Inhibition of platelet aggregation by nitric oxide donors improves with rest

It has been suggested that the platelets of patients with acute coronary syndromes (ACS) exist in a more activated state than those of patients with stable coronary heart disease (CHD) or healthy individuals. "Platelet nitrate responsiveness" (PNR) has been suggested as a measure of platelet activation, and has been shown to be reduced in both ACS and stable CHD. We examined the effect of a short period of undisturbed supine rest, an intervention aimed at reducing levels of "stress", on PNR. In 8 healthy subjects we found that 45 minutes of rest led to a highly significant reduction in platelet aggregation and increase in PNR. Our finding supports the hypothesis that stress contributes to platelet activation as reflected in reduced PNR, which may contribute to the link between acute stress and cardiovascular events. It also emphasises that standardisation of sampling conditions in vitally important in studies utilizing PNR.     

Inhibition of human blood platelet aggregation and the stimulation of nitric oxide synthesis by aspirin

Incubation of platelet-rich plasma with 80 microM aspirin that resulted in the inhibition of both the secondary phase of ADP induced platelet aggregation and prostaglandin synthesis simultaneously stimulated the production of NO in platelets. Furthermore it was found that the treatment of platelet-rich plasma either with 80 microM ibuprofen or salicylic acid, like aspirin, which inhibited the secondary phase of platelet aggregation by ADP and prostaglandin synthesis, also stimulated the production of NO in the absence of added ADP. However the inhibition of prostaglandin synthesis by ibuprofen or salicylic acid, unlike aspirin, was transient in nature. Incubation of washed platelets with any of these three compounds also stimulated NO synthesis indicating that the effect of these compounds was not mediated through plasma proteins. The in vitro effect of aspirin on the increase of NO in platelets could also be demonstrated by in vivo exposure of platelets to the compound. It was concluded that either a temporary or a lasting inhibition of prostaglandin synthesis by these inhibitors resulted in the synthesis of NO in resting platelets. Since NO is a potent inhibitor of platelet aggregation the inhibition of platelet aggregation, by these compounds may not be the consequence of the inhibition of prostaglandin synthesis alone, but could also be related, at least partly, to the stimulated synthesis of NO by these inhibitors.     

Inhibition of platelet aggregation with histamine

A sensitive in vitro technique was used to demonstrate the inhibitory effects of histamine or human blood platelets. Platelet aggregation by epinephrine was completely inhibited at 10(-3) M concentrations of histamine. Persistent elevations of cyclic AMP levels were shown to occur in the platelets when histamine was added and corresponded to the inhibitory effects on platelet aggregation. In contradistinction to the analogous inhibitory effects by histamine on other cellular elements of the hematopoietic system, this inhibitory effect on platelets could not be blocked by equimolar concentrations of either of the classes of histamine antagonists presently available. It is suggested that there may be other additional histamine receptors on the surface of membranes of platelets or that histamine mediated its inhibitory effect on platelets through a mechanism other than surface receptors.     

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.     

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s -1 ). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.     

Supine rest reduces platelet activation and aggregation

Platelet activation and aggregation are central processes in acute coronary syndromes and myocardial infarction, and are stimulated by physical and mental stress. However, it is not known if and to what extent the "ordinary" stress inherent in a person's daily routine contributes to platelet activation and aggregation. We measured platelet activation and aggregation in 12 healthy non-smokers, before and after 45 min supine rest in a calm environment. This simple manouver resulted in a highly significant fall in platelet aggregation (7.9-4.4 ohms, p <0.001) and in plasma epinephrine (35.6-22.5 ng/ml, p = 0.037), norepinephrine (392.8-202.7 ng/ml, p <0.001) and soluble P-selectin (51.9-44.7 ng/ml, p <0.001). Von Willebrand factor (86.2-80.9 IU/ml) and beta-thromboglobulin (279.1-262.4 IU/ml) did not change significantly. Our findings show that a person's ordinary daily routine contributes to platelet activation and aggregation, and that these can be reduced by supine rest. This has methodological implications for studies involving measures of platelet activation and aggregation, and also suggests a mechanism by which bed rest in a calm environment may contribute, however slightly, to the management of acute coronary syndromes.     

Supine rest reduces platelet activation and aggregation

Platelet activation and aggregation are central processes in acute coronary syndromes and myocardial infarction, and are stimulated by physical and mental stress. However, it is not known if and to what extent the "ordinary" stress inherent in a person's daily routine contributes to platelet activation and aggregation. We measured platelet activation and aggregation in 12 healthy non-smokers, before and after 45 min supine rest in a calm environment. This simple manouver resulted in a highly significant fall in platelet aggregation (7.9-4.4 ohms, p<0.001) and in plasma epinephrine (35.6-22.5 ng/ml, p = 0.037), norepinephrine (392.8-202.7 ng/ml, p<0.001) and soluble P-selectin (51.9-44.7 ng/ml, p<0.001). Von Willebrand factor (86.2-80.9 IU/ml) and beta-thromboglobulin (279.1-262.4 IU/ml) did not change significantly. Our findings show that a person's ordinary daily routine contributes to platelet activation and aggregation, and that these can be reduced by supine rest. This has methodological implications for studies involving measures of platelet activation and aggregation, and also suggests a mechanism by which bed rest in a calm environment may contribute, however slightly, to the management of acute coronary syndromes.     

Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation

The possible participation of proteases in human platelet aggregation was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of protease. Substrates used were synthetic substrates for the trypsin type as well as for the chymotrypsin type of protease. All these inhibitors and substrates inhibited platelet aggregation and serotonin release induced by ADP, collagen, epinephrine, or thrombin. In ADP- and epinephrine-induced platelet aggregation the second phase of aggregation was most efficiently inhibited. The inhibitors suppressed the formation of malondialdehyde during platelet aggregation. Release by aggregating agents of arachidonate and its metabolites from indomethacin-treated platelets as well as nontreated platelets was also inhibited. The inhibitors apperar to interact with stimulated platelets but not with unstimulated platelets. These observations suggest that the interaction of an aggregating agent with its platelet receptor activates a unique precursor serine protease that in turn activates platelet phospholipase to liberate arachidonic acid (the precursor of the potent platelet aggregating agent thromboxane A2) from platelet phospholipids.     

Testosterone modulates platelet aggregation and endothelial cell growth through nitric oxide pathway

The aim of the present study was to investigate the effect of testosterone on the modulation of cellular events associated with vascular homeostasis. In rat aortic strips, 5-20 min treatment with physiological concentrations of testosterone significantly increased nitric oxide (NO) production. The rapid action of the steroid was suppressed by the presence of an androgen receptor antagonist (flutamide). We obtained evidence that the enhancement in NO synthesis was dependent on the influx of calcium from extracellular medium, because in the presence of a calcium channel blocker (verapamil) the effect of testosterone was reduced. Using endothelial cell (EC) cultures, we demonstrated that androgen directly acts at the endothelial level. Chelerythrine or PD98059 compound completely suppressed the increase in NO production, suggesting that the mechanism of action of the steroid involves protein kinase C and mitogen-activated protein kinase pathways. It is known that endothelial NO released into the vascular lumen serves as an inhibitor of platelet activation and aggregation. We showed that testosterone inhibited platelet aggregation and this effect was dependent on endothelial NO synthesis. Indeed, the enhancement of NO production elicited by androgen was associated with EC growth. The steroid significantly increased DNA synthesis after 24 h of treatment, and this mitogenic action was blunted in the presence of NO synthase inhibitor N-nitro-l-arginine methyl ester. In summary, testosterone modulates vascular EC growth and platelet aggregation through its direct action on endothelial NO production.     

Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release

This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.     

Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling

Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.     

The influence of organic peroxides on platelet aggregation and sensitivity to nitric oxide

The effects of oxidative stress, induced by water-soluble and lipid peroxides, on platelet reactivity and platelet sensitivity to nitric oxide were investigated. Hydrogen peroxide and cumene hydroperoxide potentiated thrombin-induced platelet aggregation. In contrast, 15(S)-hydroperoxyeicosatetraenoic acid had no such effect, while 12(S)-hydroperoxyeicosatetraenoic acid inhibited platelet reactivity. All of the peroxides tested were found to decrease platelet sensitivity to nitric oxide, although the mechanisms by which the various peroxides altered platelet sensitivity to nitric oxide were different. The water-soluble peroxides opposed the actions of nitric oxide without affecting cyclic GMP levels, while 15(S)-hydroperoxyeicosatetraenoic acid caused a significant reduction in the concentration of cyclic GMP formed in response to NO. The data from this study demonstrate that water-soluble and lipid peroxides both affect platelet reactivity and regulation, but by different mechanisms. Thus, caution should be exercised when selecting peroxides to be used as models of oxidative stress.     

The influence of organic peroxides on platelet aggregation and sensitivity to nitric oxide

The effects of oxidative stress, induced by water-soluble and lipid peroxides, on platelet reactivity and platelet sensitivity to nitric oxide were investigated. Hydrogen peroxide and cumene hydroperoxide potentiated thrombin-induced platelet aggregation. In contrast, 15(S)-hydroperoxyeicosatetraenoic acid had no such effect, while 12(S)-hydroperoxyeicosatetraenoic acid inhibited platelet reactivity. All of the peroxides tested were found to decrease platelet sensitivity to nitric oxide, although the mechanisms by which the various peroxides altered platelet sensitivity to nitric oxide were different. The water-soluble peroxides opposed the actions of nitric oxide without affecting cyclic GMP levels, while 15(S)-hydroperoxyeicosatetraenoic acid caused a significant reduction in the concentration of cyclic GMP formed in response to NO. The data from this study demonstrate that water-soluble and lipid peroxides both affect platelet reactivity and regulation, but by different mechanisms. Thus, caution should be exercised when selecting peroxides to be used as models of oxidative stress.     

Inhibition of platelet aggregation by transdermal glyceryl trinitrate.

OBJECTIVE--To determine the optimum conditions for the demonstration of an antiplatelet effect of nitric oxide and to use these conditions to elucidate the effects of a transdermal glyceryl trinitrate patch on platelet aggregation in normal volunteers. METHODS--An open prospective crossover study. The effects of nitric oxide on platelet aggregation in whole blood and platelet rich plasma as stimulated by adenosine diphosphate and U46619 was assessed in the presence and absence of iloprost and MB22948. Optimum conditions for the demonstration of an antiplatelet effect of nitric oxide were then applied to whole blood from normal volunteers in the presence and absence of a transdermal glyceryl trinitrate patch. SETTING--University hospital. SUBJECTS--Eight normal volunteers. MAIN OUTCOME MEASURES--Platelet aggregation in the presence and absence of transdermal glyceryl trinitrate. RESULTS--The optimum conditions for the demonstration of an antiplatelet effect of nitric oxide in whole blood were collecting blood into a tube containing MB22948 and citrate and stimulating platelet aggregation with adenosine diphosphate in the presence or absence of iloprost. Using this method a significant effect of transdermal glyceryl trinitrate on platelet aggregation was shown (P < 0.03) in the presence and absence of iloprost. CONCLUSIONS--These results are consistent with an inhibitory effect on platelet aggregation of nitric oxide liberated by transdermal glyceryl trinitrate. Optimum test conditions are needed to show this effect. The clinical significance of the antiplatelet effect of transdermal glyceryl trinitrate is unknown.     

Inhibition of cyclooxygenase-independent platelet aggregation by low vitamin E concentration

Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 microM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 microM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A2 formation was less than 10% in samples incubated with 100 microM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 microM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.