlling the initial accumulation of platelets into thrombi at sites of vascular injury, however, is largely unexplored. Critical roles for 3Ј,5Ј-cyclic adenosine monophosphate (cAMP), cAMPdependent protein kinase (protein kinase A [PKA]), and phosphodiesterase 3A (PDE3A) in thrombus formation have been suggested in both in vivo models 6 and clinical studies of arterial thrombosis. [7][8][9][10][11][12] Cyclic AMP inhibits platelets by activating PKA, which phosphorylates several substrates important for the activation of platelets. PDE3A hydrolyzes cAMP to 5Ј-AMP. As a result, PDE3A opposes PKA-mediated platelet inhibition. Phosphorylation of several intracellular PKA substrates is associated with the inhibition of multiple platelet functions. For example, IP 3 receptor phosphorylation by PKA is proposed to downregulate calcium release from intracellular stores. 13,14 PKA phosphorylation of G ␣13 causes inhibition of the RhoA/Rho kinase pathway. 15 PKA inhibits signaling to the cytoskeleton 16,17 and may stabilize the resting cytoskeleton by phosphorylation of cytoskeletal proteins such as actinbinding protein 18 and caldesmon. 19 In addition to the phosphorylation of these intracellular substrates, PKA also inhibits other signaling events such as mitogen-activated protein kinas...

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Section: Discussionmentioning

confidence: 99%

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels

Sim

Merrill-Skoloff

Furie

et al. 2004

Blood

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“…AC represents an endogenous inhibitory mechanism of platelet activation, whose action is mediated by cAMP‐dependent activation of protein kinase A (PKA). PKA‐mediated inhibition of platelet activation involves interference with calcium release [106], phosphorylation of cytoskeletal proteins such actin binding protein (ABP) and myosin light chain kinase (MLCK) [107], as well as of the focal adhesion mediator vasodilator‐stimulated phosphoprotein (VASP) [108]. The inhibition of AC, as caused by P2Y 12 activation, therefore results in amplification of activator pathways triggered by different agonists.…”

Section: Pharmacological Modulation Of Homotypic and Heterotypic Aggrmentioning

confidence: 99%

Current concepts of platelet activation: possibilities for therapeutic modulation of heterotypic vs. homotypic aggregation

Passacquale¹,

Ferro²

2011

Brit J Clinical Pharma

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Thrombogenic and inflammatory activity are two distinct aspects of platelet biology, which are sustained by the ability of activated platelets to interact with each other (homotypic aggregation) and to adhere to circulating leucocytes (heterotypic aggregation). These two events are regulated by distinct biomolecular mechanisms that are selectively activated in different pathophysiological settings. They can occur simultaneously, for example, as part of a pro-thrombotic/pro-inflammatory response induced by vascular damage, or independently, as in certain clinical conditions in which abnormal heterotypic aggregation has been observed in the absence of intravascular thrombosis. Current antiplatelet drugs have been developed to target specific molecular signalling pathways mainly implicated in thrombus formation, and their ever increasing clinical use has resulted in clear benefits in the treatment and prevention of arterial thrombotic events. However, the efficacy of currently available antiplatelet drugs remains suboptimal, most likely because their therapeutic action is limited to only few of the signalling pathways involved in platelet homotypic aggregation. In this context, modulation of heterotypic aggregation, which is believed to contribute importantly to acute thrombotic events, as well to the pathophysiology of atherosclerosis itself, may offer benefits over and above the classical antiplatelet approach. This review will focus on the distinct biomolecular pathways that, following platelet activation, underlie homotypic and heterotypic aggregation, aiming potentially to identify novel therapeutic targets.

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“…[1][2][3][4][5][6][7] The extent of MLC 20 phosphorylation is regulated not only by protein kinases, such as Ca ϩϩ -dependent MLC kinase 8 and Ca ϩϩ -independent Rho-kinase, 9 but also by myosin phosphatase. 10,11 Platelet shape change, the earliest response induced by agonists, is a prerequisite for full platelet activation, including secretion and aggregation.…”

Section: Introductionmentioning

confidence: 99%

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Watanabe

Kataoka

et al. 2001

Blood

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Protein kinase C (PKC)–potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1δ catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A2 analog STA2 resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2–loaded platelets with no change in cytosolic Ca++. The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation of PKC by PMA induces MLC20phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA2-induced MLC20phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA2-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA2-induced MLC20 phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA2-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC20 phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase–mediated regulation of myosin phosphatase, during agonist-induced platelet secretion.

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Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets

Cited by 58 publications

References 26 publications

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels

Current concepts of platelet activation: possibilities for therapeutic modulation of heterotypic vs. homotypic aggregation

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

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