G. C. Le Breton

Direct evidence for intracellular divalent cation redistribution associated with platelet shape change

Abstract Chlortetracycline was used as a fluorescent probe to monitor shifts in divalent cation distribution when blood platelets were induced to change shape. Human platelets in their native plasma were incubated at 25°C with 50 μM chlortetracycline. It was found that when platelet shape change was stimulated by ADP or the divalent cation ionophore A 23187, a significant decrease in platelet-chlortetracycline fluorescence occurred. This fluorescence shift was consistent with the time course for the change in platelet shape. ATP, which inhibits ADP-induced shape change, also inhibited the decrease in platelet-chlortetracycline fluorescence.

Movement of sodium into human platelets induced by ADP.

1. In normal human platelets the concentrations of Na+ and K+ were 42.1 +/- 4.3 and 98.8 +/- 3.7 mequiv/l of platelet water respectively (mean +/- S.E. of 22 samples). 2. When platelet-rich plasma was incubated with 22Na+ at 37 degrees C for 2-3 h an increase in platelet Na+ concentration was found which was significant after 210 min. Platelet K+ concentration did not change significantly. The platelet 22Na+ radioactivity increased faster than did the total Na+ suggesting a Na+o-Na+ exchange process in unactivated platelets. 3. Addition of ADP to platelet-rich plasma resulted in platelet aggregation and a rapid rise (within seconds) in 22Na+-radioactivity within the platelets and after 300 s this increase diminished toward control levels. 4. Under the same experimental conditions, ADP did not bring about an increase of 36Cl- in the platelets. 5. Ouabain (10-(6) M) added to platelet-rich plasma induced an increase in Na+ concentration and 22Na+ radioactivity in the platelets, as well as a decrease in K+ concentration. ADP produced a further increase in 22Na+, which did not return toward control values, in the presence of ouabain. 6. The association of an increase in 22Na+ but not of 36Cl- accompanying aggregation by ADP suggests a selective mechanism for the movement of Na+ into platelets rather than a movement of NaCl together with water under an osmotic gradient.

Synthesis of dl-cis- and (4R,5R)-trans-7-[2,2-dimethyl-4-(phenylsulfonyl)-aminomethyl-1,3-dioxolan-5-yl]-5(Z)-heptenoic acid analogues as platelet thromboxane A2 receptor antagonist

Summary The title compounds have been synthesized and their in vitro thromboxane A2 (TxA2) receptor antagonist activity evaluated. Both cis and trans isomers (1, 2) were shown to specifically inhibit submaximal human platelet aggregation induced by 225 nM U46619 in a dose-dependent manner with an IC50 of 1 μM. The concentration of 1 and 2 required to completely block maximal aggregation induced by 3 μM U46619 was 3 μM.

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets.

A photoactive iodoarylazide derivative (I‐APA‐PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13‐azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor‐binding studies using [3H]U46619, I‐APA‐PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I‐APA‐PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding radio‐iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2‐binding protein in human platelets.

Synthesis of [17,18-3H] trans-13-azaprostanoic acid: a labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA2, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA2/PGH2 antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17, 18 3H] derivative [( 3H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17Z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH4 reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH3(4) and TXA3 (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone: A potent inhibitor of the blood platelet cyclo-oxygenase

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.

Prostaglandin H2 Causes Calcium Mobilization in Intact Human Platelets

It was originally suggested that thromboxane A2 (TxA2) production is a prerequisite for platelet activation induced by arachidonic acid (AA) addition. More recently, however, evidence has accumulated that suggests that, under the appropriate conditions, prostaglandin H2 (PGH2) can also play a role in the platelet activation process. In this regard, Needleman et al. (1976) have shown that when AA was added to washed platelets incubated with the thromboxane synthase inhibitor im-idazole (Needleman et al., 1976, 1977a,b; Moncada et al., 1976, 1977), thromboxane synthesis was blocked, but platelet aggregation was not inhibited (Needleman et al., 1977b). Based on these findings, they proposed that PGH2 is itself active in causing platelet aggregation.