Search

The present study investigated the relationship between inositol 1,4,5-trisphosphate (IP3), thromboxane (TX)A2 and ADP in IP3-induced activation of saponin-permeabilized platelets. The time course of the different responses indicated that IP3-induced Ca++ mobilization and TXA2 production preceded both aggregation and secretion. Furthermore, platelet aggregation occurred coincident with secretion. In contrast, U46619- [15(S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid] and A23187-induced aggregation was commensurate with Ca++ mobilization, and preceded the secretion response. Indomethacin and SQ29,548 ([1S-[1 alpha,2 beta(5Z),3 beta,4 alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) inhibited IP3-mediated aggregation by 100%, Ca++ mobilization by 80% and secretion by 90%. A23187 exhibited a different inhibition profile, in that only secretion was blocked (65%). U46619-induced responses were completely inhibited by SQ29,548 and unaffected by indomethacin. Pretreatment of the platelets with creatine phosphate/creatine phosphokinase (CP/CPK), which removes secreted ADP, produced 100% inhibition of IP3-induced aggregation, 90% inhibition of Ca++ mobilization and 72% inhibition of secretion. On the other hand, CP/CPK was ineffective in blocking any of the A23187-induced responses. Concerning U46619, CP/CPK produced a 30% attenuation of maximal aggregation and 78% inhibition of both Ca++ mobilization and secretion. These results in saponin-permeabilized platelets demonstrate that IP3-induced aggregation is a secretion-mediated process which requires both TXA2 and secreted ADP. Taken together, the findings suggest that IP3 is not capable of directly causing platelet aggregation, but may function in platelets to amplify an initial agonist response through TXA2 production and secretion.

The specific thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid (13-APA) reverses platelet aggregation stimulated by TXA2/PGH2 and the prostaglandin endoperoxide analog U46619. The present report demonstrates that the deaggregatory properties of 13-APA are potentiated by prostacyclin (PGI2). Human platelet-rich plasma was aggregated with U46619. Deaggregation was induced 2 min subsequent to the addition of the aggregating agent. Concentrations of 13-APA or PGI2 which induced 20 percent deaggregation were determined. Simultaneous addition of half of these concentrations resulted in 60 percent deaggregation, demonstrating that the observed response was supraadditive. Measurement of cyclic adenosine 3':5' monophosphate (cAMP) in resting or deaggregating platelets demonstrated that 13-APA itself did not stimulate cAMP production nor did 13-APA facilitate PGI2-induced increases in cAMP. In separate studies PGI2 and 13-APA were added to PRP prior to the induction of aggregation by U46619. Under these conditions, additive inhibition of aggregation was observed. Thus, it is clear that the pharmacological interaction between PGI2 and 13-APA depends upon the relative state of platelet activation.

The effects of prostaglandin F2 alpha on human blood platelet function were investigated. PGF2 alpha at 15 muM completely blocked platelet aggregation induced by 500 muM arachidonic acid or 3 muM U46619 but had no effect on aggregation induced by 7.5 muM ADP. A similar specificity of action was not obtained with either PGI2 or PGE2. Thus concentrations of PGI2 (3 nM) or PGE2 (20 muM) which inhibited U46619- induced aggregation by 100% also blocked ADP-stimulated aggregation. The inhibitory properties of PGF2 alpha were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 muM PGF2 alpha produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using induced significant increases in platelet cAMP levels. The possibility that PGF2 alpha directly interacts at the platelet TXA2/PGH2 receptor was investigated by measuring [3H]PGF2 alpha binding to isolated platelet membranes. It was found that [3H] PGF2 alpha binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF2 alpha. Furthermore, when 1000 fold excess of either the TXA2/PGH2 "mimetic' U46619 or the TXA2/PGH2 antagonist displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PFG1 alpha, azo analog 2, or TXB2, caused displacement of only 15%, 20% or 25% of the [3H] PHF2 alpha binding. Scatchard analysis indicated that [3H] PGF2 alpha has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF2 alpha of AA or U46619- induced aggregation may be mediated through interaction at the platelet TXA2/PGH2 receptor.

A series of 13-azaprostanoic acids (4a-h) and a 15-azaprostanoic acid (11a) have been prepared. Synthesis of the 15-aza derivative is based on a novel transformation of a ketone to an N-substituted ethylenamine using a formylmethylimino phosphate derivative. Several of the azaprostanoic acid derivatives were found to be potent inhibitors of platelet aggregation induced by arachidonic acid, whereas no effect was observed on ADP-induced primary aggregation, indicating blockade of the platelet arachidonic acid cascade. The compounds do not inhibit bovine cyclooxygenase activity and are postulated as acting beyond the synthesis of the prostaglandin endoperoxides. The inhibitory effect of the 13-aza series is highly sensitive to both stereochemistry and length of the amino side chain. Any deviation from the natural prostaglandin skeletal arrangement results in decreased biological activity.

A newly synthesized 13-aza derivative of prostanoic acid (13-APA) specifically inhibited human platelet aggregation induced by arachidonic acid, prostaglandin H2, or the stable endoperoxide analog (15S)-hydroxy-9 alpha,11 alpha-)epoxymethano)-prosta-5Z,13E-dienoic acid. 13-APA also inhibited [14C]serotonin release in response to arachidonic acid, ADP, or thrombin, but did not inhibit primary aggregation induced by ADP or thrombin. 13-APA completely blocked prostaglandin H2-induced aggregation in indomethacin-treated resuspended platelets but did not inhibit thromboxane synthesis. We therefore conclude that 13-APA acts as a direct antagonist of the platelet thromboxane/endoperoxide receptor.

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A2/prostaglandin H2 (TXA2/PGH2) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid , 2-(6- carboxyhexyl ) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 microM CPH. On the other hand, CPH was not an effective inhibitor of PGH2-induced aggregation. Furthermore, 100 microM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA2/PGH2 receptor). Measurement of platelet thromboxane B2 (TXB2) production demonstrated tha the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CP inhibited TXB2 formation from AA in a dose-dependent manner (0.1 microM-100 microM CPH)2. In contrast, CPH blocked TXB2 production from PGH2 only at the highest CPH concentration tested, i.e., 100 microM. These results indicate that relative to 13-APA, addition of a second nitrogen at C14 and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.

Two new azaprostanoids, a hydrazone (3) and hydrazide (4), have been prepared by the condensation of 2-(6-carboxyhexyl)cyclopentanone with n-hexylhydrazine and caproic acid hydrazide. Preliminary results with the stable hydrazide 4 indicate that it inhibits arachidonic acid (AA) induced human platelet aggregation and that, unlike 13-azaprostanoic acid (1), its site of action is at the cyclooxygenase level. Results with the unstable hydrazone derivative 3 indicate it to be a potent and time-dependent inhibitor of AA-induced human platelet aggregation, with its site of action also at the cyclooxygenase level.

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develope adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na+. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na+ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E1 Na+ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na+. In a manner analogous to the effect of ADP, polylysine also induces Na+ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na+ uptake. The increase in platelet Na+ resulting from ouabain inhibition of Na+ efflux induces an increase in the aggregation response to ADP and to epinephrine.

The mechanism by which the active metabolites of arachidonic acid (AA), i.e., thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2) induce platelet aggregation is not understood. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP, whereas other studies have proposed that this response is ADP-independent. In the present report, we used the specific TXA2/PGH2 receptor antagonist, 13-azaprostanoic acid (13-APA), and the ADP antagonist, ATP, to examine the contribution of TXA2/PGH2 or secreted ADP to aggregation. We found that 13-APA, but not ATP, deaggregates platelets stimulated by AA or U46619 (a TXA2/PGH2 mimetic). In contrast, ADP-induced aggregation was reversed in response to ATP but not to 13-APA. These results suggest that TXA2/PGH2-stimulated aggregation is mediated through TXA2/PGH2 receptor occupation. Furthermore, secreted ADP does not appear to be required for maintenance of the AA-aggregation response.