Profiling oxylipins released from human platelets activated through the GPVI collagen receptor

2021 ◽  
pp. 106607
Author(s):  
Robert E. Turnbull ◽  
Katrin N. Sander ◽  
James Turnbull ◽  
David A. Barrett ◽  
Alison H. Goodall
Keyword(s):  
Human Platelets ◽  
Collagen Receptor

Paxillin Becomes Tyrosine-Phosphorylated and Binds Csk in a GpIIb-IIIa-Dependent Manner Following GPVI-Induced Activation of Murine Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3889-3889
Author(s):  
Vipul B. Rathore ◽  
Peter J. Newman ◽  
Debra K. Newman
Keyword(s):  
Platelet Activation ◽  
Binding Proteins ◽  
Critical Role ◽  
Human Platelets ◽  
Sh2 Domain ◽  
Intramolecular Interactions ◽  
Resting Cells ◽  
Dependent Manner ◽  
Granule Secretion ◽  
Collagen Receptor

Though Src family kinases (SFKs) play a critical role in collagen-induced platelet activation, little is known about how SFK activity is regulated following exposure of platelets to collagen. In resting cells, SFKs are maintained in an inactive conformation, in part, via intramolecular interactions between their SH2 domain and a C-terminal tyrosine residue whose phosphorylation state is controlled by the C-terminal Src kinase, Csk. Access of Csk to SFKs, in turn, is regulated by recruitment of Csk, via its SH2 domain, to one or more tyrosine-phosphorylated Csk-binding proteins, which include Csk binding protein (Cbp/PAG) itself, paxillin, and its closely-related paxillin family member, Hic-5. Recent studies have shown that human platelets possess only two Csk-binding proteins: Cbp/PAG and Hic-5, and that Hic-5 can become tyrosine-phosphorylated when platelets are stimulated with a variety of platelet agonists, including, thrombin, U46619, and collagen. The purpose of the present investigation was to characterize the complement of Csk-binding proteins in murine platelets and to begin to determine their role in regulating collagen-induced platelet activation. Murine platelets, like human platelets, were found to express Hic-5, and in addition contained two other Csk-binding proteins: paxillin and leupaxin. Of these, both paxillin and Hic-5 became tyrosine phosphorylated, and paxillin was shown to be able to recruit Csk in a time-dependent manner following exposure of murine platelets to collagen and the GPVI/FcRg chain collagen receptor-specific agonist, CRP. These data suggest that the function of Hic-5 in human platelets may be performed by both Hic-5 and paxillin in mice. Finally, both paxillin tyrosine phosphorylation and Csk recruitment were blocked by agents that interfered with either platelet granule secretion or with integrin engagement, consistent with the notion that members of the paxillin family function as integrin-dependent negative feedback regulators of platelet adhesion and spreading.

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.

scholarly journals A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets

1999 ◽  
Vol 342 (1) ◽  
pp. 171-177 ◽  
Author(s):  
Jean-Max PASQUET ◽  
Régis BOBE ◽  
Barbara GROSS ◽  
Marie-Pierre GRATACAP ◽  
Michael G. TOMLINSON ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Human Platelets ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Glycoprotein Vi ◽  
Related Peptide ◽  
Lipid Species ◽  
Collagen Receptor ◽  
Phosphatidylinositol 3

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)10GCP*G]n; P* = hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca2+ ([Ca2+]i) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca2+ by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P3, but not the R28C (Arg28 → Cys) mutant which binds to PI(3,4,5)P3 with low affinity, also inhibits elevation of [Ca2+]i in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCγ2. These results demonstrate that PI 3-kinase is required for full activation of PLCγ2 by GPVI in platelets and megakaryocytes.

Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI–mediated Rap1 activation in platelets

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1409-1415 ◽  
Author(s):  
Mark K. Larson ◽  
Hong Chen ◽  
Mark L. Kahn ◽  
Anne M. Taylor ◽  
Jean-Etienne Fabre ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Independent Component ◽  
Glycoprotein Vi ◽  
Platelet Signaling ◽  
Guanosine Triphosphatase ◽  
Adp Receptor Antagonists ◽  
Rap1 Activation ◽  
Collagen Receptor

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin αIIbβ3 activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRγ but independent of another platelet collagen receptor, α2β1. Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y12 and not the P2Y1 receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y1 or the P2Y12-associated G-protein, Gαi2, indicate that human and murine platelets also have a significant P2Y12-independent component of GPVI-mediated Rap1 activation. The P2Y12-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y12-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3561-3569 ◽  
Author(s):  
Andrew C. Pearce ◽  
Jonathan I. Wilde ◽  
Gina M. Doody ◽  
Denise Best ◽  
Osamu Inoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
G Protein ◽  
Cell Types ◽  
Human Platelets ◽  
Glycoprotein Vi ◽  
Activation Motif ◽  
G Protein Coupled ◽  
Collagen Receptor

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor γ-chain–associated signaling pathway and by agglutination

Blood ◽  
2001 ◽  
Vol 98 (12) ◽  
pp. 3346-3352 ◽  
Author(s):  
David Tulasne ◽  
Barbi A. Judd ◽  
Mette Johansen ◽  
Naoki Asazuma ◽  
Denise Best ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Signaling Pathway ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Fc Receptor ◽  
Type I ◽  
Induce Platelet Aggregation ◽  
Thrombospondin 1 ◽  
Collagen Receptor ◽  
Deficient Mice

Abstract A peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys; known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (IAP), which is also known as CD47. In this study, it was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) γ chain, Syk, SLP-76, and phospholipase C γ2 in human platelets. A specific inhibitor of Src family kinases, 4-amino-4-(4-methylphenyl)-7-(t-butyl) pyrazola[3,4-d]pyrimidine, prevented phosphorylation of these proteins, abolished platelet secretion, and reduced aggregation by approximately 50%. A similar inhibition of aggregation to 4N1-1 was obtained in the presence of Arg-Gly-Asp-Ser in mouse platelets deficient in FcR γ chain or SLP-76 and in patients with type I Glanzmann thrombasthenia. These results show that 4N1-1 signals through a pathway similar to that used by the collagen receptor glycoprotein (GP) VI. The αIIbβ3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbα. Surprisingly, the ability of 4N1-1 to stimulate aggregation and tyrosine phosphorylation was not altered in platelets pretreated with anti-IAP antibodies and in IAP-deficient mice. These results show that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR γ-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbα or αIIbβ3. Neither of these pathways is mediated by IAP.

scholarly journals Isolation of Platelet Membrane Collagen Receptor

1977 ◽  
Author(s):  
A. Livne ◽  
A.L. Swanson ◽  
H. Scheuenstuhl ◽  
D. Michaeli
Keyword(s):  
Exchange Resin ◽  
Sodium Dodecylsulfate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Receptor Activity ◽  
Nonionic Detergent ◽  
Triton X ◽  
Dose Dependent ◽  
Collagen Receptor ◽  
Membrane Collagen

A fraction which competes with intact platelets for interaction with collagen was isolated from human platelets by Sepharose 2B-collagen (SC) affinity columns. Binding of platelets to SC columns, followed by lysis with a nonionic detergent and extensive wash with water (treated SC columns), reduced the columns’ capacity to subsequently bind platelets and induce serotonin release. This reduction was proportional to the number of platelets that had been applied to SC columns. Treated SC columns could be partially regenerated with solutions of high ionic strength (1 M NaCI or Tris-HCl) and most effectively with 0.3% sodium dodecylsulfate (SDS), but not with 8 M urea, 15% ethanol or 1% Triton X-100, indicating an ionic interaction. A fraction eluted with SDS from treated SC columns manifested receptor activity: when rebound to collagen it caused a dose-dependent decline in interaction of the collagen with intact platelets, as measured by binding and serotonin release. The receptor activity was sensitive to heat and was absorbed by an anionic exchange resin. When the membrane of intact platelets were labeled with 125I and lactoperoxidase, the derived receptor fraction contained a small proportion (about 1%) of the label.

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