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.

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 Use of d-erythro-sphingosine as a pharmacological inhibitor of protein kinase C in human platelets

1991 ◽  
Vol 278 (2) ◽  
pp. 387-392 ◽  
Author(s):  
W A Khan ◽  
S W Mascarella ◽  
A H Lewin ◽  
C D Wyrick ◽  
F I Carroll ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Biological Activities ◽  
Dose Dependence ◽  
Human Platelets ◽  
Kinase C ◽  
Similar Dose ◽  
Induced Aggregation

Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.

Accumulation of Unphosphorylated Calponin in the Submembranous Cytoskeletons of Arachidonic Acid-stimulated Human Platelets

1996 ◽  
Vol 75 (04) ◽  
pp. 617-622 ◽  
Author(s):  
Thomas Meyer ◽  
Christina Unterberg ◽  
Heinrich Kreuzer ◽  
Arnd B Buchwald
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Muscle Protein ◽  
Physiological Role ◽  
Protein Band ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Calpain Inhibitor ◽  
Dependent Protein

SummaryCalponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 μM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a sub-membranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.

Platelet Activating Factor (PAF) Stimulates Protein Kinase (PK) Activity In Human Platelets

1981 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer ◽  
L M Cagen ◽  
E E Muirhead
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Alkyl Ether ◽  
Dependent Protein Kinase ◽  
Proteolytic Activation ◽  
Dependent Protein ◽  
Stimulation Of

Kishimoto et al.(J.B.C.255:2273,1980) have demonstrated a Ca2+ and phospholipid-dependent protein kinase (PK) from various mammalian tissues which is markedly stimulated by the addition of unsaturated diacylglycerol. PAF, an alkyl ether analogue of phosphatidyl choline induces platelet aggregation and secretion. We investigated the ability of the C18:0 analogue (l-alkyl-2acetyl-sn-glycero-3-phosphocholine) to stimulate Ca2+ dependent PK activity in homogenates of human platelets. Two hundred ml whole blood was collected in EDTA(5mM) and EGTA(5mM). PGl2(534nM) was included to prevent platelet activation during preparation. Platelets were washed twice in 0.05M Tris containing EDTA, EGTA,and PGl2, pH 7.5. On the 3rd wash PGI2 was omitted. The platelet pellet was then resuspended in the same buffer now containing leu- peptin(0.2mM) to inhibit proteolytic activation of PK. The suspension was sonicated and centrifuged at 100,000xg for 1 hr. PK activity was assayed in the supernatant and pellet suspension by measuring the incorporation of 32P into HI histone from [γ32P]ATP. The standard reaction mixture (0.3 ml) contained 250yl supernatant or pellet suspension, HI histone(60μg), [γ32P]ATP(3nmoles),magnesium acetate (13.3mM) diolein(500ng) Ca2+ and PAF for 3 min. at 30°.Basal PK activity was 14.6pmol/min/mg protein. PAF(0.8μg) which is just saturating dose for in vitro platelet aggregation, stimulate PK activity by 70% in the supernatant but was without effect on the pellet suspension. In the absence of Ca2+ and/or diolein there was no stimulation of PK by PAF. Phosphatidyl serine(PS)(5μg) also stimulated protein kinase by 100%.Stimulation of PK by both PAF and PS occurred at endogenous platelet Ca2+ concentrations (i.e. sufficient Ca2+ added to titrate EDTA and EGTA) and at higher Ca2+ concentration (by 0.2mM.)Supernatants from platelets prepared in the absence of PGI2 were not stimulated by PAF These data show that PAF activates a Ca2+ dependent protein kinase which may mediate its effects on human platelets.

Role of Thromboxane A2 as a Mediator of Platelet-Activating-Factor-Induced Aggregation of Human Platelets

1989 ◽  
Vol 77 (1) ◽  
pp. 99-103 ◽  
Author(s):  
R. K. McCulloch ◽  
J. Summers ◽  
R. Vandongen ◽  
I. L. Rouse
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activating Factor ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Minor Role ◽  
A Minor ◽  
Induced Aggregation

1. At present it is unclear whether platelet-activating-factor (PAF)-induced aggregation is mediated by thromboxane. To obtain further information about this event we have compared the affects of aspirin on platelet aggregation and secretion induced by PAF and collagen. 2. Collagen and PAF induced aggregation and secretion in human platelets in a dose-related manner. 3. Aspirin inhibited the magnitude of both platelet aggregation and secretion induced by PAF and collagen, but the degree of inhibition was much greater for collagen. 4. Aspirin strongly inhibited the aggregation rate of collagen-induced platelet aggregation, but had no measurable effect on the rate of PAF-induced aggregation. 5. Inconsistencies reported in previous studies of the effect of aspirin on PAF-induced platelet aggregation may be explained, in part, by the doses of PAF used and the method of inactivating cyclo-oxygenase (in vitro compared with in vivo). 6. Our results suggest that the initial events of PAF-induced aggregation are independent of thromboxane A2 formation and that thromboxane A2 plays only a minor role in the later phase of PAF-induced aggregation.

A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4423-4429 ◽  
Author(s):  
Zhenyu Li ◽  
Jasna Ajdic ◽  
Martin Eigenthaler ◽  
Xiaoping Du
Keyword(s):  
Protein Kinase ◽  
Intracellular Signaling ◽  
Platelet Inhibition ◽  
Human Platelets ◽  
Predominant Role ◽  
Dependent Protein Kinase ◽  
No Donor ◽  
Vasp Phosphorylation ◽  
Dependent Protein

Abstract The vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog–induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal–responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.

scholarly journals Impaired activation of platelets lacking protein kinase C-θ isoform

Blood ◽  
2009 ◽  
Vol 113 (11) ◽  
pp. 2557-2567 ◽  
Author(s):  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Todd Getz ◽  
Yamini S. Bynagari ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Arterial Occlusion ◽  
Human Platelets ◽  
Wild Type ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Kinase C ◽  
Granule Secretion ◽  
Induced Aggregation

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-θ is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-θ–selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and α-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-θ, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-θ RACK peptide, which may contribute to the lower levels of granule secretion when PKC-θ function is lost. Furthermore, the level of JON/A binding to activated αIIbβ3 receptor was also significantly decreased in PKC-θ−/− mice compared with wild-type littermates. PKC-θ−/− murine platelets showed significantly lower agonist-induced thromboxane A2 (TXA2) release through reduced extracellular signal–regulated kinase phosphorylation. Finally, PKC-θ−/− mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl3 in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-θ isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

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