scholarly journals Glycoprotein IIIa is phosphorylated in intact human platelets

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2363-2368 ◽  
Author(s):  
LV Parise ◽  
AB Criss ◽  
L Nannizzi ◽  
MR Wardell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Transmembrane Protein ◽  
Specific Protein ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Kinase C ◽  
Glycoprotein Iiia

Abstract The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.

scholarly journals Glycoprotein IIIa is phosphorylated in intact human platelets

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2363-2368
Author(s):  
LV Parise ◽  
AB Criss ◽  
L Nannizzi ◽  
MR Wardell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Transmembrane Protein ◽  
Specific Protein ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Kinase C ◽  
Glycoprotein Iiia

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713 ◽  
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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.

scholarly journals Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase Cγ and formation of inositol phosphates

1993 ◽  
Vol 290 (2) ◽  
pp. 471-475 ◽  
Author(s):  
R A Blake ◽  
T R Walker ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Human Platelets ◽  
Functional Responses ◽  
Phosphorylation Of Proteins ◽  
Kinase C

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

Functions of Thrombin Receptors in the Reversible Distribution of Platelet Glycoprotein Ib within Activated Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3891-3891
Author(s):  
Han Yue ◽  
Jean-Max Pasquet ◽  
Alan Nurden ◽  
Ruan Changgeng
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Signal Transmission ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Platelet Surface ◽  
Kinase C ◽  
Thrombin Receptors

Abstract Platelets are critical in normal haemostasis and arterial thrombosis. As a potent activator, thrombin activates human platelets by cleavage of the protease-activated receptors PARs, exposing new amino terminus which serve as tethered peptide ligands, binding to the receptors induce transmembrane signaling. Thrombin also binds to platelet glycoprotein Ib, whose redistribution within platelet was showed to be reversible upon to thrombin or to PAR1 and PAR4 peptides. In an attempt to understand the reversible expression of platelet GPIb and cytoskeleton reorganization during platelet activation, then determine functions of thrombin receptors in GPIb redistribution. we used peptide SFLLRNPNDKYEPF (PAR1-AP, trap) and AYPGKF (PAR4-AP) for stimulating platelet at different time points (0~60minute), detected the platelet surface GPIbα and P-selectin with flowcytometry, and compared the alteration of GPIbα, actin and myosin in cytoskeleton by western-blot, then analyzed the membrane cytoskeleton followed by GPIbα immunoprecipitation. As expected, a reversible interalisation of GPIbα was obtained by PAR1 or PAR4 activation, and a transient change of actin, myosin and GPIbα /myosin GPIbα /actin association were also found in this course. These transient internalisations were apparently blocked by Cytochalasin D (inhibitor of actin polymerisation) or BAPTA/AM (calcium chelator). At the same time, ApyraseVII had weak effect on GPIbα interalisation, although the return of GPIbα to platelet surface was accelerated by this low ATP/ADPase, which quickened GPIbα dissappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during PAR1-AP activation.100nM and 10μM wortmannin were used to inhibit phosphatidylinositol 3-kinase (PI3-K) and/or myosin light chain kinase (MLCK) in our experiments. In response to both PARs activation, GPIbα interalisation was partly inhibited by 100nM wortmannin but little change for 10μM wortmannin, and a delayed restoration of surface GPIbα was observed in the presence of 10μM wortmannin upon PAR1-AP activation.. Apart from the above reagents, Ro-31-2220 (inhibitor of protein kinase C) induced a decreased GPIbα centralisation in response to PAR1 activation, and blocked the pool of GPIbα inside platelet in the latter course of PAR4 activation (p<0.05 at 10,30 min). Globally, Our study confirmed that thrombin receptors are important for platelet signal transmission, Stimulation of either receptor is sufficient to trigger platelet activation and induce GP Ib redistribution, which is correlated with cytoskeleton reorganisation, depending on the actin polymerisation and calcium mobilisation and implicate both myosin and actin. Our results also suggested critical roles of ADP, PI3-K or protein kinase C (PKC) in the GPIb redistribution.

scholarly journals Stimulatory antibody-induced activation and selective translocation of protein kinase C isoenzymes in human platelets

1995 ◽  
Vol 311 (2) ◽  
pp. 401-406 ◽  
Author(s):  
F Wang ◽  
U P Naik ◽  
Y H Ehrlich ◽  
S Osada ◽  
S Ohno ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Membrane Surface ◽  
Fold Increase ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Control Value ◽  
Kinase C ◽  
Endogenous Protein

A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces granular secretion and subsequent aggregation of human platelets. Mab.F11 recognizes a unique 32 and 35 kDa protein duplex on the platelet membrane surface, called the F11 receptor; binding of Mab.F11 to its receptor results in increased intracellular phosphorylation of P47, the known protein kinase C (PKC) substrate pleckstrin. In order to determine whether the mechanism of action of Mab.F11 involves direct activation of PKC, two types of functional assays for measuring PKC activity were performed. Measurement of PKC activity in digitonin-permeabilized platelets revealed that Mab.F11 produced a rapid, 2-3 fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser25. The increase in PKC activity induced by Mab.F11 was found to be associated with the platelet membrane; a 1.6-fold control value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, eta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstrate that in the initial stages of platelet activation by this stimulatory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PKC isoenzymes must be involved in different aspects of platelet activation. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenzymes and their specific endogenous protein substrates involved in platelet secretion and aggregation induced by platelet membrane protein antibodies.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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