Dheeraj Bhavanasi

RhoG Protein Regulates Glycoprotein VI-Fc Receptor γ-Chain Complex-mediated Platelet Activation and Thrombus Formation

Background: RhoG is a ubiquitously expressed member of the Rho family of GTPases. Results: RhoG-deficient platelets display severely impaired GPVI-dependent platelet activation. Conclusion: RhoG plays an important role in GPVI-FcRγ complex-mediated platelet activation and thrombus formation. Significance: Our study enhances the understanding of the molecular mechanisms of GPVI-FcRγ complex activation. We investigated the mechanism of activation and functional role of a hitherto uncharacterized signaling molecule, RhoG, in platelets. We demonstrate for the first time the expression and activation of RhoG in platelets. Platelet aggregation, integrin αIIbβ3 activation, and α-granule and dense granule secretion in response to the glycoprotein VI (GPVI) agonists collagen-related peptide (CRP) and convulxin were significantly inhibited in RhoG-deficient platelets. In contrast, 2-MeSADP- and AYPGKF-induced platelet aggregation and secretion were minimally affected in RhoG-deficient platelets, indicating that the function of RhoG in platelets is GPVI-specific. CRP-induced phosphorylation of Syk, Akt, and ERK, but not SFK (Src family kinase), was significantly reduced in RhoG-deficient platelets. CRP-induced RhoG activation was consistently abolished by a pan-SFK inhibitor but not by Syk or PI3K inhibitors. Interestingly, unlike CRP, platelet aggregation and Syk phosphorylation induced by fucoidan, a CLEC-2 agonist, were unaffected in RhoG-deficient platelets. Finally, RhoG−/− mice had a significant delay in time to thrombotic occlusion in cremaster arterioles compared with wild-type littermates, indicating the important in vivo functional role of RhoG in platelets. Our data demonstrate that RhoG is expressed and activated in platelets, plays an important role in GPVI-Fc receptor γ-chain complex-mediated platelet activation, and is critical for thrombus formation in vivo.

Differential dephosphorylation of the protein kinase C-zeta (PKCζ) in an integrin αIIbβ3-dependent manner in platelets.

Protein kinase C-zeta (PKCζ), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKCζ has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The T410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin αIIbβ3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKCζ was also observed in PP1cγ null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKCζ, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKCζ might be an important regulatory mechanism for platelet functional responses.

Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCv in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a G(q) inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is G(q)-, but not G₁₂/₁₃-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ- or PKCɛ-deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and G(q)-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in 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.

Protein Kinase C δ Deficiency Enhances Megakaryopoiesis and Recovery from Thrombocytopenia

Objective— We previously determined that protein kinase C &dgr; (PKC&dgr;) regulates platelet function. However, the function of PKC&dgr; in megakaryopoiesis is unknown. Approach and Results— Using PKC&dgr;-/- and wild-type littermate mice, we found that deficiency of PKC&dgr; caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKC&dgr;-/- mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKC&dgr; deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKC&dgr;-/- megakaryocytes compared with wild-type. Finally, PKC&dgr;-/- mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. Conclusions— These data suggest that PKC&dgr; is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.

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.

CGX1037 is a novel PKC isoform delta selective inhibitor in platelets

Abstract Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pre-treatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ· Furthermore, pre-treatment of platelets from PKCδ−/− mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets.

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.

Platelet Signaling: Protein Phosphorylation

Protein phosphorylation is the main mechanism by which platelet functional responses are regulated. There are myriad protein kinases in the platelet, which are classified by the residue in which they phosphorylate. Protein tyrosine kinases phosphorylate tyrosine residues, while protein serine/threonine kinases phosphorylate serine or threonine residues. Several important signaling cascades are initiated upon platelet activation. Here we will explore the relevance of phosphorylation of specific proteins important to several essential signaling cascades in platelets.

Protein Kinase C δ Deficiency Enhances Megakaryopoiesis and Recovery From ThrombocytopeniaSignificance

Objective— We previously determined that protein kinase C &dgr; (PKC&dgr;) regulates platelet function. However, the function of PKC&dgr; in megakaryopoiesis is unknown. Approach and Results— Using PKC&dgr;-/- and wild-type littermate mice, we found that deficiency of PKC&dgr; caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKC&dgr;-/- mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKC&dgr; deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKC&dgr;-/- megakaryocytes compared with wild-type. Finally, PKC&dgr;-/- mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. Conclusions— These data suggest that PKC&dgr; is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.