Lina Cipolla

Nanoparticles induce platelet activation in vitro through stimulation of canonical signalling pathways.

UNLABELLED Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)β3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. Importantly, we found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, indicating that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. FROM THE CLINICAL EDITOR In this study, nanosized particles of three virtually unrelated materials (silica, multi-walled carbon nanotubes and carbon black) were investigated regarding their effects on platelet activation and aggregation. All were found to stimulate some of the typical biochemical pathways involved in canonical platelet activation, and were found to have synergistic effects with physiologic platelet activator agonists.

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Integrin α2β1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3Kβ(KD)), but occurred normally in PI3Kγ(KD) platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)-knockout mice, and we found that intracellular Ca(2+) linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca(2+). Whereas activation of Pyk2 occurred normally in PI3Kβ(KD) platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

In the present study, we used a knockout murine model to analyze the contribution of the Ca(2+)-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin α(IIb)β(3) and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A(2) production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA(2) phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein-coupled receptors in supporting platelet aggregation and thrombus formation.

The proline-rich tyrosine kinase Pyk2 regulates platelet integrin αIIbβ3 outside-in signaling.

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 α2β1.

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase β, and Not Via CD40 and IκB Kinase α

Objective—To investigate the roles and signaling pathways of CD40L and CD40 in platelet–platelet interactions and thrombus formation under conditions relevant for atherothrombosis. Approach and Results—Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg−/−Apoe−/−) showed diminished &agr;IIb&bgr;3 activation and &agr;-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40−/−Apoe−/−) were not decreased. Using blood from Cd40lg−/−Apoe−/− and Cd40−/−Apoe−/− mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe−/− blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI–induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cbR/R platelets or by inhibiting phosphatidylinositol 3-kinase &bgr; (PI3K&bgr;). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3K&bgr;. Finally, platelets from Chuk1A/AApoe−/− mice deficient in I&kgr;B kinase &agr; (IKK&agr;), implicated in CD40 signaling to nuclear factor (NF) &kgr;B, showed unchanged responses to CD40L in aggregation or thrombus formation. Conclusions—Under atherogenic conditions, CD40L enhances collagen-induced platelet–platelet interactions by supporting integrin &agr;IIb&bgr;3 activation, secretion and thrombus growth via PI3K&bgr;, but not via CD40 and IKK&agr;/NF&kgr;B. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation.

Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets

Background: Pyk2 is abundantly expressed in platelets. Results: Pyk2 regulates thromboxane A2 generation induced by both 2-methylthio-ADP and AYPGKF. Conclusion: Pyk2 is an important functional tyrosine kinase that is activated by both G12/13 and integrin αIIbβ3 in platelets. Significance: Understanding the mechanism of activation of Pyk2 enhances our understanding of the platelet inside-out and outside-in signaling events. Proline-rich tyrosine kinase 2 (Pyk2) is activated by various agonists in platelets. We evaluated the signaling mechanism and the functional role of Pyk2 in platelets by using pharmacological inhibitors and Pyk2-deficient platelets. We found that platelet aggregation and secretion in response to 2-methylthio-ADP (2-MeSADP) and AYPGKF were diminished in the presence of Pyk2 inhibitors or in Pyk2-deficient platelets, suggesting that Pyk2 plays a positive regulatory role in platelet functional responses. It has been shown that ADP-, but not thrombin-induced thromboxane (TxA2) generation depends on integrin signaling. Unlike ADP, thrombin activates G12/13 pathways, and G12/13 pathways can substitute for integrin signaling for TxA2 generation. We found that Pyk2 was activated downstream of both G12/13 and integrin-mediated pathways, and both 2-MeSADP- and AYPGKF-induced TxA2 generation was significantly diminished in Pyk2-deficient platelets. In addition, TxA2 generation induced by co-stimulation of Gi and Gz pathways, which is dependent on integrin signaling, was inhibited by blocking Pyk2. Furthermore, inhibition of 2-MeSADP-induced TxA2 generation by fibrinogen receptor antagonist was not rescued by co-stimulation of G12/13 pathways in the presence of Pyk2 inhibitor. We conclude that Pyk2 is a common signaling effector downstream of both G12/13 and integrin αIIbβ3 signaling, which contributes to thromboxane generation.