Rachit Badolia

Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets

Background: The mechanisms by which the hemITAM receptor activates Syk are not completely understood. Results: Inhibition of PI3K or Tec family kinases abolished Syk activation downstream of CLEC-2 but not GPVI. Conclusion: PI3K is upstream of tyrosine kinase Syk in CLEC-2, but not in GPVI, activation. Significance: Understanding the differences between hemITAM and ITAM signaling helps in designing efficient drugs for thrombosis and hemostasis. Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor.

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Background: PKC regulating Syk activity has been demonstrated in other cells but is unknown in platelets. Results: PKCs regulate tyrosine phosphorylation and activity of Syk. Conclusion: PKCβ-dependent differential regulation of Syk activity is seen in human but not in murine platelets. Significance: Understanding this human pathway of platelet regulation might aid in development of anti-platelet therapy. Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets.

C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets

ADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

G q pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets

Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific Gq inhibitor, UBO-QIC, or Gq knock-out murine platelets, we demonstrate that Gq signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of Gq and identified an important role for the PLCβ-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel Gq-PLCβ-PKCα-mediated regulation of proximal CLEC-2 signaling by Gq-coupled receptors.

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

Essentials Phosphoinositide 3‐kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate‐induced platelet activation and thromboxane generation. PDK1 regulates RAF proto‐oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet‐dependent thrombosis in vivo.

IPA3 non-specifically enhances phosphorylation of several proteins in human platelets.

Abstract PAK (p21-Activation kinase), a serine-threonine protein kinase contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. This autoregulatory domain found within PAK kinase provides a unique target for chemical inhibitors. IPA3, a small molecule allosteric inhibitor of PAK activation, binds covalently to the PAK regulatory domain and prevents binding to its upstream activators. IPA3 has been used in various cells including platelets to evaluate the role of PAK in signaling. In a recent study, PAK functions in platelet aggregation and lamellipodia formation were evaluated using IPA3 as the PAK inhibitor. Herein, we investigated the specificity and selectivity of IPA3 as a PAK inhibitor in the human platelets. Stimulation of platelets pretreated with IPA3 using a PAR-4 or GPV1 agonist resulted in a concentration-dependent inhibition of aggregation, as was suggested by earlier studies. Interestingly, we found that incubation of washed human platelets with IPA3 lead to a non-specific increase in phosphorylation of several proteins in absence of any agonist. However, this phosphorylation is not sufficient for aggregation of platelets by IPA3. In summary, we demonstrate that IPA3 by itself can phosphorylate several proteins in human platelets and thus its use is not an appropriate strategy for investigating PAK function in platelets.

Syk Activity Is Dispensable for Platelet GP1b-IX-V Signaling

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein 1b-IX (GP1b-IX) leads to activation of platelets. GP1b was shown to signal via the FcRγ-ITAM (Fc Receptor γ-Immunoreceptor tyrosine-based activation motif) pathway, activating spleen tyrosine kinase (Syk) and other tyrosine kinases. However, there have been conflicting reports regarding the role of Syk in GP1b signaling. In this study, we sought to resolve these conflicting reports and clarify the role of Syk in VWF-induced platelet activation. The inhibition of Syk with the selective Syk inhibitors, OXSI-2 and PRT-060318, did not inhibit VWF-induced platelet adhesion, agglutination, aggregation, or secretion. In contrast, platelets stimulated with the Glycoprotein VI (GPVI) agonist, collagen-related peptide (CRP), failed to cause any aggregation or secretion in presence of the Syk inhibitors. Furthermore, GP1b-induced platelet signaling was unaffected in the presence of Syk inhibitors, but GPVI-induced signaling was abolished under similar conditions. Thus, we conclude that Syk kinase activity does not play any functional role downstream of GP1b-mediated platelet activation.