Platelets at the vascular interface

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Platelet signaling--blood's great balancing act

The antagonistic balance between CalDAG-GEFI and RASA3 signaling is critical for the fine-tuning of platelet adhesiveness, both in the circulation and at sites of vascular injury

RAP1-GTPase signaling and platelet function

Platelets are critical for hemostasis, i.e. the body's ability to prevent blood loss at sites of vascular injury. They patrol the vasculature in a quiescent, non-adhesive state for approximately 10 days, after which they are removed from circulation by phagocytic cells of the reticulo-endothelial system. At sites of vascular injury, they promptly shift to an activated, adhesive state required for the formation of a hemostatic plug. The small GTPase RAP1 is a critical regulator of platelet adhesiveness. Our recent studies demonstrate that the antagonistic balance between the RAP1 regulators, CalDAG-GEFI and RASA3, is critical for the modulation of platelet adhesiveness, both in circulation and at sites of vascular injury. The RAP1 activator CalDAG-GEFI responds to small changes in the cytoplasmic calcium concentration and thus provides sensitivity and speed to the activation response, essential for efficient platelet adhesion under conditions of hemodynamic shear stress. The RAP1 inhibitor RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI-dependent RAP1 activation. Upon cellular stimulation, it is turned off by P2Y12 signaling to enable sustained RAP1 activation, required for the formation of a stable hemostatic plug. This review will summarize important studies that elucidated the signaling pathways that control RAP1 activation in platelets

Phytogeographical evidence for post-glacial dispersal limitation of European beech forest species

The frequency of range sizes shows a U-shaped distribution, with 42 species occurring in < 10 regions. The highest number of beech forest species is found in the southern Alps and adjacent regions, and species numbers decrease with increasing distance from these regions. With only narrow-range species taken into consideration, secondary maxima are found in Spain, the southern Apennines, the Carpathians, and Greece. Distance to the nearest potential refuge area is the strongest predictor of beech forest species richness, while altitudinal range and soil type diversity had little or no predictive value. The clusters of narrow-range species are in good concordance with the glacial refuge areas of beech and other temperate tree species as estimated in recent studies. These findings support the hypothesis that the distribution of many beech forest species is limited by post-glacial dispersal rather than by their environmental requirements

Adoptive transfer method to study platelet function in mouse models of disease

Platelets play an important role in many physiological and pathological situations. However, the molecular mechanisms by which platelets contribute to health and disease are often ill-defined. One of the limiting factors to these studies is a fast but reliable method to generate animals with platelet-specific signaling defects. We here review recent approaches to establish an adoptive platelet transfer model in mice

A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

The dynamic regulation of ERK1 and −2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2

Extracellular Matrix Proteins in Hemostasis and Thrombosis

The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets

Transfection of human platelets with short interfering RNA.

Platelets contain mRNAs and are capable of translating mRNA into protein, and it has been previously demonstrated that platelets increase their levels of integrin β3 overtime while in blood bank storage conditions. We are unaware of prior attempts to introduce nucleic acids into platelets. Considering the potential clinical and research utility of manipulating platelet gene expression, we tested whether small interfering RNAs (siRNAs) could be transfected into normal human platelets. Multiple conditions were tested, including lipofectamine versus electroporation, different amounts of siRNA, the effect of different buffers and the presence of plasma during transfection, and the time for optimal siRNA incorporation after transfection. Using flow cytometry to assess transfection efficiency, we found that optimal transfection was obtained using lipofectamine, washed platelets, and 400 pmoles siRNA. Cell sorting of transfected platelets suggested that the incorporated siRNA was able to knockdown the level of a targeted mRNA. This is the first ever demonstration that nucleic acids can be introduced directly into platelets, and offers proof of concept for manipulating gene expression in platelets by nonviral methods. Future technical improvements may permit improving the quality and/or lifespan of stored human platelets

Emerging roles of store-operated Ca 2+ entry through STIM and ORAI proteins in immunity, hemostasis and cancer

Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway, which is defined by the fact that depletion of intracellular Ca2+ stores, mainly the endoplasmic reticulum (ER), triggers the opening of Ca2+ channels in the plasma membrane. The best characterized SOC channel is the Ca2+ release-activated Ca2+ (CRAC) channel, which was first described in cells of the immune system but has since been reported in many different cell types. CRAC channels are multimers of ORAI family proteins, of which ORAI1 is the best characterized. They are activated by stromal interaction molecules (STIM) 1 and 2, which respond to the depletion of intracellular Ca2+ stores with oligomerization and binding to ORAI proteins. The resulting SOCE is critical for the physiological function of many cell types including immune cells and platelets. Recent studies using cell lines, animal models and primary cells from human patients with defects in SOCE have highlighted the importance of this Ca2+ entry mechanism in a variety of pathophysiological processes. This review focuses on the role of SOCE in immunity to infection, allergy, hemostasis and cancer

CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia

Platelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI(−/−) (hFcR/CDGI(−/−)) were generated. Compared with controls, aggregation of hFcR/CDGI(−/−) platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI(−/−) platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT