Alan G.S. Harper

Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton

Platelet activation by potent, Ca2+‐mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from αIIbβ3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin‐mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

A role for TRPV1 in agonist‐evoked activation of human platelets

Summary.  Background: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. Objectives: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. Methods: Platelets loaded with fura‐2 or sodium‐binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5‐HT secretion was determined by fluorescence assay after conjugation with o‐phthaldialdehyde. ATP secretion was determined using luciferin‐luciferase. Results: TRPV1 was identified by Western blotting using a specific anti‐hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca2+ influx and Ca2+ release from intracellular stores, responses that were blocked in a dose‐dependent manner by the TRPV1 antagonists, 5′‐Iodo‐resiniferatoxin (5′‐Iodo‐RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na+]. Capsaicin‐evoked Ca2+ release was abolished in the absence of extracellular Na+ or by the 5‐HT2A receptor antagonist, ketanserin. Capsaicin evoked 5‐HT release from platelets, a response abolished in the absence of extracellular Na+ or by 5′‐Iodo‐RTX. Thus capsaicin‐evoked Ca2+ release appeared to be mediated by Na+‐dependent 5‐HT release. TRPV1‐dependent 5‐HT release also contributed to ADP‐ and thrombin‐evoked Ca2+ entry and release. 5′‐Iodo‐RTX reduced ADP‐ and thrombin‐evoked Ca2+ signals, effects not additive with those of ketanserin, and 5′‐Iodo‐RTX inhibited agonist‐evoked 5‐HT and ATP release. Conclusion: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis.

A role for SNAP‐25 but not VAMPs in store‐mediated Ca2+ entry in human platelets

Store‐mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non‐excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N‐ethylmaleimide‐sensitive‐factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome‐associated protein (SNAP‐25) and the vesicle‐associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP‐25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin‐ (TG) and agonist‐evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP‐25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP‐25 has a role in SMCE in human platelets.

A key role for dense granule secretion in potentiation of the Ca2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na(+)/Ca(2+) exchange in potentiation of the Ca(2+) entry elicited through the human platelet store-operated channel by controlling a Mn(2+)-impermeable Ca(2+) entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na(+)/Ca(2+) exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn(2+)-impermeable Ca(2+) entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca(2+) signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca(2+) signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N.

Summary.  Background: Most Ca2+ signaling research in platelets has relied solely on monitoring the cytosolic Ca2+ concentration ([Ca2+]cyt). Changes in [Ca2+]cyt constitute the net effect of Ca2+ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca2+ sequestration into the stores and Ca2+ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca2+ signaling difficult and subject to error. Objectives:  To validate the use of the low‐affinity Ca2+ indicator Fluo‐5N to monitor the concentration of Ca2+ in the intracellular stores ([Ca2+]st) of human platelets as a first step in developing assays for a systems‐level analysis of platelet Ca2+ signaling. Methods:  Fluo‐5N‐loaded and Fura‐2‐loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca2+]cyt and [Ca2+]st. Results:  Fluo‐5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca2+ stores and to physiologic agonists. Ca2+ reuptake inhibitors and blockers of Ca2+ release channels had the expected effects on Fura‐2 and Fluo‐5N fluorescence. Agonist‐evoked Ca2+ release was reversed by Ca2+ addition to the medium, and required intact Ca2+ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non‐SERCA Ca2+ reuptake mechanism. Evidence for a role for Ca2+‐induced Ca2+ release in agonist‐evoked responses was obtained. Conclusions:  Our data provide a validation of the use of Fluo‐5N as a method for monitoring changes in [Ca2+]st in human platelets.

Platelet Signalling: Calcium

A rise in cytosolic calcium concentration ([Ca2+]cyt) is central to platelet activation. Agonists stimulate a rise in [Ca2+]cyt through a combination of Ca2+ release from intracellular stores located in the dense tubular system (DTS) and acidic organelles as well as Ca2+ entry across the plasma membrane via several channel types. [Ca2+]cyt may be reduced by Ca2+ sequestration into the intracellular stores by sarco-endoplasmic reticulum Ca2+-ATPases (SERCAs) and via a H+-dependent mechanism, whilst Ca2+ may be removed across the plasma membrane by plasma membrane Ca2+-ATPases (PMCAs) and by Na+/Ca2+ exchangers (NCXs). Ca2+ signals are shaped by differential employment of these basic Ca2+ entry and removal processes and by Ca2+ buffers present in the platelet cytosol and other cellular compartments. In turn, Ca2+ signals can be transduced into a number of platelet responses by an array of effector proteins which may be activated in some cases by Ca2+ signals confined to specific cellular microdomains.

Islet-intrinsic effects of CFTR mutation

Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD.Summary• CF is an autosomal recessive disorder caused by mutations in the CFTR gene• The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline• Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell• Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population

Phorbol ester-evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors, not a novel non-capacitative Ca2+entry

Summary.  Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca2+entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca2+entry in human platelets for Na+/Ca2+ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca2+entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca2+signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca2+or Na+ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na+, significantly reduced PMA‐evoked Ca2+entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca2+entry. Ro‐0437626 or chelation of extracellular Ca2+ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P2X1 receptor activation but is accelerated by P2X1‐mediated Ca2+entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca2+entry results from an NCX3‐dependent dense granule secretion and subsequent P2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.

Pericellular Ca2+ recycling potentiates thrombin‐evoked Ca2+ signals in human platelets

We have previously demonstrated that Na+/Ca2+ exchangers (NCXs) potentiate Ca2+ signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca2+ removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near‐membrane indicator FFP‐18, thrombin stimulation was observed to elicit an NCX‐dependent accumulation of Ca2+ in a pericellular region around the platelets. To test whether this pericellular Ca2+ accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca2+ chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca2+ rise reduced thrombin‐evoked Ca2+ signals and dense granule secretion. Blocking Ca2+‐permeable ion channels had a similar effect, suggesting that Ca2+ exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo‐4 indicated that thrombin‐evoked rises in extracellular [Ca2+] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP‐18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca2+] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca2+].

The TRPV1 ion channel is expressed in human but not mouse platelets.

We have previously reported the expression of the TRPV1 ion channel in human platelets and have demonstrated a role for this channel in mediating agonist-evoked Ca2þ signalling in these cells [1]. Our Western blot studies have been independently reproduced by Savini et al. using a different antibody [2]. Furthermore, TRPV1 has also been identified in a human platelet proteomic study [3, 4] . These earlier studies are, however, yet to address the role that this non-selective cation channel might play in regulating platelet activity in vivo. A common strategy to assess the functions of signalling molecules in platelets has been to knockout the gene for the protein of interest in mice and then compare the responses of platelets from the knockout and wildtype animals. As a TRPV1-knockout mouse has been generated [3], we conducted pilot experiments to assess whether this channel is expressed in the platelets of wild-type mice and thus whether the knockout mouse would provide a useful model for studying the function of TRPV1 in platelets in vivo. Blood was taken by venepuncture from healthy human volunteers or by venepuncture of the abdominal vein in wildtype C57/Bl6 or Swiss mice terminally anaesthetised with ketamine (75 mg/kg) and medetomidine (1 mg/kg). After blood collection, mice were euthanized by cervical dislocation. Washed platelet suspensions from both species were prepared and the platelets were then loaded with Fura-2 by incubation with 5mM Fura-2/AM for 30 minutes at 37 C. Platelets were then collected and resuspended in Hepes-buffered saline supplemented with glucose (10 mM), bovine serum albumin (1 mg/ml) and apyrase (0.1 U/ml) [1]. In Fura-2-loaded platelets from wild-type C57/Bl6 or Swiss mice, capsaicin (100 mM), in the presence of 1 mM external Ca2þ, evoked no rise in [Ca]cyt over that observed in vehicle-treated controls (Figure 1A; n1⁄4 6). This lack of response was not due to a failure of Fura-2 loading or an ability to detect changes in [Ca]cyt as samples from all mouse platelet suspensions demonstrated changes in [Ca]cyt upon treatment with thapsigargin in the absence of extracellular Ca2þ followed by the addition of Ca2þ to the medium to elicit store-operated calcium entry (data not shown). In parallel experiments, human platelets, at the same cell density, responded to capsaicin from the same stock solution with a rise in [Ca]cyt as previously reported (Figure 1B; n1⁄4 6) [1]. We also examined whole cell lysates of murine (Swiss) and human platelets, prepared from the samples used for fluorescence experiments, for the presence of TRPV1 by Western blotting. We detected a band at around 100 kDa, consistent with the molecular weight of hTRPV1, in the human whole platelet lysates as previously reported [1, 2], but could not detect a band at around 95 kDa, as would be expected for mTRPV1 [5], in the mouse platelet lysates (Figure 1C; n1⁄4 3). The antibody was, however, able to detect a protein of around 95 kDa in mouse brain lysate (Figure 1D; n1⁄4 3). We also observed a secondary band in the mouse brain lysate, which we believe is likely to represent a truncated variant of mTRPV1 expressed in hypothalamic osmoreceptors [6]. Another N-terminal variant of TRPV1, TRPV1b, has also been reported to be present in mice [7]. The anti-TRPV1 antibody used in this study was directed to the C-terminal of TRPV1, thus our data suggest the absence of all known murine TRPV1 isoforms from mouse platelets. Recent transcriptome profiles of murine and human platelets have demonstrated low levels of TRPV1 mRNA in human platelets, whilst it appeared to be absent from murine platelets [8], supporting our observations. These results demonstrate that murine platelets lack TRPV1, and thus cannot provide a model system in which to study the physiological function of TRPV1 in platelets in vivo. However, the demonstration that these cells lack both the TRPV1 protein and responsiveness to capsaicin, even at high concentrations, does provide a cell that can be used as a negative control for screening for non-specific effects of TRPV1 agonists and antagonists in platelets. The negative results from the murine platelets also support our previous conclusion that capsaicin activates calcium signalling and serotonin secretion in human platelets through a specific effect on TRPV1 [1]. There is growing recognition of platelet involvement in a range of inflammatory conditions such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis [9]. We have previously suggested that the presence of TRPV1 in human platelets may be involved in a signalling pathway that could elicit platelet activation under these conditions [1]. The results presented here, however, suggest that the molecular identity of signalling pathways eliciting platelet activity under inflammatory conditions is likely to be different in mice. The absence of TRPV1 from murine platelets adds to a growing body of evidence of differences in both the proteomes History