Effects of tris on responses of human and rabbit platelets to aggregating agents

Despite reports that Tris [tris (hydroxymethyl)aminomethane] affects platelets, it is often used to buffer suspending media. Human or rabbit platelets were washed and resuspended in Tyrode solution containing apyrase and 0.35% albumin. Addition of 15 mM Tris partially inhibited primary aggregation induced by 10 microM ADP and inhibited aggregation and release of 14C-serotonin from prelabelled platelets stimulated with low concentrations of thrombin (0.05-0.2 U/mL), or collagen. Platelets resuspended in 15 mM Tris, 0.15 M NaCl, 0.35% albumin, pH 7.5, did not aggregate in response to 10 microM ADP whereas platelets in Tyrode-albumin aggregated extensively. Ca2+ (5 mM) did not overcome the inhibition of thrombin-induced aggregation. Tris (15 or 1.5 mM) potentiated aggregation and release induced by sodium arachidonate (20-50 microM) or the ionophore A23187 (0.6-1 microM). Pretreatment of platelets with aspirin did not prevent potentiation by A23187, indicating that it is not mediated through activation of the arachidonate pathway. The inhibitory and potentiating effects of Tris are similar to those of amino sugars, lysine, arginine and primary amines such as methylamine and cadaverine, and may represent general effects of amines on platelets. Potentiation of the effects of some aggregating agents and inhibition of others re-emphasizes the concept that there are several different mechanisms through which aggregation can occur. Tris-based buffers are unsuitable for platelet suspending media and their use as solvents for aggregating agents or inhibitors should be limited.     

Effects of enhanced P2X1 receptor Ca2+ influx on functional responses in human platelets

G-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1-10 mM. The P2X1 agonist alpha,beta-MeATP stimulated a shape change which saturated at peak [Ca2+]i of > or = 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. alpha,beta-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with alpha,beta,-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.     

A major role for P2X1 receptors in the early collagen-evoked intracellular Ca2+ responses of human platelets

In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to collagen, however the relative importance of early CCa2+ mobilisation events is unknown. We now report that selective desensitisation of P2X1 receptor activity leads to a major reduction in the initial intracellular Ca2+ responses to a wide range of collagen concentrations (0.25-2 microg ml(-1)). Peak [Ca2+](i) increases were reduced to 8.5 and 55% of control, and the maximum rate of rise was reduced to 12 and 33% of control, at low and high collagen concentrations, respectively. This P2X1-dependent acceleration and enhancement of collagen-stimulated Ca2+ responses was not observed in the absence of extracellular Ca2+. These results demonstrate a major role for ATP-gated Ca2+ influx in the early collagen-evoked Ca2+ signals and can at least partly explain the important contribution of P2X1 receptors to arterial thrombosis.     

Effect of an impermeant arginine-modifying reagent on the responses of rabbit platelets to agonists

The importance of surface arginyl residues in platelet aggregation was investigated by studying the effects of an impermeant arginine-modifying reagent, p-sulfonylphenylglyoxal (PSPG), on platelet responses to various agonists. Pretreatment of resuspended rabbit platelets with 2-15 mM PSPG resulted in complete inhibition of aggregation responses to ADP and 5-HT, and a concentration-dependent inhibition of the preceding shape change. Aggregation responses to thrombin also were inhibited in a concentration-dependent manner. The protective effects of antagonists of these three agonists (beta, gamma-methylene ATP for ADP, hirudin for thrombin and phentolamine for 5-HT) during pretreatment of platelets with PSPG indicated that intact arginine residues form part of the receptor sites for ADP and for thrombin. Arginine residues are not part of the 5-HT receptor site itself, but seem to be important for the maintenance of the functional integrity of this site.     

Purification and characterization of Ca2+-activated neutral protease inhibitor from human platelets

An endogenous inhibitor of Ca²⁺-activated neutral protease (CANP) was purified to homogeneity from the soluble fraction of human platelets by the combination of heat treatment, ammonium sulfate fractionation, ion exchange chromatography and gel filtration. The purified inhibitor was found to be a tetramer composed of identical subunits and each subunit has a molecular weight of 63 K. The purified protein exerted specific inhibition against the low Ca²⁺-requiring form of CANP (μ-CANP) purified from human platelets in the presence of micromolar concentration of Ca²⁺. The kinetic study revealed that the inhibition is non-competitive with Ki value of 3.2 × 10^?8 M.     

Alloxan-induced hyperglycemia in rabbits and the response of platelets to aggregating agents in vitro and to exposed subendothelium in vivo

Many patients with diabetes mellitus show increased platelet aggregation and prostaglandin synthesis in response to physiological agents such as ADP and collagen when their platelets are tested in platelet-rich plasma or washed platelet suspensions. However, the relationship between increased platelet aggregation in vitro and increased thrombosis in vivo is difficult to establish with certainty. We have developed an in vivo model system in rabbits which tests the response of platelets in circulating native blood to an arterial vessel wall with limited damage such as might occur in arteries of patients with diabetes mellitus. We have used this model system to investigate whether 5 to 9 weeks of alloxan-induced hyperglycemia increases platelet adhesion and aggregation on a damaged vessel wall in vivo as well as platelet aggregation in vitro. Our results show that rabbit platelet function is not affected by extreme hyperglycemia and suggest that alloxan-induced diabetes in the rabbit may not be a good model for human diabetes mellitus.     

Effects of calmodulin and protein kinase C modulators on transient Ca2+ increase and capacitative Ca2+ entry in human platelets: relevant to pathophysiology of bipolar disorder

Disturbed intracellular calcium (Ca(2+)) homeostasis has been implicated in bipolar disorder, which mechanisms may be involved in the dysregulation of protein kinase C (PKC) and calmodulin systems. In this study, we investigated a transient intracellular Ca(2+) increase induced by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase pump (SERCA), and a capacitative Ca(2+) entry followed by addition of extracellular Ca(2+), in the presence or absence of PKC/calmodulin modulators in the platelets of healthy subjects in order to elucidate the role of SERCA in Ca(2+) homeostasis and to assess how both PKC and calmodulin systems regulate the two Ca(2+) responses. Moreover, we also examined the thapsigargin-elicited transient Ca(2+) increase and capacitative Ca(2+) entry in patients with mood disorders. PKC and calmodulin systems have opposite regulatory effects on the transient Ca(2+) increase and capacitative Ca(2+) entry in the platelets of normal subjects. The inhibitory effect of PKC activation on capacitative Ca(2+) entry is significantly increased and the stimulatory effect of PKC inhibition is significantly decreased in bipolar disorder compared to major depressive disorder and normal controls. These results suggest the possibility that increased PKC activity may activate the inhibitory effect of capacitative Ca(2+) entry in bipolar disorder. However, this is a preliminary study using a small sample, thus further studies are needed to examine the PKC and calmodulin modulators on the capacitative Ca(2+) entry in a larger sample.     

Intracellular alkalinization augments capacitative Ca2+ entry in platelets

In order to elucidate the significance of intracellular alkalinization in signal transduction of platelets, we investigated the effects on capacitative Ca(2+) entry (CCE) of intracellular alkalinization that was induced by NH(4)Cl. Addition of NH(4)Cl (10 mM) to the medium resulted in an elevation of intracellular pH by about 0.35, which was eliminated by simultaneous addition of propionate (20 mM), an inducer of intracellular acidification, to the medium. CCE was induced by an extracellular addition of Ca(2+) to platelets in which Ca(2+) stores had been depleted by stimulation with thapsigargin in nominally Ca(2+)-free medium. NH(4)Cl markedly augmented CCE and subsequent platelet aggregation, both of which were abolished in the presence of SKF-96365, an inhibitor of capacitative Ca(2+) entry in non-excitable cells such as platelets. The augmentation of CCE and subsequent aggregation by NH(4)Cl was not observed in the presence of propionate or SKF-96365. Extracellular alkalosis induced by Tris also markedly augmented CCE and subsequent aggregation. These augmenting effects of extracellular alkalosis by Tris were significantly but incompletely inhibited by simultaneous addition of propionate (20 mM), which completely eliminated elevation of intracellular pH elicited by Tris. Thus, the augmenting effect of extracellular alkalosis on CCE was in part mediated by intracellular alkalosis. These findings suggest that intracellular alkalinization is a potent signal that augments CCE in platelets.     

Effect of caffeine on the processes of intracellular Ca2+ concentration regulation in isolated snail neurons

The Ca2+ indicator Fura-2 was used to measure changes of cytoplasmic free Ca2+ concentration ([Ca2+]in) in isolated neurons of the snail Helix pomatia occurring through prolonged plasma membrane depolarization. An amplitude of Ca2+ response did not practically depend on value of depolarization in the presence of 5 mmol/l of caffeine unlike normal solution, which permitted suggesting that caffeine activated calcium-dependent Ca2+ release from the intracellular stores, which was a main factor of [Ca2+]in increase during depolarization. The processes of [Ca2+]in relaxation to the rest levels were approximated monoexponentially and occurred 2 times more rapidly in caffeine than in normal solution. An increase of the [Ca2+]in relaxation rate was provided probably, by a rise in the efficiency of the intracellular Ca2+ pumps able to decrease the rest level of [Ca2+]in even lower than that one under normal extracellular solution conditions.     

Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets

Plasma membrane Ca(2+) -ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y(1176)) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y(1122) and Y(1176) can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y(1176) is a likely substrate for focal adhesion kinase (FAK), while Y(1122) is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y(1176) is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.     

Synergistic responses and receptor occupancy in rabbit blood platelets

Several observations indicate that simultaneous receptor occupancy is necessary for generation of a synergistic response of rabbit platelets to two excitatory agonists. First, an aggregatory response to adrenaline induced by prior addition of 5HT reverses rapidly unless stabilised by inhibition of 5HT uptake. The extent of response correlates with the extracellular 5HT concentration. Second, addition of an ADPase following adrenaline enhances the rate of disaggregation if the response to this agonist has been induced by prior addition of ADP. Third, disaggregation is induced, or enhanced, if an antagonist selective to the inducing agonist is added following completion of the induced aggregatory response. These data suggest marked lability in the mechanisms responsible for generation of synergistic responses.     

Conditions affecting the responses of human platelets to epinephrine

Conditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin-apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 microM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albumin-apyrase solution containing approximately 20 microM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate-dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13,177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in low voltage-activated Ca2+ channel-evoked Ca2+ responses within neurons of the thalamic paraventricular nucleus

Low voltage-activated Ca2+ channels (LVA or T-type Ca2+ channels) are crucial to burst firing and oscillations in thalamocortical relay cells and are exhibited by neurons in the paraventricular nucleus of thalamus (PVT), a dorsal midline nucleus deemed important in the neural representation of motivational behaviours. We used a functional approach (whole-cell patch-clamp electrophysiology combined with confocal laser scanning microscopy) to analyse the spatial distribution of LVA Ca2+ channel-evoked Ca2+ transients in PVT neurons. We observed that the magnitude of LVA Ca2+ channel-evoked Ca2+ transients was significantly greater in proximal dendrites (located up to 20 microm from the soma) than in the soma. In addition, the magnitudes of these Ca2+ transients varied significantly not only among different dendrites of the same cell but also within individual dendrites. These findings suggest that LVA Ca2+ channels are expressed (i) predominantly on the proximal dendrites and (ii) heterogeneously within individual dendrites of PVT neurons. The spatial characteristics of dendritic LVA Ca2+ channels in PVT neurons suggest that these channels may regulate burst firing by modulating dendritic afferent inputs.     

Cleavage of cytoskeletal proteins by two forms of Ca2+ activated neutral proteases in human platelets

Two forms of calcium activated neutral proteases (CANPs) with different affinity to Ca²⁺ were partially purified from the soluble fraction of human platelets; one (μ-CANP¹) required 6 μM Ca²⁺ and the other (m-CANP¹) did 900 μM Ca²⁺ for the respective half maximal activity. Human platelets were found to contain μ-CANP predominantly in contrast to our previous study on bovine platelets, in which μ- and m-CANP were equally distributed. Among platelet protein preparations examined for possible endogenous substrates of platelet CANPs, actin binding protein (ABP) and 230 K protein were proteolysed completely by μ- or m-CANP in the presence of the respective optimal concentration of Ca²⁺. As far as μ-CANP is concerned, this is the first demonstration of proteolysis of endogenous substrates, which implies possible involvement of μ-CANP in stimulus-linked platelet reaction coupled with an increase in intracellular Ca²⁺ to micromolar concentration. Furthermore, microtubules associated proteins (MAPs) of bovine brain was proteolysed by μ- or m-CANP of human platelet in the similar manner, indicating functional ubiquity of CANPs.     

Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

The effects of trimethyltin on the Ca+2, Mg+2 and Ca+2 + Mg+2-dependent ATPases of human neuroblastoma GM 3320

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca+2-dependent, Mg+2-independent, Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 microM), trimethyltin preferentially stimulated the Ca+2-dependent, Mg+2-independent ATPase activity while inhibiting the Ca+2 + Mg+2-ATPase activity approximately 70%. At 75 microM trimethyltin, the Ca+2 + Mg+2-dependent ATPase activity is inhibited greater than 95% while the Ca+2-dependent, Mg+2-independent activity is essentially unchanged from control activity and the Mg+2-dependent, Ca+2-independent activity is inhibited approximately 50%. At concentrations greater than 75 microM, trimethyltin significantly inhibits the Ca+2-dependent, Mg+2-independent ATPase activity. Thus, at trimethyltin concentrations of 50-75 microM, preferential inhibition of the Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities of neuroblastoma GM 3320 is achieved.     

Effect of mood stabilizing agents on agonist-induced calcium mobilization in human platelets.

The effect of mood stabilizing agents such as lithium, carbamazepine, valproic acid and clonazepam on serotonin(5-HT)- or thrombin-induced intracellular calcium (Ca) mobilization was studied in the platelets of healthy subjects using the fluorescent Ca indicator fura-2. After incubating platelet-rich plasma with these drugs for one or four hours, there was no significant difference in either basal Ca2+ concentration or 5-HT-stimulated Ca response between each agent treatment and control. 5-HT- or thrombin-induced Ca mobilization was not altered by four weeks of lithium carbonate administration in healthy volunteers. These results indicate that these mood stabilizers fail to affect the agonist-stimulated intracellular Ca mobilizing pathway either in vitro or ex vivo in the platelets of healthy subjects.     

Effect of membrane protein concentration on binding of 3H-imipramine in human platelets

Binding of 3H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing 3H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The 3H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between Kd values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies.     

Direct evidence for a role for Ca2+ in amine storage granule secretion by human platelets

Exposure of washed human platelets to a high voltage electric field renders these cells permeable to small molecules such as Ca EGTA. Such accessed platelets secrete 5-hydroxytryptamine (5HT) when challenged with 10-⁵M Ca²⁺ but do not release lactate dehydrogenase under these conditions. The secretion of 5HT induced by Ca²⁺ requires MgATP but is independent of synthesis of prostaglandin endoperoxides. Half maximal secretion of 5HT is observed in the presence of 1.9μM Ca²⁺. These data suggest a direct involvement of Ca²⁺ in platelet amine granule secretion.