scholarly journals In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1024
Author(s):  
Hyuk-Woo Kwon ◽  
Dong-Ha Lee ◽  
Man Hee Rhee ◽  
Jung-Hae Shin
Keyword(s):  
Signaling Pathways ◽  
Shape Change ◽  
Venous Blood ◽  
Medical Problem ◽  
Human Platelets ◽  
Signaling Cascades ◽  
Platelet Activity ◽  
Clot Retraction ◽  
Fibrinogen Binding

Physiological agonists trigger signaling cascades, called “inside-out signaling”, and activated platelets facilitate adhesion, shape change, granule release, and structural change of glycoprotein IIb/IIIa (αIIb/β3). Activated αIIb/β3 interacts with fibrinogen and begins second signaling cascades called “outside-in signaling”. These two signaling pathways can lead to hemostasis or thrombosis. Thrombosis can occur in arterial and venous blood vessels and is a major medical problem. Platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, controlling platelet activity is important for platelet-mediated thrombosis and cardiovascular diseases. In this study, focus on Morus alba Linn, a popular medicinal plant, to inhibit the function of platelets and found the containing component mulberroside C. We examine the effect of mulberroside C on the regulation of phosphoproteins, platelet-activating factors, and binding molecules. Agonist-induced human platelet aggregation is dose-dependently inhibited by mulberroside C without cytotoxicity, and it decreased Ca2+ mobilization and p-selectin expression through the upregulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756), and downregulation of extracellular signal-regulated kinase (ERK). In addition, mulberroside C inhibited thromboxane A2 production, fibrinogen binding, and clot retraction. Our results show antiplatelet effects and antithrombus formation of mulberroside C in human platelets. Thus, we confirm that mulberroside C could be a potential phytochemical for the prevention of thrombosis-mediated CVDs.

Epinephrine potentiates human platelet activation but is not an aggregating agent

1988 ◽  
Vol 255 (6) ◽  
pp. H1276-H1288 ◽  
Author(s):  
F. Lanza ◽  
A. Beretz ◽  
A. Stierle ◽  
D. Hanau ◽  
M. Kubina ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Membrane Fluidity ◽  
Direct Effect ◽  
Shape Change ◽  
Human Platelets ◽  
Blood Collection ◽  
Fibrinogen Binding ◽  
Serotonin Secretion

Epinephrine can in certain in vitro conditions induce the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. This study examines some functional and biochemical responses to epinephrine. Epinephrine induces the aggregation and serotonin secretion of human platelets in citrated plasma. This is not due to a direct effect of citrate itself, such as the lowering of plasma free Ca2+ but more likely to the generation of traces of thrombin during blood collection, as suggested by abrogation of these platelet responses when hirudin was added before citrate. When washed human platelets suspended in Tyrode buffer containing 2 mM Ca2+, 0.35% albumin and apyrase, and 0.1-100 microM epinephrine were used, no shape change, aggregation, or secretion of serotonin was observed, nor was the platelet ultrastructure modified. Epinephrine does not modify platelet membrane fluidity, as studied with the lipophilic fluorescent probe trimethylammonium-diphenylhexatriene. It has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+ levels measured by quin2, or protein phosphorylation. Epinephrine potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels, membrane fluidity, fibrinogen binding, or protein phosphorylation. These effects are mediated by alpha 2-adrenergic agonists and inhibited by alpha 2-adrenergic antagonists. This study shows that epinephrine alone does not induce modifications of morphology, metabolism, or function of intact and functional washed human platelets and that it cannot be considered per se as an aggregating agent. However, epinephrine interacts with alpha 2-adrenergic receptors on human platelets and potentiates biochemical and aggregatory responses induced by other platelet agonists.

scholarly journals An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

2018 ◽  
Vol 7 (11) ◽  
pp. 440 ◽  
Author(s):  
Wan Lu ◽  
Chi Chung ◽  
Ray Chen ◽  
Li Huang ◽  
Li Lien ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Phospholipase D ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Activity ◽  
Clot Retraction ◽  
First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

Thrombopoietin Activates Human Platelets and Induces Tyrosine Phosphorylation of p80/85 Cortactin

1998 ◽  
Vol 79 (01) ◽  
pp. 195-201 ◽  
Author(s):  
Guillaume Huret ◽  
Jean-Pierre Loza ◽  
Raphaël Adda ◽  
Josiane Melle ◽  
Jacques Maclouf ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Actin Polymerization ◽  
Shape Change ◽  
Human Platelets ◽  
Cortical Actin ◽  
Fibrinogen Binding ◽  
Serotonin Secretion ◽  
Acid Pathway

SummaryThe mechanism of human platelet activation by thrombopoietin (TPO) was investigated in vitro. We found that rHuTPO stimulated thromboxane A2 formation and serotonin secretion, despite the absence of shape change and aggregation. Blockade of the arachidonic acid pathway did not inhibit rHuTPO-induced platelet secretion. rHuTPO stimulated the tyrosine phosphorylation of 64, 80/85, 95, 130 and 140 kDa proteins, but phosphoproteins of 100-105 and 125 kDa obtained when platelets aggregated in the presence of thrombin were absent. rHuTPO stimulated and potentiated the thrombin-induced tyrosine phosphorylation of a 80 kDa protein identified as the cortical actin-associated protein, p80/85 cortactin. When platelets were aggregated in the presence of rHuTPO and fibrinogen, cortactin phosphorylation was enhanced as compared to rHuTPO alone. Treatment with RGDS or cytochalasin D respectively reduced or abolished cortactin tyrosine phosphorylation. This confirms the existence of fibrinogen binding-dependent and independent pools of phosphorylated cortactin, both requiring intact actin polymerization. Cytoskeleton-binding proteins may be implicated in in vitro platelet activation by rHuTPO.

ADRENALINE ACTIVATES HUMAN PLATELETS BUT IS NOT PER SE AN AGGREGATING AGENT. EFFECTS ON PLATELET MORPHOLOGY, MEMBRANEFLUIDITY, FIBRINOGEN BINDING, CYTOPLASMIC FREE CALCIUM AND PROTEIN PHOSPHORYLATION

1987 ◽  
Author(s):  
M Lanza ◽  
A Beretz ◽  
A Stierlé ◽  
D Hanau ◽  
M Kubina ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Membrane Fluidity ◽  
Direct Effect ◽  
Shape Change ◽  
Human Platelets ◽  
Free Calcium ◽  
Blood Collection ◽  
Fibrinogen Binding ◽  
Per Se

Adrenaline (Adr) is generally considered as a full agonist able to induce in vitro the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. Adr 2.5 M) induces the aggregation and secretion of 41 % of preloaded 3H-serotonin in human platelets in citrated plasma. This effect is not seen in plasma collected on 50 ATU/ml hirudin, and is due to the generation of traces of thrombin during blood collection and not to a direct effect of citrate itself, such asthe lowering of plasma free calcium. With washed human platelets suspended in Tyrode's buffer containing 2 mM Ca2+, 0.35 %albumin and apyrase, Adr (0.1 -100 M) doesnot cause shape change, aggregation or secretion of serotonin and does not modify platelet ultrastructure as judged by electron microscopy. Adr (1-100 M) does not change platelet membrane fluidity, as studied with the lipophilic fluorescent probe TMA-DPH. Adr has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+levels measured with quin2, or phosphorylation of 20 KDa or 47 KDapolypeptides, whereas all these parameters are modified after stimulation with ADP orthrombin. Adr potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels,membrane fluidity, fibrinogen binding or protein phosphorylation. This effect is also seen with alpha2-adrenergic agonists (noradrenaline, alpha-methyl noradrenaline, clonidine) and is inhibited by alpha2-adrenergicantagonists such as yohimbine. The potentiation of platelet aggregation by Adr is not modified by prior incubation of the platelets with1mM aspirin for 15 min. This study shows that Adr alone does not induce modifications ofmorphology, metabolism or function of intactand functional washed human plateletsand that Adr cannot be considered per se as an aggregating agent. However, Adr interactswith alpha2-adrenergic receptors on human plateletsand potentiates biochemical and aggregatory responses induced by other platelet agonists.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

1991 ◽  
Vol 66 (06) ◽  
pp. 694-699 ◽  
Author(s):  
Marco Cattaneo ◽  
Benjaporn Akkawat ◽  
Anna Lecchi ◽  
Claudio Cimminiello ◽  
Anna M Capitanio ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Clot Retraction ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Binding Inhibition ◽  
High Concentrations ◽  
Formalin Fixed

SummaryPlatelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrihogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPIIb/IIIa monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their K d were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

AMPK α2 subunit is involved in platelet signaling, clot retraction, and thrombus stability

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2134-2140 ◽  
Author(s):  
Voahanginirina Randriamboavonjy ◽  
Johann Isaak ◽  
Timo Frömel ◽  
Benoit Viollet ◽  
Beate Fisslthaler ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Adenosine Monophosphate ◽  
Human Platelets ◽  
Whole Body ◽  
Initial Increase ◽  
Clot Retraction ◽  
Compound C ◽  
Platelet Signaling ◽  
Upstream Kinase

Abstract The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca2+]i. Clot retraction was also impaired in platelets from AMPKα2−/− mice but not from wild-type littermates or AMPKα1−/− mice. Moreover, rebleeding was more frequent in AMPKα2−/− mice, and the FeCl3-induced thrombi formed in AMPKα2−/− mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2—by affecting Fyn phosphorylation and activity—plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.

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

2014 ◽  
Vol 111 (03) ◽  
pp. 508-517 ◽  
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.

scholarly journals Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽  
1978 ◽  
Vol 51 (6) ◽  
pp. 1065-1071 ◽  
Author(s):  
S Levy-Toledano ◽  
G Tobelem ◽  
C Legrand ◽  
R Bredoux ◽  
L Degos ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Shape Change ◽  
Antigenic Site ◽  
Human Platelets ◽  
Clot Retraction ◽  
Igg Antibody ◽  
Normal Human ◽  
Induced Aggregation ◽  
Bovine Factor

Abstract In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

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