Platelet Cyclic Adenosine Research Articles

Diindolylmethane Ameliorates Ischemic Stroke-Induced Brain Injury by Peripheral and Central Mechanisms.

Diindolylmethane (DIM), a major acid condensation product of Indole-3- carbinol, is known to inhibit platelet aggregation and thrombosis. The drugs with antiplatelet and antithrombotic activities are used to treat ischemic stroke. The present study investigated the role of DIM on platelet aggregation inhibitory properties in middle cerebral artery occluded (MCAO) rats. DIM (12.5, 25, and 50 mg/kg) was orally administered to MCAO rats for 3 days. Platelet aggregation, platelet cyclic adenosine monophosphate (cAMP), reactive oxygen species (ROS), hydrogen peroxide (H2O2), and serum cyclooxygenase (COX-1), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2), and inflammatory markers were estimated. Further brain structural and functional recovery was evaluated by measuring cerebral blood flow, neurological deficits, brain infarction, blood-brain barrier (BBB) leakage, brain water content, and histological abnormalities. DIM significantly ameliorated adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid-induced platelet aggregation by inhibiting COX-1, TXB2, and PGE2 and elevating cAMP. Further, DIM also alleviated platelet-mediated oxidative stress (ROS and H2O2) and reduced the serum inflammatory markers, tumor necrosis factor-α (TNF-α) and interleukin -6 (IL-6), and increased anti-inflammatory cytokine, IL-10, in MCAO rats. DIM treatment confers neuroprotection in MCAO rats by inhibition of platelet aggregation, platelet-mediated oxidative stress, and inflammation. Correspondingly, DIM improved cerebral blood flow and reduced neurological deficits, brain infarction, BBB leakage, brain water content, and histopathological abnormalities indicating the preservation of brain structural integrity. Thus, the present study provided preclinical evidence of DIM neuroprotection against ischemic stroke.

Abstract 11291: Ectopic Olfactory Receptor Expression in Platelets as Novel Antithrombotic Targets

Introduction: Platelets are enriched in orphan G-protein-Coupled Receptors (GPCRs) that could potentially regulate platelet reactivity. A remote study in patients with hereditary anosmia reported dysfunctional platelet responses suggesting the presence of platelet olfactory receptor (ORs) that could regulate thrombosis. Hypothesis: Platelet ORs serve as an alternative pharmacological strategy to generate platelet cyclic adenosine monophosphate (cAMP) to prevent thrombosis. Methods: Ribonucleic Acid sequencing (mRNAseq) of human megakaryocytes (MKs) was conducted. Various ORs were validated in platelets by RT-qPCR and immunoblotting (IB). Reporter cell lines with stable integration of cloned ORs identified OR ligands and later validated in adult platelets. Fluorophores for OR second messengers (Fura2 for Ca 2+ and MQAE for Cl - ) and an ELISA for cAMP were used for adult platelets. Platelet reactivity in response to OR agonists was evaluated by FACS and aggregometry, and thrombosis by biomechanical platelet activation was evaluated using ex vivo microfluidics and flow cones to simulate disturbed flow (D-flow). OR agonist injection into mice and CRISPR-Cas9 OR knockdown was conducted, with platelet reactivity and thrombosis tested as proof-of-concept. Results: Only 15 of >400 ORs in the genome were identified in MKs, and only 3 in all adult platelets (highest expression was OR2L13). A terpene family was identified as OR2L13 agonists, generating cAMP in the platelet, and structural modifications were made with IC 50 values in the low-high μM range for inhibiting agonist-induced platelet activation, ex vivo human blood thrombosis by microfluidics, and in vivo platelet activation in mice with prolonged tail bleeding. Deletion of OR2L13 (olfr168 -/- ) in mice augmented platelet reactivity with loss of OR agonist-mediated prolonged tail bleeding in olfr168 -/- . OR2L13 in human platelets is stored and trafficked by alpha granules following surface agonist stimulation and biomechanical activation. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade which operates as a negative regulator of platelet reactivity and thrombosis in response to both chemical as well as biomechanical platelet activation.

Rocuronium Has a Suppressive Effect on Platelet Function via the P2Y12 Receptor Pathway In Vitro That Is Not Reversed by Sugammadex.

Rocuronium is an aminosteroid nondepolarizing neuromuscular blocker that is widely used for anesthesia and intensive care. In this study, we investigated the effect of rocuronium on human platelet functions in vitro. The effects of rocuronium on platelet aggregation, P-selectin expression, and cyclic adenosine monophosphate (cAMP) levels in platelets were measured using an aggregometer, an enzyme immunoassay, and flow cytometry, respectively. Rocuronium inhibited ADP-induced platelet aggregation, P-selectin expression and suppression of cAMP production. These effects were not antagonized by equimolar sugammadex, a synthetic γ-cyclodextrin derivative that antagonizes rocuronium-induced muscle relaxation by encapsulating the rocuronium molecule. Morpholine, which constitutes a part of the rocuronium molecule but is not encapsulated by sugammadex, inhibited ADP-induced platelet aggregation. Vecuronium, which has a molecular structure similar to that of rocuronium but does not possess a morpholine ring, had no significant effect on ADP-induced platelet aggregation. These results indicate that rocuronium has a suppressive effect on platelet functions in vitro that is not reversed by sugammadex and suggest that this effect is mediated by blockade of the P2Y12 receptor signaling pathway via the morpholine ring of rocuronium.

Modulating platelet reactivity through control of RGS18 availability

AbstractMost platelet agonists activate platelets by binding to G-protein–coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL−/− mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm 2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Antiplatelet Agents to Prevent Recurrent Stroke

Platelet inhibitors such as aspirin, clopidogrel, and dipyridamole are common options to prevent recurrent stroke. Aspirin has been associated with a 23% lower risk for recurrence than is placebo (BMJ 2002; 324:71). Clopidogrel, an inhibitor of one of the platelet adenosine diphosphate receptors, has efficacy similar to that of aspirin in preventing recurrent events (Lancet 1996; 348:1329). Dipyridamole, a phosphodiesterase inhibitor that raises platelet cyclic adenosine monophosphate levels to inhibit platelet function, is more effective for secondary stroke prevention than is aspirin alone (JW Jun 9 2006).The last of these findings raised expectations that aspirin plus dipyridamole (A+D) would be more …

Platelet cyclic adenosine monophosphate phosphodiesterases: targets for regulating platelet-related thrombosis.

Platelets contain two cyclic adenosine monophosphate (cAMP) phosphodiesterases (PDEs) that regulate the level of cAMP, the major inhibitor of platelet activation pathways. PDE3A hydrolyzes cAMP to 5' AMP with a low K (m). PDE3A is inhibited by cyclic guanosine monophosphate (cGMP), which provides a feedback control and controls basal levels of cAMP. In contrast, PDE2A hydrolyzes both cAMP and cGMP with a high K (m), is allosterically stimulated by cGMP at moderate levels, and may control the stimulated levels of cAMP. Using affinity labeling, chemical modification, and site-directed mutagenesis of highly conserved amino acids, the amino acids required for catalytic activity and/or metal binding are H752 and H756. The singular binding sites for cAMP include N845, E971, and F972, whereas the unique amino acids interacting with cGMP are Y751, H836, H849, and D950. Residues E866 and F1004 are present in both the overlapping cGMP and cAMP sites. Two inhibitors of PDE3A are used in clinical medicine: milrinone and cilostazol. Three amino acids, Y751, D950, and F1004, show decreased sensitivity to both inhibitors (increased K (i)). These inhibitors mimic cGMP as an inhibitor of PDE3A rather than compete for cAMP binding. New nonhydrolyzable affinity labels inactivate PDE3A and are protected by Sp-cAMPS, a nonhydrolyzable substrate of the enzyme. These compounds have the potential to identify amino acids that are unique for PDE3A. An inhibitor of platelet PDE2A increases cAMP more than inhibitors of PDE3A but has much less effect on platelet activation, suggesting that these enzymes are present in different compartments of the cell.

Apolipoprotein (a) mediates the lipoprotein (a)-induced biphasic shift in human platelet cyclic AMP

Lipoproteins are known to influence platelet cyclic adenosine monophosphate (c-AMP) levels. Lipoprotein (a) (Lp(a))'s impact on platelet c-AMP levels has never been assessed. Increasing levels of purified human Lp(a) (1–100) mg/dl were incubated with washed human platelets. Lp(a) concentrations of 1–25 mg/dl resulted an initial statistically significant increase of platelet c-AMP above basal levels and decreased collagen-stimulated platelet aggregation levels. Higher concentrations progressively returned the platelet c-AMP concentrations to basal levels accompanied by further decreases in platelet aggregation. Increasing concentrations of purified apolipoprotein (a) (apo(a)) also resulted in a similar biphasic c-AMP response while Lp(a) without apo(a) was without impact. One antibody directed against apo(a) in intact Lp(a) removed the biphasic c-AMP pattern and eliminated Lp(a) platelet aggregation. Antibodies directed against apo B in intact Lp(a) gave results similar to intact Lp(a) in terms of the biphasic response of c-AMP upon platelet exposure to increasing levels of Lp(a). It is concluded that apo(a) mediates the Lp(a)-induced biphasic response in platelet c-AMP as the result of platelet exposure to increasing levels of Lp(a). The biphasic response in c-AMP assists in platelet aggregation decreases up to a concentration of 25 mg/dl Lp(a), such assistance being lost at higher Lp(a) concentrations.

Disaggregatory effects of prostaglandin E 1 , amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E 1 (PGE 1 ), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE 1 , amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 w M adenosine diphosphate (ADP) or 0.625 w g/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE 1 (0.7-10.0 w M), amrinone (106.8-801.3 w M), or milrinone (9.5-190.0 w M) was added when aggregation reached 5 m z of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC 50 ) were PGE 1 ; 3.1 - 1.2 w M, amrinone; 313.1 - 128.8 w M, milrinone; 57.1 - 20.8 w M. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6 - 5.0% for 10 w M PGE 1 , 7.8 - 2.4% for 801.3 w M amrinone, and 5.9 - 2.3% for 190.0 w M milrinone. The EC 50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67 - 13 pmoles/10 8 platelets for PGE 1 , 54 - 12 pmoles/10 8 platelets for amrinone, and 52 - 12 pmoles/10 8 platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1 - 5.2% or 11.2 - 3.1% for PGE 1 (10 w M), 22.3 - 4.1% or 15.2 - 3.2% for amrinone (801.3 w M), and 23.5 - 4.3% or 14.6 - 3.5% for milrinone (190.0 w M).

Adrenomedullin in liver transplantation and its relationship with vascular complications.

Adrenomedullin (AM) is a potent vasodilating peptide that increases rat platelet cyclic adenosine monophosphate levels and acts on endothelial cells to stimulate nitric oxide release. Both mechanisms inhibit platelet function. Considering these effects, AM may have a role in cardiovascular regulation after orthotopic liver transplantation (OLT) and could have an antithrombotic effect. The aim of the present study is to investigate plasma AM levels in the early postoperative period after OLT and their relationship with vascular complications in OLT. We measured plasma AM levels in 35 patients with cirrhosis who underwent OLT at baseline and 1, 7, and 15 days postoperatively. We found that AM levels were significantly greater in patients with cirrhosis compared with healthy subjects. Of the 35 patients, 10 had vascular complications. In these 10 patients, AM concentrations were significantly greater than those observed in the nonthrombotic group in the early postoperative period. In addition, we also noticed in the nonthrombotic group a significant increase in AM levels from baseline to day 1, then a decrease to baseline levels in the early postoperative period. Our study shows that AM might act as a new humoral factor involved in the response to surgery in OLT and is significantly associated with vascular thrombosis in OLT.

シロスタゾールによる血小板凝集抑制作用の速度論的解析とその処方設計への応用 血小板中ホスホジエステラーゼの可逆的阻害を考慮したＰＫ／ＰＤモデルの構築

Cilostazol is an antithrombotic drug expected to be useful in the treatment of chronic arterial occlusive diseases. Cilostazol is a potent inhibitor of human platelet aggregation and selectively inhibits human platelet cyclic adenosine monophosphate phosphodiesterase type III. In this paper, we describe a pharmacokinetic-pharmacodynamic model for ascertaining the antiplatelet effect of cilostazol considering the reversible inhibition of phosphodiesterase in the platelet. All data used for analysis were obtained from the literature. The estimation of average inhibition of phosphodiesterase was 59.7% following an oral administration with usual dose. A significant linear relationship between the calculated inhibitory effects on phosphodiesterase and on collagen-induced platelet aggregation was obtained (p< 0.05). Based on this finding, a new method was developed. for predicting inhibitory effects on the collagen-induced platelet aggregation following oral administration of cilostazol. This method is useful for planning a rational dosage regimen of cilostazol and for predicting the duration of antiplatelet activity after discontinuance of the drug.

Antiplatelet effects of r, s-( meso)- α, α′- bis[3-( n , n-diethylcarbamoyl) piperidino]- p-xylene ex vivo in the dog and in vivo in the mouse

1. 1. The nipecotamide α,α′-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene (A-1) is a platelet aggregation inhibitor. The meso diastereomer A-1c is superior in potency and duration to the synthetic diastereomeric mixture consisting of the R,R-, S,S-, and R,S- (meso) isomers in inhibiting collagen-induced platelet aggregation ex vivo in the dog. 2. 2. A-1c also is more potent and longer acting than A-1 in protecting mice from collagen+epinephrine-induced thromboembolic death. 3. 3. The mechanism of antiplatelet action of this compound appears to be related to its ability to prevent agonist-induced inhibition of platelet cyclic adenosine monophosphate (cAMP) levels.

Laser aggregometer studies, ATP release and thromboxane B2 release and cAMP concentration of the platelets in nephrotic syndrome

Platelet function was studied in 56 children with nephrotic syndrome, 33 were on oral prednisolone (P) treatment (group 1), while 23 were in early (< 6 months) remission (group 2): 12 on P (group 2a) and 11 not on P (group 2b), and there were 18 controls (group 3). The following tests were used: platelet aggregation with collagen in a laser rheoaggregometer; adenosine triphosphate (ATP) release: during aggregation with luciferin-luciferase in a lumiaggregometer; thromboxane B2 (TXB2) release: by radio-immunoassay; platelet cAMP concentration: by binding assay. The changes in plasma cholesterol (C) and triglycerides (TG) were compared with the platelet aggregation results. Patients in group 1 and 2 exhibited significantly higher aggregability, TXB2 release and ATP release in response to collagen than those in group 3 (p < 0.01), but there was no difference between groups 1 and 2 or groups 2a and 2b. Some differences were observed between the histological groups. Patients with IgA and SLE nephropathy displayed higher aggregability than those with minimal change nephrotic syndrome in remission (p < 0.05). The highest level was in membranous nephropathy. The platelet cyclic adenosine monophosphate (cAMP) concentration was significantly lower in groups 1 and 2 than in group 3 (p < 0.001). No differences were observed between groups 1 and 2 or between groups 2a and 2b. Plasma C and TG levels did not show any correlation with the platelet aggregation. The increased platelet aggregation, TXB2 release and ATP release and decreased cAMP concentration in the platelets may play a role in the pathogenesis of nephrotic syndrome which remains altered in early remission. Oral P and plasma lipids did not change the platelet function significantly.

Flow cytometric studies of platelet responses to shear stress in whole blood.

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates ("large" refers to particles larger than 10 microns in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1- 14C]oleate to individual phospholipids of platelets

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1- 14C]oleate resulting in esterification of [1- 14C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1- 14C]oleate and esterification of [1- 14C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.

Iloprost and echistatin protect platelets during simulated extracorporeal circulation

Temporary, reversible inhibition of platelets during cardiopulmonary bypass is an attractive strategy to protect platelets and normalize postoperative bleeding times. Iloprost, an analogue of prostacyclin, and the disintegrins reversibly inhibit platelets by different mechanisms. We tested the hypothesis that reduced doses of iloprost and either echistatin, a natural disintegrin, or RO43-5054, a peptidomimetic, in combination provide better platelet protection than any drug alone during simulated extracorporeal circulation. Thirty-five recirculation studies using fresh, heparinized human blood in an extracorporeal perfusion circuit that contained a 0.45-m2 spiral coil membrane oxygenator were performed. Iloprost, but neither echistatin nor RO43-5054, increased platelet cyclic adenosine monophosphate. Combinations of iloprost and either fibrinogen receptor antagonist at reduced doses submaximally increased platelet cyclic adenosine monophosphate. Platelet adhesion and release of beta-thromboglobulin antigen was completely inhibited by combinations of the two classes of drugs, but only partially inhibited by each drug alone. Combinations of drugs also completely inhibited platelet aggregation to adenosine diphosphate; these platelets retained full sensitivity to adenosine diphosphate after 90 minutes of recirculation when drugs were removed by gel filtration. We conclude that combinations of iloprost and a fibrinogen receptor antagonist at doses that are unlikely to produce clinical side effects completely inhibit platelet activation and preserve platelet function during in vitro extracorporeal circulation.

Changes in cardiovascular risk profile during the cessation of smoking

The present study evaluated the intraindividual changes of serum lipids, blood pressure, adrenergic stimulation, and platelet reactivity in chronic smokers undergoing controlled smoking cessation assisted by nicotine chewing gum. One hundred twenty-one healthy smokers, who had smoked at least 20 cigarettes a day for at least 5 years, were included in the study. Serum lipids, blood pressure, catecholamine concentrations in serum and urine, platelet volume and platelet function in vitro were assessed during an initial phase of continued smoking, after stopping smoking during nicotine gum chewing, and after abstaining from both smoking and gum chewing. After a median of 11 weeks of chewing nicotine gum, 52 persons (43%) remained abstinent from smoking and chewing for at least 12 weeks. In these successful study participants, low-density lipoprotein (LDL)-cholesterol levels decreased significantly by a mean of 7.5 mg/dL (5.6%). High-density lipoprotein (HDL)-cholesterol increased by 5 to 6 mg/dL during the first weeks after ceasing to smoke, but levels at the end of study were only 2.1 mg/dL (3.4%) higher than baseline (nonsignificant difference). Triglyceride levels did not change during smoking cessation. While systolic blood pressure remained unchanged, diastolic blood pressure increased significantly by a mean of 3.5 mm Hg. For LDL-cholesterol, HDL-cholesterol, and systolic and diastolic blood pressure, there was an inverse correlation between the respective baseline values and the observed changes during the cessation of smoking. The urinary excretion of adrenaline and noradrenaline decreased upon quitting. The volume of platelets became significantly less during smoking cessation, but aggregation induced by adenosine diphosphate (ADP) and collagen and thromboxane synthesis in vitro remained uninfluenced. The platelet cyclic adenosine monophosphate (cAMP) response to stimulation of adenylate cyclase with prostaglandin E 1 showed marked enhancement upon quitting. The changes observed intraindividually during smoking cessation largely correspond to the differences between smokers and nonsmokers described in epidemiologic studies. For serum lipids and blood pressure, a significant dependence of the changes on the respective values at baseline may indicate greater benefit for persons at higher risk. Decreased platelet volume and increased susceptibility of platelets to antiaggregatory prostaglandin E 1 indicate a favorable influence of quitting on platelet reactivity.

Laser aggregometer studies, ATP release and thromboxane B 2 release and cAMP concentration of the platelets in nephrotic syndrome

Platelet function was studied in 56 children with nephrotic syndrome, 33 were on oral prednisolone (P) treatment (group 1), while 23 were in early (< 6 months) remission (group 2): 12 on P (group 2a) and 11 not on P (group 2b), and there were 18 controls (group 3). The following tests were used: platelet aggregation with collagen in a laser rheoaggregometer; adenosine triphosphate (ATP) release: during aggregation with luciferin-luciferase in a lumiaggregometer; thromboxane B 2 (TXB 2) release: by radio-immunoassay; platelet cAMP concentration: by binding assay. The changes in plasma cholesterol (C) and triglycerides (TG) were compared with the platelet aggregation results. Patients in group 1 and 2 exhibited significantly higher aggregability, TXB 2 release and ATP release in response to collagen than those in group 3 (p < 0.01), but there was no difference between groups 1 and 2 or groups 2a and 2b. Some differences were observed between the histological groups. Patients with IgA and SLE nephropathy displayed higher aggregability than those with minimal change nephrotic syndrome in remission (p < 0.05). The highest level was in membranous nephropathy. The platelet cyclic adenosine monophosphate (cAMP) concentration was significantly lower in groups 1 and 2 than in group 3 (p < 0.001). No differences were observed between groups 1 and 2 or between groups 2a and 2b. Plasma C and TG levels did not show any correlation with the platelet aggregation. The increased platelet aggregation, TXB 2 release and ATP release and decreased cAMP concentration in the platelets may play a role in the pathogenesis of nephrotic syndrome which remains altered in early remission. Oral P and plasma lipids did not change the platelet function significantly.

Alprostadil: An Effective Antiplatelet Agent for Calves

Aspirin and other nonsteroidal, antiinflammatory drugs inhibit human platelets, but their effect as an antiplatelet agent on bovine platelets is not certain. Since calves are used for cardiovascular implant research, need exists for an effective antiplatelet agent for this animal model. After screening a number of potential antiplatelet drugs for calves, Alprostadil (prostaglandin E1) appeared to be the most promising. Alprostadil was administered (intravenous continuous drip) to 8 calves. The concentration of the drug administered was gradually increased until 50% inhibition of platelet aggregation was obtained. Platelet function, blood pressure, body temperature, and hematologic parameters were closely monitored. For in vitro evaluation, Alprostadil was added to human or bovine blood, and the platelet aggregation and cyclic adenosine monophosphate levels were measured. Alprostadil inhibited both bovine and human platelets, although bovine platelets were relatively more responsive to this drug. At an infusion rate of approximately 0.20 micrograms/kg/min in vivo, Alprostadil showed 50% inhibition of platelet aggregation with slightly decreased blood pressure (7 +/- 6 mm Hg) but no adverse effects. Complete reversal of in vivo platelet inhibition was noted within 24 h after cessation of drug administration. This short half-life and the lack of significant adverse effects make Alprostadil an attractive antiplatelet agent for calves with cardiovascular implants.

Effect of Prostacyclin on Platelet Intracellular Free Calcium Concentration of Hypertensive and Normotensive Humans

Platelet intracellular free calcium concentration ([Ca2+]i) has been reported to be increased in essential hypertensive patients (EHT) as compared with normotensive controls (NT). Prostacyclin (PGI2), which influences cellular Ca2+, has been reported to be reduced in EHT. This study tested the hypothesis that the resting level of platelet [Ca2+]i in humans is influenced by PGI2. We also investigated the role of PGI2 in regulating platelet [Ca2+]i of 28 EHT subjects compared to 28 NT controls. Platelet [Ca2+]i was measured using the fluorescent Ca2+ probe fura-2 under control conditions and a 10-min preincubation with PGI2. Simultaneous measurement of platelet cyclic-adenosine 3':5'-monophosphate (cAMP) was performed by radioimmunoassay. The resting level of platelet [Ca2+]i was significantly higher in EHT than in NT (32.7 +/- 1.4 v 28.3 +/- 0.9 nmol/L; P < .01). PGI2 from 30 nM to 1 mumol/L lowered the resting level of platelet [Ca2+]i in a dose-dependent manner (EHT -22.2 +/- 2.4, NT -22.9 +/- 2.3%, 1 mumol/L PGI2); however, no significant difference in platelet [Ca2+]i was observed between NT and EHT. While prostacyclin induced a transient rise in platelet cAMP, the magnitude of PGI2-induced cAMP level was similar between the two groups. These results do not support the hypothesis that endogenous PGI2 activity contributes to the increased level of platelet [Ca2+]i in EHT, although PGI2 incubation lowered the resting level of platelet [Ca2+]i.