Adenosine Triphosphate Secretion Research Articles

Purinergic signalling

The Chief Editor of Acta Physiologica earned his degree at the ‘I. Physiologisches Institut’ in Heidelberg. He is right now writing these lines in Hessische Str. 3–4 in Berlin. Who cares? And what does this have to do with purines in physiology, a topic so hot that our journal dedicated a whole issue to it (Fredholm & Verkhratsky 2010)? Little did I know at the time in Heidelberg that the enormous desk I was sitting at had once been the desk of Albrecht Karl Martin Leonhard Kossel (Had I known, our laboratory dogs would probably not have been allowed to relieve their bowels there as often). Kossel discovered the nitrogenous compounds purine and pyrimidine in the resolvent of the nucleic acid. He separated two different purines: adenine and guanine and three different pyrimidines: thymine (separated firstly), cytosine and uracil (Anon 2007, http://www.nobelkepu.org.cn/english/physiology/134556.shtml). For this, Kossel was awarded the Nobel Prize in 1910. In the late 19th century, Kossel joined the famous physiologist Emil Du Bois-Reymond in Berlin. And as you may already anticipate, their laboratory was situated in the building I am now sitting. Looking out of the window to the left, the previous Chemistry Department appears. There, in the Chemistry Department, once sat Hermann Emil Fischer, who coined the name ‘purine’ (purum uricum) in 1884 and was awarded the Nobel Prize in 1902. Finally, in 1929, Hans Karl Heinrich Adolf Lohmann discovered ATP. Naturally, his office in Berlin was just down the hall. Realizing all this can cause acute academic insignificance syndrome! Also in 1929, Alan Drury and Albert Szent-Györgyi proposed that purines are signalling molecules. Geoffrey Burnstock, the man who built the concept of adenosine triphosphate (ATP) as a transmitter molecule, and contributed to the special issue in Acta Physiologica (Burnstock et al. 2010), was born, naturally, in 1929. Purinergic signalling is a true strength of Acta Physiologica (Oxford). As pointed out by Fredholm & Verkhratsky (2010), only water is involved in a higher number of chemical reactions than ATP. Given its high concentration in the cytosol, it does not surprise that ATP acts also as a signalling molecule. When cells take harm, surrounding ATP concentration increases. ATP then acts as an extracellular signal, such as in the gut (Kaunitz & Akiba 2011) or in the fish gill when hypotonic shock occurs. In this situation, epithelial release of ATP and NaCl secretion is stimulated via purinergic receptors (Marshall 2011). Interestingly, specific ATP sensitive receptors, such as the dorsal root ganglion neurones, inhibit cAMP by a G protein-coupled pathway, suggesting a neuronal expression of a plasmalemmal cAMP receptor (Mamenko et al. 2010). ATP seems so fundamental that even neural stem cell migration involves this pathway (Grimm et al. 2010). Adenosine is a second basic purinergic signalling molecule acting almost universally in the organism (Aliagas et al. 2010, Nilsson et al. 2010, Ribeiro & Sebastiúo 2010, Wang et al. 2010, Yang et al. 2010). It results from ATP degradation. Any increase in energy consumption and, hence, ATP hydrolysis will increase cytosolic adenosine concentration. Adenosine signals ATP breakdown, intra- or extracellularly, thereby providing crucial information for complex organisms and may even help regulate apoptosis (Mlejnek & Dolezel 2010). In addition to adenosine, uridine adenosine tetraphosphate (Jankowski et al. 2005, Tolle et al. 2010, Schuchardt et al. 2011), diadenosine tetraphosphate (Schluter et al. 1994, Hoyle & Pintor 2010) and UDP have important roles as signalling molecules (Harden et al. 2010). All these purines are important for cardiovascular physiology (Hansen et al. 2010, Andersen et al. 2011) and pathophysiology (Waldenstrom et al. 2010). Take for instance the kidney, which is a crucial organ for blood pressure control (Guyton 1991). The kidney controls blood pressure by regulating volume and electrolytes, among other mechanisms. ATP supplies the energy to the plasma membrane pumps. Thus, it does not surprise that electrolyte reabsorption is regulated by purinergic receptors (Novak 2011), which help control renal perfusion (Dahl et al. 2011) and sodium homeostasis (Jankowski et al. 2011). In particular, it seems that the renal medulla and its blood supply by the vasa recta determine the long-term blood pressure (Cowley et al. 1995, Makino et al. 2002, Mattson 2003). It seems that nucleotides released from adjacent tubules play an important role in determining vasa recta blood flow (Crawford et al. 2011). To close the circle, Burnstock’s interests in purinergic signalling started as he took up his first postdoctoral post with Feldberg (Burnstock 2009). Of course Feldberg’s laboratory was here in the Physiological Institute in Berlin (Hessische Str. 3–4). Sadly, he was dismissed in 1933 during the Nazi purge of Jewish scientists and only thanks to the aid of Hill, he could relocate to Britain’s National Institute for Medical Research where he welcomed the young Burnstock.

Role of BK channels in hypertension and potassium secretion

This review summarizes recent studies of hypertension associated with a defect in renal K excretion due to genetic deletions of various components of the large, Ca-activated K channel (BK), and describes new evidence and theories regarding K secretory roles of BK in intercalated cells. Isolated perfused tubule methods have revealed the importance of BK in flow-induced K secretion. Subsequently, mice with genetically deleted BK subunits revealed the complexities of BK-mediated K secretion. Deletion of BKα results in extreme aldosteronism, hypertension, and an absence of flow-induced K secretion. Deletion of the BKβ1 ancillary subunit results in decreased handling of a K load, increased plasma K, mild aldosteronism and hypertension that is exacerbated by a high K diet. Deletion of BKβ4 (β4KO) leads to insufficient K handling, high plasma K, fluid retention, but with milder hypertension. Fluid retention in β4KO may be the result of insufficient flow-induced secretion of adenosine triphosphate (ATP), which normally inhibits epithelial Na channels (ENaCs). Classical physiological analysis of electrolyte handling in knockout mice has enlightened our understanding of the mechanism of handling K loads by renal K channels. Studies have focused on the different roles of BK-α/β1 and BK-α/β4 in the kidney. BKβ1 hypertension may be a 'three-hit' hypertension, involving a K secretory defect, elevated production of aldosterone, and increased vascular tone. The disorders observed in BK knockout mice have shed new insights on the importance of proper renal K handling for maintaining volume balance and blood pressure.

The Biliary HCO3 − Umbrella: A Unifying Hypothesis on Pathogenetic and Therapeutic Aspects of Fibrosing Cholangiopathies

This review focuses on the hypothesis that biliary HCO(3)(-) secretion in humans serves to maintain an alkaline pH near the apical surface of hepatocytes and cholangiocytes to prevent the uncontrolled membrane permeation of protonated glycine-conjugated bile acids. Functional impairment of this biliary HCO(3)(-) umbrella or its regulation may lead to enhanced vulnerability of cholangiocytes and periportal hepatocytes toward the attack of apolar hydrophobic bile acids. An intact interplay of hepatocellular and cholangiocellular adenosine triphosphate (ATP) secretion, ATP/P2Y- and bile salt/TGR5-mediated Cl(-)/ HCO(3)(-) exchange and HCO(3)(-) secretion, and alkaline phosphatase-mediated ATP breakdown may guarantee a stable biliary HCO(3)(-) umbrella under physiological conditions. Genetic and acquired functional defects leading to destabilization of the biliary HCO(3)(-) umbrella may contribute to development and progression of various forms of fibrosing/sclerosing cholangitis.

Platelet aggregation responses vary over a period of time in healthy controls

Platelet aggregation study is performed to investigate platelet function abnormality. A normal healthy control sample is usually run with the patient sample as a quality control measure. At our institution, we observed variations in platelet aggregation responses in our normal repeat controls. Therefore, we analysed aggregation parameters in these controls. Whole blood aggregation studies were performed with adenosine diphosphate (ADP), arachidonic acid (AA), collagen and ristocetin. Adenosine triphosphate (ATP) secretion was also measured simultaneously by leuciferin-leuciferase reaction. During a 5-year period, a total of 86 studies were performed on seven controls. Aggregations were within the acceptable range in 67% of the time. Collagen was the most affected agonist in our study. On five occasions, four controls had subnormal aggregations with two agonists. All abnormal responses were hypoaggregation except for two who had hyperaggregation with collagen and AA. Only one out of seven controls was always normal. In the presence of a subnormal control result, a new control was run before releasing the patient's platelet aggregation results. These findings suggest that many physiological factors, other than medications, may affect platelet function even in normal individuals. Therefore, a repeat study at a later date to demonstrate a reproducible abnormality would be prudent before labeling a patient's platelets abnormal.

The Similarity Between Aspirin Treatment and the Lack of Response to Epinephrine and the Frequency of Aspirin Resistance in Healthy Males

The lack of response of platelets against epinephrine has been discovered with a frequency of 14% to 40% in previous studies. There are studies that have demonstrated the effect of aspirin on platelets may resemble the lack of response to epinephrine. In this study, the extent of the effects of aspirin treatment on aggregation and secretion in healthy males with a lack of response to epinephrine and the frequency of aspirin resistance were investigated. Blood samples were collected at the beginning and at the end of a 10-day aspirin usage in 52 healthy males. Epinephrine, adenosine diphosphate (ADP), collagen, arachidonic acid (AA) and thrombin aggregations, and adenosine triphosphate (ATP) secretion were studied. Participants were assigned to nonresponder (<20%), semiresponder (20%-60%), and responder (>60%) groups, depending on their maximum aggregation responses to epinephrine. Participants who displayed an aggregation to AA at the end of the aspirin treatment were accepted to be aspirin resistant. Of the 52 participants, 4 were found to be nonresponders and 3 of 52 of the participants were found to be semiresponders. Although the lack of response to epinephrine and aspirin treatment displayed similarities in aggregations using epinephrine, ADP, collagen, and thrombin, they differed in aggregations using AA and for ATP secretion. The ratio of aspirin resistance was determined to be 4:52. The observation of AA aggregation in the participants with a lack of response to epinephrine demonstrates that epinephrine nonresponse cannot substitute aspirin treatment. The fact that aspirin resistance is observed in healthy males supports the view that aspirin resistance exists even before the first usage.

Human Platelet Aggregation and Degranulation Is Induced In Vitro by L-Thyroxine, but Not by 3,5,3′-Triiodo-L-Thyronine or Diiodothyropropionic Acid (DITPA)

The endogenous thyroid hormones L-thyroxine (T(4)) and 3,5,3'-triiodo-L-thyronine (T(3)) induce angiogenesis via an endothelial cell iodothyronine receptor on integrin alphaVbeta3. This receptor also exists on platelets. Diiodothyropropionic acid (DITPA) and GC-1, a noniodinated thyroid hormone analog, also induce angiogenesis. Here we examined the effects of iodothyronines (L-T(4) vs L-T(3)) and analogs DITPA and GC-1 on human platelet function. Subthreshold aggregation of platelets obtained from healthy human donors was induced with collagen. Platelet activation (proaggregation) and adenosine triphosphate (ATP) secretion (degranulation) induced by L-T( 4), L-T(4)-agarose, L-T(3), DITPA, or GC-1 were determined simultaneously. Platelet aggregation and ATP secretion induced by a subthreshold level of collagen were enhanced 3-fold by either L-T(4) or L-T( 4)-agarose (0.01 micromol/L) as compared to control, whereas, L-T( 3), DITPA, or GC-1 had no effect under the same conditions. The platelet proaggregatory and degranulation effects of L-T(4) were blocked by the alphavbeta3 antagonist XT199 (0.1 micromol/ L) and by tetraiodothyroacetic acid (tetrac; 0.1 micromol/L). Tetrac inhibits binding of thyroid hormone analogs to the receptor on alphavbeta3 and lacks thyromimetic activity at this site; thus, the proaggregatory action of L-T(4) likely involves the cell surface receptor on integrin alphavbeta3. The thyroid hormone receptor (TR) on human platelets but not endothelial cells distinguishes among iodothyronines, reflecting quantitative differences in integrin sites on endothelial cells and platelets or qualitative differences in the phospholipids/protein microenvironment of endothelial and platelet membranes that can affect integrin function. Additional studies in different populations with larger sample sizes are warranted to determine the impact of the current findings on clinical interventions.

Aggregation and Microparticle Production Through Toll-like Receptor 4 Activation in Platelets From Recently Menopausal Women

Bacterial infection may increase risk for thrombosis and atherosclerosis. Human platelets express toll-like receptor 4 (TLR4), the receptor for gram-negative bacterial lipopolysaccharide (LPS). Experiments were designed to evaluate direct, acute effects of TLR4 activation on aggregation, secretion, and generation of prothrombogenic microparticles in vitro on platelets derived from healthy women at risk for development of cardiovascular disease because of their hormonal status. Platelet-rich plasma from recently menopausal women was incubated with ultrapure Escherichia coli LPS in the absence or presence of antibodies that neutralize the human TLR4. Incubating platelets with LPS (100 ng/mL) for 5 minutes decreased aggregation and dense granule adenosine triphosphate secretion induced by thrombin receptor agonist peptide (TRAP) but not by adenosine diphosphate or collagen. The antibody to TLR4 blocked this effect of LPS. TLR4 activation increased phosphorylation of p38 mitogen-activated protein kinase and decreased production of prothrombotic phosphatidylserine and P-selectin-positive microparticles in response to TRAP. Therefore, acute, direct activation of TLR4 reduces platelet reactivity to TRAP stimulation in vitro. Increased thrombotic and cardiovascular risk with bacterial infection most likely reflects the sum of TLR4 activation on other blood and vascular cells to release proinflammatory cytokines/chemokines, which indirectly affect platelet reactivity.

Effect of Iron Therapy on Platelet Function among Iron-Deficient Women with Unexplained Menorrhagia

This study was performed to evaluate the effect of iron therapy on platelet function among women with unexplained menorrhagia in order to better understand possible interactions between iron deficiency anemia and platelet behavior and menorrhagia. Platelet aggregation and adenosine triphosphate (ATP) release induced by 5.0 mM adenosine diphosphate (ADP), 0.5 mM arachidonic acid (AA), 1.0 mg/ml ristocetin and 2 μg/ml collagen were studied by whole-blood platelet lumi-aggregometer in 50 menorrhagic women before and after oral iron therapy and in 22 women of the control group. There was a significant increase in AA- induced platelet aggregation (p < 0.05) and a decrease in ristocetin-induced platelet aggregation (p < 0.01) after treatment. Pre- and posttreatment platelet aggregation responses to ADP and collagen were not significantly different (p > 0.05). Pre- and posttreatment platelet secretion responses to all agonists disclosed no significant difference (p > 0.05). There was no significant difference between the study group after treatment and the control group in respect to platelet aggregation and ATP secretion values induced by all agonists (p > 0.05). We conclude that iron deficiency anemia in women causes AA-induced platelet dysfunction, which may give rise to increased menstrual blood loss and can be reversed by iron repletion.

Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

Preservation of platelet aggregation and dense granule secretion during extended storage of blood samples in the presence of a synthetic dual inhibitor of factor Xa and thrombin

The clinical significance of platelet function tests may be limited by the use of citrate anticoagulant. We examined the influence of BAPA, a dual inhibitor of factor Xa and thrombin, on platelet responsiveness to agonists when measured between 2 and 48 h after venipuncture. Blood samples from 24 healthy individuals were anticoagulated with citrate or BAPA. Impedance platelet aggregation (IPA) and adenosine triphosphate (ATP) release induced by ADP, collagen and thrombin-receptor activating peptide (TRAP) were determined in whole blood after a storage period between 2 and 48 h after venipuncture. Citrate resulted in significantly reduced collagen or TRAP-induced IPA and ATP release when measured 32 h and 48 h after blood collection. ADP-induced IPA and ATP release in citrated blood dropped significantly between 8 and 24 h. The length of storage of BAPA-anticoagulated blood samples over 48 h had no significant influence on platelet response to collagen and TRAP. In BAPA-anticoagulated blood, ADP-induced IPA and ATP secretion in whole blood were maintained over a storage period of 32 h. No difference in ADP-induced ATP secretion in whole blood anticoagulated with citrate and BAPA was observed, but it was largely suppressed in BAPA-anticoagulated platelet rich plasma. IPA and ATP release remain stable for at least 32 h when whole blood is anticoagulated with a dual inhibitor of factor Xa and thrombin. This would allow shipment of samples for platelet function testing on the following day. Platelet secretion studies in whole blood may include platelet activation by ADP when BAPA is used as anticoagulant.

Estrogen, inflammation, and platelet phenotype

Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

Ursodeoxycholic Acid Stimulates Cholangiocyte Fluid Secretion in Mice via CFTR-Dependent ATP Secretion

Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study. Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl(-) channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl(-) and Ca(2+) efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot. UDCA stimulated fluid secretion and Cl(-) efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca(2+)]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR-KO cholangiocytes. UDCA-stimulated [Ca(2+)]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice. Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca(2+)]i increase and PKC activation stimulates Cl(-) efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.

Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile

Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca(2+) can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca(2+) release channel in cholangiocytes. Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca(2+) was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms. Both cAMP and Ca(2+) agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate.

Genetic and pharmacologic evidence that Rac1 GTPase is involved in regulation of platelet secretion and aggregation.

Rac1 GTPase, a member of the Ras-related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function. To rigorously define the role of Rac1 in platelet regulation. We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function. Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Rac1 species and phosphorylation of the Rac effector, p21-activated kinase; (ii) expression of P-selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5'-diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A(2). NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac1 for normal platelet function, the bleeding times in Rac1(-/-) mice or mice given NSC23766 were significantly prolonged. Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1 knockout or Rac1 inhibitor-treated mice.

The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters

Secretory granules of chromaffin cells and neurons co-store and release, by exocytosis, the acidic soluble protein chromogranin A (human, CHGA; rodent, Chga) along with catecholamines, neuropeptides and adenosine triphosphate (ATP). CHGA serves as a pro-protein and upon proteolytic cleavage it generates active peptides, including catestatin (human CHGA352-372), first discovered in adrenal medullary chromaffin granules. Studies in our laboratory demonstrated that catestatin acts at the nicotinic acetylcholine receptor to inhibit catecholamine secretion. However, the specificity of catestatin to exert nicotinic-cholinergic antagonism among its co-transmitters is not clearly known, nor is the potential effect of catestatin on multiple vesicle co-transmitters understood. Here we probed the specificity of catestatin's actions among its co-transmitters: catecholamines, ATP, and neuropeptide Y (NPY). We studied the effects of each transmitter on exocytotic secretion of its co-transmitters from PC12 chromaffin cells, stimulating secretion by triggering physiological pathways at multiple sites. We observed that, among chromaffin granule co-transmitters, only catestatin and NPY inhibited catecholamine release induced by nicotinic-cholinergic stimulation; catestatin was more than tenfold more potent than NPY in this setting. We also stimulated norepinephrine secretion by other chromaffin cell agonists: catestatin blocked norepinephrine release induced by nicotine, but not by other agents (such as membrane depolarization) acting at later stages in the secretory pathway, nor by agents acting on other receptor classes. By contrast, NPY acted less specifically, blocking norepinephrine release triggered by either nicotine or membrane depolarization. Catestatin inhibited nicotinic-cholinergic co-release of all classes of chromaffin granule co-transmitters: catecholamines, chromogranins, neuropeptides, and ATP. Naturally occurring variants of human catestatin (Gly364Ser and Pro370Leu) exhibited parallel changes in potency to inhibit secretion of catecholamines and ATP. We conclude that, among the chromaffin granule co-transmitters, catestatin acts as the most specific and potent inhibitor of physiological pathway (nicotinic-cholinergic) stimulated secretion. Furthermore, catestatin generally inhibits nicotinically triggered exocytotic release of multiple co-transmitters from chromaffin granules. The results have physiological and pharmacological implications for co-transmission in the sympathochromaffin system.

A Prospective, Randomized Platelet-Function Study of Heparinized Oxygenators and Cardiotomy Suction

The purpose of this study was to determine if substitution of a heparin-coated oxygenator and salvaged autologous blood for cardiotomy suction would improve platelet function. A prospective, randomized trial. A large academic medical center. Sixty adult patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass (CPB). Patients were randomized into 1 of 4 groups in a 2 x 2 factorial design by oxygenator (heparinized v nonheparinized) and blood salvage during CPB (cardiotomy suction v salvaged autologous blood). Primary outcome measures were platelet function, glass-bead retention, platelet dense-body adenosine triphosphate secretion, platelet-rich plasma (PRP) aggregometry, Plateletworks platelet-function analyzer (Helena Laboratories Corp, Allen Park, MI), and platelet count. Secondary outcome measures were chest-tube drainage and allogeneic blood transfusion requirements. All platelet-function tests except thrombin-receptor activator peptide-induced PRP aggregometry showed a reduction in platelet function during and immediately after CPB (all p < 0.05). The only statistically significant difference in platelet-function tests between the groups was the glass-bead assay at 5 minutes before discontinuation of CPB (p < 0.05). This difference resolved 10 minutes after protamine administration. There were no differences between the groups in the amount of blood transfused, chest-tube drainage, and routine laboratory test results. The authors concluded that the effects of these changes to the CPB circuit were small and inconsequential in this cohort of patients.

Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an αIIbβ3- and aggregation-independent manner

AbstractBinding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes αIIbβ3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWF–mediated agglutination activates αIIbβ3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)– and Ca2+-dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain–containing leukocyte protein 76 (SLP-76), phospholipase Cγ2 (PLCγ2), linker for activation of T cells (LAT), or Fc receptor γ-chain (FcRγ-chain) were used for these studies. LAT and FcRγ-chain were found not to be required for agglutination-driven TxA2 production or activation of αIIbβ3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCγ2, and protein kinase C (PKC).

The TUBB1 Q43P functional polymorphism reduces the risk of cardiovascular disease in men by modulating platelet function and structure

AbstractThe discoid form of platelets is maintained by a marginal band of tightly coiled microtubules. β1-tubulin is the major isoform within platelet and megakaryocyte microtubules. In 24.2% of 33 unrelated inherited macrothrombocytopenia patients and in 10.6% of 272 subjects of a healthy population a P for Q substitution in β1-tubulin was found in the highly conserved residue 43. Heterozygous carriers of the Q43P variant showed a reduced platelet protein β1-tubulin expression. Transfection of green fluorescent protein (GFP)-tagged Q43P β1-tubulin in megakaryocytic MEG01 cells resulted in a disturbed tubulin organization. Electron microscopy revealed enlarged spherocytic platelets with a disturbed marginal band and organelle-free zones. In addition, platelets with the Q43P β1-tubulin variant had reduced adenosine triphosphate (ATP) secretion, thrombin receptor activating peptide (TRAP)-induced aggregation and collagen adhesion. The prevalence of the Q43P β1-tubulin variant was also 2 times higher (odds ratio, [OR] = 2.1;95% confidence interval [CI], 1.22-3.59) among control subjects than among patients with cardiovascular disease (10.4% versus 5.2%, P < .001). By analyzing this protective factor in men and women separately, this association was only found in men. This study thus presents the functional consequences of the platelet Q43P β1-tubulin substitution that is frequent in the healthy population and may protect men against arterial thrombosis.

Platelet Characteristics Change With Aging: Role of Estrogen Receptor

Estrogen receptor beta (betaER) is the predominant estrogen receptor in platelets. Experiments were designed to define phenotypic changes in platelets with aging following deletion of betaER (betaERKO). Blood was collected from wild-type and betaERKO female mice at 4-7 (young) and 24-25 (aged) months of age. In young animals, total number of platelets, number of platelets containing RNA (reticulated platelets), aggregation, dense body adenosine triphosphate secretion, and alpha granular secretion were the same in both groups. With aging, total number of platelets decreased but reticulated platelets increased in betaERKO mice; aggregation and dense granule adenosine triphosphate secretion decreased whereas basal expression of fibrinogen receptors increased with age in wild-type and betaERKO mice. Basal expression of P-selectin and annexin V binding increased with aging only in betaERKO mice; thrombin did not increase expression in these mice. Therefore, deletion of betaER is associated with specific platelet functions, which are expressed only with age-associated reproductive senescence.

Expression and function of syndecan-4 in human platelets

Platelet recruitment crucially depends on amplification systems provided by autocrine and paracrine factors such as adenosine diphosphate. In inflammatory states, consumption of coagulation proteins, such as antithrombin aggravates the procoagulant state. In this study, we report that platelets express syndecan-4, an antithrombin-binding cell surface heparan sulphate proteoglycan, whose ligation with antithrombin inhibits activated platelet-dependent superoxide anion release from neutrophils by the limitation of adenosine diphosphate and adenosine triphosphate secretion in activated platelets. Adenosine triphosphate-induced platelet aggregation is reduced after treatment of platelets with antithrombin, which is reversed by blockade of syndecan-4. We further observed that antithrombin limits CD40 ligand expression in adenosine diphosphate-activated platelets and inhibits the shedding of syndecan-4 from activated platelets. Syndecan-4 appears to be directly involved in regulating platelet aggregation as anti-syndecan-4 antibody augments platelet aggregation. We suggest that antithrombin might exert beneficial effects in disseminated intravascular coagulation by reducing platelet activation, observed as inhibited CD40 ligand expression, syndecan-4 shedding, and adenosine diphosphate- and adenosine triphosphate-release from activated platelets with subsequent inhibition of neutrophil respiratory burst. From these data it is concluded that syndecan-4 may play important roles in the regulation of inflammatory effects of platelets.