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

The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

Introduction  Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods  Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results  In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion  ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies

Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands

Background  Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods  Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H2DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results  Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts. Conclusion  Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils

Fragility of Information Cascades: An Experimental Study using Elicited Beliefs

This paper examines the occurrence and fragility of information cascades in laboratory experiments. One group of low informed subjects make predictions in sequence. In a matched pairs design, another set of high informed subjects observe the decisions of the first group and make predictions. According to the theory of information cascades (Bikhchandani, Hirshleifer, and Welch, 1992), if initial decisions coincide, an information cascade should occur: it is rational for subsequent players with low quality information to follow the observed pattern regardless of their private information. However, an information cascade should be fragile: it is always rational for subsequent players with high quality information to follow their private information. In line with existing experiments on information cascades, we find some evidence that low informed subjects follow the herd when it is rational, and this herding behavior occurs more frequently if there is a pronounced imbalance. The main finding of this paper is that information cascades are not fragile. We find strong evidence that highly informed subjects follow the herd regardless of their private information. In accordance with those observations we show, by explicitly eliciting subjects' beliefs about the state, that beliefs are not constant in the number of previous decisions that coincide, whether or not an information cascade already occurred. Subjects' behavior can be understood with a statistical model that allows for the possibility of errors in earlier decisions.Information cascades, elicited beliefs, experimental economics

The effect of short-term refrigeration on platelet responsiveness

Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2–6 °C) or at RT (20–24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30–40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1–2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage

Basic characteristics of PB samples and WB units during storage.

<p>Basic characteristics of PB samples and WB units during storage.</p

P2X1 function remains stable in washed platelets and in PRP.

<p>The figures show the calcium induced fluorescence curves in Fluo-4AM loaded washed platelets (A) and in platelets from PRP (B) after stimulation with the P2X1 agonist α,β-MeATP at different time points after preparation as indicated. Mean fluorescence curves of five independent experiments are presented (relative fluorescence units, R.F.U.).</p

The surface expression of platelet purinergic receptors show a biphasic variation in washed platelets after preparation.

<p>Washed platelets (3 x 10⁸ per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and P2X1 (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p < 0.05 (compared to 0 min).</p