Thrombin-stimulated Platelet Aggregation Research Articles

Caged nitric oxide. Stable organic molecules from which nitric oxide can be photoreleased.

We report the synthesis and testing of a series of "caged" nitric oxide compounds that are stable indefinitely in oxygen-containing solutions until photolyzed by ultraviolet irradiation, whereupon they release nitric oxide (NO) with quantum yields of delta 8% for 3a (CNO-1) and delta 2% for compounds 3b-e (CNO2-5). After a flash, NO release is complete within 5 ms, so that precise temporal control of NO release is possible. NO donor 3d (CNO-4) includes two carboxylate negative charges at physiological pH, which reduce membrane permeability and enable photolytic generation of NO to be selectively confined to either extracellular or intracellular compartments. Esterification of these carboxyls with acetoxymethyl groups produces 3e (CNO-5), which is membrane-permeant and intracellularly hydrolyzable. Therefore, large populations of intact cells can be conveniently intracellularly loaded with "caged" NO donor 3d by incubation with 3e (CNO-5). The biological efficacy of these NO donors and their absolute dependence on UV-irradiation was demonstrated by inhibition of thrombin-stimulated platelet aggregation. Extracellular hemoglobin blocked the effects of NO generated outside but not inside platelets, verifying the sidedness of the NO donors and the limited spatial range of NO action. These molecules should permit precise spatial, temporal, and concentration control of NO release for investigation of its important biological functions.

Reducing atherogenic risk in hyperlipemic humans with flax seed supplementation: a preliminary report.

The effect on serum lipids of a flax seed supplement consisting of three slices of flax seed-containing bread and 15 g of ground flax seed was studied in 15 hyperlipemic subjects on long-term intake (800 IU/day) of vitamin E. The flax seed, which was high in alpha-linolenic acid and fiber, and which has been reported to lower serum cholesterol in elderly subjects, was provided in a 3-month feeding trial. Serum total and low-density lipoprotein cholesterol levels were reduced significantly; high-density lipoprotein cholesterol did not change during flax seed consumption. Thrombin-stimulated platelet aggregation decreased with the supplement. Serum lipid oxidation products decreased significantly during the washout period.

Modulation of rat mast cell reactivity by IL-1 beta. Divergent effects on nitric oxide and platelet-activating factor release.

Release of inflammatory mediators by mast cells can be modulated by certain cytokines and by nitric oxide. An in vitro platelet aggregation bioassay was used to assess the effects of interleukin-1 beta (IL-1 beta) on the release of platelet-activating factor and nitric oxide from resting or ionophore-activated peritoneal mast cells (PMC) from rat. PMC spontaneously released a substance that inhibits thrombin-stimulated platelet aggregation. The activity of this substance is abolished by addition of hemoglobin to the platelet suspension and augmented by preincubation of the PMC with L-arginine, suggesting that it is nitric oxide. Within minutes, IL-1 beta concentration-dependently (1 pg/ml-100 ng/ml) enhanced the release from activated PMC of nitric oxide, as measured by its ability to inhibit thrombin-induced platelet aggregation, and as confirmed with a biochemical assay for nitrite. This action of IL-1 beta was inhibited by pretreatment of PMC with a calmodulin antagonist (calmidazolium), an IL-1 receptor antagonist, or either of two nitric oxide synthase inhibitors (L-NAME and LY-83583). IL-1 beta also inhibited the release of platelet-activating factor from PMC through a nitric-oxide-dependent mechanism. These results demonstrate that IL-1 beta is a potent and rapid-acting modulator of mast cell reactivity, stimulating nitric oxide release while inhibiting the production of platelet-activating factor.

Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata.

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to homogeneity by a combination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molecular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an IC50 of approximately 50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen-stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhibits collagen-dependent aggregation in plasma. At a higher concentration of moubatin (> 1 microM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits platelet activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleavage of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity.

On the relationship between the inhibition of thrombin stimulated aggregation and thromboxane formation in isolated platelets treated with betaadrenoceptor blocking drugs

Thromboxane B 2 (TXB 2) formation in isolated , thrombin-stimulated rat platelets was time dependent and appeared after 5 s of incubation. Betaadrenoceptor blocking (BAB) drugs inhibited thrombin-stimulated TXB 2 formation in the following rank order of potency: metipranolol ≈ alprenolol ≈ propranolol > oxprenolol > practolol. Atenolol was ineffective in inhibiting TXB 2 production in stimulated platelets. The inhibition of thrombin-stimulated TXB 2 formation by BAB drugs correlated with their inhibitory effect on thrombin-stimulated platelet aggregation, arachidonic acid liberation from membrane phospholipids and with their membrane fluidisation. The higher was the liposolubility of the betaadrenoceptor blocking drugs investigated, the higher was their inhibition of stimulated TXB 2 formation. Hydrophilic, selective atenolol and practolol revealed slight or no inhibitory effect on stimulated thromboxane production.

Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp(a) concentrations

The purpose of this study was to compare the relative effect of n − 3 fatty acids on plasma lipids and platelet function in normolipemic subjects n − 8 with plasma Lp(a) levels > 30 mg/dl and normolipemic subjects n − 7 without detectable plasma Lp(a) concentrations. Six weeks of dietary supplementation (3.8 g EPA and 2.9 g DHA/d) significantly reduced ( P < 0.005) plasma TGs in both groups whereas no changes of plasma TC, LDL-C, HDL-C, and Lp(a), respectively, were found. Collagen- or thrombin-stimulated platelet aggregation and collagen- or thrombin-induced TXB 2 generation from platelets decreased by approx. 45% in Lp(a)-negative and Lp(a)-positive platelet donors after a 6 week dietary intake. Four more weeks without n − 3 supplementation restored the pretreatment values of TGs, platelet aggregability and TXB 2 release. The biophysical properties of platelets from normolipemics with and without high plasma Lp(a) concentrations revealed a similar structural order of platelets at 37°C using DPH, TMA-DPH, or 6-AS as fluorescent probes. Also similar temperature-dependent changes in platelet fluidity from 37°C to 17°C were observed in platelet preparations from Lp(a)-positive and Lp(a)-negative subjects. However, no subtle changes in the structural order of platelets due to nutrient intakes were found in all subjects ( n = 15, 19–28 yrs) using fluorescence polarization technique. The present data suggest a similar in vitro platelet behaviour from normolipemic subjects with and without high plasma levels of Lp(a) (which is considered a risk for premature atherosclerosis) in contrast to platelet aggregability and platelet fluidity in certain hyperlipidemic stages.

Differences among betaadrenoceptor blocking drugs in modifying platelet aggregation and arachidonic acid liberation under thrombin stimulation

The dose-dependent inhibition of thrombin stimulated platelet aggregation due to betaadrenoceptor blocking drugs followed the rank order of potency:propranolol >alprenolol > metipranolol and correlated with arachidonic acid ( 3H-AA) liberation. Atenolol which slightly potentiated stimulated aggregation increased also the liberation of 3H-AA from membrane phospholipids of isolated platelets. Stimulation of platelets resulted in decreased 3H-AA incorporation into phosphatidylcholine, phosphatidylinositol and phosphatidic acid and increased incorporation into phosphatidylethanolamine and phosphatidylserine. A 1-prenolol, metipranolol and propranolol enhanced the incorporation of 3H-AA into phosphatidylcholine of stimulated platelets.

Cimetidine and ketotifen inhibit stimulated aggregation and histamine liberation from rabbit blood platelets

The effect of cimetidine and ketotifen was studied on thrombin-stimulated rabbit platelet aggregation as well as on histamine and serotonin liberation. Both drugs inhibited the stimulation of platelets in a dose-dependent way. The effect of ketotifen on stimulated aggregation was about twice that of cimetidine. Stimulated histamine and serotonin liberation was more inhibited by ketotifen than by cimetidine. Both drugs inhibited the liberation of serotonin to a greater extent than that of histamine.

Anti-(Arg-Gly-Asp-Ser) antibody and its interaction with fibronectin, fibrinogen and platelets

An antibody population recognizing the sequence Arg-Gly-Asp-Ser (RGDS) in fibronectin, anti-(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different anti-fibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I-fibronectin to anti-(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 microM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long-range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti-(RGDS)N antibodies purified from anti-fibronectin serum had a strong inhibitory effect on thrombin-stimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin-stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.

The effect of carbonic anhydrase inhibition on the velocity of thrombin-stimulated platelet aggregation under physiological conditions

We have studied the effect of ethoxzolamide, a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10 −6 M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10 −11 M and 10 −10M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO 2 from the platelet rich plasma. These results suggest that the intracellular CO 2 hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.