Critical role for platelet Ral GTPases in regulating venous thrombosis in mice.

Background: Deep vein thrombosis is a major cause of morbidity and mortality globally. While its pathophysiology is complex, increasing evidence suggests a more prominent role for platelets than previously suspected. Genetic deletion of Ral GTPases, RalA and RalB, conditionally in mouse platelets (RalAB double knockout [DKO]), results in a near complete defect in P-selectin externalization upon activation, while other platelet activation responses and arterial thrombosis are preserved.

Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis.

The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets.

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets.

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8.

Small GTPases in platelet membrane trafficking.

Our understanding of fundamental biological processes within platelets is continually evolving. A critical feature of platelet biology relates to the intricate uptake, packaging and release of bioactive cargo from storage vesicles, essential in mediating a range of classical (haemostasis/thrombosis) and non-classical (regeneration/inflammation/metastasis) roles platelets assume. Pivotal to the molecular control of these vesicle trafficking events are the small GTPases of the Ras superfamily, which function as spatially distinct, molecular switches controlling essential cellular processes.

Loss of the mitochondrial kinase PINK1 does not alter platelet function.

PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson's disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice.

Do platelets promote cardiac recovery after myocardial infarction: roles beyond occlusive ischemic damage.

Our understanding of platelet function has traditionally focused on their roles in physiological hemostasis and pathological thrombosis, with the latter being causative of vessel occlusion and subsequent ischemic damage to various tissues. In particular, numerous in vivo studies have implicated causative roles for platelets in the pathogenesis of ischemia-reperfusion (I/R) injury to the myocardium. However, platelets clearly have more complex pathophysiological roles, particularly as a result of the heterogeneous nature of biologically active cargo secreted from their granules or contained within released microparticles or exosomes.

Platelets Protect Cardiomyocytes from Ischaemic Damage.

Platelets are classically known for their roles in bleeding control and occlusive thrombus formation causing ischaemic tissue damage. Recently non-classical roles for platelets have been described, many of which may be mediated by the heterogeneous cargo that platelets secrete from granular stores upon activation. Using an model of ischaemic injury to ventricular cardiomyocytes, we observed that platelets, through secreted factors, delayed the rate of cardiomyocyte death during ischaemia.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.

Objective: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase 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 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 on activation.

Approach And Results: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages.

Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

Background: We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.

Aims: To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.

SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production.

The functional role of platelets in the regulation of angiogenesis.

Functionally, platelets are primarily recognized as key regulators of thrombosis and hemostasis. Upon vessel injury, the typically quiescent platelet interacts with subendothelial matrix to regulate platelet adhesion, activation and aggregation, with subsequent induction of the coagulation cascade forming a thrombus. Recently, however, newly described roles for platelets in the regulation of angiogenesis have emerged.

Shear stress is a positive regulator of thimet oligopeptidase (EC3.4.24.15) in vascular endothelial cells: consequences for MHC1 levels.

Aims: Thimet oligopeptidase (TOP, endopeptidase EC3.4.24.

Stabilization of brain microvascular endothelial barrier function by shear stress involves VE-cadherin signaling leading to modulation of pTyr-occludin levels.

Blood-brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE-cadherin can transmit shear signals to tight junction occludin with consequences for pTyr-occludin and barrier function.

Down-regulation of neprilysin (EC3.4.24.11) expression in vascular endothelial cells by laminar shear stress involves NADPH oxidase-dependent ROS production.

Neprilysin (NEP, neutral endopeptidase, EC3.4.24.