Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y₁₂ inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.     

Dual P2Y 12 receptor signaling in thrombin-stimulated platelets--involvement of phosphoinositide 3-kinase beta but not gamma isoform in Ca2+ mobilization and procoagulant activity

During thrombus formation, thrombin, which is abundantly present at sites of vascular injury, activates platelets in part via autocrine-produced ADP. We investigated the signaling pathways by which thrombin and ADP in synergy induced platelet Ca(2+) elevation and procoagulant activity, and we monitored the consequences for the coagulation process. Even at high thrombin concentration, autocrine and added ADP enhanced and prolonged Ca(2+) depletion from internal stores via stimulation of the P2Y(12) receptors. This P2Y(12)-dependent effect was mediated via two distinct signaling pathways. The first is enhanced Ca(2+) mobilization by the inositol 1,4,5-trisphosphate receptors due to inhibition of protein kinase A. The second pathway concerns prolonged activation of phosphoinositide 3-kinase (PI3-K) and phospholipase C. Experiments with phosphoinositide 3-kinase isoform-selective inhibitors and p110gamma deficient platelets demonstrated that the phosphoinositide 3-kinase beta and not the phosphoinositide 3-kinase gamma isoform is responsible for the prolonged Ca(2+) response and for the subsequent increases in procoagulant activity and coagulation. Taken together, these results demonstrate a dual P2Y(12)-dependent signaling mechanism, which increases the platelet-activating effect of thrombin by prolongation of Ca(2+) elevation, thereby facilitating the coagulation process.     

Adenosine analogues as inhibitors of P2Y12 receptor mediated platelet aggregation

Modified adenosine derivatives may lead to the development of P2Y(12) antagonists that are potent, selective, and bind reversibly to the receptor. Analogues of 2',3'-trans-styryl acetal-N6-ureido-adenosine monophosphate were prepared by modification of the 5'-position. The resulting analogues were tested for P2Y(12) antagonism in a platelet aggregation assay.     

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.     

Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides

We studied the actions of receptor-activating peptide analogues (PAR4APs), modeled on the proteolytically-revealed tethered ligand sequence of murine proteinase-activated receptor-4 (PAR4), in a rat platelet aggregation assay. The PAR4APs GYPGKF-NH2 (GY-NH2) and AYPGKF-NH2 (AY-NH2) were able to cause aggregation with EC50 values of about 40 microM and 15 microM, respectively. The reverse human PAR4 sequence (VQGPYG-NH2, YG-NH2) and the PAR1AP SFLLR-NH2, did not cause aggregation. In contrast, trans-cinnamoyl-YPGKF-NH2 (tcY-NH2) did not cause aggregation but blocked aggregation caused by GY-NH2, AY-NH2, and thrombin without affecting ADP-mediated aggregation. We conclude that in contrast to the PAR1AP, the PAR4APs GY-NH2 and AY-NH2 activate rat platelets via a PAR4-related receptor and that peptide analogues modeled on the PAR4 tethered activating sequence can serve as useful agonist and antagonist probes for assessing the consequence of activating PAR4 either by PAR4APs or thrombin in rat tissue preparations.     

Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.     

Inhibition of ADP-induced intracellular Ca2+ responses and platelet aggregation by the P2Y12 receptor antagonists AR-C69931MX and clopidogrel is enhanced by prostaglandin E1

P2Y(12) antagonists such as clopidogrel and AR-C69931MX inhibit aggregation by antagonizing the effects of ADP at P2Y(12) receptors on platelets. Agents such as PGE(1) also inhibit aggregation by stimulating adenylate cyclase to produce cAMP, which interferes with Ca(2+) mobilization within the cell. Since one facet of P2Y(12) receptors is that they mediate inhibition of adenylate cyclase by ADP, it might be expected that P2Y(12) antagonists would interact with PGE(1). We have explored the effects of PGE(1) and AR-C69931MX singly and in combination on ADP-induced intracellular Ca(2+) ([Ca(2+)](i)) responses and aggregation. PGE(1) alone caused parallel dose-dependent inhibition of [Ca(2+)](i) and aggregation responses. AR-C66931MX alone caused only partial inhibition of [Ca(2+)](i) despite a marked inhibitory effect on aggregation. Combinations of PGE(1) with AR-C66931MX were found to act in synergy to reduce both [Ca(2+)](i) and aggregation. This effect was confirmed in patients with acute coronary syndromes by studying the inhibitory effects of PGE(1) on [Ca(2+)](i) and aggregation before and after clopidogrel. In summary, we have shown that P2Y(12) antagonists interact with natural agents such as PGE(1) to provide more effective inhibition of [Ca(2+)](i) and platelet aggregation. This would contribute to the effectiveness of P2Y(12) antagonists as antithrombotic agents in man.     

P2Y receptor subtypes evoke different Ca2+ signals in cultured aortic smooth muscle cells

Adenine and uridine nucleotides evoke Ca(2+) signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca(2+) signals are unresolved. Cytosolic Ca(2+) signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca(2+) indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca(2+) signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca(2+) from intracellular stores via inositol 1,4,5-trisphosphate (IP(3)) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca(2+) signals were independent of the Na(+)/Ca(2+) exchanger and were probably mediated by store-operated Ca(2+) entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca(2+) signals in cultured aortic smooth muscle cells using the same intracellular (IP(3) receptors) and Ca(2+) entry pathways (store-operated Ca(2+) entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca(2+) signal evoked by each P2Y receptor subtype.     

Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y₁₂ antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.     

Activation of sodium-proton exchange is not a prerequisite for Ca2+ mobilization and aggregation in human platelets

Recently it has been suggested [(1987) Nature 325, 456-458; (1987) FEBS Lett. 212, 123-126] that the activation of Na+/H+ exchange is a prerequisite for platelet aggregation and the development of the Ca2+ signal. As direct evidence for the role of the Na+/H+-exchange pathway the inhibition of the Ca2+ signal by EIPA, a specific inhibitor of Na+/H+ exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 microM) completely block Na+/H+ exchange while EIPA inhibits aggregation or Ca2+ mobilization only in concentrations 100-times greater than 1 microM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+ exchange, does not affect Na+/H+ exchange in platelets but blocks aggregation and Ca2+ mobilization. We conclude that while Na+/H+ exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+ signal and aggregation.     

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca²⁺ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca²⁺ levels ([Ca²⁺]_i). The order of potency followed ATP = UTP > ADP = UDP. ATP‐induced rise in [Ca²⁺]_i was suppressed by U73122 and suramin, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), suggesting the functional expression of G protein‐coupled P2Y₂ receptors. RT‐PCR and immunohistochemical studies also showed the expression of P2Y₂ receptors. [Ca²⁺]_i response to UTP changed with cell density. The UTP‐induced rise in [Ca²⁺]_i was greatest at high density. V_max (maximum Ca²⁺ response) and EC₅₀ (agonist concentration that evokes 50% of V_max) suggest that the amount and property of P2Y₂ receptors were changed by cell density. Note that UTP induced Ca²⁺ oscillation at only medium cell density. Pharmacological studies indicated that UTP‐induced Ca²⁺ oscillation required Ca²⁺ influx by store‐operated Ca²⁺ entry. Carbenoxolone, a gap junction blocker, enhanced Ca²⁺ oscillation. Immunohistochemical and quantitative real‐time PCR studies revealed that proliferating cell nuclear antigen (PCNA)‐positive cells declined but the mRNA expression level of the P2Y₂ receptor increased as cell density increased. Co‐application of fetal calf serum with UTP induced Ca²⁺ oscillation at high cell density. These results suggest that the different patterns observed for [Ca²⁺]_i mobilization with respect to cell density may be associated with cell cycle progression. J. Cell. Physiol. 219: 372–381, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.     

Protease-activated receptor-1 (PAR1) and PAR2 but not PAR4 mediate relaxations in lower esophageal sphincter

Protease-activated receptor-1 (PAR1), PAR2 and PAR4 activation can alter the gastrointestinal motility. To investigate effects mediated by PARs in the lower esophageal sphincter, we measured contraction or relaxation of transverse strips from the guinea-pig lower esophageal sphincter caused by PAR1 (TFLLR-NH2 and SFLLRN-NH2), PAR2 (SLIGKV-NH2 and SLIGRL-NH2) and PAR4 peptide agonists (GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2) as well as PAR protease activators (thrombin and trypsin). In resting lower esophageal sphincter strips, TFLLR-NH2 and SFLLRN-NH2 caused moderate concentration-dependent relaxation whereas thrombin did not cause any relaxation or contraction. Furthermore, in carbachol-contracted strips, TFLLR-NH2 and SFLLRN-NH2 caused marked whereas thrombin caused mild concentration-dependent relaxation. These indicate the existence of PAR1 mediating relaxation. Similarly, in resting lower esophageal sphincter strips, trypsin caused moderate concentration-dependent relaxation whereas SLIGRL-NH2 and SLIGKV-NH2 did not cause any relaxation or contraction. In addition, in carbachol-contracted strips, trypsin caused marked whereas SLIGRL-NH2 and SLIGKV-NH2 caused mild concentration-dependent relaxation. These indicate the existence of PAR2 mediating relaxation. The relaxant response of thrombin, TFLLR-NH2, trypsin and SLIGKV-NH2 was insensitive to atropine or tetrodotoxin, suggesting a direct effect. The relaxant response of trypsin was not affected by apamin, charybdotoxin, indomethacin and capsaicin but was attenuated by NG-nitro-L-arginine methyl ester, indicating involvement of NO. FSLLR-NH2, a PAR1 control peptide, and VKGILS-NH2, a PAR2 control peptide, as well as all three PAR4 peptide agonists, GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2, did not cause any relaxation or contraction. Taken together, these results demonstrate that PAR1 and PAR2 but not PAR4 mediate relaxations in the guinea-pig lower esophageal sphincter.     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

Optimization of 2-phenyl-pyrimidine-4-carboxamides towards potent,orally bioavailable and selective P2Y12 antagonists for inhibition of platelet aggregation

2-Phenyl-pyrimidine-4-carboxamide analogs were identified as P2Y₁₂ antagonists. Optimization of the carbon-linked or nitrogen-linked substituent at the 6-position of the pyrimidine ring provided compounds with excellent ex vivo potency in the platelet aggregation assay in human plasma. Compound 23u met the objectives for activity, selectivity and ADMET properties.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle

Background

The multisubunit (α_1S,α₂-δ, β_1a and γ₁) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca²⁺ channel. To more fully investigate the function of the γ₁ subunit in these two processes, we produced mice lacking this subunit by gene targeting.

Results

Mice lacking the DHPR γ₁ subunit (γ₁ null) survive to adulthood, are fertile and have no obvious gross phenotypic abnormalities. The γ₁ subunit is expressed at approximately half the normal level in heterozygous mice (γ₁ het). The density of the L-type Ca²⁺ current in γ₁ null and γ₁ het myotubes was higher than in controls. Inactivation of the Ca²⁺ current produced by a long depolarization was slower and incomplete in γ₁ null and γ₁ het myotubes, and was shifted to a more positive potential than in controls. However, the half-activation potential of intramembrane charge movements was not shifted, and the maximum density of the total charge was unchanged. Also, no shift was observed in the voltage-dependence of Ca²⁺ transients. γ₁ null and γ₁ het myotubes had the same peak Ca²⁺ amplitude vs. voltage relationship as control myotubes.

Conclusions

The L-type Ca²⁺ channel function, but not the SR Ca²⁺ release triggering function of the skeletal muscle dihydropyridine receptor, is modulated by the γ₁ subunit.     

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid