Platelet-activating Antagonists Research Articles

Effects of hormones on platelet aggregation.

Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

Traitement par la thrombopoïétine du syndrome hématopoïétique induit par une irradiation létale

Treatment with thrombopoietin of the hematopoietic syndrome induced by lethal irradiation. The therapeutical efficacy of thrombopoietin, an haematopoietic cytokine, has been evaluated on improvement of haematopoietic syndrome, haematopoietic recovery and survival in C57BL6J mice following total body irradiation with a 60 Co source (7-10 Gy). A single administration of 0.3 μ g thrombopoietin 2 h after an irradiation of 9 Gy increased the 30-day survival. The survival enhancement was related to an accelerated haematopoietic recovery for all lineages and a decrease in bacterial infections. However, when the administration was delayed to 24 h after irradiation, thrombopoietin had a lower efficiency on both survival and haematopoietic recovery. We observed that half of the thrombopoietin-treated mice died between the 2nd and the 6th month after irradiation. This long-term mortality was likely to be due to thrombosis formation. In fact, we have demonstrated that thrombopoietin activates platelets which in turn enhance thrombosis formation in irradiated mice. In order to avoid thrombosis, we administered ticlopidine, an antagonist of platelet activation, to mice before irradiation. Almost all of the 9 Gy irradiated mice which received both thrombopoietin and ticlopidine survived up to 6 months, reaching the same efficiency than a bone marrow graft. In summary, we have shown that thrombopoietin treatment enhances survival of lethally irradiated mice in relation with protection from haematopoietic syndrome. Nonetheless, an anti-platelet drug might be associated with thrombopoietin to avoid thrombosis formation which might be lethal on a long-term basis.

The P2Y1 Receptor Is Necessary for Adenosine 5′-Diphosphate–Induced Platelet Aggregation

The human P2Y1 receptor heterologously expressed in Jurkat cells behaves as a specific adenosine 5'-diphosphate (ADP) receptor at which purified adenosine triphosphate (ATP) is an ineffective agonist, but competitively antagonizes the action of ADP. This receptor is thus a good candidate to be the elusive platelet P2T receptor for ADP. In the present work, we examined the effects on ADP-induced platelet responses of two selective and competitive P2Y1 antagonists, adenosine-2'-phosphate-5'-phosphate (A2P5P) and adenosine-3'-phosphate-5'-phosphate (A3P5P). Results were compared with those for the native P2Y1 receptor expressed on the B10 clone of rat brain capillary endothelial cells (BCEC) and for the cloned human P2Y1 receptor expressed on Jurkat cells. A2P5P and A3P5P inhibited ADP-induced platelet shape change and aggregation (pA2 = 5) and competitively antagonized calcium movements in response to ADP in fura-2-loaded platelets, B10 cells, and P2Y1-Jurkat cells. In contrast, these compounds had no effect on ADP-induced inhibition of adenylyl cyclase in platelets or B10 cells, whereas known antagonists of platelet activation by ADP such as Sp-ATPalphaS were effective. These identical signaling responses and pharmacologic properties suggest that platelets and BCEC share a common P2Y1 receptor involved in ADP-induced aggregation and vasodilation, respectively. This P2Y1 receptor coupled to the mobilization of intracellular calcium stores was found to be necessary to trigger ADP-induced platelet aggregation. The present results, together with data from the literature, also point to the existence of another as yet unidentified ADP receptor, coupled to adenylyl cyclase and responsible for completion of the aggregation response. Thus, the term, P2T, should no longer be used to designate a specific molecular entity.

Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers: Evidence for endoperoxide/thromboxane A 2 receptor blockade

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11α,9α-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619)-, 12- O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A 2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP 2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)TMQ was 40- and 22-fold more potent than S(−)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP 2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(−)-isomer respectively. In contrast to S(−)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [ 3H]U46619 binding to endoperoxide/thromboxane A 2 receptor sites in washed platelets. In other experiments, S(−)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(−)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A 2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [ R(+)-TMQ S(−)-TMQ] antagonist of endoperoxide/thromboxane A 2 receptor sites in platelets. Additionally, S(−)-TMQ was more potent than R(+)-TMQ as an antagonist of platelet activation by inducers of protein kinase C activity and calcium mobilization. Thus, the isomers of TMQ may also interfere by a yet undefined mechanism with calcium-regulated cytosolic processes in platelets.

Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3-aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2',3'-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5'-p-fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2',3'-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of High-Affinity (Kd 0.35 μmol/L) and Low-Affinity (Kd 7.9 μmol/L) Platelet Binding Sites for ADP and Competition by ADP Analogues

AbstractSteady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3H]ADP bound to two sites with a small amount (-5% of total) of nonspecific binding: 410,000 ± 40,000 sites of low affinity (Kd 7.9 ± 2.0 μmol/L) and 160,000 ± 20,000 sites of high affinity (Kd 0.35 ± 0.04 μmol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 μmol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3-aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 μmol/L and 200 μmol/L, respectively. The partial agonist 2‘ x2018;,3’- dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 μmol/L and 49 μmol/L. respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 μmol/L) = ATP (Kd 0.45 μmol /L) » AMP (Kd 360 μmol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuri-benzene sulfonate (Ki 250 μmol/L) but only very weakly by 5’-p-f1uorosulfonylbenzoyladenosine (K, 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 μmol/L), 2-MeS ADP (200 μmol/L) and 2‘ x2018;,3’-dialdehyde ADP (270 μmol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 μmol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50, -100 μmol/L) but not by collagen, thrombin, or ionophore A23187. These studies (a) provide accurate parameters for the binding of ADP to fixed platelets in the absence of complications due to metabolism and secretion, (b) demonstrate that both high-affinity (Kd 0.35 μmol/L) and low-affinity (Kd 7.9 μmol/L) binding sites are present and that both are accessible to ADP and C-2-substituted ADP analogues; and (c) suggest that the high-affinity binding site corresponds to the receptor modulating platelet activation by ADP. These results help to define possible platelet receptors for ADP and may provide a useful indicator system for evaluating the interaction of agonists and antagonists with platelets.

Arachidonate is an antagonist of platelet activation by the endoperoxide analog U46619

In the presence of cyclooxygenase inhibitors, arachidonate inhibits platelet activation by the endoperoxide analog U46619. The degree of inhibition depends on the concentrations of U46619 and arachidonate. The mechanism apparently does not involve arachidonate metabolism, because (i) pre-incubation did not enhance inhibition, (ii) inhibition of lipoxygenase did not prevent inhibition, and (iii) eicosatetraynoate and dihomo-γ-linolenate were also inhibitory. Under similar conditions, activation of platelets by other agonists was only slightly inhibited.