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 αIIb ß3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets. SUMMARY: Background Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS-induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored. Objectives To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis. Results We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI-FcRγ immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor, in an Src-dependent manner. GPVI-induced platelet aggregation, integrin αIIb ß3 activation, and α-granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal-regulated kinase, were inhibited in TC21-deficient platelets ex vivo. In contrast, these responses were normal in TC21-deficient platelets following stimulation with P2Y, protease-activated receptor 4 and C-type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI-FcRγ-ITAM-specific. TC21 was required for GPVI-induced activation of Rap1b. TC21-deficient mice did not show a significant delay in injury-induced thrombosis as compared with wild-type controls; however, thrombi were unstable. Hemostatic responses showed similar effects. Conclusions TC21 is essential for GPVI-FcRγ-mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway.

Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo. SUMMARY: Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3ß. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1-/- mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.

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. GSK3ß 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.