Nancy Ostrowski

Effects of verapamil on platelet aggregation, ATP release and thromboxane generation

We examined the effects of verapamil on platelet function. Verapamil (0.5 micrograms/ml) in this study inhibited platelet aggregation induced by threshold amounts of ADP, AA, and epinephrine. With higher concentrations of aggregating stimuli, verapamil caused a dose-dependent inhibition of aggregation. Verapamil reduced the extent of epinephrine-induced primary wave, but not that by ADP. Ristocetin-induced aggregation was not affected at any concentration of verapamil. Platelet ATP release induced by AA and ADP was also inhibited. In vitro platelet TXA2 generation was inhibited by verapamil in concentrations lower than those required for inhibition of aggregation.

Comparison of umbilical vein models for measurement of relative prostacyclin and thromboxane production

There is growing evidence that blood vessels generate TXA2 in addition to PGI2. We examined effluents from continuously perfused human umbilical vein and supernatants from umbilical vein rings for TXB2 and 6-keto-PGF1 alpha measurements (stable metabolites of TXA2 and PGI2, respectively). TXB2 and 6-keto-PGF1 alpha were identified in all samples. 6-keto-PGF1 alpha to TXB2 ratio was higher in intact vein effluents than in the venous ring supernatants (112:1 and 28:1, respectively, P less than 0.01). Arachidonate stimulation increased 6-keto-PGF1 alpha and TXB2 levels similarly in the intact vein effluent. In contrast, stimulation of the venous rings resulted in a relatively larger increase in TXB2 than in 6-keto-PGF1 alpha. This caused 6-keto-PGF1 alpha to TXB2 ratio to decline (p less than 0.01). The identity of TXB2 was confirmed in several different ways. These data suggest that 1) human umbilical veins produce TXA2 in addition to PGI2, 2) TXA2 release is more by venous rings than by the intact vein probably reflecting contribution from non-endothelial layers, and 3) arachidonate stimulation causes relatively greater release of TXA2 than of PGI2 from the venous rings, whereas release of PGI2 and TXA2 is similar from the intact vein.

Calcium Blocker Diltiazem Inhibits Platelet Activation and Stimulates Vascular Prostacyclin Synthesis

The effects of slow channel calcium blocker diltiazem on platelet aggregation and on the generation of vasoactive prostanoids, thromboxane A2 and prostacyclin were examined. Diltiazem, in therapeutic concentrations (50–200 ng/ml), inhibited human platelet activation induced by cumulative sub-threshold concentrations of calcium ionophore A 23187 plus ADP or epinephrine. However, platelet activation induced by cumulative effects of ADP plus ephinephrine was inhibited by diltiazem only in very high concentrations (>5 μg/ml). These data indicate that platelet aggregation mediated only through calcium flux is inhibited by diltiazem in therapeutic concentrations. In other experiments, diltiazem significantly potentiated prostacyclin release from human umbilical veins. These effects of diltiazem may contribute to efficacy of this compound in ischemic heart disease.

Platelet function and biosynthesis of prostacyclin and thromboxane A2in whole blood after aspirin administration in human subjects

Small doses of aspirin have been shown to inhibit platelet thromboxane A2 while sparing vascular prostacyclin synthesis. Because leukocytes generate prostacyclin and participate in thrombosis along with platelets, the effects of three different doses of aspirin (40, 325 and 650 mg) on platelet function as well as on endogenous biosynthesis of thromboxane A2 and prostacyclin in whole blood were examined. In normal volunteers given a single 40 mg dose of aspirin, platelet aggregation and adenosine triphosphate release were inhibited for 24 hours. In contrast, platelet function was inhibited for 4 to 7 days in volunteers given 325 or 650 mg of aspirin. Platelet and whole blood generation of thromboxane A2 was inhibited less than 60% by the 40 mg dose, but almost completely by both the 325 and 650 mg doses. Likewise, whole blood generation of prostacyclin was inhibited 70% by the 40 mg dose and over 90% by the larger doses. Inhibition of thromboxane A2 as well as of prostacyclin was evident for 4 days with the 40 mg dose and for 7 days with the larger doses. Decreases in whole blood thromboxane A2 and prostacyclin with any dose of aspirin were of similar magnitude. These data indicate that aspirin in doses of 40 to 650 mg inhibits platelet function and biosynthesis of thromboxane A2 and prostacyclin in whole blood in human beings in a dose-dependent fashion.

Comparative effects of nitroglycerin and nitroprusside on prostacyclin generation in adult human vessel wall.

The precise mechanism of vasodilatory actions of nitroso-compounds is not clear. It has been suggested that these drugs might modulate release of the vasodilator, prostacyclin, from cultured endothelial cells and bovine arteries or potentiate actions of prostacyclin. This study was designed to examine the effects of nitroglycerin and nitroprusside on prostacyclin release from adult human vasculature. Saphenous vein ring preparations were incubated with nitroglycerin or nitroprusside and arachidonic acid, the substrate for prostacyclin. Vascular rings incubated with nitroglycerin released significantly more prostacyclin (measured as 6-keto-prostaglandin F1 alpha, a stable hydrolysis product of prostacyclin by radioimmunoassay) compared with the control vascular rings (p less than 0.02). This increase was observed at the therapeutic concentrations of nitroglycerin (5 to 10 ng/ml). However, incubation of saphenous vein rings with nitroprusside in concentrations as high as 1 microgram/ml was not associated with any increase in prostacyclin release. Prior incubation of vascular rings with the cyclooxygenase blocker, indomethacin, inhibited nitroglycerin-induced prostacyclin release. Incubation of vascular rings with the selective thromboxane A2 blocker, OKY 1581, resulted in additional prostacyclin release with nitroglycerin treatment, presumably by inhibiting vessel wall-generated thromboxane A2. Nitroprusside had no significant effect on prostacyclin release from indomethacin-treated or OKY 1581-treated vascular rings. This study suggests significant stimulatory effects of nitroglycerin, but not of nitroprusside, on prostacyclin release from human saphenous vein. Nitroglycerin-induced prostacyclin release may be an important mechanism of its antiischemic actions in human subjects.

Influence of propranolol and 4-hydroxypropranolol on platelet aggregation and thromboxane A2 generation.

We examined in vitro effects of propranolol and its major metabolite, 4‐hydroxypropranolol, on human platelets. Both propranolol and its 4‐hydroxy metabolite had no significant direct effect on either platelet aggregation or TXA2 generation in therapeutic concentrations. Higher concentrations (≥10 μg/ml), however, reduced both parameters of platelet function. When propranolol and 4‐hydroxypropranolol were incubated in combination with platelets in equal concentrations, there were synergistic effects on platelet aggregation and TXA2 generation. Neither propranolol nor 4‐hydroxypropranolol inhibited platelet aggregation or TXA2 synthesis in sonicated platelets at any concentration, indicating that intact platelet membrane is necessary for their action.

Increased plasma concentrations of prostacyclin metabolite 6-keto-PGF1α in essential hypertension

To evaluate the role of the vasoactive prostaglandins prostacyclin and thromboxane A2 in essential hypertension, the stable metabolites 6-keto-PGF1 alpha and thromboxane B2, respectively, were measured in plasma before and after therapy in 7 patients. During the placebo phase, plasma 6-keto-PGF1 alpha levels were significantly greater than normal. Plasma thromboxane B2 levels were not statistically different from those in normal subjects. After intravenous administration of labetalol to the point of blood pressure reduction, neither plasma 6-keto-PGF1 alpha nor thromboxane B2 values changed. With prolonged oral labetalol therapy and concurrent regulation of blood pressure, a significant decrease in plasma 6-keto-PGF1 alpha levels occurred while thromboxane B2 values remained unaltered. Elevation of plasma 6-keto-PGF1 alpha in untreated hypertensive subjects suggests that enhanced vessel wall prostacyclin synthesis may be a protective mechanism to prevent organ damage. As blood pressure is controlled this increase is no longer needed, and prostacyclin generation returns to normal.

Arterial prostacyclin generation is decreased in patients with malignant bone tumors

Arterial production of PGI2 was measured in patients with malignant bone tumors and compared to that in arteries from subjects with benign bone tumors and others without tumor. Arteries from all study subjects exhibited spontaneous and arachidonate‐induced PGI2 release. Arterial production of PGI2 as measured by 6–keto‐PGF1a was in the normal range in patients with benign tumors. In contrast, arteries from patients with malignant tumors had lower PGI2 release. Deficient arterial PGI2 production in patients with malignant tumor was present both at rest and upon stimulation of blood vessels with arachidonate. Decreased PGI2 generation could relate to tumor attachment to the endothelium, penetration of the vessel walls and subsequent metastasis.

Effect of different amounts of arachidonic acid on vessel wall-generated PGI2 and TXA2.

It has been suggested that low concentrations of AA may have vasoprotective and high concentrations vaso-damaging effects. To relate these effects to vascular generation of PGI2 and TXA2, we incubated human umbilical vein rings with AA (0, 0.01, 0.1, 1 and 2 mM) and examined the supernates for 6-keto-PGF1 alpha and TXB2. Low concentrations of AA (0.01 and 0.1 mM) caused preferential and maximal PGI2 release, whereas higher concentrations (1 and 2 mM) resulted in marked and preferential increase in TXA2 release. Disequilibrium in vascular PGI2 and TXA2 release towards the latter may relate to vaso-damaging effects of high concentrations of AA.

Stimulation of vessel wall prostacyclin by selective thromboxane synthetase inhibitor oky 1581

Human umbilical veins have been shown to produce small amounts of TXA2 in addition to PGI2. We measured relative TXA2 and PGI2 production by umbilical veins in the presence of selective TXA2 synthetase inhibitor OKY 1581. OKY 1581 treatment resulted in inhibition of TXA2 but a marked increase in PGI2 release, which may relate to diversion of cyclic endoperoxides to PGI2 pathway or to removal of a feedback control at the level of AA mobilization.