The proline-rich tyrosine kinase Pyk2 regulates platelet integrin αIIbß3 outside-in signaling.

BACKGROUND: The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2ß1. OBJECTIVE: In this study we have investigated the involvement of Pyk2 in integrin αIIbß3 outside-in signaling in human and murine platelets. METHODS: We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen. RESULTS: Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbß3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbß3 engagement selectively stimulated the ß-isoform of PI3K (PI3Kß), and that, as for Pyk2, PI3Kß activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kß were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbß3 engagement triggered the association of the PI3Kß regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets. CONCLUSIONS: These results identify a novel pathway of integrin αIIbß3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen.

Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets.

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid ß-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.

Altered cytoskeleton organization in platelets from patients with MYH9-related disease.

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor.

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments.

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.

Hydrolysis of NADP+ by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2'-phosphate.

CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD+ and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP+ by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP+ resulted in the rapid and time-dependent accumulation of ADP-ribose 2'-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2'-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP+ glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2'-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2'-phosphate did not result in the formation of ADP-ribose 2'-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2'-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD+, displays exclusively a NADP+ glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2'-phosphate.

Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation.

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.