Mechanisms of the protease-induced aggregation of human platelets

The role of phospholipase C (an enzyme involved in the metabolism of inositol-containing phospholipids), cyclooxygenase and lipoxygenase (the enzymes of arachidonic acid metabolism), and adenylate cyclase and phosphodiesterase (the enzymes of cyclic adenosine 3,5-monophosphate (cAMP) metabolism) in the mechanisms of the aggregation of human platelets induced by the serine protease in low concentrations (thrombin 0.5 mkg per ml, trypsin 1 mkg per ml, and alpha-chymotrypsin 10 mkg per ml) have been investigated with the use of the inhibitor analysis. The effect of neomycin sulfate (phospholipase C inhibitor), indometacin (cyclooxygenase inhibitor), and nordihydrogvayaretic acid (lipoxygenase inhibitor) on protease-induced increase in the content of calcium cations in platelet plasma has been studied. The results of the inhibitor analysis indicated that the enzymes of metabolism of inositol-containing phospholipids, arachidonic acid, and cAMP are involved in the mechanisms of the protease-induced platelet aggregation. The increase in the content of calcium ions, associated with the protease-induced activation of phospholipase C, in cytoplasm may play an important role in the mechanisms of platelet aggregation.     

Human blood platelets possess specific binding sites for C1q

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.     

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.     

Effect of ozone exposure on contractile activity and chemoreactivity of uterus horns longitudinal muscles of nonpregnant rats

With the purpose of studying the mechanism of ozone action on uterus smooth muscles it was investigated the influence of ozone-content (approximately 0.50 mkg/ml) Krebs' solution or its 10- and 100-fold dissolution on contractile activity and beta-adrenoreactivity of 56 longitudinal strips of uterus horns of 17 nonpregnant rats. Ozone at concentration approximately 0.50 mkg/ml (but not in concentration of approximately 0.05 and approximately 0.005 mkg/ml) reversibly raised frequency, amplitude and total contractile activity of intact myometrium strips, and also fast and reversibly reducel its beta-adrenoreactivity, i.e. decreased of inhibitory action of adrenaline (10(-8), 10(-7), 10(-6) g/ml), but did not change uterostimulatory effect of acetylcholine (10(-6) g/ml) and oxiyocin (5 x 10(-4) ME/ml), what evident about specificity of ozone beta-adrenoblokate effect. Ozone (approximately 0.50 and 0.05 mkg/ml) did not change ov value of potassium contracture of myometrium strips which was depolarized by hyperpotassium (60 mM KCL) Krebs' solution, but reduced inhibitory action of adrenaline (10(-8) g/ml). The question is being discussed about mechanisms of ozone beta-adrenoblocade actions, about clinical role of this phenomenon, and the possibility of using beta-adrenoreceptor sensibilizators direct action (histidine, tryptophan, tyrosine, trimetazidin and mildronat) at ozonotherapy with the purpose reduction of its negative effects.     

In vitro induction of numerical changes in the karyotype of normal and transformed Djungarian and Chinese hamster cells]

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.     

C1q (c1) receptor on human platelets: inhibition of collagen-induced platelet aggregation by C1q (C1) molecules.

Hemolytically active human C1q incubated with EA before the addition of complement inhibited the immune hemolysis. On the contrary, heat-inactivated preparation (30 min 56 degrees C) was ineffective. Preincubation of EA with bovine collagen also resulted in a decreased hemolysis. When aggregation was measured by a turbidimetric method in citrated human platelet-rich plasma, it was found that hemolytically active human C1q (C1) alone does not induce platelet aggregation. However, in its presence the platelets failed to aggregate or exhibited a significantly reduced aggregation response to bovine collagen. The inhibition by C1q depended on the preincubation time with platelets. Heat treatment (30 min 56 degrees C) destroyed the inhibitory action of C1q (C1). The effect of C1q proved to be highly specific because different C1q preparations at their inhibitory doses in collagen-induced platelet aggregation did not influence the response to other aggregating agents (bovine thrombin, ADP, horse anti-human thymocyte globulin, goat anti-baboon platelet antiserum). The results prove that collagen and C1q are capable of binding to the same site(s); namely, to those of EA and human platelets; furthermore, they suggest the presence of a receptor for C1q (C1) on human platelets.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

A meta-analysis and genome-wide association study of platelet count and mean platelet volume in african americans

Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N?=?16,388) with p<5×10(-8) of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p?=?9.1×10(-9)), 7q11 (rs13236689, CD36, p?=?2.8×10(-9)), 10q21 (rs7896518, JMJD1C, p?=?2.3×10(-12)), 11q13 (rs477895, BAD, p?=?4.9×10(-8)), and 20q13 (rs151361, SLMO2, p?=?9.4×10(-9)). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N?=?14,909) and one (11q13) in Hispanic Americans (N?=?3,462). For MPV (N?=?4,531), genetic variants in 3 regions were significant at p<5×10(-8), two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis.     

Effects of bopindolol on platelet function in hypertension at rest and during exercise

The effects of bopindolol, a new nonselective beta-blocking agent, on platelet function have been studied in 10 male hypertensive patients given the drug (1 mg/day) in turn for eight weeks. Bopindolol significantly (p less than 0.01) decreased the bicycle exercise- (1.5 W/kg body weight for 6 minutes) induced increase in platelet aggregation. During bopindolol-treatment both the slope and the height of the platelet aggregation response curve were moderately decreased at rest before exercise and significantly (p less than 0.05) decreased at rest after exercise. During exercise the slope amounted to 75.4 +/- 44 degrees before and to 70.8 +/- 5.3 degrees after therapy (p less than 0.01), the height to 64.0 +/- 11.9% before and to 58.1 +/- 14.7% (p less than 0.05) after therapy. Furthermore, bopindolol significantly increased the exercise-induced decrease in platelet sensitivity to PGI2 (p less than 0.05; IC-50-value: 2.10 +/- 0.47 vs 1.88 +/- 0.31 ng/ml) and PGD2 (p less than 0.05; IC-50-value: (19.88 +/- 2.10 vs 18.57 +/- 1.63 ng/ml). Bopindolol also significantly (p less than 0.05) decreased the exercise-induced elevation in serum-TXB2 (244.9 +/- 35.2 vs 237.3 +/- 27.2 ng/ml) and plasma-TXB2 (15.7 +/- 6.3 vs 13.1 +/- 3.7 pg/ml). The platelet count, the plasma levels of 6-oxo-PGF1 alpha, beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) were not affected by bopindolol. It is concluded that bopindolol favourably affects platelet function, in that it lowers exercise-induced platelet aggregation and TXB2-formation in therapeutical doses and increases platelet sensitivity to antiaggregatory prostaglandins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanidinoacetate-N-methyltransferase: location in mammalian retina and rat Harderian gland]

Homogenates of retinal external segments of rat, rabbit, beef and hen and of rat Harderian gland were found to possess a considerable activity of guanidineacetate-N-methyltransferase (GAMT, E.C. 2.1.1.2), comparable with the enzyme activity in liver, pancreas and testis. Permanent UV-illumination of rats (from 1 day to 1 week) resulted in the decrease of GAMT activity in retina and especially in Harderian gland. Caffeine (10(-4) M) and papaverine (10(-7) M) activated GAMT in retina and rat Harderian gland, while cycloheximide, a protein synthesis inhibitor (0.5-2 mkg/ml), eliminated caffeine-stimulated GAMT activity. Histamine (0.3 mkg/ml) inhibited GAMT activity both in retina and Harderian gland. A decrease of GAMT activity in retina, liver and testis of rat and an increase of the enzyme activity in rat pancreas and Harderian gland were observed in the presence of Mg2+ (5 mM). Physiological importance of GAMT and creatine in mammalian retina and rat Harderian gland is discussed.     

Effect of opioid peptides on oxygen-dependent microbicidity of peripheral blood neutrophils

Investigation ofopioid peptide effect on the production of reactive oxygen species by neutrophils in non-fractionated leukocyte suspension and in purified fraction of peripheral blood neutrophils is disclosed in this work. It was determined that selective delta- and micro-agonists of peptide origin stimulated the spontaneous and suppressed 15 mkg/ml zymosan-induced LDCL (luminol-dependent chemiluminescence) reaction of neutrophils in leukocyte suspension. beta-endorphin was found to render less marked suppressive action on 15 mkg/ml zymosan-induced LDCL, and delta2-agonist deltorphin 2 promoted 15 mkg/ml zymosan-induced LDCL only toward the 25 minutes of the experiment. beta-endorphin and selective d- and m- agonists did not affect the spontaneous and suppressed 15 mkg/ml and 150 mkg/ml zymosan-induced neutrophil LDCL. Therefore, opioid peptides play essential role in the process of direct and indirect regulation of oxygen-dependent system of neutrophil granulocyte bactericidal activity.     

The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.     

Mitomycin C induced structural changes in heterochromatic regions of human chromosomes 1, 9, 16 and Y

Aberrations and variations in the heterochromatic blocks of chromosomes 1, 9, 16 and Y were found under the influence of mitomycin C in cultured lymphocytes of peripheral human blood. Lymphocytes were cultured during 96 hours, mitomycin C in final concentration of 0.3 mkg/ml was present in the culture during the latest 24 hours of culturing. Different changes in the heterochromatic regions of chromosomes were found in approximately 30% of cells: in 6.3% of cells mitotic chiasmata were indicated. In 9.5% of cells isolocus breaks were observed in heterochromatic region of chromosome 1 in segment 1q11. In the latter case this may be a fragile site detected under the influence of mitomycin C on the lymphocytes.     

Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation.

The effects of aqueous extracts of raw and boiled garlic and onions were studied in vitro on the collagen-induced platelet aggregation using rabbit and human platelet-rich plasma. A dose dependant inhibition of rabbit platelet aggregation was observed with garlic. Onion also showed dose-dependent inhibitory effects on the collagen-induced platelet aggregation but this inhibition was of a lesser magnitude compared to garlic when related to dose. The concentration required for 50% inhibition of the platelet aggregation for garlic was calculated to be approximately 6.6 mg ml(-1) plasma, whereas the concentration for onion was 90 mg ml(-1) plasma. Boiled garlic and onion extracts showed a reduced inhibitory effect on platelet aggregation. Garlic but not onion significantly inhibits human platelet aggregation in a dose-dependent fashion. The potency of garlic in inhibiting the collagen-induced platelet aggregation is approximately similar to that of rabbit platelets (8.8 mg ml(-1) produced 50% inhibition of platelet aggregation). The results of this study show that garlic is about 13 times more potent than onion in inhibiting platelet aggregation and suggest that garlic and onion could be more potent inhibitors of blood platelet aggregation if consumed in raw than in cooked or boiled form.     

Inhibition of thrombocyte aggregation by antioxidants

The effects of antioxidants (3-hydroxypyridines, 5-hydroxypyrimidines, hindered phenols) on platelet aggregation were studied. All the compounds under study possessed low anti-aggregation activity against indometacin-sensitive aggregation (activation with arachidonic acid, 50 M). Half-maximal inhibition of aggregation was achieved at a concentration similar to that of the compounds used (10(-3) M in cases of indomethacin-insensitive aggregation, platelet activation by thrombine 1.5 mu/ml and Ca2+-ionophore A23187 1.5 g/ml). 4-methyl-2.6-ditretbutyl phenol (BHT) in the concentration range of 10(-5)-4 X 10(-5) M inhibited and in the concentration range of 4 X 10(-5)-10(-4) M activated indomethacin-sensitive aggregation. The latter effect was not observed in the absence of Ca2+ ions in the incubation medium. It is concluded that the effects of the antioxidants studied on platelet aggregation were due to their non-specific action on platelet membranes.     

Analgos of riboflavin, lumiflavin and alloxazine derivatives. II. Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis]

The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites. They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin. Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied. Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain. An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively. Riboflavin (1 mkg/ml) recovers the growth of bacteria. The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin. 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected. 150 mutants over-synthetize riboflavin.     

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.     

A spectrophotometric method for following initial rate kinetics of blood platelet aggregation

A double-beam recording spectrophotometer was used to assay platelet aggregation. Agonist-induced turbidity changes, at 540 nm, in dilute suspensions of platelets (1 ml, 6-8 X 10(7) platelets) were recorded differentially against a reference cuvette, also containing platelets, as a function of time. The curves obtained showed downward pen deflections (decrease of turbidity) abolished by preincubation with the aggregation inhibitor, citrate. The turbidity decrease occurred simultaneously with microscopically determined single platelet recruitment into aggregates and its initial slope (r0) was a linear function of platelet concentration upto approximately 1.5 X 10(8) per ml. At a fixed platelet concentration the r0 values of ADP-induced aggregation of calf platelet-rich plasma varied as a hyperbolic function of ADP concentration at both 32 degrees C and 37 degrees C. The kinetic data at 37 degrees C were comparable to those, in the literature, obtained by following single platelet recruitment into aggregates. The increase in turbidity due to ADP-induced shape-change (6 +/- 1%, mean +/- S.E.M., n = 7) measured in the present study was substantially greater than that (3%) measured in the aggregometer.     

Plasma thromboxane B2 levels and thromboxane B2 production by platelets are increased in patients during spinal and epidural anesthesia

Concentrations of thromboxane (Tx) B2 in plasma and its production by platelets were measured in 20 spinal and 10 epidural anesthesia patients scheduled for small operations in the lower extremities. The main metabolite of prostacyclin, 6-keto-PGF1 alpha and prostaglandin (PG) E2 in plasma were also determined. Plasma TxB2 and TxB2 production by platelets increased during both spinal and epidural anesthesia. Plasma TxB2 levels also remained elevated 1 h after anesthesia. The plasma concentrations of 6-keto-PGF1 alpha and PGE2 did not change during spinal or epidural anesthesia. In in vitro studies, only low concentrations of lidocaine (0.5-1.0 micrograms/ml) and bupivacaine (0.5-3.0 micrograms/ml) increased platelet TxB2 production. In platelet rich plasma, neither lidocaine nor bupivacaine in concentrations of 0.5-3.0 micrograms/ml caused constant changes in ADP-induced platelet aggregation, but they inhibited it in toxic concentrations (12 micrograms/ml). The results suggest that the increased TxB2 plasma levels and platelet TxB2 production during regional anesthesia are not caused by local anesthetics itself but by other factors, e.g. tissue trauma. In clinically found concentrations, local anesthetics do not cause any constant changes in platelet aggregation.     

Regulation of the activity of Escherichia coli deo-operon structural genes: the mutation mapped within the operon boundaries and affecting drm and pup gene activity]

The mutant AIR38 is isolated from Escherichia coli K-12 strain deficient in thymidilate synthetase and deoxyriboaldolase (HfrH, thy, dra)--by selection for low thymine requirement on the medium containing inosine as the carbon source. Under the conditions mentioned the mutant AIR38 (thy, dra) grows at low thymine concentration (2 mkg/ml), and is uncapable to grow in the presence of thymidine (40 mkg/ml). Dra+ derivatives of the AIR38 do no catabolize inozine in the presence of thymidine as well. The mutation AIR38 is mapped within the deo-operon between drm and pup mutation markers. The levels of phosphodeoxyribomutase and purine nucleoside phosphorylase in cell extracts of AIR38 are 2.5-6-fold decreased. In transductional experiments with phage P1 and the mutant AIR38 as recipient the delayed haploidization of merozygotes dra+, AIR+/dra, AIR38, thy and the dominant expression of the sensitivity to thymidine in the presence of inosine as the carbon source are observed. It is supposed that the mutation AIR38 affects the structural gene of purine nucleoside phosphorilase by altering the mode of interaction of this enzyme with the membrane under the conditions of thymine starvation.