New carbamoylpiperidines as human platelet aggregation inhibitors

A series of 3-carbamoylpiperidines (nipecotamides) are designed, synthesized and tested for their inhibitory action against adenosine diphosphate (ADP)-induced aggregation of human platelets. A structure-activity analysis of the bis(nipecotamido)aralkane type showed that a substituent on the piperidine ring should preferably be an amide and that the electronegativity of the carbonyl oxygen and the orientation of the amide group affected activities. Based on the knowledge of factors influencing platelet activation and aggregation, a nitric ester moiety which could release nitric oxide (NO) in situ, is incorporated into the nipecotamide structure. These compounds exhibit increased activity compared to those having no -ONO2 function. They also show stereoselectivity, with the meso isomer being approximately twice as potent as the synthetic diastereomeric mixture. Replacement of the -ONO2 function with hydroxyl, ester or alkyl groups considerably diminishes aggregation-inhibitory potential. Nipecotamides are shown here to inhibit the basal and collagen-induced rise in platelet inositol trisphosphate (IP3) levels, as well as phosphoinositide turnover. A comprehensive mechanism of action is proposed taking earlier results into consideration.     

P66Shc mediates increased platelet activation and aggregation in hypercholesterolemia

Background and hypothesis

Hypercholesterolemia leads to a prothrombotic phenotype. Platelet hyperactivity associated with hypercholesterolemia has been attributed, in part, to oxidative stress. P66Shc is a well-known determinant of cellular and organismal oxidative stress. However, its role in platelet biology is not known. We hypothesized that p66Shc mediates platelet hyperactivation and hyperaggregation in hypercholesterolemia.

Methods and results

P66Shc was expressed in both human and mouse platelets, as determined by qRT-PCR and immunoblotting. Mouse platelet p66Shc expression was upregulated by hypercholesterolemia induced by high-fat diet feeding. Compared to wild-type mice, high-fat diet-induced p66Shc expression in platelets was suppressed in transgenic mice expressing a short hairpin RNA targeting p66Shc (p66ShcRNAi). High-fat diet feeding of wild-type mice amplified surface P-selectin expression on platelets stimulated by the thrombin receptor agonist protease-activated receptor-4 (PAR4), and increased aggregation of platelets induced by thrombin. These exaggerated platelet responses induced by high-fat diet feeding were significantly blunted in p66ShcRNAi mice. Finally, thrombin-stimulated platelet reactive oxygen species were suppressed in p66ShcRNAi mice.

Conclusions

Hypercholesterolemia stimulates p66Shc expression in platelets, promoting platelet oxidative stress, hyperreactivity and hyperaggregation via p66Shc.     

Cultured astrocytoma cells inhibit platelet aggregation by releasing a nitric oxide-like factor

Cultured astrocytoma cells were tested for their ability to generate a nitric-oxide like factor using platelet aggregation as a bioassay. Incubation of astrocytoma cells with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation which was proportional to the number of astrocytoma cells added. The inhibition was potentiated by superoxide dismutase (SOD) and prevented by oxyhaemoglobin (oxyHb). The inhibitory activity of astrocytoma cells was also prevented by the NO biosynthesis inhibitor NG-monomethyl-L-arginine (MeArg), an effect reversed by co-incubation with L-arginine (L-Arg) but not D-arginine (D-Arg). These results demonstrate that astrocytoma cells release, independent of added agonist, a factor with the same pharmacological profile as NO, which is likely to be derived from L-arginine.     

Inhibition of ADP-induced platelet aggregation by reduced factor Xa

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.     

Cooperativity between platelet-activating factor and collagen in platelet aggregation

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF), by thin-layer chromatography, phospholipase A2 digestion, inhibition by a specific antagonist, CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all.     

Prolactin as a luteotrophin

This review summarizes evidence suggesting a direct luteotrophic role for the hypophyseal hormone prolactin (PRL). This direct role consists of the capability to stimulate progesterone synthesis in vitro, the capability to maintain the membrane fluidity and receptors for luteinizing hormone and the capability to import substrate for progesterone synthesis. The time required for PRL-induced luteotrophic events is in the order of hours and sometimes days, and it appears that the effects are not associated with acute intracellular changes. The relatively slow responses and the stimulation of specific protein synthesis by PRL in target tissues other than the ovary suggest that PRL may function primarily through activation of the genome. PRL may induce the synthesis of specific luteal proteins, including enzymes for the regulation of intracellular substrate pools, membrane receptors for LH, or receptor proteins for lipoproteins, a major extracellular source of substrate.     

Arachidonic-acid-induced platelet aggregation is increased in male but not in female smokers

Arachidonic-acid-induced platelet aggregation was studied in 40 healthy drug-free subjects aged 18–43 years. Aggregation was elicited by lower concentrations of arachidonic acid in smoking men (n=10) than in non-smoking men (n=10)(p<0.05). No significant difference was found between smoking and non-smoking females. Adenosine-diphosphate-induced platelet aggregation was not increased in smokers. There was no difference between smokers and non-smokers with respect to mean platelet volume and count within each sex. Females and males differed regarding platelet count in whole blood and platelet sensitivity to arachidonic acid; both differences were secondary to the difference in hematocrit.The parallelism between an increased tendency to aggregation after arachidonic acid stimulation and an increased rate of myocardial infarction in male smokers, and the absence of both these phenomena in female smokers, might be of pathophysiological significance.     

Effect of prostacyclin on platelet-activating factor induced rabbit platelet aggregation

In this paper, the effect of prostacyclin (PGI₂) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI₂ and PGI₂-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of ³H-serotonin (³H-5HT).     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

The equation for platelet aggregation rate

A platelet aggregation model in shear flow taking into account the kinetics of intercellular fibrinogen bond formation limited by aggregated platelets rotation time was considered. For this consideration the average duration of platelets interaction in flow with shear rate value G is shown to be pi/4G. One fibrinogen bond is sufficient to form a solid aggregate between two platelets. The equation for single platelets disappearance rate concerned with intercellular fibrinogen bond formation, stochastic character of bond distribution in collided platelets and hydrodynamically controlled interaction time was obtained. The Hill's approximation for the obtained aggregation rate dependences was suggested and appropriate constants were determined. The qualitative criterion of platelets aggregating systems behavior was introduced.     

Evidence for the presence of a new plasma factor which acts synergistically to ADP induced platelet aggregation

The existence of a new factor (AF) in mice acting synergistically with known proaggregatory stimuli has been suggested by the present study in the plasma of mice challenged with intravenous collagen and adrenaline. Indomethacin, nordihydroguaretic acid (NDGA), BW 755C, phentolamine, cimetidine and ketanserin could not block the response of AF. Activity of the factor remained unaltered after treatment with pancreatic phospholipase A2, collagenase, CP/CPK, trypsin and heparin. Fractionation of the plasma indicated the presence of AF in acetone precipitate. Activity was destroyed by pronase and it was lost after dialysis and charcoal treatment. Existence of such a factor which is heat resistant, is of low molecular weight and is proaggregatory in nature in the thrombotic mice plasma and which requires calcium ions for the expression of its activity, has not been reported earlier.     

Effects of platelet activating factor antagonists on arachidonic acid-induced platelet aggregation and TXA2 formation

The effects of the PAF receptor antagonists WEB 2086, WEB 2170, BN 50739 and BN 52021 on AA-induced platelet aggregation (PA) and TXA2 formation were investigated in comparison with the TXA2 synthetase inhibitor HOE 944 and the TXA2 receptor antagonist BM 13.177. All PAF antagonists tested were weak inhibitors of AA-induced PA and TXA2 formation (IC50 values between 80 and 2,737 mumol/l). HOE 944 was effective in concentrations 2-3 orders of magnitude lower than PAF antagonists in inhibiting TXA2 generation. These results imply that the inhibition of TXA2 formation is of minor relevance for the actions of the investigated PAF antagonists in AA-induced PA.     

Effect of prostacyclin on platelet-activating factor induced rabbit and platelet aggregation

In this paper, the effect of prostacyclin (PGI2) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI2 and PGI2-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of 3H-serotonin (3H-5HT).     

A rate equation for blood platelet aggregation

A kinetic scheme for agonist-induced aggregation of blood platelets was formulated in terms of the kinetics of agonist interaction with the nonaggregable, discoid platelets, formation of aggregable forms by shape-change reactions, and interactions among shape-changed forms. Taking into account the relative magnitudes of the rate constants of the different steps and assuming aggregation to be by hydrophobic forces, an equation similar in form to the Michaelis-Menten equation was derived to characterize aggregation kinetics. The kinetic formulation could account for several empirical observations and may be used to interpret kinetic effects of antiplatelet drugs more informatively than at present.     

Glycine reduces platelet aggregation

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl^? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.     

Polyamines and platelet aggregation

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polycythemia rubra vera. Spermidine and spermine inhibit $\text{in}$$\text{vitro}$ plate-let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents. The proposal is made that these organic cations may modulate $\text{in}$$\text{vivo}$ platelet agglutinability.     

Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y₁₂ antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.     

Cinnamtannin B-1 as an antioxidant and platelet aggregation inhibitor

Cinnamtannin B-1 is a naturally occurring trimeric A-type proanthocyanidin, present in a limited number of plants, which exhibits a large number of cellular actions mostly derived from its antioxidant properties. Cinnamtannin B-1 modulates several biological processes such as changes in cytosolic free Ca(2+) concentration, endogenous reactive oxygen species generation, protein tyrosine phosphorylation and platelet aggregation. Proanthocyanidins, such as cinnamtannin B-1, have been reported to exert antitumoral activity mediated by a selective proapoptotic action in a number of tumoral cell lines associated with antiapoptotic activity in normal cells. The opposite effects of proanthocyanidins in normal and tumoral cells suggest that these compounds might be the base for therapeutic strategies directed selectively against tumoral cells. In addition, cinnamtannin B-1 shows antithrombotic actions through inhibition, in platelets, of endogenous ROS generation, Ca(2+) mobilization and, subsequently, aggregation. This has been reported to be especially relevant in platelets from diabetic patients, where cinnamtannin B-1 reverses both platelet hypersensitivity and hyperactivity. Considering the large number of cellular effects of cinnamtannin B-1 the development of therapeutic strategies for thrombotic disorders or certain types of cancer deserves further studies. This review summarizes the current knowledge on the actions and relevance of the signalling pathways modulated by cinnamtannin B-1.     

Prolactin receptor signaling during platelet activation.

Prolactin is a newly recognized platelet coactivator that functions through potentiation of ADP-induced platelet activation. However, the possible association between hyperprolactinemia and venous thromboembolism (VTE) has not been systematically investigated up to now; prolactin signaling mechanisms in platelets still need to be elucidated. In this study, plasma prolactin levels in healthy subjects and patients with VTE were determined, demonstrating that patients with VTE and no other congenital risk factors had significantly increased plasma prolactin levels. Moreover, prolactinoma patients demonstrated a higher incidence of VTE than the general population. To elucidate the molecular mechanisms for the development of venous thrombosis, prolactin receptor signaling during platelet activation was investigated with a focus on ADP-stimulated G-protein-regulated signaling pathways. The short isoform of prolactin receptors was detected on platelets. Signaling through this receptor, although not directly linked to Gq-proteins, substitutes for Gq-protein regulated signaling pathways involved in platelet activation. We identified protein kinase C, a well-established signaling molecule in platelet activation, as a target molecule for prolactin signaling pathways in human platelets. Our findings indicate that hyperprolactinemia may be an important novel risk factor for VTE, suggesting that its thrombogenic effect may be mediated through enhanced platelet reactivity. Revealing the molecular mechanisms of prolactin signaling will allow the design of new antithrombotic therapies.     

Normal platelet adhesiveness and aggregation in congenital PTA or Hageman factor deficiency

Platelet adhesiveness and aggregation were studied in two patients with congenital factor XI deficiency and in a patient with congenital factor XII deficiency. A normal aggregation pattern was observed in every instance, regardless of the aggregating agent. The same was true for platelet adhesiveness. It is concluded that factor XI and factor XII play no role in platelet aggregation and adhesiveness.