Response Of Human Platelets Research Articles

Platelet aggregation by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex: heterogenous responses in platelet-rich plasma but not in washed platelets

Platelet aggregation by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex: heterogenous responses in platelet-rich plasma but not in washed platelets

Time-dependent translocation of the alpha and beta 1 isotypes of protein kinase C in human platelets in response to phorbol ester stimulation.

Conference Abstract| February 01 1997 Time-dependent translocation of the α and β1 isotypes of protein kinase C in human platelets in response to phorbol ester stimulation Jacqueline M. Kew; Jacqueline M. Kew 1Centre of Pharmacognosy, School of Pharmacy, University of London, 29–39 Brunswick Square, London, WCIN IAX, U.K. Search for other works by this author on: This Site PubMed Google Scholar W. Jonathan Ryves; W. Jonathan Ryves *Dept. of Neuroscience, Institute of Psychiatry, Denmark Hill, London, SE5 8AF, U.K. Search for other works by this author on: This Site PubMed Google Scholar Fred J. Evans Fred J. Evans 1Centre of Pharmacognosy, School of Pharmacy, University of London, 29–39 Brunswick Square, London, WCIN IAX, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (1): 44S. https://doi.org/10.1042/bst025044s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation Jacqueline M. Kew, W. Jonathan Ryves, Fred J. Evans; Time-dependent translocation of the α and β1 isotypes of protein kinase C in human platelets in response to phorbol ester stimulation. Biochem Soc Trans 1 February 1997; 25 (1): 44S. doi: https://doi.org/10.1042/bst025044s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: C-cytosolic, P-particulate or membrane, PKC-protein kinase C, SDS-PAGE-sodium dodecylsulphate-polyacrylamide gel electrophoresis, TPA-12-O-tetradecanoylphorbol-13-acetate, Q-cytoskeletal This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

Interplay between milrinone and adenosine in the inhibition of human platelet response

1. 1. In this study, we investigated the influence of the isotropic agent and coronary vasodilator milrinone on platelet aggregation and intracellular levels of 3′,5′ cyclic adenosine monophosphate (cAMP) in human platelet-rich plasma (PRP) and whole blood (WB). Furthermore, we evaluated the influence of milrinone on the effects of adenosine, which reduces the platelet aggregation through an elevation of intraplatelet cAMP levels. 2. 2. Milrinone decreased the platelet aggregation in response to agonists in both PRP and WB. A dose-dependent increase of intraplatelet cAMP levels was demonstrated: this result is in accordance with an effect on platelet phosphodiesterases. 3. 3. Milrinone at low concentration and adenosine exerted additive effects on platelet aggregation and intraplatelet cAMP levels. 4. 4. An interplay between milrinone and adenosine was shown in WB. Furthermore, dipyridamole, which prevents the uptake of endogenous adenosine, markedly enhanced the milrinone antiaggregating effect, whereas the adenosine receptor blocker, theophylline, decreased it. 5. 5. The present data provide evidence that milrinone modulates the platelet function through an influence on intraplatelet levels of cAMP and it is able to interplay with substances stimulating adenylyl cyclase. 6. 6. The interplay between milrinone and adenosine in the inhibition of the human platelet function could be effective during milrinone administration in the treatment of heart failure, when blood adenosine levels are significantly increased. These milrinone effects could be advantageous from a therapeutic point of view, since patients with heart failure are at risk of thrombosis and ischemic heart disease.

Effects of exogenous gangliosides on intracellular Ca2+ mobilization and functional responses in human platelets.

Gangliosides, highly expressed in the outer leaflet of plasma membranes, mediate a variety of biological processes, including cell-cell and cell-matrix interactions. We examined the effects of exogenous gangliosides on intracellular Ca2+ mobilization and functional responses in human platelets. Gangliosides (GM3 and GM1) induced rapid and reversible elevation of intracellular Ca2+ in fura2-loaded platelets in a concentration-dependent manner. The Ca(2+)-mobilizing effect of gangliosides was not mimicked by deN-acetyl-GM3, lactosylceramide, or free sialic acid, suggesting that structural integrity as ganglioside is essential for this effect. GM3 and GM1 also induced platelet shape change by themselves and elicited aggregation in combination with epinephrine. Our observations suggest the involvement of ganglioside-activated platelets in atherosclerosis, in view of the high observed ganglioside levels in atherosclerotic lesions of human aorta.

C 6-ceramide maintains elevated cytosolic calcium levels in activated platelets

The effects of cell-permeable C 2 and C 6-ceramides on human platelet responses were investigated. In thrombin-activated platelets, C 6 (5–30 μM) potentiated Ca 2+ mobilization and Ca 2+ influx, and decreased the rate of removal of Ca 2+ from cytosol. The effect of C 2 was not significant. Phorbol ester or calyculin A inhibition of thrombin-induced rises in platelet [Ca 2+] i was attenuated by C 6. Assays show that C 6 either prolonged the generation, or retarded the metabolism of inositol trisphosphates. Previous studies indicate that protein kinase C (PKC) acts in a negative feedback manner by inhibiting phosphatidylinositol breakdown, accelerating inositol trisphosphate metabolism, and increasing Ca 2+ pump activity. C 6 may counter these PKC effects indirectly. The synthetic ceramides inhibited platelet aggregation weakly and had no effect on pleckstrin (p47) phosphorylation. Recently we reported that C 2 but not C 6 inhibits superoxide generation and store-regulated Ca 2+ influx in neutrophils at similar concentrations. Cellular differences in ceramide metabolism or ceramide-sensitive enzymes and their substrates may account for the disparate results.

Differentiation of the two forms of GPIb functioning as receptors for alpha-thrombin and von Willebrand factor: Ca2+ responses of protease-treated human platelets activated with alpha-thrombin and the tethered ligand peptide.

Previous results have shown that both GPIb and the seven transmembrane domain receptor (STDR) are required for optimal thrombin-induced platelet activation (Greco et al., 1996). Limited degradation (approximately 10%) of GPIb and the STDR by elastase reduced the Ca2+ response to 0.5 nM alpha-thrombin by only 10% whereas Serratia marcescens metalloprotease reduced the Ca2+ response by 80% and fully abrogated high-affinity thrombin binding and aggregation. vWF/ristocetin-induced agglutination was only slightly reduced (20%) while Ca2+ and aggregation response to higher thrombin concentrations were retained. At increasing elastase and Serratia protease concentrations, degradation of the STDR proceeded from the amino-terminal domain, but Ca2+ responses to the tethered ligand peptide SFLLRNPNDKYEPF were not affected by either protease. These results show that both putative thrombin receptors are susceptible to protease degradation and suggest that Serratia protease is able to differentiate the GPIb-mediated events associated with thrombin activation from those associated with ristocetin-induced agglutination.

Heterogeneity of the aggregation response of human platelets to arginine vasopressin

Previous reports have alluded to variability in the aggregation response of normal human platelets to the neuropeptide arginine vasopressin (AVP). Since it has not been well documented, the current studies were undertaken to characterize this response. AVP (1-100 nM) produced a concentration-dependent aggregation response. Although the aggregation response to 100 nM AVP did not correlate with age or sex, there was a bimodal response distribution based on the presence or absence of a second wave of aggregation. In kinetic studies, the apparent km of AVP was 18.3 +/- 5.4 nM. There was a significant inverse relationship between the maximal aggregation response to 100 nM AVP and the km (r = -0.82). One hundred nanomolar AVP increased the intracellular calcium concentration of platelets by 406 +/- 120 nM in calcium free buffer and by 658 +/- 233 nM in the presence of 1.0 mM CaCl2. The aggregation response to 100 nM AVP correlated most strongly with the transmembrane influx of calcium (r = 0.84). In individuals whom 100 nM AVP was able to generate a second wave of aggregation, the selective protein kinase C inhibitor bis-indolylmaleimide significantly decreased the platelet aggregation response. Thus, there is significant heterogeneity in the aggregation response of normal human platelets to AVP. Based on our kinetic studies and the effects of PKC inhibition on the aggregation response to AVP, we would hypothesize that the variability of the aggregation response of normal human platelets to AVP is related to a polymorphism of the platelet AVP V1 receptor.

1- O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine—A methoxy ether lipid inhibiting platelet activating factor-induced platelet aggregation and neutrophil oxidative metabolism

Whether or not two alkylglycerols could initiate a functional response in human platelets or modify responses induced by platelet activating factor (PAF) was evaluated. It was found that 1–100 μM 1- O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine (Et-16-OCH 3) induced platelet aggregation but 1- O-hexadecyl- sn-glycerol (chimyl alcohol; CA) did not. Et-16-OCH 3-induced platelet aggregation was abolished by pretreatment with the PAF receptor antagonist WEB 2086. While CA had no effect on platelet aggregation induced by PAF, pretreatment with Et-16-OCH 3 (0.1 μM or higher) significantly inhibited platelet aggregation induced by PAF, but had no effect on aggregation caused by ADP, thrombin or phorbol myristate acetate (PMA). A receptor binding study using radiolabelled [ 3H]WEB 2086 showed that Et-16-OCH 3 exerts its actions through interaction with the PAF receptor. Moreover, Et-16-OCH 3 inhibited neutrophil chemiluminescence responses induced by PAF, but not reactions to PMA or a formyl peptide. Finally, 1 μM Et-16-OCH 3 induced a rise in the intracellular calcium concentration in platelets equal to that induced by PAF and also had an calcium ionophore-like effect at 100 μM. Thus, this study shows that Et-16-OCH 3 is both a potent inducer of platelet aggregation and an inhibitor of PAF-induced platelet aggregation and neutrophil chemiluminescence, through interaction with the PAF receptor.

Endothelin influences pH i of human platelets through protein kinase C mediated pathways

Stimulation of human platelets with endothelin raises cytosolic pH (pH i). In order to determine whether this effect is mediated via protein kinase C and Na +-H + linked pathways, the effects of staurosporine and calphostin C (protein kinase C inhibitors) and 5-(N,N-hexamethylene) amiloride (Na +-H + exchange blocker) on endothelin-induced pH i responses in human platelets were assessed. In addition, platelet endothelin receptor subtypes were determined pharmacologically using the selective ET A receptor antagonist BQ-123 and the ET B receptor agonist sarafotoxin S6c. pH i was measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets obtained from 15 healthy subjects. Endothelin-1 at a fixed concentration of 10 −9 M significantly increased pH i from 7.11 ± 0.01 ([H +] i = 77 ± 0.9 nM) to 7.19 ± 0.04 ([H +] i = 64 ± 0.9 nM) (p<0.01). The pH i-stimulating effect of endothelin-1 was inhibited by 10 −7 M staurosporine, calphostin C and 5-(N,N-hexamethylene) amiloride. BQ-123 (10 −7 M) abolished the pH i responses to endothelin-1, whereas sarafotoxin S6c had no effect on platelet pH i. These data suggest that in vitro effects of endothelin-1 on platelet pH i are receptor-mediated via a pathway involving protein kinase C. Platelet endothelin receptors appear to be of the ET A subtype.

F17. Increased cytosolic calcium and α-granule release of human platelets in response to fluid shear stress

F17. Increased cytosolic calcium and α-granule release of human platelets in response to fluid shear stress

Two different responses in human platelets stimulated by calcium ionophores

Two different responses in human platelets stimulated by calcium ionophores

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

Effects of extracellular divalent cations on responses of human blood platelets to adenosine 5′-diphosphate

The effects of extracellular divalent cations on the responses of human platelets to adenosine 5′-diphosphate (ADP) and on its inhibition by the competitive antagonist adenosine 5′-triphosphate (ATP) were investigated. Two responses were studied, shape change and the inihibition of prostaglandin E 1 (PGE 1)-stimulated adenylate cyclase, and experiments were carried out in the presence of divalent cations (Ca 2+ and Mg 2+, 1 mM) or in their absence. For each response there was a small leftward shift of the concentration-response curve to ADP in the absence of divalent cations compared to that in their presence, and this leftward shift disappeared when the results were plotted in terms of ADP 3− rather than total ADP concentration. The shape change results were, however, complicated by a reduction in the maximal response to ADP in the absence of divalent cations. For each response there was also a marked increase in the p A 2 value of ATP in the absence of divalent cations compared to that in their presence, and this difference disappeared if the results were calculated in terms of ATP 4− instead of total ATP. These results suggest that the human platelet ADP receptor, in common with other receptors for adenine nucleotides, recognises predominantly the uncomplexed forms of ADP and ATP as ligands.

Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors

The mechanism underlying the synergistic inhibition of platelet activation by cGMP- and cAMP-elevating vasodilators was investigated using washed human platelets and platelet-rich plasma. With both types of human platelet preparations, low concentrations of sodium nitroprusside increased the cAMP-elevating potency of low concentrations of prostaglandin E 1 (PG-E 1). Using threshold concentrations of both sodium nitroprusside and PG-E 1, the NO-donor potentiated the effect of PG-e 1 with respect to the phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein (VASP) at serine 157. In contrast, threshold concentrations of cell-membrane permeant selective activators of the platelet cGMP-dependent protein kinase or the cAMP-dependent protein kinase had only additive effects on VASP serine 157 phosphorylation in washed human platelets. The data demonstrate that low intracellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-dependent protein kinase present in this cell type. This study provides the first evidence that the simultaneous activation of both cGMP-and cAMP-dependent protein kinase results in additive effects on VASP serine 157 phosphorylation, whereas the supra-additive effects observed with the combination of sodium nitroprusside and PG-E 1 are due to cGMP-mediated inhibition of type III phosphodiesterase. VASP phosphorylation at serine 157 may be an important component underlying the synergistic inhibition of human platelets by cGMP-and cAMP-elevating agents.

Comparison of Phorbol Ester-induced Responses in Rabbit and Human Platelets and Difference in Shape Change

Phorbol 12,13-dibutyrate (PDBu) induced aggregation and ATP release of washed human and rabbit platelets in a concentration-dependent manner. The EC(50) for PDBu-induced aggregation of washed human and rabbit platelets were 67.6 ± 3.3 and 32.7 ± 5.9 nM, respectively. PDBu and l-oleoyl-2-acetylglycerol (OAG) also caused a shape change in rabbit platelets detected by microscopic and turbidimetric techniques. Both 20K and 47K proteins of rabbit platelets were phosphorylated by PDBu treatment. All the PDBu-induced responses of human and rabbit platelets were blocked by a protein kinase C inhibitor, staurosporine, in a concentration-dependent manner. In both human and rabbit platelets, PDBu induced no phosphoinositide breakdown, caused no increase of [Ca(2+)](f) and induced no formation of TxB(2). Indomethacin, BN52021, nitroprusside and cytochalasin B did not inhibit, whereas verapamil, prostaglandin E, and EGTA inhibited PDBu-induced aggregation of washed human and rabbit platelets. Apyrase and creatine phosphate/creatine phosphokinase inhibited PDBu-induced aggregation, but did not affect the shape change or 20K myosin light chain phosphorylation of rabbit platelets. In Ca(2+)-free Tyrode's solution containing 1 mM EGTA, PDBu induced a shape change in rabbit platelets and this effect was inhibited by staurosporine and cytochalasin B. It is concluded that PDBu and OAG induce a shape change in rabbit platelets probably through phosphorylation of 20K myosin light chain.

Insulin increases the aggregation response of human platelets to ADP

Insulin increases the aggregation response of human platelets to ADP

Initiation of RVD response in human platelets: mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2.

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A3. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A3 formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca(2+)-insensitive phospholipase A2, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A2 inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 microM, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mM) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTA-AM (100 microM) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP beta S (1 microM) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A3; (v) NaF, 1 mM, induces hepoxilin A3 formation, tested by the RVD reconstitution assay; and (vii) GDP beta S inhibits hepoxilin A3 formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A3 formation in human platelets.

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.

Antiplatelet protease, kistomin, selectively cleaves human platelet glycoprotein Ib

Kistomin, a metalloprotease purified from venom of Calloselasma rhodostoma, dose- and time-dependently prolonged the latent period of aggregation and inhibited ATP secretion of human washed platelets stimulated by thrombin. It inhibited aggregation induced by low concentrations of thrombin (≤ 0.2 U/ml) whereas it had only slight effect on aggregation induced by high concentrations of thrombin (≥ 0.5 U/ml). Meanwhile it also inhibited ristocetin-induced platelet aggregation in a dose- and time-dependent manner. It significantly inhibited cytosolic calcium rise of Quin 2 — loaded platelets, completely blocked thromboxane B 2 formation, and blocked [ 3H]inositol phosphates formation of [ 3H]myoinositol loaded platelets stimulated by 0.1 U/ml of thrombin. Kistomin inhibited significantly thromboxane formation but not [ 3H]inositol phosphates formation of platelets stimulated by a high concentration of thrombin (1 U/ml). Incubation of platelets with kistomin resulted in a selective cleavage of platelet membrane glycoprotein Ib as revealed by SDS/PAGE stained by periodic acid/Schiff reagent. These results suggested that thrombin activates platelets at least through two receptors/or effectors-mediated events. In addition to glycoprotein Ib, other surface membrane component(s) (e.g., the seven transmembrane domain thrombin receptor) may also be important in regulating the biochemical events of human platelets in response to thrombin. However, the extent and rate of platelet aggregation stimulated by low concentrations of thrombin (≤ U/ml) are closely related with intactness of glycoprotein Ib.

Inhibiting effect ofYersinia pestis protein factor on the hormone-stimulated response of human platelets

Inhibiting effect ofYersinia pestis protein factor on the hormone-stimulated response of human platelets