Determination of ATP and ADP Secretion from Human and Mouse Platelets by an HPLC Assay

Papen, Michael von; Gambaryan, Stepan; Schütz, Claudia; Geiger, Jörg

doi:10.1159/000350294

Cited by 26 publications

(15 citation statements)

References 38 publications

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“…This assay is widely used as an in vivo assessment of the hemostatic action of platelets in rodents and is considered a general approach to assess primary hemostasis. Bleeding time is mainly determined by the interaction between platelets and damaged vessel wall leading to the hemostatic plug formation 49,50 . The prolonged bleeding time we observed in mice whose tails were immersed in CxD7L1 matched the research of other authors who linked the presence of an ADP platelet receptor deficiency with extended bleeding time in mice 51 .…”

Section: Discussionmentioning

confidence: 99%

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

et al. 2020

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During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the Nor the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.

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Section: Discussionmentioning

confidence: 99%

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

et al. 2020

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“…The aggregability of platelets can be assessed by quantitative determination of ATP exocytosis [ 21 ]. WB samples containing low, physiological or high levels of albumin were centrifuged at 150 g for 12 min in order to obtain platelet rich plasma (PRP) samples.…”

Section: Methodsmentioning

confidence: 99%

Anticoagulant action of low, physiologic, and high albumin levels in whole blood

et al. 2017

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Albumin is the most abundant plasma protein. Critical illness is often associated with altered, predominately decreased, serum albumin levels. This hypoalbuminaemia is usually corrected by administration of exogenous albumin. This study aimed to track the concentration-dependent influence of albumin on blood coagulation in vitro. Whole blood (WB) samples from 25 volunteers were prepared to contain low (19.3 ± 7.7 g/L), physiological (45.2 ± 7.8 g/L), and high (67.5 ± 18.1 g/L) levels of albumin. Haemostatic profiling was performed using a platelet function analyzer (PFA) 200, impedance aggregometry, a Cone and Platelet analyzer (CPA), calibrated automated thrombogram, and thrombelastometry (TEM). Platelet aggregation-associated ATP release was assessed via HPLC analysis. In the low albumin group, when compared to the physiological albumin group, we found: i) shortened PFA 200-derived closure times indicating increased primary haemostasis; ii) increased impedance aggregometry-derived amplitudes, slopes, ATP release, as well as CPA-derived average size indicating improved platelet aggregation; iii) increased TEM-derived maximum clot firmness and alpha angles indicating enhanced clot formation. TEM measurements indicated impaired clot formation in the high albumin group compared with the physiological albumin group. Thus, albumin exerted significant anticoagulant action. Therefore, low albumin levels, often present in cancer or critically ill patients, might contribute to the frequently occurring venous thromboembolism.

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“…Adenosine diphosphate (ADP), rapidly released by activated platelets 1 and also present in plasma in low amounts 1 derived from cellular adenosine triphosphate (ATP) (erythrocytes, endothelial cells), enhances platelet activation by virtually any other stimulant to complete platelet aggregation and thrombus formation. 2 It binds specifically to the G-protein-coupled receptors P2Y 1 and P2Y 12 , 3 stimulating intracellular phosphorylation-based pathways via the G-proteins G qa and G ia , respectively.…”

Section: Introductionmentioning

confidence: 99%

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

Beck

Geiger²,

Gambaryan

et al. 2017

Blood

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114

160

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Key Points• Temporal profiles of .4000 phosphopeptides after stimulating human platelets (a) with ADP and (b) consecutively with ADP and Iloprost.• Reciprocal phosphorylation profiles of ADP and Iloprost point to central players of platelet homeostasis.Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin aIIbb3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPaseactivating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3A Ser312 , CALDAG-GEFI Ser587 , ENSA Ser109 ), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. (Blood. 2017;129(2):e1-e12)

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Determination of ATP and ADP Secretion from Human and Mouse Platelets by an HPLC Assay

Cited by 26 publications

References 38 publications

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

Anticoagulant action of low, physiologic, and high albumin levels in whole blood

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

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