P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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Effects of ovariectomy on aggregation, secretion, and metalloproteinases in porcine platelets

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00958.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. H1679-H1685 ◽

Cited By ~ 19

Author(s):

Muthuvel Jayachandran ◽

Whyte G. Owen ◽

Virginia M. Miller

Keyword(s):

Platelet Aggregation ◽

Vessel Wall ◽

Atp Release ◽

Ovarian Hormones ◽

Reticulated Platelets ◽

Tissue Inhibitors Of Metalloproteinase ◽

Atp Secretion ◽

Sexually Mature ◽

Wall Interactions ◽

Expression And Activity

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.

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Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/7071987 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Muhammad Irfan ◽

Tae-Hyung Kwon ◽

Dong-Ha Lee ◽

Seung-Bok Hong ◽

Jae-Wook Oh ◽

...

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Thrombus Formation ◽

Calcium Mobilization ◽

Potential Candidate ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Atp Secretion ◽

Antithrombotic Effects ◽

Epimedium Koreanum Nakai

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin αIIbβ3 activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin αIIbβ3-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin αIIbβ3 activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

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ATP secretion in the male reproductive tract: essential role of CFTR

The Journal of Physiology ◽

10.1113/jphysiol.2012.230581 ◽

2012 ◽

Vol 590 (17) ◽

pp. 4209-4222 ◽

Cited By ~ 29

Author(s):

Ye Chun Ruan ◽

Winnie W. C. Shum ◽

Clémence Belleannée ◽

Nicolas Da Silva ◽

Sylvie Breton

Keyword(s):

Essential Role ◽

Reproductive Tract ◽

Male Reproductive Tract ◽

Atp Secretion

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Glycoprotein V-Deficient Platelets Have Undiminished Thrombin Responsiveness and Do Not Exhibit a Bernard-Soulier Phenotype

Blood ◽

10.1182/blood.v94.12.4112 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4112-4121 ◽

Cited By ~ 69

Author(s):

Mark L. Kahn ◽

Thomas G. Diacovo ◽

Dorothy F. Bainton ◽

Francois Lanza ◽

JoAnn Trejo ◽

...

Keyword(s):

Wild Type ◽

Spontaneous Bleeding ◽

Atp Secretion ◽

Platelet Receptor ◽

High Concentrations ◽

And Function ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor ◽

Adhesion Of Platelets

Abstract Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.

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LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS

10.1055/s-0038-1643550 ◽

1987 ◽

Author(s):

R Malmgren

Keyword(s):

Platelet Adhesion ◽

Rank Order ◽

Shape Change ◽

Dense Granule ◽

Dependent Manner ◽

Equal Degree ◽

Atp Secretion ◽

Ic50 Values ◽

Dose Dependent ◽

Platelet Shape

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.

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The relation between peak [Ca2+]i signals, platelet aggregation and ATP secretion induced by increasing concentrations of archidonic acid

Thrombosis Research ◽

10.1016/0049-3848(92)90573-s ◽

1992 ◽

Vol 65 ◽

pp. S130

Author(s):

A. Dumitrescu ◽

L. Hasselmark ◽

R. Malmgren

Keyword(s):

Platelet Aggregation ◽

Atp Secretion ◽

Archidonic Acid

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PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Thrombosis and Haemostasis ◽

10.1160/th13-06-0484 ◽

2014 ◽

Vol 111 (03) ◽

pp. 508-517 ◽

Cited By ~ 30

Author(s):

Carol Dangelmaier ◽

Bhanu Kanth Manne ◽

Elizabetta Liverani ◽

Jianguo Jin ◽

Paul Bray ◽

...

Keyword(s):

Protein Kinase ◽

Protein A ◽

Human Platelets ◽

Clot Retraction ◽

Functional Responses ◽

Atp Secretion ◽

Dependent Protein ◽

Induced Aggregation

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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