Convulxin Induces Platelet Activation by a Tyrosine-Kinase-Dependent Pathway and Stimulates Tyrosine Phosphorylation of Platelet Proteins, Including PLCγ2, Independently of Integrin αIIbβ3

1998 ◽  
Vol 353 (2) ◽  
pp. 239-250 ◽  
Author(s):  
Ivo M.B. Francischetti ◽  
Faika A. Ghazaleh ◽  
Ricardo A.M. Reis ◽  
Célia R. Carlini ◽  
Jorge A. Guimarães
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Integrin Αiibβ3 ◽  
Dependent Pathway ◽  
Platelet Proteins

scholarly journals A Collagen-Like Peptide Stimulates Tyrosine Phosphorylation of syk and Phospholipase Cγ2 in Platelets Independent of the Integrin α2β1

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1235-1242 ◽  
Author(s):  
Judith Asselin ◽  
Jonathan M. Gibbins ◽  
Marcus Achison ◽  
Young Han Lee ◽  
Laurence F. Morton ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Protein Tyrosine Phosphorylation ◽  
Direct Role ◽  
Amino Acid Code ◽  
Helical Peptide ◽  
Platelet Receptor ◽  
Dependent Pathway

AbstractActivation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.

scholarly journals Involvement of Hic-5 in platelet activation: integrin αIIbβ3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2

2001 ◽  
Vol 355 (3) ◽  
pp. 691-697 ◽  
Author(s):  
Makoto OSADA ◽  
Tsukasa OHMORI ◽  
Yutaka YATOMI ◽  
Kaneo SATOH ◽  
Shigemi HOSOGAYA ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Focal Adhesion ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Cell Periphery ◽  
Integrin Αiibβ3 ◽  
Tyrosine Kinase 2 ◽  
Exogenous Addition

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A2 analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin αIIbβ3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on αIIbβ3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on αIIbβ3-mediated outside-in signalling, through association with Pyk2.

A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1648-1648
Author(s):  
Ilaria Canobbio ◽  
Lucia Stefanini ◽  
Gianni F. Guidetti ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Lipid Rafts ◽  
Platelet Activation ◽  
Human Platelets ◽  
G Protein Coupled Receptors ◽  
Weak Stimulation ◽  
High Concentrations ◽  
G Protein Coupled

Abstract The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

2021 ◽  
Author(s):  
Leila Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R. Dilks ◽  
Mattia Bordoli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Blood ◽  
2021 ◽  
Author(s):  
Leila Denise Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R Dilks ◽  
Shihui Guo ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

scholarly journals Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 522-530 ◽  
Author(s):  
A Robinson ◽  
J Gibbins ◽  
B Rodriguez-Linares ◽  
PM Finan ◽  
L Wilson ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Receptor Agonist ◽  
Binding Proteins ◽  
Human Platelets ◽  
Sh2 Domain ◽  
Stable Complex ◽  
Glutathione S Transferase ◽  
Sh3 Domains

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

The Protein Tyrosine Kinase-Dependent Pathway Mediates the Effect of K Intake on Renal K Secretion

Physiology ◽  
2005 ◽  
Vol 20 (2) ◽  
pp. 140-146 ◽  
Author(s):  
Dao-Hong Lin ◽  
Hyacinth Sterling ◽  
Wen-Hui Wang
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Collecting Duct ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Protein Tyrosine ◽  
K Secretion ◽  
Dependent Pathway

Dietary K intake plays an important role in the regulation of K secretion: a decrease stimulates and an increase suppresses kidney expression of protein tyrosine kinase (PTK), which plays a role in regulating Kir1.1 (ROMK), which is responsible for K secretion in the cortical collecting duct (CCD) and K recycling in the thick ascending limb. Tyrosine phosphorylation of ROMK channels increases with low dietary K and decreases with high dietary K. Moreover, stimulation of tyrosine phosphorylation of ROMK1 enhances ROMK1 internalization and reduces the K channel number in the cell surface in the CCD.

scholarly journals Bruton’s Tyrosine Kinase Inhibitors Impair FcγRIIA-Driven Platelet Responses to Bacteria in Chronic Lymphocytic Leukemia

2021 ◽  
Vol 12 ◽  
Author(s):  
Leigh Naylor-Adamson ◽  
Anisha R. Chacko ◽  
Zoe Booth ◽  
Stefano Caserta ◽  
Jenna Jarvis ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Platelet Activation ◽  
Infection Risk ◽  
Lymphocytic Leukemia ◽  
Bruton’S Tyrosine Kinase ◽  
Immune Functions ◽  
Bruton's Tyrosine Kinase ◽  
Integrin Αiibβ3 ◽  
Target Effects

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton’s tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

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