Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

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Objective Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including PDI and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation and thrombus formation. In this study we examined for the first time the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets and identified the cellular events responsible for their movement to the platelet surface upon activation. Approach and Results Immunofluorescence microscopy (IFM) imaging was used to localize PDI and ERp57 in murine and human megakaryocytes at various developmental stages. IFM and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and SERCA3. IFM and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin), and in the presence or absence of membrane fusion mediated by Munc 13-4 (absent in platelets from Unc13dJinx mice). Conclusions Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes, and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not SNARE/Munc 13-4-dependent vesicular-plasma membrane fusion.

The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor 37,43Gap27 on Cx40−/− mouse platelets and of the Cx40 inhibitor 40Gap27 on Cx37−/− mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.

Objective-Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. Approach and Results-Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner.It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagencoated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. Conclusions-This

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In the presence of a previous history of CS, patients with antepartum diagnosis of placenta previa are considered to be at a greater risk for having placenta accreta. The risk increases with the increase in the number of previous CS. Patients with placenta previa accreta have a significantly higher incidence of PPH and are more likely to undergo emergency hysterectomy.

Background and PurposeThe discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function.Experimental ApproachThe ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α‐granule secretion and calcium mobilization assays were performed using platelet‐rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Key ResultsNobiletin was shown to suppress a range of well‐established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)‐stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator‐stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Conclusions and ImplicationsThis study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.