Annexin V binding to platelets is agonist, time and temperature dependent

Ramström, Sofia; O‘Neill, Sarah; Dunne, Eimear; Kenny, Dermot

doi:10.3109/09537101003660564

Cited by 22 publications

(20 citation statements)

References 25 publications

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“…In agreement with the previous reports (Dale et al , 2002; Keuren et al , 2005; Ramstrom et al , 2010), there are three possible ways to initiate platelet activation so that two subpopulations with different PS expression will be ultimately produced: via either GPVI, PAR1, or PAR4. The sizes of the subpopulations can be additionally modulated by adenosine 5′disposphate acting via the P2Y12 receptor, while thromboxane A2 signalling and the P2Y1 receptor do not appear to contribute significantly (Kotova et al , 2008).…”

Section: Discussionsupporting

confidence: 90%

“…Comparative contribution of PAR1 and PAR4 to thrombin‐induced procoagulant activity has been a subject of some controversy. Different reports suggested either that PAR1 is predominant and determines the effects of thrombin (Andersen et al , 1999; Keuren et al , 2005; London et al , 2004), or that PAR1 has a minor role, if any (Dicker et al , 2001), or that PAR1 and PAR4 are comparable (Ramstrom et al , 2010), or that even stimulation of both receptors cannot explain the effects of thrombin (Dorsam et al , 2004). These studies differ by the platelet isolation method (unless performed directly in whole blood), the type and degree of platelet activation (with or without stimulation of collagen receptors), the methods of receptor stimulation/inhibition, and the methods of procoagulant activity characterization (PS detection or coagulation factor activation).…”

Section: Discussionmentioning

confidence: 99%

“…Specifically, identification of signal transduction pathways that determine platelet segregation into subpopulations is of particular interest. Several studies have investigated the roles of thrombin (Keuren et al , 2005; Ramstrom et al , 2010) and adenosine 5′diphosphate (Kotova et al , 2008) receptors subtypes in this phenomenon. There have also been studies on calcium signalling in subpopulations formed after stimulation with the PAR1 agonist SFLLRN (London et al , 2006) or thrombin with collagen (Keuren et al , 2005).…”

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confidence: 99%

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Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

et al. 2012

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Summary Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

et al. 2012

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“…Studies by Bevers et al in 1982 [5] clearly identified that collagen is more potent, but that the combination of collagen and thrombin greatly potentiated the capability of platelets to support thrombin formation [5]. The small percentage of phosphatidylserine (PS)-positive platelets reported in this paper are well in accordance with other studies [14,27,28], where only a small percentage of platelets turn PS positive even in response to high concentrations of thrombin, although it should be noticed that PS exposure determined by annexin V binding could vary considerably as a result of variations in experimental conditions [29]. The important contribution by collagen is also evident in this paper, as the combination of thrombin and collagenrelated peptide in Fig.…”

supporting

confidence: 91%

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Lindahl

Macwan

Ramström

2016

Journal of Thrombosis and Haemostasis

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To cite this article: Lindahl TL, Macwan AS, Ramstr€ om S. Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets. J Thromb Haemost 2016; 14:1639-41.See also French SL, Arthur JF, Lee H, Nesbitt WS, Andrews RK, Gardiner EE, Hamilton JR. Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood. This issue, pp 1642-54.Thrombin is the most important enzyme in coagulation and also a potent activator of platelets. It activates platelets via N-terminal cleavage of protease-activated receptors (PARs) 1 and 4. Although structurally similar, both PARs have distinct and complementary roles; for example, PAR1 is more sensitive than PAR4 to low concentrations of thrombin [1], whereas PAR4 gives rise to a more prolonged calcium mobilization after activation compared with a transient 'spike' by PAR1 [2]. Moreover, PAR4 is more important for clot lysis resistance [3]. After activation, a subpopulation of the platelets become procoagulant by allowing the formation of coagulation factor complexes on their surface. They do not participate in aggregation but facilitate generation of even more thrombin and, consequently, amplification of the response [4]. Experimental data suggest that collagen exposed during vessel damage in combination with thrombin is the most potent trigger for formation of procoagulant platelets [5], but the signaling processes regulating the formation of the aggregatory and procoagulant platelet subpopulations are still not known in detail.The article by Hamilton and co-workers in this issue of JTH [6] shows for the first time that activation of PAR4 is crucial for the thrombin-induced activation of platelets in an in vitro thrombus model; that is, platelets adhered on a collagen surface in a flow chamber system. Two tools were essential for this study, a rabbit polyclonal antibody directed towards the cleavage site for thrombin at the N-terminal of PAR4 and a fluorescence resonance energy transfer-based thrombin activity sensor linked to an anti-CD41a antibody, to prevent washing out by the flowing blood, which was invented by Welsh et al. [7]. The anti-PAR4 antibody only attenuated the peak of the thrombin-induced cytosolic calcium but abolished the sustained elevation of cytosolic calcium; thus PAR4 was responsible for the sustained elevation, in accordance with previous studies [8][9][10][11][12]. Presumably this sustained elevation is essential for the creation of the negatively charged surface and the procoagulant activity, which is supported by the known very strong effect in this direction by the calcium ionophore A23187 (see Fig. 4G in [6] and [13]).The relative importance of PAR1 and PAR4 for different aspects of platelet activation is far from clear; published studies have shown various, even contradicting, results. To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to st...

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“…This causes integrin activation, granule secretion and an increase in intracellular Ca 2+ [18,19,21,22]. Activation of GPVI is also highly important for the exposure of procoagulant phosphatidylserine [23][24][25].…”

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Formation and Relevance of Platelet Subpopulations

Södergren¹

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α α-granules are the most abundant of the secretory granules, with 50-80 α-granules per platelet. The contents of the α-granules are produced by the megakaryocyte or taken up by the megakaryocyte or platelet through endocytosis. Endocytosed proteins found in the α-granule include fibrinogen and coagulation factor (F) V. αgranules contain both membrane proteins, where αIIbβ3 and P-selectin (CD62P) Platelets have two major ADP receptors, P2Y1 and P2Y12, where P2Y12 is the more abundant. The role of P2Y1 and P2Y12 is, similar to the TP receptor, to reinforce platelet activation induced by other agonists having caused dense granule release. P2Y1 and P2Y12 are both GPCRs. P2Y1 couples to Gq and mediates platelet shape change and reversible aggregation. P2Y12 couples to Gi, resulting in intracellular Ca 2+ mobilization via inhibition of adenylate cyclase. P2Y12 is the target for several common anti-platelet drugs, such as clopidogrel and ticagrelor [28]. Upon vessel damage, the extracellular matrix (ECM) becomes exposed. This contains platelet activating substances such as collagen, vWF, fibronectin, laminin and thrombospondin. If the shear rate of the damaged vessel is high, as in arteries, platelet adhesion is mediated through binding of vWF to GPIb-IX-V, whereas this interaction is not essential at lower shear rates, as in veins. Platelets in circulation bind to vWF, immobilized at the site of vessel damage. The GPIb-IX-V receptor complex has a fast on-and off-rate, allowing the platelet to roll along the vWF multimers. This slows down the platelets enough to allow adhesion, via αIIbβ3, and α2β1 [17-19], figure 1A.

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Annexin V binding to platelets is agonist, time and temperature dependent

Cited by 22 publications

References 25 publications

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Formation and Relevance of Platelet Subpopulations

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