Relationship between intracellular calcium-dependent process and protein-tyrosine phosphorylation in human platelets: studies of platelets from a patient with defective A23187-induced platelet aggregation

SummaryWe have investigated the intracellular mechanisms involved in microtubular remodelling by thrombin and its possible involvement in platelet aggregation and secretion. Platelet stimulation with thrombin induces a time- and concentration-dependent regulation of the microtubular content, which was found to be maximally effective at the concentration 0.1 U/ml. Thrombin (0.1 U/ml) evoked an initial decrease in the microtubule content detectable at 5 seconds (sec) and reached a minimum 10 sec after stimulation.The microtubular content then increased, exceeding basal levels again approximately 30 sec after stimulation. Inhibition of tyrosine phosphatases using vanadate abolished thrombin-induced microtubular depolymerisation while inhibition of tyrosine kinases by methyl-2,5-dihydroxycinnamate prevented microtubule polymerisation.Thrombin activates the cytosolic Brutons tyrosine kinase (Btk) and Src proteins. Inhibition of Btk or Src by LFM-A13 or PP1, respectively, abolished thrombin-induced microtubular polymerisation, while maintaining intact its ability to induce initial depolymerisation. Microtubular disruption by colchicine significantly reduced thrombin induced platelet aggregation and ATP secretion. Similar results were observed after inhibition of microtubular disassembly by paclitaxel.These findings indicate that thrombin induces microtubular remodelling by modifying the balance between protein tyrosine phosphorylation and dephosphorylation. The former seems to be required for microtubular polymerisation, while tyrosine dephosphorylation is required for microtubular depolymerisation. Both, initial microtubular disassembly and subsequent polymerisation are required for thrombin-induced platelet aggregation and secretion in human platelets.

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Wortmannin inhibits store-mediated calcium entry and protein tyrosine phosphorylation in human platelets

FEBS Letters ◽

10.1016/0014-5793(96)00130-5 ◽

1996 ◽

Vol 381 (3) ◽

pp. 249-251 ◽

Cited By ~ 13

Author(s):

Susanne Jenner ◽

Richard W. Farndale ◽

Stewart O. Sage

Keyword(s):

Tyrosine Phosphorylation ◽

Human Platelets ◽

Calcium Entry ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

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Calcium store depletion in dimethyl BAPTA-loaded human platelets increases protein tyrosine phosphorylation in the absence of a rise in cytosolic calcium

Experimental Physiology ◽

10.1113/expphysiol.1994.sp003762 ◽

1994 ◽

Vol 79 (2) ◽

pp. 269-272 ◽

Cited By ~ 41

Author(s):

P Sargeant ◽

RW Farndale ◽

SO Sage

Keyword(s):

Tyrosine Phosphorylation ◽

Cytosolic Calcium ◽

Human Platelets ◽

Protein Tyrosine Phosphorylation ◽

Calcium Store ◽

Protein Tyrosine ◽

Store Depletion

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Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells

FEBS Letters ◽

10.1016/0014-5793(95)00808-m ◽

1995 ◽

Vol 370 (1-2) ◽

pp. 127-130 ◽

Cited By ~ 27

Author(s):

Lori A. Semenchuk ◽

Joseph Di Salvo

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Intracellular Calcium ◽

Tyrosine Phosphorylation ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

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Protein-Tyrosine Phosphorylation and p72 syk Activation in Human Platelets Stimulated with Collagen Is Dependent upon Glycoprotein Ia/IIa and Actin Polymerization

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650337 ◽

1996 ◽

Vol 75 (04) ◽

pp. 648-654 ◽

Cited By ~ 26

Author(s):

Naoki Asazuma ◽

Yutaka Yatomi ◽

Yukio Ozaki ◽

Ruomei Qi ◽

Kenji Kuroda ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Actin Polymerization ◽

Human Platelets ◽

Dependent Manner ◽

Protein Tyrosine Phosphorylation ◽

Concentration Dependent Manner ◽

Protein Tyrosine ◽

Collagen Receptors ◽

Fibrinogen Binding ◽

Adp Release

SummaryIn human platelets treated with acetylsalicylic acid, collagen induced protein-tyrosine-phosphorylation of several proteins. The major 75 kDa band included cortactin and autophosphorylated p72 syk . p72 syk activity rapidly increased upon collagen stimulation, whereas p60c-src activation was below detectable levels. A combination of inhibitors to remove the effects of extracellular and intracellular Ca2+, released ADP, and fibrinogen binding to GPIIb/IIIa delayed and attenuated the major 75 kDa band. By contrast, p72 syk activation was not inhibited by these treatments. Cytochalasin D completely inhibited protein tyrosine phosphorylation and p72 syk activation. It also potently inhibited aggregation and [Ca2+]i elevation. Anti-GPMIa/IIa MoAb in a concentration-dependent manner partially attenuated protein tyrosine phosphorylation and p72 syk activation. Its inhibitory effects on intracellular Ca2+ mobilization, release of intracellular granule contents, and aggregation also were partial. No tyrosine kinase activity was coprecipitated with GPIa/IIa. These results suggest that p72 syk activation lies upstream of protein tyrosine phosphorylation, Ca2+ mobilization, ADP release, thromboxane A2 production and aggregation. GPIa/IIa plays a key role in p72 syk activation induced by collagen, but other collagen receptors may work in synergy to fully activate p72 syk . Actin polymerization is a prerequisite for both p72 syk activation and other intracellular signal transduction pathways.

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