Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4246-4253 ◽  
Author(s):  
Lynn S. Quek ◽  
Jean-Max Pasquet ◽  
Ingeborg Hers ◽  
Richard Cornall ◽  
Graham Knight ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fc Receptor ◽  
Inhibitory Pathway ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Granule Secretion ◽  
Activation Motif ◽  
Additional Role ◽  
Feedback Pathway

Abstract Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor γ (FcRγ chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn−/−platelets, tyrosine phosphorylation of FcRγ chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn−/−platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and α-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn−/−lyn−/−double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLCγ2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway.

Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4246-4253 ◽  
Author(s):  
Lynn S. Quek ◽  
Jean-Max Pasquet ◽  
Ingeborg Hers ◽  
Richard Cornall ◽  
Graham Knight ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fc Receptor ◽  
Inhibitory Pathway ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Granule Secretion ◽  
Activation Motif ◽  
Additional Role ◽  
Feedback Pathway

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor γ (FcRγ chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn−/−platelets, tyrosine phosphorylation of FcRγ chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn−/−platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and α-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn−/−lyn−/−double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLCγ2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway.

scholarly journals Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

2004 ◽  
Vol 383 (3) ◽  
pp. 581-588 ◽  
Author(s):  
Gavin E. JARVIS ◽  
Denise BEST ◽  
Steve P. WATSON
Keyword(s):  
Tyrosine Phosphorylation ◽  
Chain Complex ◽  
Fc Receptor ◽  
Adapter Protein ◽  
Wild Type ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Src Homology 2 ◽  
Src Homology ◽  
Collagen Receptor

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1.

scholarly journals Bleeding diathesis in mice lacking JAK2 in platelets

2021 ◽  
Vol 5 (15) ◽  
pp. 2969-2981
Author(s):  
Nathan Eaton ◽  
Saravanan Subramaniam ◽  
Marie L. Schulte ◽  
Caleb Drew ◽  
David Jakab ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Cytokine Signaling ◽  
Hematopoietic Stem ◽  
Shear Rates ◽  
Granule Secretion ◽  
Static Conditions ◽  
Activation Motif

Abstract The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells, such as hematopoietic stem cells and megakaryocytes (MKs). Individuals expressing the somatic JAK2 V617F mutation commonly develop myeloproliferative neoplasms (MPNs) associated with venous and arterial thrombosis, a leading cause of mortality. The role of JAK2 in hemostasis remains unclear. We investigated the role of JAK2 in platelet hemostatic function using Jak2fl/fl Pf4-Cre (Jak2Plt−/−) mice lacking JAK2 in platelets and MKs. Jak2Plt−/− mice developed MK hyperplasia and splenomegaly associated with severe thrombocytosis and bleeding. This notion was supported by failure to occlude in a ferric chloride carotid artery injury model and by a cremaster muscle laser-induced injury assay, in which Jak2Plt−/− platelets failed to form stable thrombi. Jak2Plt−/− platelets formed thrombi poorly after adhesion to type 1 collagen under arterial shear rates. Jak2Plt−/− platelets spread poorly on collagen under static conditions or on fibrinogen in response to the collagen receptor GPVI-specific agonist, collagen-related peptide (CRP). After activation with collagen, CRP, or the CLEC-2 agonist rhodocytin, Jak2Plt−/− platelets displayed decreased α-granule secretion and integrin αIIbβ3 activation or aggregation, but showed normal responses to thrombin. Jak2Plt−/− platelets had impaired intracellular signaling when activated via GPVI, as assessed by tyrosine phosphorylation. Together, the results show that JAK2 deletion impairs platelet immunoreceptor tyrosine-based activation motif signaling and hemostatic function in mice and suggest that aberrant JAK2 signaling in patients with MPNs affects GPVI signaling, leading to hemostatic platelet function.

Role of Erythrocytes in Signal Transduction Mechanisms of Recruiting Platelets: Effects on Platelet Tyrosine Phosphorylation and Cytoskeletal Reorganization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3887-3887
Author(s):  
Juana Valles ◽  
Maria T. Santos ◽  
Antonio Moscardo ◽  
Maria A. Dasi ◽  
Justo Aznar
Keyword(s):  
Signal Transduction ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Receptor Activation ◽  
Cytoskeletal Reorganization ◽  
Functional Responses ◽  
Transduction Mechanisms ◽  
Platelet Releasate ◽  
Biochemical Mechanisms

Abstract Platelet activation induces the release of granular components and metabolic products. This platelet releasate is a complex physiological agonist that upon interaction with other platelets induces platelet recruitment and thrombus growth. The agonistic potency of the platelet releasate is increased when platelets are stimulated in the presence of intact erythrocytes (RBCs) (Santos et al J. Clin Invest1991; 87: 571; Valles et al Blood1991;78:154). The result is enhanced recruitment, α IIbβ3 receptor activation and P-selectin exposure in recruiting platelets (Valles et al Blood2002;99:3978). Receptor-mediated platelet activation initiates mechanisms of signal transduction that culminate in platelet functional responses. The biochemical mechanisms regulating the effects of cell-releasates in promoting platelet recruitment have not yet been elucidated. The aims of this study are: a) to characterize the effects of releasates from collagen-stimulated platelets on protein tyrosine phosphorylation (PTP), cytoskeletal reorganization and translocation to cytoskeleton of proteins in platelets being tested for recruitment; and b) to study the effects of platelet-erythrocyte interactions in these processes. Washed platelets (WP) or WP+RBCs (Htc. 40%) were stimulated with fibrillar collagen (1μg/mL) and rapidly centrifuged to obtain a cell-free collagen-free releasate within 1 min. An aliquot of this releasate was used as platelet agonist and the proaggregatory response (recruitment) was monitored by optical aggregometry as in the references above. For kinetic studies the reaction was halted at 15–180 sec., and PTP, cytoskeletal reorganization and cytoskeletal-associated tyrosine phosphorylated substrates were evaluated (Santos et al Circulation2000; 102:1924–1930). Our results demonstrate for the first time that the platelet releasate induces PTP in the platelets tested for recruitment. This effect is remarkably amplified with releasates from platelet-erythrocyte mixtures especially at earlier time points. Of interest is the strong FAK phosphorylation induced by this releasate, a kinase thought to play a role in αIIbβ3 clustering and focal adhesion formation. The greater potency of the platelet-erythrocyte releasate was also evident in cytoskeletal reorganization (actin, ABP, talin). The platelet-erythrocyte releasate induced more PTP and cytoskeletal reorganization than 20 m M ADP. Cytoskeletal reorganization and translocation of tyrosine phosphorylated substrates to the cytoskeleton induced by the platelet-erythrocyte releasate were quicker than those induced by 1 U/ml thrombin and were of comparable final intensity. Interestingly, erythrocytes markedly enhanced translocation of FAK kinase and αIIbβ3 to the platelet cytoskeleton. The data reveal new biochemical mechanisms regulating platelet recruitment and demonstrate the remarkable role of erythrocytes in the signal transduction mechanisms of platelets during the recruitment process, which is a limiting step in mural thrombus formation. (Grant FIS 03/0270).

nPKC Epsilon Negatively Regulates Platelet Functional Responses

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2855-2855
Author(s):  
Yamini Saraswathy Bynagari ◽  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Donna Woulfe ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granule ◽  
Dependent Manner ◽  
P2y1 Receptor ◽  
Wild Type ◽  
Functional Responses ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(𝛉), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

scholarly journals Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cγ2 regulation in platelets

2002 ◽  
Vol 364 (3) ◽  
pp. 755-765 ◽  
Author(s):  
Peter WONEROW ◽  
Achim OBERGFELL ◽  
Jonathan I. WILDE ◽  
Régis BOBE ◽  
Naoki ASAZUMA ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Membrane Domains ◽  
Adapter Protein ◽  
Glycoprotein Vi ◽  
Receptor Signalling ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Collagen Receptor ◽  
Lines Of Evidence

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin αIIbβ3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cγ2 (PLCγ2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCγ2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates αIIbβ3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCγ2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and αIIbβ3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCγ2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

scholarly journals LAT Is Required for Tyrosine Phosphorylation of Phospholipase Cγ2 and Platelet Activation by the Collagen Receptor GPVI

1999 ◽  
Vol 19 (12) ◽  
pp. 8326-8334 ◽  
Author(s):  
Jean-Max Pasquet ◽  
Barbara Gross ◽  
Lynn Quek ◽  
Naoki Asazuma ◽  
Weiguo Zhang ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Major Pathway ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Protein Receptor ◽  
Thrombin Activation ◽  
Granule Secretion ◽  
A Minor ◽  
Collagen Receptor

ABSTRACT In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cγ2 (PLCγ2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcγRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCγ2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCγ2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin αIIbβ3 in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCγ2, leading to downstream responses such as α-granule secretion and activation of integrin αIIbβ3. The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCγ2. We propose a model in which LAT and SLP-76 are required for PLCγ2 phosphorylation but are regulated through independent pathways downstream of Syk.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function

2021 ◽  
Author(s):  
Tony J. Zheng ◽  
Elizabeth R. Lofurno ◽  
Alexander R. Melrose ◽  
Hari Hara Sudhan Lakshmanan ◽  
Jiaqing Pang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Glycoprotein ◽  
Critical Systems ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Platelet Signaling ◽  
Granule Secretion ◽  
And Function ◽  
Btk Inhibitors

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

scholarly journals Platelet-associated IgAs and impaired GPVI responses in platelets lacking WIP

Blood ◽  
2009 ◽  
Vol 114 (21) ◽  
pp. 4729-4737 ◽  
Author(s):  
Hervé Falet ◽  
Michael P. Marchetti ◽  
Karin M. Hoffmeister ◽  
Michel J. Massaad ◽  
Raif S. Geha ◽  
...  
Keyword(s):  
Platelet Function ◽  
Actin Assembly ◽  
Irreversible Loss ◽  
Interacting Protein ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Granule Secretion ◽  
And Function ◽  
Syndrome Protein

Abstract The role of the Wiskott-Aldrich syndrome protein (WASp) in platelet function is unclear because platelets that lack WASp function normally. WASp constitutively associates with WASp-interacting protein (WIP) in resting and activated platelets. The role of WIP in platelet function was investigated using mice that lack WIP or WASp. WIP knockout (KO) platelets lack WASp and thus are double deficient. WIP KO mice have a thrombocytopenia, similar to WASp KO mice, resulting in part from enhanced platelet clearance. Most WIP KO, but not WASp KO, mice evolved platelet-associated immunoglobulins (Ig) of the IgA class, which normalize their platelet survival but diminish their glycoprotein VI (GPVI) responses. Protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in defects in α-granule secretion, integrin αIIbβ3 activation, and actin assembly. The anti-GPVI antibody JAQ1 induces the irreversible loss of GPVI from circulating platelets in wild-type mice, but not in WIP KO mice that bear high levels of platelet-associated IgAs. Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling and function in WIP KO mice.

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