Bhanu Kanth Manne

PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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.

Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2)

Background: Fucoidan was tested as a novel drug for hemophilia, but its effect on platelets has not been studied. Results: We show that fucoidan activates human and mouse platelets and that activation is blocked in CLEC-2 knock-out murine platelets. Conclusion: Fucoidan is a novel agonist for the CLEC-2. Significance: Understanding the effects of fucoidan on platelets can help in designing efficient drugs for hemophilia. 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.

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.

Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets.

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

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.

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials RAS proteins are expressed in platelets but their functions are largely uncharacterized. TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo. TC21 regulates platelet aggregation by control of αIIbβ3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Identification of novel Syk-independent functional roles of FcγRIIa in platelet outside-in signaling using transgenic mice expressing human FcγRIIa

Platelet outside-in signaling is a complex and dynamic process in which integrin αIIbβ3 transmits signals to the actin cytoskeleton. An important part of the process is the activation of tyrosine kinases from the Src family kinases (SFK) and spleen tyrosine kinase (Syk). Src, which is constitutively bound to β3 cytoplasmic tail, becomes activated upon integrin clustering [1, 2] and transmits signals which recruit and activate Syk kinase [3]. Syk is a tyrosine kinase that contains two Src-homology 2 domains (SH2). Due to the known interaction of Syk-SH2 domains with immunoreceptor tyrosine-based activation motifs (ITAMs), Syk was thought to be a kinase involved exclusively in classical immunoreceptor signaling. Subsequently, Syk was identified as an important mediator of integrin signaling [4, 5] but the crucial question of how the SH2 domains of Syk were engaged in an integrin signaling context remained elusive. In an elegant study, Woodside et al. [6] described the direct binding of Syk to the β3 tail and proposed a model in which Syk was activated independently of ITAM domains. However, blocking FcγRIIa with mAb IV.3 or using platelets from a patient with an autoantibody-induced shedding of GPVI that expressed reduced levels of a crippled FcγRIIa receptor, Boylan et al. [7] proposed a different model that identifies FcγRIIa as the ITAM-bearing adaptor required for Syk activation in platelet outside-in signaling. Our group has identified c-Cbl as an important component of αIIbβ3-mediated signaling. In human platelets, we identified two different tyrosines on c-Cbl (Y700 and Y774) as Syk substrates [8]. We observed significantly lower levels of Syk phosphorylation in murine platelets compared to human platelets spread over fibrinogen [8]. As murine platelets do not express FcγRIIa, this result led us to rationalize that indeed FcγRIIa is required for platelet outside-in signaling and drives the observed difference in Syk phosphorylation between human and murine platelets [8]. Nonetheless, the precise role of FcγRIIa in outside-in signaling remained to be determined, as murine platelets do spread over fibrinogen and some Syk phosphorylation occurs upon fibrinogen binding. Also, a significant difference in platelet spreading over fibrinogen exists between WT and Syk –/– platelets [3]. Zhi et al. [9] published a more detailed description of the role of FcγRIIa in thrombus formation in vitro and in vivo. The present work extends these observations by demonstrating that, at least in part, the role of FcγRIIa in platelet outside-in signaling is Syk-independent and that murine platelets have an outside-in signaling mechanism that differs from human in the requirement for FcγRIIa. Integrin outside-in signaling regulates functional responses such as cell spreading and clot retraction. Figure 1(A1) shows an image of platelets spreading over fibrinogen from WT versus FcγRIIa transgenic mice [10]. Quantitative analysis demonstrates a significant difference (*P ≤ 0.05) in the area of spread platelets expressing human FcγRIIa versus WT at 45 min. Additionally, clot retraction analysis identified a novel role for FcγRIIa in the retraction process. Platelets from FcγRIIa transgenic mice retract faster than their WT counterparts (Figure 1(A2)) demonstrating enhanced retraction kinetics that is visible at 45 and 90 min. Using pharmacological inhibitors, our previous work has identified a role for Syk kinase in platelet spreading but not in clot retraction in human platelets [8]. In agreement with this, platelets from FcγRIIa transgenic mouse treated with the Syk inhibitor OXSI-2 (Figure 1(A2)) retracts with the same kinetics as control platelets. In control experiments, we have ascertained that OXSI-2 inhibits Syk using PLCγ2 phosphorylation as a readout in transgenic platelets spreading on fibrinogen (not shown). Together, we conclude that FcγRIIa aids in clot retraction independently of its association with Syk. The results shown in Figures 1(A1) and (A2) demonstrate that FcγRIIa is playing a pivotal role in platelet outside-in signaling. Indeed, the enhanced functional responses are reflected in the phosphorylation state of Syk Y525/526 (activation marker). Figure 1(B1) shows a representative western blot analysis of Syk phosphorylation at 20 and 45 min of platelet spreading over a fibrinogen-coated surface. Densitometry of three different experiments indicates that outside-in signaling-mediated Syk phosphorylation is higher in FcγRIIa transgenic platelets versus WT (*P ≤ 0.05). To further investigate the signaling events mediated by FcγRIIa upon fibrinogen binding, we analyzed downstream substrates of Syk. As seen on Figure 1(B2) and (B3) PLCγ2 and c-Cbl Y774 exhibited enhanced phosphorylation (*P ≤ 0.05) in Correspondence: Satya P Kunapuli, Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Rm. 414 MRB, 3420 N. Broad St., Philadelphia, PA 19140, USA. E-mail: spk@temple.edu http://www.tandfonline.com/iplt ISSN: 0953-7104 (print), 1369-1635 (electronic)