Joerg Geiger

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFκB-IκBα-PKAc complex. We demonstrate mRNA and protein expression for most of the NFκB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IκBα, and conversely, IκBα was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IκBα, disruption of an IκBα-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBα phosphorylation, PKA-IkBα dissociation, and VASP phosphorylation, and potentiated integrin αIIbβ3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFκB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets In Vitro and In Vivo/Ex Vivo Studies

Background and Purpose— Dipyridamole and in particular dipyridamole in combination with low-dose aspirin are very effective in preventing recurrent stroke. However, the mechanism(s) underlying this dipyridamole effect have not been elucidated. Since dipyridamole inhibits the cGMP-specific phosphodiesterase type V in vitro, we hypothesized and tested whether therapeutically relevant dipyridamole concentrations enhance NO/cGMP-mediated effects in intact human platelets studied ex vivo. Methods— Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established marker of NO/cGMP effects in human platelets, was quantified by phosphorylation-specific antibodies and Western blots. Serotonin secretion and thromboxane synthase activity were determined by fluorometric quantification of derivatized serotonin and synthase products, respectively. Results— Endothelium-derived factors such as NO and prostaglandin I2 are known to elevate both cGMP and cAMP levels with concomitant platelet inhibition and VASP phosphorylation. In our in vitro experiments, therapeutically relevant concentrations (3.5 &mgr;mol/L) of dipyridamole amplified only cGMP-mediated VASP phosphorylation due to the NO donor sodium nitroprusside, but not cAMP-mediated effects. Furthermore, thromboxane synthase activity and serotonin secretion, events important for initial platelet activation, were inhibited by sodium nitroprusside, an effect also enhanced by dipyridamole, demonstrating the functional relevance of these observations. Finally, the ex vivo enhancement of NO/cGMP effects was also observed with platelets obtained from healthy volunteers treated with extended-release dipyridamole. Conclusions— Under therapeutically relevant conditions, dipyridamole enhances platelet inhibition by amplifying the signaling of the NO donor sodium nitroprusside. These data support the concept that enhancement of endothelium-dependent NO/cGMP-mediated signaling may be an important in vivo component of dipyridamole action.

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors

Abstract Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. Methods: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. Results: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Conclusions: Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

Reciprocal regulation of human platelet function by endogenous prostanoids and through multiple prostanoid receptors

Platelets are permanently exposed to a variety of prostanoids formed by blood cells or the vessel wall. The two major prostanoids, prostacyclin and thromboxane act through well established pathways mediated by their respective G-protein coupled receptors inhibiting or promoting platelet aggregation accordingly. Yet the role of other prostanoids and prostanoid receptors for platelet function regulation has not been thoroughly investigated. We aimed at a comprehensive analysis of prostanoid effects on platelets, the receptors and pathways involved and functional consequences. We analyzed cAMP formation and phosphorylation of proteins pivotal to platelet function as well as functional platelet responses such as secretion, aggregation and phosphorylation. The types of prostanoid receptors contributing and their individual share in signaling pathways were analyzed and indicated a major role for prostanoid IP1 and DP1 receptors followed by prostanoid EP4 and EP3 receptors while prostanoid EP2 receptors appear less relevant. We could show for the first time the reciprocal action of the endogenous prostaglandin PGE2 on platelets by functional responses and phosphorylation events. PGE2 evokes stimulatory as well as inhibitory effects in a concentration dependent manner in platelets via prostanoid EP3 or EP4 and prostanoid DP1 receptors. A mathematical model integrating the pathway components was established which successfully reproduces the observed platelet responses. Additionally we could show that human platelets themselves produce sufficient PGE2 to act in an autocrine or paracrine fashion. These mechanisms may provide a fine tuning of platelet responses in the circulating blood by either promoting or limiting endogenous platelet activation.

Robust workflow for iTRAQ-based peptide and protein quantification.

Quantitative proteomics has become a routinely used technique to globally compare protein content and expression profiles of biological samples, for instance after differential stimulation. In this context, chemical stable isotope-based labeling techniques, such as ICAT and iTRAQ, have been successfully applied in a large variety of studies. Since iTRAQ labels are isobaric, quantitation is conducted on the MS/MS level. Consequently, up to eight samples can be multiplexed and quantified in a single experiment without increasing sample complexity. Here, we present a robust workflow to conduct iTRAQ quantification of biological samples such as human platelets, which comprises (a) an adequate sample preparation procedure, (b) an optimized tryptic digestion protocol, (c) SPE desalting and subsequent peptide labeling using a 4-plex iTRAQ labeling kit, and (d) fractionation of the obtained peptide mixture by strong cation exchange chromatography.

The oligopeptide DT-2 is a specific PKG I inhibitor only in vitro, not in living cells

BACKGROUND AND PURPOSE cGMP is involved in the regulation of many cellular processes including cardiac and smooth muscle contractility, aldosterone synthesis and inhibition of platelet activation. Intracellular effects cGMP are mediated by cGMP‐dependent PKs, cGMP‐regulated PDEs and cGMP‐gated ion channels. PKG inhibitors are widely used to discriminate PKG‐specific effects. They can be divided into cyclic nucleotide‐binding site inhibitors such as Rp‐phosphorothioate analogues (Rp‐cGMPS), ATP‐binding site inhibitors such as KT5823, and substrate binding site inhibitors represented by the recently described DT‐oligopeptides. As it has been shown that Rp‐cGMPS and KT5823 have numerous non‐specific effects, we analysed the pharmacological properties of the oligopeptide (D)‐DT‐2 described as a highly specific, membrane–permeable, PKG inhibitor.

Multifaceted effects of arachidonic acid and interaction with cyclic nucleotides in human platelets

INTRODUCTION Arachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state. MATERIALS AND METHODS We explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability. RESULTS Arachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death. CONCLUSIONS While arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.

Specific PKG inhibitors: do they really exist?

Background Cellular cGMP effects can be mediated by a number of effectors including cGMP-dependent protein kinases (PKGs), cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMPgated channels. Pharmacological inhibitors of PKG are often used to discriminate between these diverse cGMP effects. Currently used PKG inhibitors can be divided into three classes: cyclic nucleotide binding site inhibitors like Rp-phosphorothioate analogs, ATP binding site inhibitors like KT5823, and substrate binding site inhibitors like the recently described DT-oligopeptides. However, several studies have observed no PKG inhibition by KT5823 in intact cells [1] or by Rp-cGMPS analogs in smooth muscle cells [2], as well as unspecific (PKG-independent) effects of Rp-cGMPS analogs in platelets [3]. Results We tested the inhibitory effects of (D)-DT2 and DT3 on PKG and its effects on intact cells. Our data show that (D)-DT2 selectively inhibited 2 nM purified PKG Ia and Ib with an IC50 of 8n M, and that up to 1µ M (D)-DT2 did not inhibit PKG II or PKA. In broken platelet cell experiments, PKG activity was inhibited by (D)-DT2 starting at 5 µM, with complete inhibition at 20 µM, but we also observed inhibition of PKA activity at these concentrations. However, concentrations of up to 200 µM of compounds failed to inhibit PKG activity (assessed by phosphorylation of the established PKG substrates VASP, PDE5 and GRP2) in intact human platelets, rat mesangial cells and neonatal mouse cardiac myocytes. It should be noted that the measured PKG concentration is about 7 µM in platelets and 0.1 – 0.5 µM in all other tested cells. (D)-DT2 effects on platelet function did not correlate with PKG activity. Preincubation of platelets with 10 nM (D)-DT2 strongly inhibited thrombin-induced platelet aggregation and calcium mobilization, whereas it potentiated these effects in collagen-stimulated platelets. Conclusion Interpretations of results based on PKG inhibitors require caution. None of the commercially available PKG inhibitors should be used without control experiments in intact cells since they may have unpredictable functional effects not mediated by PKG activity.