Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2)

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.

G6f-like is an ITAM-containing collagen receptor in thrombocytes

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.

Critical role for an acidic amino acid region in platelet signaling by the HemITAM (hemi-immunoreceptor tyrosine-based activation motif) containing receptor CLEC-2 (C-type lectin receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α(2) β(1)

Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.

Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

CLEC-2 activates Syk through dimerization

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.

The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.

Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

Differential roles of the PKC novel isoforms, PKCδ and PKCε, in mouse and human platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2.

The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP-76 in response to weak activation of GPVI and CLEC-2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads-independent pathway of platelet activation downstream of LAT.

Globular adiponectin induces platelet activation through the collagen receptor GPVI-Fc receptor gamma chain complex.

We identify gAd as a novel ligand for GPVI that stimulates tyrosine kinase-dependent platelet aggregation. Our data raise the possibility that gAd may promote unwanted platelet activation at sites of vascular injury.

Minimal regulation of platelet activity by PECAM-1.

PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, alphaIIbbeta3, even though both receptors signal through Src-kinase regulation of PLCgamma2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCgamma2. Studies using PECAM-1 knockout-mice and cross-linking of PECAM-1 using specific antibodies demonstrated a minor inhibitory role on platelet responses to the above three receptors and also under some conditions to the G-protein agonist thrombin. The degree of inhibition was considerably less than that produced by PGI2, which elevates cAMP. There was no significant difference in thrombus formation on collagen in PECAM-1-/- platelets relative to litter-matched controls. The very weak inhibitory effect of PECAM-1 on platelet activation relative to that of PGI2 indicate that the Ig-receptor is not a major regulator of platelet activation. PECAM-1 has been reported to have contrasting effects on platelet activation. The present study demonstrates a very mild or negligible effect on platelet activation in response to stimulation by a variety of agonists, thereby questioning the physiological role of the immunoglobulin receptor as a major regulator of platelet activation.