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Biomedical subjects

W Siess

Publications and source records attributed to W Siess.

At least 37 records · Page 2Linked to original sources
PubMed1995

The association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium.

The integrin alpha IIb beta 3-mediated redistribution of the tyrosine kinases pp125FAK and pp60Src and the small GTP-binding proteins CDC42Hs and Rap1B from the membrane skeleton to the cytoskeleton was found to be reversible: upon prolonged platelet aggregation (up to 15 min) induced by the thrombin-receptor activating peptide (TRAP) these signalling proteins dissociated from the cytoskeleton and reappeared in the membrane skeleton. Addition of the extracellular Ca2+ chelator EGTA and the intracellular Ca2+ chelator BAPTA/AM 30 s after TRAP allowed platelet aggregation and the association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton, but prevented their dissociation from the cytoskeleton. The results indicate that the prolonged elevation of cytosolic Ca2+ in stimulated platelets leads to the dissociation of signalling proteins from the cytoskeleton.

Amino Acid Sequence↗
PubMed1995

Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost.

Human platelets suspended in plasma or buffer were incubated with low concentrations of the nitric oxide (NO)-donor 3-morpholino-syndnonime (SIN-1; 100 nM to 1 microM) and the stable prostacyclin analogue iloprost (50 or 100 pM) and analyzed for cyclic nucleotide levels and protein phosphorylation. SIN-1 and iloprost synergistically stimulated the phosphorylation of rap1B and the 50 kDa vasodilator-stimulated phosphoprotein. SIN-1 stimulated platelet cyclic GMP and cAMP-levels and enhanced the increase in cyclic AMP elicited by iloprost. It was found that the mechanism underlying the synergistic phosphorylation of the 50 kDa protein and rap1B was different: synergistic phosphorylation of the 50 kDa protein seemed to be mediated by activation of both protein kinases A and G, whereas the synergistic rap1B phosphorylation could be attributed entirely to activation of protein kinase A. Measurement of rap1B phosphorylation might be a useful tool to monitor the action of systemically applied prostacyclin-analogues and nitrovasodilators in pharmacological studies.

Autoradiography↗
PubMed1995

Platelet shape change induced by thrombin receptor activation. Rapid stimulation of tyrosine phosphorylation of novel protein substrates through an integrin- and Ca(2+)-independent mechanism.

Activation of human platelets by the peptide YFLLRNP has been shown to induce shape change but not secretion, Ca2+ mobilization, or pleckstrin phosphorylation (Rasmussen, U.B., Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J.P., and Pavirani, A. (1993) J. Biol. Chem. 268, 14322-14328). YFLLRNP was added to washed human platelets that had been pretreated with EGTA at 37 degrees C or preincubated with the fibrinogen receptor antagonist RGDS to preclude the activation of the integrin alpha IIb beta 3 (fibrinogen receptor). YFLLRNP induced shape change and stimulated the tyrosine phosphorylation of proteins of 62, 68, and 130 kDa within 7 s. Tyrosine phosphorylation of these proteins reached maximum levels (2-3-fold) 15-30 s after addition of YFLLRNP and decreased subsequently. The chelation of intracellular Ca2+ by BAPTA-AM decreased basal tyrosine protein phosphorylation but did not inhibit the increase of tyrosine phosphorylation of P62, P68, and P130 or the shape change induced by YFLLRNP. Preincubation of platelets with the tyrosine kinase inhibitors genistein or tyrphostin A23 completely inhibited platelet shape change and protein tyrosine phosphorylation induced by YFLLRNP. The inactive structural analogs daidzein and tyrphostin A1 were barely inhibitory. P62, P68, and P130, which exhibited increased tyrosine phosphorylation upon stimulation with YFLLRNP, were found in the cytoskeleton. P130 was not identical to vinculin or the focal adhesion kinase pp125FAK. The results indicate that stimulation of G-protein-coupled thrombin receptors rapidly induces protein tyrosine kinase activation through a Ca(2+)- and integrin-independent mechanism. Protein tyrosine kinase activation and tyrosine phosphorylation of novel protein substrates seem to play an essential role in the induction of platelet shape change.

Amino Acid Sequence↗
PubMed1995

Interaction of antiplatelet drugs in vitro: aspirin, iloprost, and the nitric oxide donors SIN-1 and sodium nitroprusside.

The interaction of three antiplatelet drugs was studied in vitro: aspirin, an inhibitor of the cyclooxygenase pathway of platelet activation; iloprost, a stable analog of prostacyclin that increases platelet cAMP; and the nitrix oxide donors SIN-1 and sodium nitroprusside (SNP), which both raise platelet cGMP. Platelet adhesion and aggregation evoked by collagen/ADP were measured in anticoagulated blood under physiological flow conditions using the new Thrombostat. Aggregation was also measured in platelet-rich plasma (PRP) upon stimulation by a low (2.5 micrograms/ml) and high (20 micrograms/ml) dose of collagen, ADP, or thrombin-receptor activating peptide (TRAP). We found a synergism between iloprost and aspirin in inhibiting platelet adhesion/aggregation in flowing blood and aggregation of PRP stimulated by collagen. The mean inhibitory concentrations (IC50) of iloprost in the presence of aspirin were much lower (0.7 nM and 0.5 nM in flowing blood and low-dose collagen-stimulated PRP, respectively) than in the absence of aspirin (3 and 3.6 nM, respectively). Synergism between SIN-1 and aspirin was observed in inhibiting platelet activation in flowing blood but was much less pronounced in inhibiting collagen-induced aggregation of PRP. SIN-1/SNP and iloprost synergistically inhibited the aggregation of PRP induced by collagen as well as platelet adhesion/aggregation in blood. We found that two protein substrates of cAMP- and cGMP-dependent protein kinases, rap1B and a 50 kD protein, were associated with the functional synergism between SIN-1 and iloprost and were synergistically phosphorylated by platelet treatment with both iloprost and SIN-1. Platelet inhibition by SIN-1, iloprost, and aspirin was synergistic when measured in blood. In contrast, only additive effects of SIN-1 and iloprost were observed when platelet aggregation was measured in aspirin-treated PRP stimulated by ADP, TRAP, or collagen. Our study defines the basis for a more effective antiplatelet therapy using a combination of cGMP- and cAMP-elevating and cyclooxygenase-inhibiting drugs. The results also emphasize the importance of using various methods for the evaluation of antiplatelet drugs.

Adenosine Triphosphate↗
PubMed1995

The Thrombostat system. A useful method to test antiplatelet drugs and diets.

UNLABELLED: The use of platelet inhibitory drugs, like aspirin, has resulted in a significant reduction of thrombotic complications in primary and secondary prevention of heart attacks. To find more effective substances or better drug combinations, inhibition of primary hemostasis in vitro (Thrombostat system) was investigated, with different drugs and fish diet, using small samples (1 ml) of anticoagulated (Na- citrate 3.8%, 1/9) human blood. RESULTS: 1. In the presence of 1 mM aspirin, which had no effect on bleeding volume, only 0.6 nM iloprost were necessary to show a 50% inhibition, in contrast to 2.5 nM without aspirin. 2. At aspirin concentrations of 1 mM, 50% inhibition of primary hemostasis could be achieved with 20 microM SIN-1, or with 7 microM SIN-1 together with iloprost (500 pM). The same effect was seen only with very high doses of SIN-1 (1000 microM) alone. 3. For 50% inhibition of primary hemostasis in vitro, RGDS concentrations were reduced from 250 microM to 160 microM when blood was pretreated with 1 mM aspirin and to 75 microM when 500 pM iloprost were added additionally. 4. Japanese fishermen (eating 270 g fish/day) demonstrated significantly longer in-vivo bleeding times and in-vitro bleeding volumes (6.49 min/224 microliters), respectively, as compared to Japanese farmers (90 g fish/day, 4.85 min/137 microliters). 5. In Japanese subjects in-vivo bleeding times correlated with in-vitro bleeding volumes (0.69). The Thrombostat system proved to be a sensitive method to detect synergistic effects of various antiplatelet drugs in vitro and of a platelet inhibitory diet ex vivo.

Bleeding Time↗
PubMed1995

Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators.

Rap1B, a ras-like protein expressed in high concentrations in human platelets, serves as a substrate for protein kinase A (PKA) and, eventually, protein kinase G (PKG). We measured rap1B phosphorylation by autoradiography of 32P-labeled proteins in platelets prelabeled with [32P]-orthophosphate. Platelets coincubated with histamine-stimulated human umbilical vein endothelial cells (EC) showed increased phosphorylation of the 50-Kd vasodilator-stimulated phosphoprotein (VASP) of 2.6 +/- 0.5-fold maximally and of rap1B of 17.5 +/- 7.1-fold maximally (mean +/- SE, n = 4). Incubation of platelets with prostacyclin (PGI2), the PGI2-analogue iloprost (ILO), the nitric oxide (NO) donors SIN-1 or sodium nitroprusside (SNP) showed greater concentration-dependent phosphorylation of rap1B than of VASP. Phosphorylation of rap1B had a slow time course and was irreversible in contrast to that of VASP, which was rapid and reversible. Phosphorylation of rap1B was dependent on an increase of platelet cyclic AMP and/or cyclic GMP. Very small concentrations of ILO (50 pM), PGI2 (1 nM), and SIN-1 (100 nM) increased rap1B phosphorylation. Rap1B phosphorylation could also be detected by Western blot after incubation of platelet-rich plasma (PRP) with ILO or SIN-1. Measurement of platelet rap1B phosphorylation is a novel tool that allows monitoring of the action of labile (PGI2, NO) and more stable (ILO, SIN-1, SNP) platelet inhibitors and vasodilators that increase intracellular cyclic AMP and cyclic GMP. Determination of rap1B phosphorylation by Western blot opens new possibilities of measuring platelet-EC interactions in clinical studies and of monitoring the action of systemically applied PGI2 analogues and nitrovasodilators in pharmacologic studies.

Arginine↗
PubMed1995

Thrombin receptor-activating peptide sensitizes the human endothelial thrombin receptor.

Receptor-operated effects of alpha-thrombin and of the thrombin receptor-activating peptide TRAP14 on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura 2-loaded endothelial cells. Experiments with hirudin showed that alpha-thrombin-induced Ca2+ influx requires the continuous presence of active alpha-thrombin. YFLLRNP, known to antagonize alpha-thrombin- and TRAP7-induced [Ca2+]i transients in platelets, did not antagonize [Ca2+]i transients in response to alpha-thrombin and TRAP14 in human umbilical vein endothelial cells (HUVEC). Repetitive short-term stimulations with alpha-thrombin desensitized [Ca2+]i transients to subsequent stimulations with either alpha-thrombin or TRAP14. In contrast, repeated short-term stimulations with TRAP14 sensitized [Ca2+]i transients to subsequent stimulations with either agonist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP14. The results indicate distinct characteristics of platelet and endothelial thrombin receptors and suggest that alpha-thrombin and TRAP14 activate the receptor differently. It appears that receptor desensitization occurs independently of TRAP14 binding and, hence, tethered ligand binding to and activation of the receptor. Persistent receptor desensitization after alpha-thrombin seems to depend on both alpha-thrombin binding to the hirudin-like receptor domain and the irreversible proteolytic cleavage of the receptor. It does not involve the TRAP14/tethered ligand binding site of the receptor. TRAP14 primes the receptor by a mechanism mediated by Ca2+ influx.

Calcium↗
PubMed1995

Mildly oxidized LDL induces platelet aggregation through activation of phospholipase A2.

Native LDL and LDL oxidized under various conditions were compared in terms of their ability to activate platelets. Native LDL did not induce platelet shape change or aggregation, even at high concentrations (2 mg protein/mL). LDL was mildly oxidized with either CuSO4 (mox-LDL) or 3-(N-morpholino)sydnonimine (SIN-1-LDL). Analysis of mox-LDL and SIN-1-LDL showed a small increase of dienes (E234nm from 0.28 +/- 0.04 to 0.55 +/- 0.09, mean +/- SD) and thiobarbituric acid-reactive substance (from 0 to 10.6 +/- 1.5 nmol/mg, mean +/- SEM), no change in apo B electrophoretic mobility, and a minor (12% to 30%) decrease in polyunsaturated fatty acid content. Interestingly, this small oxidative modification of LDL dramatically changed its effect on platelets. Irreversible aggregation and secretion were induced by a threshold concentration of 0.4 mg protein/mL. In contrast, LDL thoroughly oxidized with CuSO4 (ox-LDL) did not aggregate platelets. Although mox-LDL was depleted in antioxidants (alpha- and gamma-tocopherol, alpha- and beta-carotene, and other carotenoids), incubation of mox-LDL with exogenous alpha-tocopherol did not reverse its ability to induce platelet aggregation and secretion. Preincubation of platelets with the cyclooxygenase inhibitor aspirin or the phospholipase A2 inhibitors trifluoperazine, quinacrine, 4-bromophenacyl bromide, and propranolol completely prevented platelet aggregation and secretion caused by mox-LDL or SIN-1-LDL. These results indicate that mildly oxidized LDL activates platelets through a phospholipase A2/cyclooxygenase-dependent pathway. The complete inhibition of mox-LDL-induced platelet aggregation by aspirin could contribute to its beneficial effect in cardiovascular disease.

Adenosine Triphosphate↗
PubMed1994

Thrombin-induced Ca2+ influx and protein tyrosine phosphorylation in endothelial cells is inhibited by herbimycin A.

During endothelial cell activation, alpha-thrombin elicits both Ca2+ release from internal stores and influx of external Ca2+ across the plasma membrane. The mechanisms of alpha-thrombin-induced Ca2+ entry into endothelial cells are unclear. Therefore, effects of the specific tyrosine kinase inhibitor herbimycin A on protein tyrosine phosphorylation and on intracellular Ca2+ transients were studied in alpha-thrombin-stimulated human umbilical vein endothelial cells. alpha-Thrombin caused significant tyrosine phosphorylation of mainly two proteins and evoked typical biphasic changes of free cytosolic Ca2+ concentration. We show that 24 h pretreatment with herbimycin A inhibited alpha-thrombin-induced endothelial protein tyrosine phosphorylation. Moreover, herbimycin A significantly attenuated alpha-thrombin-induced Ca2+ influx but not release from internal stores. The data suggest that protein tyrosine phosphorylation by alpha-thrombin is involved in the regulation of alpha-thrombin-induced Ca2+ influx into endothelial cells.

Benzoquinones↗
PubMed1994

Lovastatin inhibits receptor-stimulated Ca(2+)-influx in retinoic acid differentiated U937 and HL-60 cells.

Lovastatin was used to study the role of isoprenylated proteins on stimulus-induced increase of cytosolic Ca2+ in retinoic acid-differentiated U937 and HL-60 cells. Preincubation of the cells with lovastatin for 11-24 h reduced the Ca(2+)-influx induced by PAF of FMLP. The maximal decrease was 60% in U937 cells and 40% in HL-60 cells. The ID50s of lovastatin in U937 and HL-60 cells were 5 microM and 15 microM, respectively. Lovastatin did not inhibit Ca(2+)-discharge from intracellular stores. Addition of mevalonate to lovastatin-treated cells completely reversed the inhibition of PAF- and FMLP-stimulated Ca(2+)-mobilization. Immunoreactivity of ras-like proteins was decreased in membranes and increased in the cytosol of U937 cells by 1 day treatment with lovastatin. We conclude that isoprenylated proteins are involved in the regulation of receptor-stimulated Ca(2+)-entry of differentiated HL-60 and U937 cells.

Calcium↗
PubMed1994

Integrin-dependent protein dephosphorylation on tyrosine induced by activation of the thrombin receptor in human platelets.

Activation of human platelets by thrombin or a thrombin receptor-activating peptide (TRAP) resulted in a decrease in tyrosine phosphorylation of two proteins, P38 and P140. Preincubation of platelets with the tyrosine phosphatase inhibitor orthovanadate prevented the tyrosine dephosphorylation of P38 and P140, and reduced platelet aggregation induced by thrombin receptor activation. When platelets were stimulated under conditions that precluded the activation of glycoprotein IIb/IIIa (dissociation of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (preincubation of platelets with RGDS), tyrosine dephosphorylation of P38 and P140 was not observed. The results indicate that protein tyrosine phosphatase stimulation (a) occurs during platelet activation induced by a physiological stimulus, (b) is a positive regulatory signal for platelet aggregation and (c) is dependent on the activation of the integrin alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1994

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biologically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entry into human umbilical vein endothelial cells but not its discharge from intracellular stores as determined spectrofluorometrically by changes of intracellular calcium concentration in fura-2-loaded cells. Concordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F1 alpha and thromboxane B2 in a dose-dependent manner. To assess the functional significance of endothelial formation of prostacyclin and EDRF to platelets, the cAMP- and cGMP-dependent phosphorylation of two platelet proteins, rap1B and a 50-kD vasodilator-stimulated phosphoprotein (VASP), was analyzed in coincubation experiments of endothelial cells with platelets. Autacoids released by histamine-stimulated endothelial cells caused the phosphorylation of rap1B and VASP in platelets, which was only partly inhibited by either indomethacin or NG-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A2 had no effect on protein kinase C- and calcium-dependent phosphorylation of platelet proteins. The results demonstrate that blockade of receptor-mediated calcium entry by SK&F 96365 markedly reduced the release of biologically active prostacyclin and EDRF from endothelial cells. Thus, calcium influx but not calcium release from intracellular stores plays a critical role in the receptor-stimulated formation and liberation of prostacyclin and EDRF in endothelial cells.

Animals↗
PubMed1993

Activation of the cloned platelet thrombin receptor decreases the pertussis-toxin-dependent ADP-ribosylation of the membrane and soluble inhibitory guanine-nucleotide-binding-alpha proteins. Inhibition by the prostacyclin analog, iloprost.

Thrombin binds at least to two sites of the platelet surface; to the recently cloned thrombin receptor [Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. (1991) Cell 64, 1057-1068] and to glycoprotein Ib. In the present study, the decrease of pertussis-toxin-dependent ADP-ribosylation of membrane and soluble inhibitory guanine-nucleotide-binding alpha (Gi alpha) proteins was measured after platelet stimulation with a thrombin-receptor-activating peptide (TRAP), and compared to stimulation with thrombin. Stimulation of intact platelets with TRAP decreased the pertussis-toxin-dependent ADP-ribosylation of the major membrane 41-kDa Gi alpha protein and the minor soluble 40 kDa Gi alpha protein recently described in platelets [Gennity, J. M. & Siess, W. (1991) Biochem. J. 279, 643-650]. The kinetics and extent of the decrease of pertussis-toxin-dependent ADP-ribosylation after stimulation of TRAP were similar to the effect of thrombin. The decrease of pertussis-toxin-dependent ADP-ribosylation of the soluble Gi alpha protein was more pronounced and observed at lower agonist concentrations than the decrease of the membrane Gi alpha protein. Desensitization of the thrombin receptor by incubating platelets with a low concentration of TRAP reduced the subsequent decrease of pertussis-toxin-dependent ADP-ribosylation of Gi alpha proteins, evoked by TRAP or thrombin. Platelet stimulation with gamma-thrombin that does not bind to glycoprotein Ib also showed a decrease in the pertussis-toxin-dependent ADP-ribosylation of the soluble and membrane Gi alpha proteins. Treatment of platelets with the stable prostacyclin analog, iloprost, reduced the decrease of pertussis-toxin-dependent ADP-ribosylation of Gi alpha proteins induced by TRAP or thrombin. Among other platelet stimuli tested (endoperoxide/thromboxane analog U44619, collagen, ADP, vasopressin), only U44619 decreased the pertussis-toxin-dependent ADP-ribosylation of the soluble and membrane Gi alpha proteins to a degree comparable to TRAP. It is concluded that the thrombin-induced activation of both the membrane and soluble Gi alpha proteins in platelets occurs via stimulation of the recently cloned thrombin receptor and is independent of the binding of thrombin to glycoprotein Ib. Furthermore, the coupling thrombin receptor/Gi protein is reduced by intracellular cAMP.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Phosphorylation of rap1B by protein kinase A is not involved in platelet inhibition by cyclic AMP.

The functional consequence of cyclic AMP-dependent phosphorylation of rap1B for stimulus-induced platelet activation is not known. Platelets were pretreated with the stable prostacyclin-analogue iloprost and resuspended in plasma without iloprost. Western blot analysis showed that rap1B was completely converted into its phosphorylated form in the iloprost-pretreated platelets. Surprisingly, the platelets that contained phosphorylated rap1B were found to respond fully to activation by a wide variety of stimuli: aggregation upon stimulation by collagen, phorbol ester, vasopressin, ADP, epinephrine, and ATP-secretion from dense granules induced by collagen, thrombin-receptor activating peptide, vasopressin and phorbol ester were unchanged as compared to control. The results indicate that cyclic AMP-dependent phosphorylation of rap1B does not play a role in the inhibition of the various signal transduction pathways that lead to platelet aggregation and dense granule secretion.

Adenosine Diphosphate↗
PubMed1992

Platelet signal transduction mechanisms induced by eicosanoids.

Platelets are in many ways involved in the pathogenesis of artherosclerosis and the development of coronary heart disease, and play a role in acute myocardial infarction (8). They are also an useful model to study cellular activation mechanisms: their isolation from peripheral venous blood is simple and rapid and their physiological responses such as shape change, aggregation and secretion can be easily measured (10). This chapter is focused on our results on the mechanisms of platelet activation and inhibition induced by eicosanoids.

Blood Platelets↗
PubMed1991

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets.

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.

Adenosine Diphosphate Ribose↗