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Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke.5-HT transporter knockout mice (5Htt-/-) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.In 5Htt-/- platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt-/- platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt-/- mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt-/- mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation--platelet adhesion and aggregation--have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84(-/-) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84(-/-) mice. In vivo, Cd84(-/-) mice exhibited unaltered hemostatic function and arterial thrombus formation.These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.

This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.

Twinfilins are evolutionarily conserved regulators of cytoskeletal dynamics. They inhibit actin polymerization by binding both actin monomers and filament barbed ends. Inactivation of the single twinfilin gene from budding yeast and fruit fly results in defects in endocytosis, cell migration, and organization of the cortical actin filament structures. Mammals express three twinfilin isoforms, of which twinfilin-1 and twinfilin-2a display largely overlapping expression patterns in non-muscle tissues of developing and adult mice. The expression of twinfilin-2b, which is generated through alternative promoter usage of the twinfilin-2 gene, is restricted to heart and skeletal muscles. However, the physiological functions of mammalian twinfilins have not been reported. As a first step towards understanding the function of twinfilin in vertebrates, we generated twinfilin-2a deficient mice by deleting exon 1 of the twinfilin-2 gene. Twinfilin-2a knockout mice developed normally to adulthood, were fertile, and did not display obvious morphological or behavioural abnormalities. Tissue anatomy and morphology in twinfilin-2a deficient mice was similar to that of wild-type littermates. These data suggest that twinfilin-2a plays a redundant role in cytoskeletal dynamics with the biochemically similar twinfilin-1, which is typically co-expressed in same tissues with twinfilin-2a.