Local and systemic effects of Na+/K+ ATPase inhibition

The positive inotropic and electrophysiological effects of cardiac glycosides on cardiac muscle are mediated through inhibition of Na⁺/K⁺ ATPase by binding to a specific extracytoplasmic site of the a-subunit of this enzyme. The inhibition of Na⁺/K⁺ ATPase affects ionic flux and produces direct local effects on cardiac contractility, electrical excitability and conduction, but also profound systemic effects mainly as a result of haemodynamic changes. These effects are responsible for beneficial therapeutic as well as toxic effects.
Inhibition of Na⁺/K⁺ ATPase results in potentiation of K⁺ loss from cells and Na⁺ entry into cells, so consequently affects action potential generation and propagation. This also underlines the potentiation of certain effects of cardiac glycosides by hypokalemia and hypomagnesaemia, and the effects of changes in calcium homeostasis on the cardiac glycoside pharmacodynamics. Furthermore, inhibition of Na⁺/Ca⁺⁺ exchange enhances Ca⁺⁺ mobilization and promotes contractility. These effects (locally and systemically) differ greatly, depending on the haemodynamic status and myocardial oxygen supply.
Cardiac glycosides have less affinity for Na⁺/K⁺ ATPases at other sites (e.g. skeletal muscle), but some extracardiac effects (vascular effects, effects on colour vision, CNS and autonomic effects, renal effects) may be related to Na⁺/K⁺ ATPase inhibition.     

Erythrocyte Na+–H+ exchanger kinetics and Na+–Li+ countertransport activity in essential hypertensive patients

The authors measured Na⁺–H⁺ exchanger kinetics together with Na⁺–Li⁺ countertransport V _max in the erythrocytes of 21 subjects with essential hypertension and 16 normotensive control subjects. Na⁺–H⁺ exchanger V _max appeared to be increased in patients with essential hypertension, while the Na⁺–H⁺ exchanger affinity for intracellular proton sites ( K _50%) proved to be unchanged and the index of cooperativity among intracellular proton binding sites as measured by Hill's coefficient (Hill's n ) decreased as compared with normotensive control subjects. Na⁺–Li⁺ countertransport V _max appeared to be higher in patients with essential hypertension than in control subjects. The authors were unable to find any correlations between Na⁺–H⁺ exchanger kinetic parameters and metabolic variables such as parameters of insulin resistance and plasma lipids. On the basis of the data obtained, erythrocyte Na⁺–H⁺ exchanger activity was found to be abnormal in two kinetic variables in essential hypertensive patients and showed no simple linear correlations with the main variables of glucose metabolism, plasma lipids, renin or aldosterone.     

Relevance of erythrocyte Na+/Li+ countertransport measurement in essential hypertension, hyperlipidaemia and diabetic nephropathy: a critical review

In this review the usefulness of the measurement of erythrocyte Na⁺/Li⁺ countertransport (Na⁺/Li⁺ CT) activity is evaluated. In particular, the association between enhanced erythrocyte Na⁺/Li⁺ CT activity and essential hypertension, hyperlipidaemia and diabetic nephropathy is discussed. The conclusion of this review is that elevated erythrocyte Na⁺/Li⁺ CT activity is associated with essential hypertension and hyperlipidaemia. A relationship between Na⁺/Li⁺ CT activity and diabetic nephropathy is less evident. Despite a significant link of Na⁺/Li⁺ CT activity with hypertension and hyperlipidaemia, the diagnostic significance of Na⁺/Li⁺ CT activity is low. This is due to the large overlap between the results of control subjects and patients. The factors that contribute to this broad range are discussed in detail.     

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery

Summary— The involvement of large conductance Ca²⁺-activated K⁺ channels (BK) and ATP-sensitive K⁺ (K_ATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca²⁺]_i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca²⁺]_i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca²⁺]_i with force in the presence of serotonin at different extracellular Ca²⁺ concentration ([Ca²⁺]_o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca²⁺-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.     

Inhibitory effects of okadaic acid on rat uterine contractile responses to different spasmogens

Summary— In the present study, we examined the effects of okadaic acid, a selective inhibitor of type 1 and 2A protein phosphatases, on the mechanical responses evoked by oxytocin, K⁺- and Na+-modified solutions and ouabain in estrogen-primed rat myometrium. Oxytocin elicited a rapid, phasic contraction followed by rhythmic oscillations. The phasic response was partially resistant to the absence of external Ca²⁺. Okadaic acid (1 μM) and the L-type calcium channel blocker nifedipine (1 μM) abolished the oscillatory component and reduced the initial, phasic response to about 80% of the control response. High K⁺ (60 mM) solution, ouabain (1 mM), K⁺-free medium and low Na⁺ (25 mM) solution induced extracellular Ca²⁺-dependent biphasic responses composed by an early rapid (KCl, ouabain and K⁺-free solution) or slower developed (25 mM Na⁺ solution) phasic contraction followed by a sustained increase in tension. Okadaic acid and nifedipine, alone or in combination, abolished or decreased similarly the contractile response evoked by these stimulants. The okadaic acid- and nifedipine-insensitive responses to ouabain, K⁺-free and low Na⁺ solution were enhanced by increasing the extracellular concentration of Ca²⁺ in the medium and were inhibited in a dose-dependent manner by amiloride (0.05–0.5 mM). These data suggest that, in estrogen-primed rat uterus, dephosphorylating mechanisms by OA-sensitive protein phosphatases play an important role in regulating myometrial contractions elicited by Ca²⁺ entry through voltage-sensitive Ca²⁺ channels.     

Effect of serotonin and calcium in normal and sensitized guinea pig isolated trachea

Summary— Tracheal strips from normal and actively sensitized guinea pigs were studied to determine the responses to serotonin (5-hydroxytryptamine, 5-HT; 1 nM – 0.1 mM) and ouabain (0.1 μM – 0.1 mM), and the effects of increasing the extracellular calcium (Ca_o) concentration on tonic contractions elicited by 5-HT. Sensitized trachea exhibited an increased responsiveness and sensitivity to 5-HT and ouabain. Increases in Ca_o to achieve final concentrations of 5, 10 and 20 mM caused concentration-related relaxations of normal and sensitized tissues contracted to a similar plateau level with 5-HT. Inhibition of the Na⁺/K⁺-ATPase by ouabain (10 μM) reversed the effects of Ca_o from relaxation to contraction in normal and sensitized tissues contracted with 5HT. Sensitized preparations showed reduced relaxations in response to Ca_o (10–20 mM), and sensitized, ouabain-treated, trachea showed augmented contractions to Ca_o (10–20 mM) when compared to normal tissues. These results demonstrate a decreased membrane-stabilizing effect of Ca_o in sensitized trachea and the implication of the Na⁺/K⁺-ATPase in the regulation of membrane stability by Ca_o, suggesting a possible relevance to those mechanisms underlying airway hyperreactivity.     

Tonic block of the Na+ current in single atrial and ventricular guineapig myocytes, by a new antiarrhythmic drug, Ro 22-9194

Summary— Ro 22-9194 reduced the Na⁺ current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22-9194 had a higher affinity to the inactivated state Na⁺ channels (Kd₁ = 3.3 μM in atrial myocytes, Kd₁ = 10.3 μM in ventricular myocytes) than to those in the rested state (Kd_R = 91 μM in atrial myocytes, Kd_R = 180 μM in ventricular myocytes), which indicated that Ro 22-9194 had a higher affinity to the Na⁺ channels in atrial myocytes than in ventricular myocytes. Ro 22-9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22-9194 more strongly inhibited the Na⁺ channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na⁺ channels in ventricular myocytes.     

Atrial Membranes Contain Nucleoside Diphosphate Kinase (NDPK) Activity: Its Role in Regulation of Muscarinic K+ Channels

Guinea pig or rabbit atrial muscarinic K⁺ channels in cell-free inside-out patches ran be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg²⁺,^1,2 This ATP-dependent activation is compatible with the existence of a membrane-bound nudeos de diphosphate kinase (NDPK), which directly phosphorylates G_K-bound GDP. We show that this ATP-dependent activation is also possible in frog atria] cells, and that atrail membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphogphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation o/muscarinic K⁺ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.     

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells

Summary— The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K⁺, and on voltage-dependent Na⁺ and Ca²⁺ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 μM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 μM) did not. In dispersed cells, both tacrine (1 μM-1 mM) and physostigmine (1 μM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K⁺ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1–100 μM) or physostigmine (1 μM-1 mM). In fura-2 loaded cells, tacrine (100 μM) almost abolished [Ca²⁺]i rise induced by veratridine, but only slightly reduced that evoked by high K⁺. In voltage-clamped cells, tacrine (300 μM) depressed the voltage-dependent Na⁺ and Ca²⁺ currents by about 93% and 69%, and physostigmine (300 μM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na⁺ channels rather than the Ca²⁺ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.     

Effects of K+ channel inhibitors and antagonists on NS-004 evoked relaxations in guinea-pig isolated trachea

Summary— The smooth muscle relaxant responses to NS-004, an activator of charybdotoxin-sensitive, large conductance Ca²⁺-dependent K⁺ channels (BKCa) were studied on the basal spontaneous tone in guinea-pig trachea in vitro. The sensitivity of these responses to a range of K⁺ channel inhibitors and antagonists were also evaluated. NS-004 (0.1–30 μM) evoked concentration-related relaxations (pIC₅₀ 5.48 ± 0.13) on the spontaneous tone in guinea-pig tracheal rings, suspended in Krebs bicarbonate solution, with a maximum response not different to that to aminophylline (1 μM). Charybdotoxin (0.03 and 0.1 μM) or iberiotoxin (0.1 μM) significantly displaced the NS-004 concentration-response curve to the right of control with no change in maximum response. In contrast, glibenclamide (1.0 μM), apamin (0.1 μM) and dofetilide (1.0 μM) each failed to modify the responses to NS-004 on spontaneous tone in guinea-pig trachea. These results suggest that relaxations in guinea-pig tracheal smooth muscle to the substituted benzimidazolone, NS-004, involve the activation of BK_Ca channels.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L.)

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V-ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3-day-old feeding adult Amblyomma americanum (L.) female ticks. The 5' end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V-ATPase decrease the rate of dopamine-stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na⁺, K⁺ ATPase, indicating that a V-ATPase may participate in the mechanism of salivary fluid secretion in A. americanum , but the volume of saliva secreted is more dependent on an active Na⁺, K⁺ ATPase.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg⁻¹, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg⁻¹) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg⁻¹) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.     

Effects of cyclooxygenase inhibitors, prostaglandins F2α and I2, on isolated coeliac and basilar arteries of alloxan-diabetic dogs

Abstract. The effects of prostaglandin synthetase inhibitors PGF_2α and PGI₂ on the tone of isolated basilar and coeliac arteries were studied in healthy and alloxan-diabetic dogs. PGF_2α (1 μmol l^-1) produced a significantly higher tone in diabetic basilar arteries (1·15 ± 0·16 mN) than in normal cerebral vessels (0·7 + 0·10 mN). By contrast, the contractile responses of normal and diabetic coeliac arteries to PGF_2α did not differ. The cyclooxygenase inhibitors indomethacin (3 μmol l^-1) and suprofen (0·58 μmol l^-1) potentiated the PGF_2α-evoked contractions in all of the vessels studied. The percent potentiation was greater (50–60%) in the basilar arteries from alloxan-treated dogs than in normal basilar vessels (22–30%). There was not such a difference between diabetic and normal coeliac arteries. Prostacyclin produced a concentration-related relaxation in the presence of indomethacin or indomethacin + PGF_2α. The relaxant potencies of PGI₂ were similar in the vessels from metabolically healthy and diabetic dogs. The IC₅₀ values for PGI₂ were 11·6 ± 1·3 and 11·8 ± 1·8 nmol l^-1 in normal and diabetic basilar arteries, respectively; they were 25·4 ± 3·2 and 26·2 ± 3·9 nmol l^-1 in control and alloxan-treated coeliac vessels. These results indicate that normal and diabetic vessels may have differential reactivity to cyclooxygenase inhibitors, this difference being dependent on the vascular region.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

Imaging of Ca2+-induced cytoplasmic Ca2+ responses in normal and pathological parathyroid cells

Ca²⁺-induced changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) were studied in bovine and normal and pathological human parathyroid cells using digital image analysis of fura-2-loaded cells. When raising external Ca²⁺ from 0.5 to 3.0 mmol L⁻¹, about 95% of all cells reacted rapidly and simultaneously with sustained elevation of [Ca²⁺]_i. In approximately two out of three bovine parathyroid cells, normal human cells and cells from most patients with hyperparathyroidism (HPT) the sustained phase was preceded by an overshooting [Ca²⁺]_i transient. The proportion of cells with such a transient was decreased in cells from severe cases of uraemic parathyroid hyperplasia only. However, pathological human cells from adenomas and normal-sized glands associated with adenomas, as well as cells from primary and uraemic hyperplasias, had lower peak and sustained levels than normal human and bovine cells. The results indicate that both normal and pathological parathyroid cells exhibit heterogeneity in their [Ca²⁺]_i responses to elevation of external Ca²⁺. The Ca²⁺-induced [Ca²⁺]_i transients and the sustained elevations are attenuated in pathological human parathyroid cells. However, the presence of the overshooting transient represents physiological variability rather than being a consequence of the pathophysiology associated with HPT.     

Comparative Absorption of Ferri-Haemoglobin-59Fe/Ferro-Haemoglobin-59Fe and 59Fe3+/59Fe2+ in Humans with Normal and Depleted Iron Stores*

Abstract. The intestinal ⁵⁹Fe absorption from ferri- and ferro-haemogIobin-⁵⁹Fe and ⁵⁹Fe³⁺ and ⁵⁹Fe⁺ was calculated from whole body-⁵⁹Fe-retention measurements in subjects with normal and depleted iron stores. A ferri-haemoglobin-⁵⁹Fe/ferro-haemoglobin-⁵⁹Fe absorption ratio of 1.03 ±0.11 was observed for the absorption of ferri-haemoglobin-⁵⁹Fe (8.6± 0.77%) and ferro-haemogIobin-⁵⁹Fe (8.7±0.94%) in persons with normal iron stores. Depletion of iron stores caused a slight but significant higher rise of ferri-haemoglobin-⁵⁹Fe absorption (22 ± 1.7%) than the increase of ferro-haemoglobin-⁵⁹Fe absorption (18 ±0.9%) so that the absorption ratio was 1.24±0.073.—This remarkable iron valence independence of haemoglobin iron absorption is in considerable contrast to the well-established valence dependence of inorganic iron absorption which favours ferrous iron absorption especially with rising iron doses. The ⁵⁹Fe³⁺/⁵⁹Fe²⁺ absorption ratio for a diagnostic 0.56 mg Fe dose increased from 0.43 in subjects with normal iron stores to 0.74 in persons with depleted iron stores, whereas this absorption ratio was augmented only from 0.21 to 0.28 for the therapeutic 50 mg Fe-dose.—The different influence of iron valence on iron absorption from inorganic and haemoglobin iron supports other evidence for the existence of two separate mechanisms for ferrous iron and haem iron absorption in humans.     

Changes in Potassium Content of Erythrocytes during Exercise in Man

Abstract. Factors affecting erythrocyte K⁺ content and plasma electrolytes during light exercise were examined in 8 normal subjects and 27 patients with chronic obstructive lung disease. The patients were classified into bronchitis, emphysema and intermediate groups. Arterial blood was obtained during rest, after 2, 5, and 7 min. of exercise on a bicycle ergometer (30 km/h without mechanical brake), and at 10 and 20 min. during recovery for analysis of PO2, PCO₂, plasma H, Na⁺, K, and Cl^-, erythrocyte K⁺ content and whole blood lactate. Plasma H increased significantly in all subjects after 2 min. of exercise and P_CO2 rose in normal, bronchitis, and emphysema groups during exercise, whereas PO2 did not change significantly. Plasma K rose promptly after the beginning of exercise and remained at high values during exercise. The rapid rise in P_CO2 was associated with a significant decrease of calculated erythrocyte K in patients with chronic bronchitis. Although changes in plasma H were dependent on both P_CO2 and lactate, erythrocyte K⁺ changes were significantly related only to changes in P_CO2. These results indicate that the decrease in erythrocyte K⁺ is mainly controlled by changes in arterial P_CO2 during light exercise in patients with chronic bronchitis.     

Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Summary.  The integrin αvβ3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates αvβ3 activity in platelets, we measured the effect of the P2Y₁ antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y₁₂ antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y₁ and P2Y₁₂ was required to activate αvβ3. The reducing agent dithiothreitol and Mn²⁺ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates αvβ3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, αvβ3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Gα_q- and Gα_i-coupled receptors converge to release cytoskeletal constraints on αvβ3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for αvβ3-mediated platelet adhesion.