Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Weak inhibition of protein kinase C coupled with various non-specific effects make sphingosine an unsuitable tool in platelet signal transduction studies

The effects of sphingosine, the newly described inhibitor of the enzyme protein kinase C, on human platelet activation, were studied in order to gain further information on the role of protein kinase in platelet responses. Concentrations of the drug (5-20 microM) which had little effect on protein kinase C activation as measured by the phosphorylation of the 45 kDa and 20 kDa protein substrates induced by phorbol 12-myristate 13-acetate (PMA) and thrombin, strongly inhibited platelet aggregation induced by these agonists, as well as aggregation induced by ADP and ionomycin, which caused no detectable protein kinase C activation or 5-hydroxy[14C]tryptamine[( 14C]5HT) secretion. At approx. 10-fold higher concentrations (150-200 microM), sphingosine had significant inhibitory effects on PMA and thrombin-induced 45 kDa and 20 kDa protein phosphorylation. However, at these high concentrations, the drug caused extensive membrane damage/leakiness as suggested by the substantial release of [14C]5HT and [3H]adenine from pre-loaded platelets (50-70% release of both markers), and the total quenching of quin2 fluorescence by Mn2+ in the presence of the drug. Due to the increased membrane leakiness in the presence of the drug, an apparent potentiation of agonist-induced intracellular Ca2+ elevations in quin2-loaded platelets, as well as an increase in quin2 fluorescence with the drug alone (more than 50 microM) were also observed. Despite this, however, thrombin-induced [3H]arachidonate release was severely reduced in the presence of sphingosine, underlining the inhibitory effects at the membrane level. It is concluded that the weak, if any, inhibitory effects on protein kinase C at concentrations not affecting membrane integrity, as well as the inhibitory effects of sphingosine on platelet aggregation, make it an unsuitable compound as a tool for studies on platelet stimulus-response coupling.     

Direct evidence for the modulation of human platelet cytosolic free Ca2+ by intracellular cyclic AMP produced with a photoactivatable derivative

We have previously reported that intraplatelet "cyclic AMP jumps" produced with newly synthesized photoactivatable cyclic AMP analogue, inhibited washed rat platelet aggregation and serotonin release as induced by thrombin. Using the same approach on human platelets, thrombin-induced platelet aggregation was dose-dependently inhibited only when a flash was delivered. The mechanism of action of intraplatelet cyclic AMP as resulting from photolysis could be by controlling the level of cytosolic Ca2+. In order to test this hypothesis, the same protocol was used on human platelets preloaded with the internal Ca2+ fluorescent indicator, Quin 2, we found that the extent and the rate of the rise of the cytosolic Ca2+ induced by thrombin were dramatically decreased, in the presence of the photoactivatable cyclic AMP, only following photoirradiation. In addition, the flashes were produced, in the presence of photoactivatable cyclic AMP, after the thrombin-induced rise of internal Ca2+ had reached its peak. In these conditions, photoirradiation caused a rapid fall in fluorescence. These experiments provide the first direct evidence that intracellular cyclic AMP is involved in the control of platelet cytosolic Ca2+ by inhibition of its mobilization and by stimulation of its sequestration.     

Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Influence of sodium conductances on platelet activation

The effects of extracellular Na+ and tetrodotoxin on resting membrane potential, cytosolic free Ca2+ levels and aggregation of human platelets have been studied. Neither the decrease in extracellular Na+-concentration (from 140 mmol/l to 0 mmol/l) nor the addition of tetrodotoxin (10(-7) to 10(-5) mol/l) modified the platelet membrane potential. Zero extracellular Na+ concentration or the presence of tetrodotoxin in the medium inhibited platelet aggregation; however, K+-depolarized platelets showed an unchanged aggregation induced by ADP or thrombin in media with zero or low extracellular Na+ concentrations or in the presence of tetrodotoxin. Moreover, zero extracellular Na+ concentration or tetrodotoxin inhibited calcium mobilization in platelets during activation induced by thrombin. Hence, voltage-dependent activation linked to Na+ influx appears to be necessary for ADP- and thrombin-induced platelet aggregation under control conditions. Mechanisms for the role of Na+ conductances in platelet function are discussed.     

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.     

Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex]

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.     

Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin.

We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.     

Evidence for a role of NA+/H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.