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
BackgroundDirect interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets.MethodsTo test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate.ResultsWe show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite.ConclusionsAll our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.
BACKGROUND AND PURPOSEStimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. EXPERIMENTAL APPROACHThe effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. KEY RESULTSRiociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50-100 μM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. CONCLUSIONS AND IMPLICATIONSRiociguat inhibits platelet activation in whole blood only at concentrations above 50 μM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered. AbbreviationsCTEPH, chronic thromboembolic pulmonary hypertension; FDA, Food and Drug Administration; LaSca, low-angle scattering; PAH, pulmonary arterial hypertension; sGC, soluble guanylyl cyclase BJP British Journal of Pharmacology
A number of direct thrombin inhibitors are successfully used clinically and experimentally as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants in certain blood collection tubes for the analysis of platelet function. A series of platelet function tests have emerged to measure adequate responses to antiplatelet therapy. For comparative and practical reasons, it would be of advantage to use the same anticoagulant in blood collection tubes for different methods, e.g. thrombin inhibitors. However, there are little data on the effects of thrombin inhibitors on platelet signalling pathways that could influence results. We examined the applicability of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets. We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin containing blood samples. In consequence, PRI levels were highly variable compared to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation which contributes to deviations in PRI measurements. These effects of thrombin inhibitors on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate the presence of an important additional platelet-based mechanism for the reduction of thrombus formation and thromboembolism by thrombin inhibitors.
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