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Biomedical subjects

W Siess

Publications and source records attributed to W Siess.

At least 55 records · Page 3Linked to original sources
PubMed1991

Thrombin inhibits the pertussis-toxin-dependent ADP-ribosylation of a novel soluble Gi-protein in human platelets.

A new G-protein was detected in human platelets which was ADP-ribosylated in a pertussis-toxin-dependent manner, was located in the supernatant of saponized platelets and was of a slightly lower molecular mass (40 kDa) than platelet membrane Gi alpha. This soluble ADP-ribosylated protein was immunoprecipitated by an antiserum to Gi alpha, but not by one to Go alpha. Prior thrombin stimulation of platelets led to an inhibition of the ADP-ribosylation of this protein. This inhibition was evident even under conditions which abolished the thrombin-stimulated inhibition of membrane Gi alpha ADP-ribosylation. These results indicate that the platelet thrombin receptor is coupled to two structurally and functionally distinct Gi alpha proteins: a major Gi alpha protein present in platelet membranes, and a minor Gi alpha protein detectable in the platelet soluble fraction.

Adenosine Diphosphate Ribose↗
PubMed1990

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition.

Exposure of human platelets to prostacyclin (PGI2), iloprost or prostaglandin E1 (PGE1) elicits the cyclic AMP-dependent phosphorylation of proteins of 22, 24, 30, 39, 50, 60 and 250 kDa (P22, P24 etc.). P22 was recently identified as rap 1B, a ras-like protein, and P24 was shown to be the beta-chain of glycoprotein Ib. We found that cyclic AMP-dependent phosphorylation of all proteins except P22 was maximal 1 min after exposure of platelets to PGI2, iloprost or PGE1; maximal phosphorylation of P22 occurred after 45 min of incubation. Inhibition of thrombin-induced platelet activation required only a 30 s incubation with PGI2 or iloprost; at this time phosphorylation of P22 was only slightly increased. Although at maximal concentrations PGI2 was more potent than PGE1 in inhibiting thrombin-induced platelet activation, no difference in the degree and the kinetics of cyclic AMP-dependent protein phosphorylation was found. Platelets that had been preincubated and washed in the presence of PGE1 and later resuspended in the absence of PGE1 responded fully to activation by thrombin despite maximal phosphorylation of P22 and P24. Furthermore, addition of PGI2 to PGE1-washed platelets prevented thrombin-induced platelet activation, but did not evoke further phosphorylation of P22 or P24. Phosphorylation of P39 and P50 correlated better with PGI2-induced inhibition of platelet activation. In experiments in which PGE1-induced inhibition of platelet activation was overcome by the addition of thrombin, no dephosphorylation of proteins phosphorylated by cyclic AMP-dependent kinases was observed. These experiments indicate that: (a) phosphorylation of rap 1B and glycoprotein Ib is not related to platelet inhibition by cyclic AMP; (b) phosphorylation of other proteins such as P39 and P50 probably plays a role in mediating cyclic AMP-dependent platelet inhibition; (c) reactions other than cyclic AMP-dependent protein phosphorylation may participate in platelet inhibition by cyclic AMP.

Alprostadil↗
PubMed1990

Rap1-B is phosphorylated by protein kinase A in intact human platelets.

Agonists that increase cAMP levels in platelets promote the phosphorylation of a 24 kDa GTP-binding protein that is immunoreactive with a monoclonal antibody (M90) to the H-ras p21 protein. Evidence is presented which indicates that this protein is rap-1b, not rap1-a as previously suggested (Ohmori, T., Kikuchi, A., Yamamoto, K., Kawata, M., Kondo, J. and Takai, Y. (1988) Biochem. Biophys. Res. Commun. 157, 670-676). The amino acid sequence of labeled peptides obtained by proteolytic cleavage of the purified phosphorylated protein was identical with that of rap-1b. Furthermore, a comparison of the kinetics of phosphorylation of synthetic peptides corresponding to the C-terminal region of rap-1a and rap-1b proteins indicated that rap-1b is the preferred substrate for phosphorylation by cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1989

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.

Adenosine Diphosphate Ribose↗
PubMed1989

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

Calcimycin↗
PubMed1989

Epinephrine and the Ca2+ ionophore A23187 synergistically induce platelet aggregation without protein kinase C activation.

Aspirin-pretreated, 32P-prelabeled, washed human platelets resuspended in a buffer containing apyrase and 2% plasma were exposed to epinephrine and the Ca2+ ionophore A23187. Epinephrine potentiated platelet aggregation (not secretion), the production of [32P]phosphatidic acid and myosin light chain phosphorylation induced by A23187. No phosphorylation of the 40 kDa protein, the substrate of protein kinase C, was observed. We conclude that G1-protein activation evoked by epinephrine and Ca2+ mobilization caused by A23187 represents a novel synergism for platelet aggregation and that protein kinase C activation, under these conditions is not needed for platelet aggregation.

Calcimycin↗
PubMed1989

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C.

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.

Adenosine Diphosphate↗
PubMed1988

Direct comparison of the effects of nitroprusside, SIN 1, and various nitrates on platelet aggregation and soluble guanylate cyclase activity.

We have directly compared the effects of the nitrates isosorbide-5-mononitrate, nitroglycerin and isosorbide dinitrate and of the nitric oxide-containing sodium nitroprusside and 3-morpholino-sydnonimine (SIN 1) as well as of the bioinactive precursor of SIN 1, molsidomine, on platelet activating factor-induced platelet aggregation and activation of soluble guanylate cyclase. The effects of these agents on the aggregation and on soluble guanylate cyclase activity of human platelets were closely correlated. Whereas nitroprusside and SIN 1 were very potent inhibitors of aggregation and activators of soluble guanylate cyclase in micromolar concentrations, the other drugs were effective only at millimolar concentrations. Preincubation of platelets with cysteine did not or only slightly increase the ability of isosorbide-5'-mononitrate and isosorbide dinitrate to inhibit aggregation, but a clear increase was observed after preincubation with nitroglycerin. These data support the concept that cyclic GMP is the mediator of nitric oxide-induced inhibition of platelet aggregation and indicate that nitrates cannot directly inhibit aggregation or be converted to nitric oxide-containing agents by a specific mechanism in platelets. The data also suggest that SIN 1 and nitroprusside, but not or only to a certain degree the nitrates, can be considered as exogenous endothelium-derived relaxing factors.

Adult↗
PubMed1988

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.

Adenosine Diphosphate↗
PubMed1987

Phorbol esters sensitize platelets to activation by physiological agonists.

Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist-induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist-induced myosin light chain phosphorylation.

Adenosine Diphosphate↗
PubMed1986

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists.

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.

Adenosine Triphosphate↗
PubMed1986

Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets.

The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4-monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D-arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1-receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation.

Arachidonate Lipoxygenases↗
PubMed1985

Evidence for the formation of inositol 4-monophosphate in stimulated human platelets.

Human platelets were prelabeled with [3H] inositol and exposed to thrombin or vasopressin. The radioactive inositol monophosphates were separated by high-performance liquid chromatography and identified by cochromatography with unlabeled standard substances. Radioactive inositol 1-monophosphate (Ins 1-P) and inositol 4-monophosphate (Ins 4-P) were detected in unstimulated platelets and accumulated in response to thrombin or vasopressin. Ins 4-P as well as Ins 1-P increased after the formation of inositol 1,4-bisphosphate (Ins 1,4-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). Lithium augmented the accumulation of Ins 1-P and Ins 1,4-P2 in stimulated platelets, and also of Ins 4-P in platelets stimulated by vasopressin, but not by thrombin. The results indicate that Ins 1,4-P2 formed in stimulated platelets is partly degraded to Ins 4-P. The significance of Ins 4-P as a marker molecule for the study of inositol phosphate metabolism in stimulated cells is discussed.

Blood Platelets↗
PubMed1985

Thrombin induces the rapid formation of inositol bisphosphate and inositol trisphosphate in human platelets.

Human platelets prelabeled with [3H]inositol were exposed to thrombin. The aqueous soluble inositol phosphates were separated by anion exchange column chromatography, paper chromatography or high-performance liquid chromatography, and identified by cochromatography with authentic standard substances. Thrombin immediately induces the rapid formation of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of inositol-1-monophosphate and inositol-2-monophosphate occurs later after a time lag of 10 sec. The results indicate that the phospholipase C induced polyphosphoinositide hydrolysis rather than the phosphatidylinositol hydrolysis is the triggering event for platelet activation, and support the concept of inositol 1,4,5-trisphosphate as putative second messenger.

Blood Platelets↗