Plant-based Food Cyanidin-3-Glucoside Modulates Human Platelet Glycoprotein VI Signaling and Inhibits Platelet Activation and Thrombus Formation.

Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 µM) of Cy-3-g. The expression of activated integrin αIIbß3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-µM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-µM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-µM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-µM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk-linker for activation of T cells (LAT)-SLP76 (Syk: -39.08%, LAT: -32.25%, SLP76: -40.00%) and the expression of Lyn (-31.89%), Fyn (-36.27%), and phospholipase C-γ2 (-39.08%).Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gαs Signaling in Platelets.

OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.

Alumina nanocomposites: a comparative approach highlighting the improved characteristics of nanocomposites for phosphopeptides enrichment.

The work is based on the comparative study of metal oxide nanocomposites based on alumina in combination with two transition metal oxides (zirconia and titania) and two lanthanide oxides (ceria and lanthanum oxide). The choice is based on specific aims, i.e. to improve the limitations of individual metal oxides in phosphopeptide enrichment. The nanocomposites have shown improved phosphopeptide enrichment efficiency in comparison to the individual metal oxides. Alumina-zirconia show enhanced mono-phosphorylated peptide enrichment than ZrO2 whereas alumina-titania has better recovery of mono- and multi-phosphorylated peptides in comparison to individual TiO2. Alumina-ceria and alumina-lanthanum oxide overall enrich higher number of phosphopeptides. The alumina nanocomposites show higher selectivity and sensitivity for spiked ß-casein in BSA (1:1000) and diluted ß-casein digest (10 femtomole), respectively. Through the transition metal oxide nanocomposites, number of phosphoproteins from human serum are identified while this number is highest in case of alumina-lanthanum oxide nanocomposite. Thus the enrichment is affected by the choice of metal oxide in the nanocomposite based enrichment strategies.

Dietary resistant maltodextrin ameliorates testicular function and spermatogenesis in streptozotocin-nicotinamide-induced diabetic rats.

This study investigated the effect of resistant maltodextrin (RMD) on reproduction in streptozotocin (STZ)-nicotinamide-induced type 2 diabetic male rats. Forty male rats were induced with diabetes by a single intraperitoneal injection of STZ (50 mg kg(-1)) and nicotinamide (100 mg kg(-1)). Five groups were analysed in total: normal, diabetic rats without RMD, diabetic rats with RMD 1.2 g per 100 g diet (1×), with RMD 2.4 g per 100 g (2×), and with RMD 6.0 g per 100 g (5×). The groups of diabetic rats with the RMD supplement, compared to those without supplement, showed improved plasma glucose control, attenuated insulin resistance and recovery of testosterone level and spermatogenesis stage. The STZ-nicotinamide-induced diabetes mellitus (DM) caused a significant reduction in serum testosterone, testis androgen receptor (AR), steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) protein, but a statistical recovery in each of these was observed in the 5× group. TUNEL-positive cells were observed in the diabetic without RMD group, and RMD treatment reduced apoptotic germ cells. The expression of Bax/Bcl2 was induced in the diabetic group and also significantly reduced in the 5× group. Dietary RMD may improve metabolic control in STZ-nicotinamide-induced diabetic rats and attenuate hyperglycaemia-related impaired male reproduction and testicular function.

CPU86017-RS attenuate hypoxia-induced testicular dysfunction in mice by normalizing androgen biosynthesis genes and pro-inflammatory cytokines.

AIM: Downregulation of androgen biosynthesis genes StAR (steroidogenic acute regulatory) and 3ß-HSD (3ß-hydroxysteroid dehydrogenase) contributes to low testosterone levels in hypoxic mice and is possibly related to increased expression of pro-inflammatory cytokines in the testis. The aim of this study is to investigate the effects of CPU86017-RS that block Ca(2+) influx on hypoxia-induced testis insult in mice. METHODS: Male ICR mice were divided into 5 groups: control group, hypoxia group, hypoxia group treated with nifedipine (10 mg/kg), hypoxia groups treated with CPU86017-RS (60 or 80 mg/kg). Hypoxia was induced by placing the mice in a chamber under 10%±0.5% O2 for 28 d (8 h per day). The mice were orally administered with drug in the last 14 d. At the end of experiment the testes of the mice were harvested. The mRNA and protein levels of StAR, 3ß-HSD, connexin 43 (Cx43), matrix metalloprotease 9 (MMP9), endothelin receptor A (ET(A)R) and leptin receptor (OBRb) were analyzed using RT-PCR and Western blotting, respectively. The malondialdehyde (MDA), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acid phosphatase (ACP) levels were measured using biochemical kits. Serum testosterone concentration was measured with radioimmunoassay. RESULTS: Hypoxia significantly increased the MDA level, and decreased the LDH, ACP and SDH activities in testes. Meanwhile, hypoxia induced significant downregulation of StAR and 3ß-HSD in testes responsible for reduced testosterone biosynthesis. It decreased the expression of Cx43, and increased the expression of MMP9, ETAR and OBRb, leading to abnormal testis function and structure. These changes were effectively diminished by CPU86017-RS (80 mg/kg) or nifedipine (10 mg/kg). CONCLUSION: Low plasma testosterone level caused by hypoxia was due to downregulation of StAR and 3ß-HSD genes, in association with an increased expression of pro-inflammatory cytokines. These changes can be alleviated by CPU86017-RS or nifedipine.

In vivo modulation of 4E binding protein 1 (4E-BP1) phosphorylation by watercress: a pilot study.

Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80 g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297 nm following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8 h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.

Effects of polyunsaturated omega-3 fatty acids on responsiveness to dual antiplatelet therapy in patients undergoing percutaneous coronary intervention: the OMEGA-PCI (OMEGA-3 fatty acids after pci to modify responsiveness to dual antiplatelet therapy) study.

OBJECTIVES: The purpose of this study was to investigate whether omega-3 polyunsaturated fatty acids (PUFAs) are able to modify platelet responsiveness to dual antiplatelet therapy in stable coronary artery disease patients undergoing percutaneous coronary intervention (PCI). BACKGROUND: Although previous studies have suggested antiplatelet properties of omega-3 polyunsaturated fatty acids, it is unknown whether they can enhance platelet inhibition on standard aspirin and clopidogrel treatment. METHODS: The OMEGA-PCI (OMEGA-3 Fatty Acids After PCI to Modify Responsiveness to Dual Antiplatelet Therapy) study was an investigator-initiated, prospective, single-center, double-blind, placebo-controlled, randomized study. Patients receiving standard dual antiplatelet therapy (aspirin 75 mg/day and clopidogrel 600 mg loading dose followed by 75 mg/day) were randomly assigned to receive the addition of 1 g of omega-3 ethyl esters (n = 33) or placebo (n = 30) for 1 month. Platelet function was measured serially by light transmission aggregometry (adenosine diphosphate and arachidonic acid [AA] were used as agonists) and assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein at baseline, 12 h, 3 to 5 days, and 30 days after randomization. RESULTS: The P2Y(12) reactivity index was significantly lower, by 22.2%, after 1 month of treatment with omega-3 polyunsaturated fatty acids compared with placebo when used in addition to dual antiplatelet therapy (p = 0.020). Maximal platelet aggregation induced by 5 and 20 micromol/l adenosine diphosphate was lower by 13.3% (p = 0.026) and 9.8% (p = 0.029), respectively, after 1 month of treatment with omega-3 polyunsaturated fatty acids compared with placebo. Platelet aggregation after AA stimulation was low and did not change significantly throughout the study. There were no cases of aspirin resistance during follow-up that was suggestive of good compliance with the medication. CONCLUSIONS: The addition of omega-3 ethyl esters to the combination of aspirin and clopidogrel significantly potentiates platelet response to clopidogrel after percutaneous coronary intervention.

Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb(3+)-tiron complex as a fluorescence probe.

A sensitive method based on the fluorescence quenching effect of the Tb(3+)-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus (ALP) released from fish plasma. The detection limit was 5.4 ng/ml (S/N = 2), and the relative standard deviation of the quenching effect (6 replicates) was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.

Inflammation and carnitine in hemodialysis patients.

The bioincompatibility of dialytic systems along with the loss of antioxidant substances via the dialysis may contribute to peripheral blood mononuclear cell (PBMC) activation and the production of inflammatory mediators, such as cytokines, oxygen radicals, and complement fragments, that may sustain a state of chronic microinflammation responsible for the pathogenesis of a variety of diseases, including atherosclerosis, anemia, and malnutrition. Moreover, during hemodialysis (HD), oxidative stress may influence several intracellular signaling enzymes, including some stress-activated kinases, such as jun-N-terminal kinase (JNK), potentially leading to PBMC activation and proinflammatory cytokine production. Recent reports suggest that L-carnitine may play an important role in balancing antioxidative systems. Therefore, we sought to evaluate the effect of L-carnitine supplementation on the PBMC responses to oxidative stress induced by different HD membranes. We observed in PBMC from cellulosic (C)-treated patients an increase in the amount of intracellular tyrosine-phosphorylated proteins and a striking activation of JNK, as compared with synthetic (S)-treated patients. On the contrary, 3 months of L-carnitine supplementation significantly lowered intracellular levels of phosphorylated proteins and JNK activity in PBMC from C-treated patients. In addition, after 180 minutes of HD, a significant decrease in global plasma antioxidant capacity was found, particularly in C-treated patients, whereas L-carnitine supplementation improved plasma antioxidant capacity levels in these patients. These observations were also confirmed by in vitro experiments, showing the ability of L-carnitine to reduce the JNK activation in normal PBMC exposed to different amounts of hydrogen peroxide. In conclusion, the uremic milieu is characterized by an enhanced inflammatory response and an increased oxidant load, affecting lipids, carbohydrates, and proteins. Regular L-carnitine supplementation in HD patients can improve cellular defense against chronic inflammation and oxidative stress, most likely by modulating the specific signal transduction cascade activated by an overproduction of proinflammatory cytokines and oxidative stress.

[Effects of siwu tang on protein expression of bone marrow of blood deficiency mice induced by irradiation].

OBJECTIVE: To observe the effects of siwu tang on protein expression of bone marrow of blood deficiency mice and provide the theoretical and experimental basis for understanding the molecular mechanism of blood enriching function of siwu tang. METHOD: Blood deficiency mice were established by using 3.5 Gy 60Co gamma-ray. With proteomic technologies including two-dimensional electrophoresis, image analysis, in-gel digestion, peptide mass fingerprinting and bioinformatics the proteins of bone marrow of blood deficiency mice were isolated, analyzed, and identified. RESULT: Siwu tang could reverse 10 up-regulated and 4 down-regulated proteins of blood deficiency mice bone marrow. Seven of the proteins were likely to be lymphocyte specific protein 1, proteasome 26S ATPase subunit 4, hematopoietic cell protein-tyrosine phosphatase, glyceraldehyde-3-phosphate dehydrogenase, growth factor receptor binding protein 14 and lgals12 respectively. CONCLUSION: Siwu tang can regulate the protein expression of bone marrow of blood deficiency mice and thus promote the growth and differentiation of hematopoietic cells and then exert its effects on blood enriching function.

Serum levels of matrix extracellular phosphoglycoprotein (MEPE) in normal humans correlate with serum phosphorus, parathyroid hormone and bone mineral density.

Matrix extracellular phosphoglycoprotein (MEPE), a member of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family, is primarily expressed in normal bone and has been proposed as a phosphaturic factor because of high expression and secretion in oncogenic hypophosphatemic osteomalacia tumors. In order to begin to address the role of MEPE in normal human physiology, we developed a competitive ELISA to measure serum levels of MEPE. The ELISA was used to characterize the distribution pattern in a population consisting of 114 normal adult subjects. The mean value of MEPE was 476 +/- 247 ng/ml and levels decreased significantly with increasing age. MEPE levels were also significantly correlated with serum phosphorus and parathyroid hormone (PTH). In addition, MEPE levels correlated significantly with measures of bone mineral density in the femoral neck and total hip in a subset of 50 elderly subjects. The results are consistent with MEPE being involved in phosphate and bone metabolism in a normal population.

Dietary branched-chain amino acids suppress the expression of pancreatic amylase mRNA in rats.

Regulators for pancreatic amylase were examined. Rats were fed ad libitum a 20% amino acid (AA) mixture diet (Con), a 60% AA diet (HA), a branched-chain amino acid (BCAA)-rich diet (BC), or a diet supplemented with AA other than BCAA (OA) for 7 d, or fed the Con, HA, BC diets or diets supplemented with individual BCAA. Activity and mRNA levels of pancreatic amylase in the BC and HA groups were lower than those in the Con and OA groups. Leucine and isoleucine contributed to these effects of the BC diet. The mRNA levels correlated with individual pancreatic BCAA concentrations but not with plasma insulin level. In conclusion, dietary BCAA, especially leucine and isoleucine, may reduce amylase mRNA and activity in rats.

Effect of three triterpenoid compounds isolated from root bark of Aralia elata on stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation of p47(phox) and p67(phox) to cell membrane in human neutrophil.

BACKGROUND: Root bark of Aralia elata is used as a folk medicine for neurasthenia, rheumatism, diabetes, hepatitis virus and spasm of the stomach in China, Japan and Russia. METHODS: The effect of three triterpenoid compounds isolated from root bark of A. elata on stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation of p47(phox) and p67(phox) to cell membrane was investigated. The three compounds examined were Elatoside A, Elatoside C, and Tarasaponin V. RESULTS: When the cells were preincubated with these compounds, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was suppressed in a low concentration range. However, the superoxide generation was significantly enhanced by 40 micromol/l triterpenoid, and was again suppressed in the higher concentration range. In the case of superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), the compounds had no obvious effect on the superoxide generation in low concentration but suppressed that at 40 micromol/l. These compounds also efficiently suppressed the superoxide generation induced by arachidonic acid (AA) at 10 micromol/l. In parallel to the effect on the fMLP-induced superoxide generation, these compounds suppressed fMLP-induced tyrosyl phosphorylation and the translocation to membrane of cytosolic compounds, p47(phox) and p67(phox) at 10 and 80 micromol/l but not at 40 micromol/l. CONCLUSIONS: Triterpenoid saponins examined in this study effect stimulus-induced superoxide generation and tyrosyl phosphorylation and translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner, and may have some pharmaceutical applications.

Up-regulation of nucleolin mRNA and protein in peripheral blood mononuclear cells by extracellular-regulated kinase.

The signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2-2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3'-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production.

Biochemical characterization of 60S acidic ribosomal P proteins from porcine liver and the inhibition of their immunocomplex formation with sera from systemic lupus erythematosus (SLE) patients by glycyrrhizin in vitro.

The three casein kinase II (CK-II) phosphate acceptors (p35, p17 and p15) in the Superdex CK-II fraction prepared from a 1.5 M NaCl extract of porcine liver were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC) as a heterocomplex associated with CK-II. Determination of the N-terminal amino acid sequences and immunological tests confirmed that these three CK-II phosphate acceptors belong to the family of 60S acidic ribosomal proteins (P0, P1 and P2). Three polyphenol-containing anti-oxidant compounds [catechin, epigallocatechin gallate (EGCG) and quercetin] inhibited CK-II activity (phosphorylation of these ribosomal P proteins) in a dose-dependent manner in vitro. Quercetin (ID50 = approx. 50 nM) was found to be an effective CK-II inhibitor. In contrast, CK-II activity was significantly stimulated by lower doses (0.3-3 microl) of GL, but was inhibited at high doses above 30 microM. As expected, GL at high doses above 200 microM inhibited the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera from patients with systemic lupus erythematosus (SLE). These results suggest that (i) a GL-affinity column is useful for effective purification of 60S acidic ribosomal P proteins from various mammalian cells as a heterocomplex associated with CK-II; and (ii) a relative high dose of GL may prevent the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera of SLE patients.

Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors.

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations.

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.

The nature of the phosphorus linkage in component I, a phosphoprotein isolated from the blood serum of DES-treated cockerels.

The nature of the phosphorus linkage in electrophoretic component I of the blood serum of DES-treated cockerels was investigated by enzymatic and partial acid and alkali dephosphorylation. The C-P bond was excluded because this bond is stable to 6 N HCl at 110 degrees C. for 20 hours, and all the phosphorus was released under these conditions. No phosphorus was released when diesterase, pyrophosphatase and 0.25 N HCl were employed, and this excludes the diester, pyrophosphate and N-P bonds. The presence of the O-P bond was supported by the release of phosphate by the phosphatases and 0.25 N NaOH. The hydrolysate produced upon hydrolysis with pronase and papain contained phosphoserine. No phosphothreonine was present.