The voltage-gated K channel Kv1.3 modulates platelet motility and αβ integrin-dependent adhesion to collagen.

Kv1.3 is a voltage-gated K-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood.

Why do platelets express K channels?

Potassium ions have widespread roles in cellular homeostasis and activation as a consequence of their large outward concentration gradient across the surface membrane and ability to rapidly move through K-selective ion channels. In platelets, the predominant K channels include the voltage-gated K channel Kv1.3, and the intermediate conductance Ca-activated K channel KCa3.

Ca waves coordinate purinergic receptor-evoked integrin activation and polarization.

Cells sense extracellular nucleotides through the P2Y class of purinergic G protein-coupled receptors (GPCRs), which stimulate integrin activation through signaling events, including intracellular Ca mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca waves and fibrinogen binding to the platelet integrin αβ (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y and P2Y generated a series of Ca transients that each induced a rapid, discrete increase in fibrinogen binding.

Development of a P2X1-eYFP receptor knock-in mouse to track receptors in real time.

A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes.

A major interspecies difference in the ionic selectivity of megakaryocyte Ca-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01.

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes.

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets.

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.

Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions.

The platelet receptors glycoprotein (Gp)VI, integrin αβ and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca ([Ca]) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca] measurements using confocal imaging.

Evidence for shear-mediated Ca entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

The role of mechanosensitive (MS) Ca-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches.

Calcium Signalling through Ligand-Gated Ion Channels such as P2X1 Receptors in the Platelet and other Non-Excitable Cells.

Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca(2+) whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes.

Regulation of Pannexin-1 channel activity.

Pannexin-1 (Panx1) forms anion-selective channels with a permeability up to 1 kDa and represents a pathway for the release of cytosolic ATP. Several structurally similar connexin (Cx) proteins have been identified in platelets and shown to play roles in haemostasis and thrombosis. More recently, functional Panx1 channels have been demonstrated on the surface of human platelets [Taylor et al.

Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+ signaling and ADP-evoked platelet aggregation.

Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiologic or pathophysiologic responses. For example, during hemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1 and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously been reported that P2X1 receptors amplify P2Y1-evoked Ca(2+) responses in platelets, but the underlying mechanism and influence on function is unknown.

Connexin40 regulates platelet function.

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.

A role for platelet TRPC channels in the Ca2+ response that induces procoagulant activity.

After vascular injury, platelets are rapidly activated by collagen and other agonists, causing them to adhere and aggregate to prevent blood loss. In addition, phosphatidylserine (PS) exposure on the platelet surface accelerates thrombin formation by the coagulation pathway. Thrombin is a potent platelet agonist and converts fibrinogen to fibrin, thereby stabilizing the platelet plug.

Heat shock protein 90 inhibitors reduce trafficking of ATP-gated P2X1 receptors and human platelet responsiveness.

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70-85% by the selective HSP90 inhibitor geldanamycin (2 μM, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

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 examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair.

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.

Background And Objective: Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Platelet Ca(2+) responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca(2+) stores.

Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation.

Purification of native bone marrow megakaryocytes for studies of gene expression.

Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41.

The P2X1 receptor and platelet function.

Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca(2+), leading to shape change, movement of secretory granules and low levels of α(IIb)β(3) integrin activation.

Extracellular Ca(2+) modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation.

ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca(2+) [(Ca(2+) )(o) ]. Lowering [Ca(2+) ](o) paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A(2) production. This study examined the role of ectonucleotidases in the [Ca(2+) ](o) -dependence of platelet activation.

P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation.

P2X1 receptors for ATP contribute to signalling in a variety of cell types and following stimulation undergo rapid desensitisation (within 1 s), and require approximately 5 min to recover. In HEK293 cells P2X1 receptors C-terminally tagged with enhanced green fluorescent protein (P2X1-eGFP) were predominantly expressed at the cell surface. Following > 90% photo-bleaching of P2X1-eGFP within a 6 microm(2) circle at the cell surface fluorescence recovery after photo-bleaching (FRAP) was fit with a time constant of approximately 60 s and recovered to approximately 75% of pre-bleach levels.

Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors in Xenopus oocytes.

Lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) play important roles in a variety of physiological and pathophysiological processes, including cell proliferation, angiogenesis, central nervous system development and carcinogenesis. Whilst many ion channels and transporters are recognized to be controlled by a change in cell membrane potential, little is known about the voltage dependence of other proteins involved in cell signalling. Here, we show that the InsP(3)-mediated Ca(2+) response stimulated by the endogenous LPA GPCR in Xenopus oocytes is potentiated by membrane depolarization.