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.

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.

scholarly journals Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets

2019 ◽  
Vol 20 (21) ◽  
pp. 5430
Author(s):  
Ildikó Beke Debreceni ◽  
Gabriella Mezei ◽  
Péter Batár ◽  
Árpád Illés ◽  
János Kappelmayer
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Thrombin Generation ◽  
Platelet Adhesion ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Activation Loop ◽  
Clot Retraction ◽  
Lines Of Evidence

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.

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.

Regulation of Syk Function by Ubiquitination in Human Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 627-627
Author(s):  
Carol A. Dangelmaier ◽  
Patricia Quinter ◽  
Jianguo Jin ◽  
Alexander Y. Tysgankov ◽  
Satya P. Kunapuli ◽  
...  
Keyword(s):  
Protein Modification ◽  
Kinase Inhibitors ◽  
Specific Activity ◽  
Human Platelets ◽  
Integral Function ◽  
Kinase Assay ◽  
Translation Control ◽  
Venom Protein ◽  
Platelet Receptor

Abstract Platelets have an integral function in maintaining hemostasis. Signaling pathways are strictly regulated to ensure the cessation of bleeding without precipitating a thrombotic episode. An important initial physiological step in this process is the interaction of platelets with freshly exposed subendothelial collagen after vascular injury. A major platelet receptor involved is the GPVI/FcRγ-chain complex. Upon activation of this receptor, the immunoreceptor tyrosine-based activation motif (ITAM) present in the FcRγ-chain is phosphorylated by Src family kinases. This causes the tyrosine kinase Syk to bind to the ITAM where it is autophosphorylated, initializing a signaling cascade that activates a number of proteins including PLCγ2, PI-3 kinase and small G proteins. Syk appears to play an early pivotal role in GPVI/FcRγ-chain signal transduction and its regulation is crucial. Recent studies have shown that platelet aggregation, in response to collagen-related peptide (CRP), is potentiated in c-Cbl knockout mice. c-Cbl, a protein containing an E3 ligase responsible for substrate recognition for ubiquitin, appears to regulate numerous cytoplasmic kinases in other cell systems. This protein modification has emerged as one of the most common regulatory processes in all eukaryotes, second only to possibly phosphorylation. It appears to target proteins for proteosomal degradation although recently other mechanisms have been described, ranging from protein kinase activation to translation control. In the work presented here, we demonstrate that Syk associates with c-Cbl after GPVI/FcRγ-chain activation and therefore explored the possibility that Syk is ubiquitinated during GPVI/FcRγ-chain stimulation in human platelets. We have found that Syk is rapidly ubiquitinated upon activation by collagen, CRP and the snake venom protein convulxin, but not thrombin. PP2 and SU6656, two Src kinase inhibitors, prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases and probably requires Syk phosphorylation. The ubiquitination of Syk did not cause any apparent degradation of the protein as evidenced by the lack of effect of proteosomal and lysosomal inhibitors. We have been able to separate ubiquitinated Syk from its non-ubiquitinated counterpart. We have used an in vitro kinase assay to compare the activity of ubiquitinated Syk to non-ubiquitinated Syk. Surprisingly, when we compared the specific activity of the two Syk fractions, we found that the ubiquitinated Syk appeared to be about five-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that the majority (75%) of the active Syk is in the ubiquitinated fraction. In addition we found that Syk is not ubiquitinated in c-Cbl deficient mice. We therefore propose that c-Cbl plays a regulatory role in GPVI/FcRγ-chain stimulation through ubiquitination of Syk. The fact that 75% of active Syk is ubiquitinated suggests that ubiquitination plays an essential role in the regulation of its function. A possible function of ubiquitination may be targeting Syk to appropriate signaling molecules.

scholarly journals Role of AMP-activated protein kinase during postovulatory aging of mouse oocytes†

2020 ◽  
Vol 103 (3) ◽  
pp. 534-547
Author(s):  
Guang-Yi Sun ◽  
Shuai Gong ◽  
Qiao-Qiao Kong ◽  
Zhi-Bin Li ◽  
Jia Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Species Difference ◽  
Adenosine Monophosphate ◽  
Cytoplasmic Calcium ◽  
Ampk Activation ◽  
Mouse Oocytes ◽  
Compound C ◽  
Oocyte Aging ◽  
Amp Activated Protein Kinase

Abstract Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cyclic adenosine monophosphate (cAMP) decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

Distinct localization and function of1,4,5IP3 receptor subtypes and the1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3412-3422 ◽  
Author(s):  
Samer S. El-Daher ◽  
Yatin Patel ◽  
Ashia Siddiqua ◽  
Sheila Hassock ◽  
Scott Edmunds ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Adenosine Monophosphate ◽  
Human Platelets ◽  
Surface Proteins ◽  
Receptor Subtypes ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Receptor Antibodies

Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.

Distinct localization and function of1,4,5IP3 receptor subtypes and the1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3412-3422 ◽  
Author(s):  
Samer S. El-Daher ◽  
Yatin Patel ◽  
Ashia Siddiqua ◽  
Sheila Hassock ◽  
Scott Edmunds ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Adenosine Monophosphate ◽  
Human Platelets ◽  
Surface Proteins ◽  
Receptor Subtypes ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Receptor Antibodies

Abstract Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.

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.

scholarly journals IMMUNOREACTIONS INVOLVING PLATELETS

1958 ◽  
Vol 107 (5) ◽  
pp. 697-710 ◽  
Author(s):  
N. Raphael Shulman
Keyword(s):  
Complement Fixation ◽  
Human Platelets ◽  
Antibody Activity ◽  
Human Endothelial Cells ◽  
Clot Retraction ◽  
Specific Factors ◽  
The Stability ◽  
Increased Susceptibility

Quantitative aspects of platelet agglutination and inhibition of clot retraction by the antibody of quinidine purpura were described. The reactions appeared to depend on formation of types of antibody-quinidine-platelet complexes which could fix complement but complement was not necessary for these reactions. Complement fixation was at least 10 times more sensitive than platelet agglutination or inhibition of clot retraction for measurement and detection of antibody activity. Although it has been considered that antibodies of drug purpura act as platelet lysins in the presence of complement and that direct lysis of platelets accounts for development of thrombocytopenia in drug purpura, the present study suggests that attachment of antibody produces a change in platelets which is manifested in vitro only by increased susceptibility to non-specific factors which can alter the stability of platelets in the absence of antibody. The attachment of antibody to platelets in vivo may only indirectly affect platelet survival. In contrast to human platelets, dog, rabbit, and guinea pig platelets, and normal or trypsin-treated human red cells did not agglutinate, fix complement, or adsorb antibody; and intact human endothelial cells did not fix complement or adsorb antibody. Rhesus monkey platelets were not agglutinated by the antibody but did adsorb antibody and fix complement although their activity in these reactions differed quantitatively from that of human platelets. Cinchonine could be substituted for quinidine in agglutination and inhibition of clot retraction reactions but quinine and cinchonidine could not. Attempts to cause passive anaphylaxis in guinea pigs with the antibody of quinidine purpura were not successful.

Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets

2003 ◽  
Vol 89 (01) ◽  
pp. 122-131 ◽  
Author(s):  
Tina Wan ◽  
Martin Zabe ◽  
William Dean
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Tyrosine Residues ◽  
Phosphorylation Motif ◽  
C Terminus ◽  
Activation Sequence

SummaryPlasma membrane Ca2+-ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y1176) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y1122 and Y1176 can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y1176 is a likely substrate for focal adhesion kinase (FAK), while Y1122 is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y1176 is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.

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