Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs.

The goal of this study was to clarify that blockade of adenosine receptors during myocardial ischemia causes further reductions in coronary blood flow due to platelet aggregation. Coronary perfusion pressure in 47 open-chest dogs was reduced such that coronary blood flow decreased to one fifth of the control value; thereafter, coronary perfusion pressure was maintained at the low levels. During hypoperfusion, coronary flow was kept low but constant with a massive release of adenosine. When 8-phenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow (18 +/- 2 ml/100 g/min) gradually decreased at 5-10 minutes of ischemia and reached almost zero at 20 minutes. Three minutes after the onset of ischemia, before further reduction of coronary flow, the microscopic examination revealed the existence of thromboembolization in the small coronary arteries, and the number of platelets in the regional coronary venous blood were significantly decreased, indicating that a further reduction of coronary flow due to treatment with 8-phenyltheophylline is attributed to thromboembolism caused by platelet aggregations. This reduction of coronary flow and formation of thromboembolism were inhibited by the treatments with dibutyryl cAMP, forskolin, and yohimbine, indicating that this thromboembolization during a lack of adenosine activity is due to platelet aggregation and that platelet aggregation caused by 8-phenyltheophylline is triggered by stimulation of alpha 2-adrenoceptors by released norepinephrine during ischemia. We demonstrate that adenosine, generated endogenously in response to ischemia, inhibits platelet aggregation. The finding that adenosine is not merely a vasodilator but that it also regulates thrombosis has major implications for designing new strategies of myocardial salvage.     

Correlation of membrane anisotropy with function in subpopulations of human blood platelets*

Summary. Human blood platelets were fractionated on a discontinuous Percoll gradient into high density (HD), intermediate (ID), and low density (LD) platelets. The subpopulations were characterized with regard to [¹⁴C]serotonin uptake and release, cAMP content, aggregation, and membrane anisotropy, which is inversely related to membrane fluidity. Membrane anisotropy, which was high in LD platelets, was found to decrease with increasing density (LD> ID > HD). LD platelets showed significant lower cAMP levels and [¹⁴C]serotonin uptake than the total platelet population (TPP) and ID and HD platelets. Upon ADP and serotonin stimulation the cAMP content was reduced in all platelet populations with the exception of HD platelets in which cAMP was unchanged. Upon thrombin stimulation the cAMP content was reduced only in TPP and LD platelet population and it was increased in HD platelet population. Thrombin activation changed the anisotropy only in LD platelets. Thrombin at a concentration of 0·001 U/ml reduced whereas 0·01 and 0·05 U thrombin/ml increased the membrane anisotropy significantly. As compared with TPP and the other subpopulations, LD platelets were most sensitive upon ADP and thrombin stimulated [¹⁴C]serotonin release as well as upon ADP, serotonin and thrombin induced aggregation. The findings suggest that the differing functional abilities of the platelet subpopulations are correlated to the various membrane anisotropies observed in these fractions.     

Effects of Ethanol on Human Platelets Stimulated with Platelet-Activating Factor, a Biologically Active Ether Phospholipid

The interaction between ethanol and 1-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet activating factor, PAF) was addressed using platelets obtained from normal nonalcoholic volunteers. Ethanol at concentrations of 20 to 100 mM inhibited PAF activation of human platelets. Ethanol inhibited prominently the second or arachidonic acid metabolite dependent wave of platelet aggregation, which occurs with human platelets in citrated plasma. It also inhibited serotonin release and thromboxane A2 formation associated with this secondary phase of aggregation. Ethanol did not readily inhibit the primary wave of PAF-induced aggregation. The incorporation of PAF into platelets or metabolism of PAF was not influenced by up to 100 mM ethanol. Since ethanol inhibited only the secondary response, a direct interaction between PAF, ethanol, and a platelet PAF receptor is unlikely. The effect of ethanol on PAF-induced platelet aggregation shows a selectivity similar to that demonstrated by other investigators for epinephrine and adenosine diphosphate.     

Disaggregatory effects of prostaglandin E 1 , amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E 1 (PGE 1 ), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE 1 , amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 w M adenosine diphosphate (ADP) or 0.625 w g/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE 1 (0.7-10.0 w M), amrinone (106.8-801.3 w M), or milrinone (9.5-190.0 w M) was added when aggregation reached 5 m z of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC 50 ) were PGE 1 ; 3.1 - 1.2 w M, amrinone; 313.1 - 128.8 w M, milrinone; 57.1 - 20.8 w M. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6 - 5.0% for 10 w M PGE 1 , 7.8 - 2.4% for 801.3 w M amrinone, and 5.9 - 2.3% for 190.0 w M milrinone. The EC 50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67 - 13 pmoles/10 8 platelets for PGE 1 , 54 - 12 pmoles/10 8 platelets for amrinone, and 52 - 12 pmoles/10 8 platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1 - 5.2% or 11.2 - 3.1% for PGE 1 (10 w M), 22.3 - 4.1% or 15.2 - 3.2% for amrinone (801.3 w M), and 23.5 - 4.3% or 14.6 - 3.5% for milrinone (190.0 w M).     

Relation between the Inhibition of Aggregation and the Concentration of cAMP in Human and Rat Platelets

Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Inhibitory mechanisms of kinetin,a plant growth-promoting hormone,in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 μM) also concentration-dependently inhibited intracellular Ca²⁺ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 μg/ml). Kinetin (70 and 150 μM) significantly inhibited thromboxane A₂ formation stimulated by collagen (1 μg/ml) and arachidonic acid (60 μM) in human platelets. In addition, kinetin (70 and 150 μM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 μM). Rapid phosphorylation of a platelet protein of molecular weight (M_r) 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 μg/ml). This phosphorylation was inhibited by kinetin (70 and 150 μM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na⁺/H⁺ exchanger. This leads to lower intracellular Ca²⁺ mobilization, followed by inhibition of TxA₂ formation and then increased cyclic AMP formation, followed by a further inhibition of the Na⁺/H⁺ exchanger, ultimately resulting in markedly decreased intracellular Ca²⁺ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.     

Antiplatelet activity of 3-butyl-6-bromo-1(3H)-isobenzofuranone on rat platelet aggregation

This study aimed to explore the impact of 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) on rat platelet aggregation induced by arachidonic acid (AA). Anti-platelet activities in vitro and ex vivo in rat platelets and the possible mechanism were also investigated. The 50% inhibitory concentration (IC₅₀) of Br-NBP was 84 μM in washed platelet added AA (final concentration, 780 μM) in vitro, meanwhile intravenous injection of Br-NBP also potently inhibited platelet aggregation ex vivo. Br-NBP significantly restrained thromboxane B₂ formation and Ca²⁺ mobilization caused by AA, but failed to regulate 6-keto-prostaglandin F_1α production and malonaldehyde content. Treatment of Br-NBP improved cAMP, cGMP levels and NO synthesis but prevented serotonin secretion and PF₄ release. Results suggested that Br-NBP inhibited rat platelet aggregation and that the anti-platelet activity was related to both arachidonic acid cascade and cGMP-NO signal path.     

Effect of quinidine on platelets

The effects of quinidine on platelets over a wide range of concentrations of the drug were studied. It was found that quinidine at high concentrations (1 and 1.5 mM) induced platelets to undergo profound ultrastructural changes and 14C-serotonin release. In addition, the drug at these concentrations caused marked inhibition of platelet aggregation mediated by adenosine diphosphate, adrenaline, collagen and arachidonate but had no effect on ristocetin-induced agglutination. However, quinidine at 10 microM (a pharmacological concentration) had only a variable inhibitory effect on adrenaline-induced platelet aggregation.     

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.     

Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation

Abstract

Prostacyclin (PGI₂) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI₂. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI₂ and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI₂-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI₂ were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI₂. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI_2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.     

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo. In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A₂ receptor antagonist (5 μM), a stable prostacyclin (0.55 nM), a stable prostaglandin E₁ (141 nM) and a phosphodiesterase inhibitor (100 μM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

In vitro platelet abnormality in adenosine deaminase deficiency and severe combined immunodeficiency.

The platelets of an infant with severe combined immune deficiency and adenosine deaminase deficiency showed markedly diminished responses to ADP-induced aggregation in vitro. This abnormality was corrected by the addition of purified adenosine deaminase in vitro. Exogenous adenosine added to platelet-rich plasma caused markedly prolonged inhibition of ADP-induced aggregation. This was shown by isotopic studies to be due to slow clearance of adenosine and hence persistence of this nucleoside. Direct assay for adenosine deaminiase in plasma and platelet lysates of the patient confirmed the very low activity of this enzyme. Raised cAMP levels were demonstrated in his platelets. The deranged adenosine metabolism and raised cAMP in the platelets of this child with severe combined immunodeficiency may explain the altered response to ADP. Despite the in vitro platelet aggregation abnormality, the patient had no clinical evidence of impaired hemostasis.     

Epinephrine potentiation of arachidonate-induced aggregation of cyclooxygenase-deficient platelets

Evaluation of platelet physiology, biochemistry, ultrastructure, and function in a young woman without history of hemorrhagic problems revealed that her platelets were deficient in cyclooxygenase activity. Her citrate platelet-rich plasma (C-PRP) responded monophasically when stirred with aggregating agents in the same manner as aspirin-treated normal C-PRP, but could be irreversibly aggregated by high concentrations of thrombin, collagen, and ADP. Her platelets did not aggregate when stirred with AA at concentrations as high as 2 mM. Ultrastructure and levels of serotonin and adenine nucleotides were normal. Amounts of ¹⁴C-AA released after stirring with thrombin were similar to normal cells. However, evaluation of prostaglandin synthesis after stirring with ¹⁴C-AA revealed no evidence of endoperoxide or thromboxane production, although products of the lipoxygenase pathway were produced in normal amounts. Aggregation in response to AA was completely corrected after mixing with 10% normal C-PRP. However, equal volumes of her C-PRP and normal aspirin-treated C-PRP did not respond to AA, whereas 10% normal platelets combined with aspirin-treated cells corrected aggregation to AA. Since epinephrine pretreatment corrects the response of dog platelets that are not aggregated by AA, we evaluated the influence of epinephrine on her platelets. Preexposure to 5 μM epinephrine, a concentration that gave only primary waves of aggregation, resulted in normalization of her response to AA, even though correction was not associated with the generation of endoperoxides or thromboxanes. The results may explain why patients with platelet cyclooxygenase deficiency have mild or absent bleeding symptoms.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

A monoclonal anti-human platelet antibody: a new platelet aggregating substance

A monoclonal anti-human platelet antibody, TP82, is described, which caused irreversible aggregation of platelets in association with the release of adenosine triphosphate or [14C] serotonin, and which inhibited ristocetin-induced agglutination. Immunofluorescence assay showed that the antibody binds to platelets, megakaryocytes, and common acute lymphoblastic leukemia cells. The antibody (IgG1) immunoprecipitated a polypeptide of 23,000 daltons with an isoelectric point of about 7.0. The aggregation induced by the purified antibody and/or F(ab')2 fragments occurred in platelet-rich plasma and with washed platelets, but not with formalin-fixed washed platelets. TP82- induced aggregation was completely inhibited by disodium ethylendiaminotetraacetate, diltiazem, W-7, PGE1, and several metabolic inhibitors. At a concentration of apyrase or CP/CPK, which inhibited adenosine 5-diphosphate-induced aggregation. TP82-induced aggregation was only partially affected. Thrombin was not required for the antibody- mediated effects, since two thrombin inhibitors failed to block the reaction. The antibody, at least at a high concentration, induced platelet aggregation by a mechanism almost independent of thromboxane A2 formation, since cyclooxygenase inhibitors had little inhibitory effect on aggregation. TP82 monoclonal antibody is a new platelet- aggregating substance that interacts with a low-molecular-weight binding site on the platelet membrane.     

Sodium fluoride mimics effects of both agonists and antagonists on intact human platelets by simultaneous modulation of phospholipase C and adenylate cyclase activity

Using intact human platelets, we studied the effect of sodium fluoride (NaF) on platelet aggregation and release reaction and correlated the functional changes to intracellular events specific for either agonist- induced or antagonist-induced platelet responses. At lower concentrations, with a peak activity between 30 and 40 mmol/L, NaF induced aggregation and release of adenosine 5'-triphosphate (ATP) that was associated with increased formation of inositol phosphates, a rise in cytosolic free Ca2+, and phosphorylation of 20-kd and 40-kd proteins. At NaF concentrations greater than 40 mmol/L, aggregation and ATP release decreased dose-dependently in parallel with a decrease in Ca2+ mobilization, whereas neither inositol phosphate formation nor 40- kd protein phosphorylation was reduced. At these concentrations, NaF caused a dose-dependent transient rise in platelet cyclic adenosine 3',5'-monophosphate (cAMP) levels that was sufficient to account for the observed reduction in Ca2+ mobilization, aggregation, and ATP release. Stimulated cAMP levels started declining rapidly within 30 seconds of addition of NaF, however. Similarly, prostacyclin (PGI2)- induced cAMP accumulation was temporarily enhanced but subsequently suppressed by NaF, suggesting either stimulation of a cAMP phosphodiesterase or delayed inhibition of adenylate cyclase. Evidence for the latter was provided by the finding that NaF pretreatment of platelets resulted in partial inhibition of PGI2-stimulated cAMP formation in the presence of the cAMP phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (MIX). We conclude that NaF exerts a dual (stimulatory and inhibitory) effect on adenylate cyclase in intact platelets that is accompanied by simultaneous activation of a phosphoinositide-specific phospholipase C; in addition, a cAMP phosphodiesterase may be activated.     

P2Y₁₂ and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y?? and EP3 receptors, act as antagonists at G(i)-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G(i)-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y?? antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA? mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI?, iloprost, PGD?, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE? and PGE? as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y?? antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.