John C. Kostyak

Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization

Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in β1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.

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.

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.

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.

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.

PKCδ 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.