Anna Kobsar

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.

The effect of immunoadsorption with the Immusorba TR-350 column on coagulation compared to plasma exchange.

Plasma exchange (PE) and immunoadsorption with the Immusorba TR‐350 column (IA) are used to remove autoantibodies from plasma in acute neurological autoimmune disorders. The impact of IA on coagulation and on low molecular weight heparin (LMWH) levels in comparison with PE was investigated.

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.

Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis‐derived platelet concentrates

Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)‐dependent signaling systems in stored PLTs was investigated.

Increasing susceptibility of nitric oxide-mediated inhibitory platelet signaling during storage of apheresis-derived platelet concentrates.

Storage of platelets (PLTs) affects PLT integrity and functionality, a process named the PLT storage lesion. Normal PLT function essentially depends on the balanced interaction of activating and inhibitory signaling pathways. As there are poor data on the alterations of inhibitory signaling during storage of PLT concentrates, this study investigates the modulation capability of the cyclic nucleotide–mediated inhibitory pathways by use of the nitric oxide donor diethylamine diazenium diolate (DEA/NO).

Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets.

Background Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied. Objectives Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation. Results In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets. Conclusion In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.

Specific inhibitory effects of the NO donor MAHMA/NONOate on human platelets

Nitric oxide (NO) is a physiological inhibitor of platelet function and has vaso-dilating effects. Therefore, synthesized NO releasing agents are used e.g. in cardiovascular medicine. The aim of this study was to characterise specific effects of the short living agent MAHMA/NONOate, a NO donor of the diazeniumdiolate class, on human platelets. Whole blood was obtained from healthy volunteers. In washed human platelets, the MAHMA/NONOate induced phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and cyclic nucleotide production were studied by Western Blot and by enzyme immunoassay kits. Agonist induced aggregation was measured in platelet rich plasma. Paired Student׳s t-test was used for statistical analysis. MAHMA/NONOate significantly stimulated platelet VASP phosphorylation in a concentration dependent manner and increased intracellular cGMP, but not cAMP levels, transiently. ODQ, a specific inhibitor of the soluble guanylyl cyclase, completely prevented VASP phosphorylation induced by low MAHMA/NONOate concentrations (5nM-15nM). The effects of higher concentrations (30-200nM) were only partially inhibited by ODQ. MAHMA/NONOate reduced platelet aggregation induced by low doses of agonists (2µM ADP, 0.5µg/mL collagen, 5µM TRAP-6) in a concentration dependent manner. MAHMA/NONOate leads to a rapid and transient activation of platelet inhibitory systems, accompanied by decreased platelet aggregation induced by low dose agonists. At low MAHMA/NONOate concentrations, the effects are cGMP dependent and at higher concentrations additionally cGMP independent. The substance could be of interest for clinical situations requiring transient and subtotal inhibition of platelet function.

The role of agonist-induced activation and inhibition for the regulation of purinergic receptor expression in human platelets

INTRODUCTIONnAdenosine diphosphate (ADP) as physiological activator of human platelets mediates its effects via three purinergic receptors: P2Y1, P2Y12 and P2X1. The inhibition of P2Y12 is used pharmacologically to suppress aggregation underlining the physiological significance of this receptor. Since the regulation of purinergic receptor expression has not thoroughly been investigated yet, this study analyzed the content of purinergic receptors on the platelet surface membrane upon activation and inhibition.nnnMATERIALS AND METHODSnThe surface expression of purinergic receptors was measured by flow cytometry using two different polyclonal antibodies as basal values and after incubation with thrombin receptor activating peptide (TRAP-6) or with inhibitors DEA/NO, MAHMA/NO or Prostaglandin E1 (PGE1). Western blot analysis was used to confirm inhibitory effects.nnnRESULTSnBoth investigated antibodies revealed a significant increase of purinergic receptor expression upon TRAP-6 stimulation. The NO donors, DEA/NO and MAHMA/NO, did not influence basal or TRAP-6 stimulated values. PGE1 did not affect basal receptor expression, but diminished TRAP-6 stimulated purinergic receptor expression in a dose-dependent manner.nnnCONCLUSIONSnIn summary, TRAP-6 induced platelet activation leads to an elevation of purinergic receptor expression. In contrast to other surface ligands, this effect is not suppressed by cGMP-mediated inhibition, but almost completely abrogated by enhanced cAMP-mediated signaling as induced by PGE1.

Evaluation of dose-dependent effects of the proteasome inhibitor bortezomib in human platelets

Platelets express key proteins of the proteasome system and contain protein ubiquitination pathways. The functional role of the proteasome system in platelets, however, is still subject of studies. In addition to its role as anticancer drug, the potent and selective proteasome inhibitor bortezomib can be used for experimental proteasome research. Since it is mandatory to know exact dose-effect relationships, we intended to evaluate dose-dependent specific bortezomib effects on basal and on agonist-induced proteasome activitiy, on levels of poly-ubiquitinated proteins and on platelet aggregation. In washed platelets, unstimulated or stimulated with different agonists and pre-incubated with various bortezomib concentrations, the proteasome activity was determined by a fluorometric assay. The levels of poly-ubiquitinated proteins were assessed by an immunoassay kit. Platelet aggregation was measured by light transmission aggregometry in platelet-rich-plasma. Platelet agonists stimulate both, the proteasome activity and the accumulation of poly-ubiquitinated proteins in platelets. Bortezomib inhibits the basal and the agonist induced proteasome activity and increased the content of poly-ubiquitinated proteins in a concentration dependent manner. Bortezomib concentrations in the nM-range causing complete blockade of platelet proteasome activity do not affect agonist induced platelet aggregation, indicating that the level of platelet proteasome activity is not directly linked with the induction of platelet aggregation. Bortezomib in the µM-range may tamper platelet aggregation, possibly due to unspecific and toxic effects.

Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates.

BACKGROUNDnThe storage of platelets affects platelet integrity and functionality, a process named platelet storage lesion (PSL). Reduced adenosine diphosphate (ADP)-induced platelet aggregation is a typical manifestation of PSL. However, the role of ADP receptors in this context has not been evaluated yet. The aim of this study was, therefore, to investigate surface expression and function of the purinergic receptors P2Y1, P2Y12 and P2X1 in stored platelet concentrates.nnnMATERIAL AND METHODSnPlatelets were obtained from venous whole blood and from apheresis-derived platelet concentrates stored for 0, 2 and 5 days. Purinergic receptor expression was measured by flow cytometry and western blot analysis. Receptor function was determined by calcium-induced fluorescence (P2Y1 and P2X1) or by flow cytometric measurement of the platelet reactivity index (P2Y12).nnnRESULTSnThe basal surface expression and total content of purinergic receptors remained unchanged throughout storage. After an initial reduction during apheresis, P2X1-mediated calcium flux was maintained, whereas the P2Y1-mediated increase of calcium flux gradually decreased during the course of storage. In contrast, the platelet reactivity index was comparable in freshly obtained and stored platelets.nnnDISCUSSIONnThe function of the P2Y12 receptor is maintained during storage of apheresis-derived platelet concentrates. However, the impairment of P2X1 and especially of P2Y1 receptor function indicated by decreased receptor-mediated calcium flux is an important mechanism contributing to reduced ADP responsiveness of stored platelets.