Low-dose Btk inhibitors selectively block platelet activation by CLEC-2

Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

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Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

Blood ◽

10.1182/blood.v68.2.565.565 ◽

1986 ◽

Vol 68 (2) ◽

pp. 565-570 ◽

Cited By ~ 16

Author(s):

RW Colman ◽

WR Figures ◽

LM Scearce ◽

AM Strimpler ◽

FX Zhou ◽

...

Keyword(s):

Platelet Activation ◽

Shape Change ◽

Thromboxane A2 ◽

Adenosine Diphosphate ◽

Platelet Surface ◽

Prostaglandin Endoperoxide ◽

Fibrinogen Binding ◽

Low Concentrations ◽

Absolute Requirement

Abstract The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.

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Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets

Blood ◽

10.1182/blood-2004-12-4866 ◽

2005 ◽

Vol 106 (2) ◽

pp. 550-557 ◽

Cited By ~ 50

Author(s):

Swaminathan Murugappan ◽

Haripriya Shankar ◽

Surya Bhamidipati ◽

Robert T. Dorsam ◽

Jianguo Jin ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Thromboxane A2 ◽

Human Platelets ◽

Time Dependent ◽

Dependent Manner ◽

Src Tyrosine Kinase ◽

Functional Implications

Abstract Thrombin has been known to cause tyrosine phosphorylation of protein kinase C δ (PKCδ) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCδ and the role of this event in platelet function. PKCδ was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCδ did not occur in Gαq-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5′-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ′, N ′-tetraacetic acid), suggesting a role for Gαq pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCδ. Inhibition of tyrosine phosphorylation or the kinase activity of PKCδ dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCδ in a calcium- and Src-family kinase–dependent manner in platelets, with functional implications in thromboxane A2 generation.

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

Blood ◽

10.1182/blood.v106.11.1648.1648 ◽

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.

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The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽

10.1182/blood.v108.11.3904.3904 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3904-3904

Author(s):

Samantha Baldassarri ◽

Alessandra Bertoni ◽

Paolo Lova ◽

Stefania Reineri ◽

Chiara Sarasso ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Cannabinoid Receptors ◽

Thromboxane A2 ◽

Human Platelets ◽

Dependent Manner ◽

Cb2 Receptor ◽

Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

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Human group II 14 kDa phospholipase A2 activates human platelets

Biochemical Journal ◽

10.1042/bj3270259 ◽

1997 ◽

Vol 327 (1) ◽

pp. 259-265 ◽

Cited By ~ 12

Author(s):

János POLGÁR ◽

Ruth M. KRAMER ◽

Suzane L. UM ◽

Joseph A. JAKUBOWSKI ◽

Kenneth J. CLEMETSON

Keyword(s):

Phospholipase A2 ◽

Platelet Activation ◽

Heparan Sulphate ◽

Thromboxane A2 ◽

Inhibitory Effect ◽

Human Platelets ◽

Time Dependent ◽

Human Group ◽

Group Ii ◽

Induced Aggregation

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low μg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

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Studies on the Effect of Platelet Inhibitors on Platelet Adhesion to Collagen and Collagen-Induced Human Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661310 ◽

1985 ◽

Vol 53 (03) ◽

pp. 337-342 ◽

Cited By ~ 7

Author(s):

S Krishnamurthi ◽

V V Kakkar

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Antiplatelet Agents ◽

Calmodulin Antagonist ◽

Release Reaction ◽

Low Concentrations

SummaryThe effect of pyridoxal 5’-phosphate (PALP) and trifluoperazine (TFPZ), the calmodulin antagonist, on in vitro platelet adhesion to collagen and collagen-induced platelet activation was studied using platelet-rich-plasma (PRP) or washed platelets (WPL). Platelet aggregation and [14C]-5HT release induced by “threshold” or low concentrations of collagen (0.6 μg/ ml) in PRP were completely abolished by PALP (24 mM), TFPZ (250 μM) as well as indomethacin (10 μM). At higher concentrations of collagen (10–15 μg/ml) in PRP and WPL, the use of stirred and unstirred platelets treated with collagen enabled a distinction to be made between aggregation and adhesion- mediated release reaction. Platelet aggregation and the aggregation-mediated release reaction induced by these concentrations of collagen in stirred platelets were completely abolished by PALP, TFPZ and indomethacin although neither adhesion to collagen nor the adhesion-mediated release reaction of unstirred platelets was significantly affected by these inhibitors. Interestingly, both adhesion and the adhesion-mediated release reaction were abolished by concentrations of PALP 10–40 fold higher than those required to abolish aggregation. Collagen-induced platelet aggregation, but not platelet adhesion, was inhibited in resuspended platelets pretreated with PALP and NaBH4 indicating a separation in the membrane sites involved in aggregation and adhesion. The results further emphasize the distinction between adhesion and aggregation-mediated events with regards to collagen with the latter being more susceptible to inhibition by antiplatelet agents such as PALP and TFPZ.

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The Roles of Akt1 and Akt2 in GPIb-IX-Mediated Platelet Activation Signaling.

Blood ◽

10.1182/blood.v110.11.3635.3635 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3635-3635

Author(s):

Hong Yin ◽

Aleksandra Stojanovic ◽

Nissim Hay ◽

Xiaoping Du

Keyword(s):

Shear Stress ◽

Signaling Pathway ◽

Platelet Activation ◽

Platelet Adhesion ◽

Thromboxane A2 ◽

Human Platelets ◽

Integrin Activation ◽

Akt Inhibitor ◽

Platelet Spreading ◽

Inside Out

Abstract The platelet von Willebrand factor (VWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. To understand the downstream signaling pathway of GPIb-IX signaling, we investigated the roles of PI3K effector kinases, Akt1 and Akt2, in VWF/GPIb-IX-induced platelet activation. VWF/GPIb-IX-induced platelet aggregation was impaired in Akt1- or Akt2-knockout mouse platelets and in human and mouse platelets treated with an Akt inhibitor, SH-6. GPIb-IX-mediated platelet stable adhesion to VWF under shear stress was also inhibited in mouse platelets deficient in Akt1 or Akt2, and in human platelets treated with SH-6. Interestingly, while deficiency of Akt1 or Akt2 caused nearly complete inhibition of stable platelet adhesion to VWF under shear stress, stable platelet adhesion was only partially reduced in platelets treated with both P2Y1 and P2Y12 ADP receptor antagonists, A3P5P and 2MeSAMP or thromboxane A2 pathway inhibitor, aspirin or Syk inhibitor, piceatannol. Therefore, Akt1 and Akt2 are important in early GPIb-IX signaling independent of Syk, ADP or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of either Akt1 or Akt2 diminished platelet spreading on VWF, but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both VWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1 or Akt2 deficiency diminished VWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by low concentration of exogenous cGMP, indicating that Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway. In conclusion, both Akt1 and Akt2 mediate VWF/GPIb-IX-induced signaling pathway leading to platelet activation and the consequent stable platelet adhesion, spreading and aggregation.

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Thromboxane A2 Signaling Regulates Heterogeneous Platelet Activation Following Laser-Induced Injury In Mouse Cremaster Arterioles

Blood ◽

10.1182/blood.v122.21.1055.1055 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1055-1055

Author(s):

Jian Shen ◽

Fei Yang ◽

Yujun Shen ◽

Ying Yu ◽

Timothy J. Stalker ◽

...

Keyword(s):

Platelet Activation ◽

Vascular Injury ◽

Low Dose ◽

Thromboxane A2 ◽

Dose Aspirin ◽

Low Dose Aspirin ◽

Platelet Accumulation ◽

Cox 1 ◽

Platelet Mass

Abstract Background A recent study demonstrated that platelet accumulation following vascular injury in vivo is hierarchically organized resulting in a structure comprised of a core of fully-activated platelets that is overlaid with an unstable shell of less activated platelets (Stalker et al, Blood, 2013). This structure results from different elements of the platelet signaling network giving rise to regions that differ in platelet activation state, packing density, and stability. It was thus proposed that regional differences in platelet activation reflect regional differences in the distribution of platelet agonists. This provides new insights into heterogeneous platelet activation during platelet accumulation in vivo. Thromboxane A2 (TxA2), a dominant prostanoid product of cyclooxygenase 1 (COX-1) generated in platelets, plays an important role in the maintenance of vascular hemostasis and is a major therapeutic target of anti-platelet therapy. But its contribution to the regional architecture of a platelet mass is unknown. Approach To determine the contribution of TxA2 activity to the hierarchical organization of a thrombus, multicolor intravital microscopy was used to observe platelet accumulation and activation in thromboxane A2 receptor knockout (TP-/-) and low dose aspirin treated WT mice following laser-induced injury in mouse cremaster arterioles. Results TP-/- mice showed reduced total platelet (CD41) accumulation following vascular injury, consistent with a previous report (Yu et al, Sci Transl Med, 2012). The peak CD41 area in TP-/- mice was significantly reduced relative to WT mice (p=0.004). Interestingly, the core area of the thrombus in which the platelets are fully activated (P-selectin+), was not significantly different in TP-/- compared to WT during thrombus formation. This suggests that TxA2 signaling via the TP receptor primarily influences platelet recruitment and retention in the outer shell region of a platelet mass, but not full platelet activation in the core region. Aspirin inhibits TxA2 production through acetylation of COX-1, and is widely used as both primary and secondary prevention of cardiovascular diseases. We treated WT mice with aspirin in their drinking water (30 mg/L) for more than 1 week to mimic the effect of low dose aspirin treatment in humans (Yu et al, J Clin Invest, 2005). Similar to our findings in TP-/- mice, we found that aspirin treatment reduced total platelet accumulation following laser-induced injury in vivo (p<0.05). The decrease in peak platelet accumulation caused by aspirin was observed in the shell region at early time points (up to 2 min post-injury). In contrast to our findings in the TP-/- mice, low dose aspirin also resulted in reduced platelet activation and core region formation at later timepoints (p<0.05), suggesting that COX-1 may contribute to full platelet activation independent of TP receptor signaling. Conclusion Our studies show for the first time the role of TxA2 signaling in producing the hierarchical structure of a platelet mass formed in response to vascular injury. Our data indicate that TxA2signaling is critical for recruitment and/or retention of platelets prior to robust platelet activation including alpha granule secretion. These findings further highlight the importance of discrete spatial localization of platelet agonists within an evolving platelet plug in order to achieve the optimal hemostatic response. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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Low concentrations of sodium azide specifically inhibit a thromboxane A2 pathway in human platelets

Thrombosis Research ◽

10.1016/0049-3848(92)90180-i ◽

1992 ◽

Vol 66 (2-3) ◽

pp. 101-110 ◽

Cited By ~ 4

Author(s):

Eric Rubinstein ◽

Irene Urso ◽

Roger C. Carroll

Keyword(s):

Sodium Azide ◽

Thromboxane A2 ◽

Human Platelets ◽

Low Concentrations

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

Blood ◽

10.1182/blood.v88.2.522.bloodjournal882522 ◽

1996 ◽

Vol 88 (2) ◽

pp. 522-530 ◽

Cited By ~ 43

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.

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