English

On calcium fluorophore’s impact in platelet signaling studies

Observation of calcium signaling in platelets - blood cells designed to be involved in stopping bleeding and forming blood clots - is an important part of fundamental research in hemostasis. However, such a study is possible only with the use of calcium fluorophores - small molecules that penetrate the platelet membrane due to their hydrophobic -AM part, which is then hydrolyzed by cytosol esterases. In this work, we consider the phenomenon of inhomogeneous loading of calcium fluorophores into platelets.

We used platelets from healthy adult donors loaded with various fluorescent probes (CalBryte590, DiOC6 (3), Fura Red, Fluo-4 and CellTracker Violet BMQC) and immobilized on antibodies to CD31 in parallel plane flow chambers. Total internal reflection fluorescence (TIRF) microscopy was used for observations.

We demonstrated that all studied probes are loaded heterogeneously, with 30% platelets being loaded with a probe 2-6 times higher than the population median value. Using the CalBryte590 probe as an example, we have shown that a decrease in the incubation temperature, the addition of Pluronic 127 to the incubation medium, or membrane cholesterol depletion significantly reduces the heterogeneity of the probe distribution in the population. By looking at platelet activation from the surface, we have shown that the probability of experiencing strong activation, as measured by the intensity of calcium oscillations, correlates with the amount of probe in the platelet.

Thus, we conclude that the type of fluorophore used and the conditions of its loading into platelets can significantly affect the results of experiments on the observation of calcium signaling in platelets.

Overall scheme of the assay. First, whole pre-processed blood is perfused through the flow chamber for 2.5 minutes. Then Tyrode's buffer is perfused through the chamber for 5 minutes to wash away unattached cells. Then, depending on the protocol, the microscopy video is recorded for 5-10 minutes.
3 720
0
#platelets#calcium fluorophores#membrane#fluorescent probes#intracellular signaling

Platelet functional responses and signalling: the molecular relationship. Part 2: receptors.

Small, non-nuclear cells, platelets, are primarily designed to form aggregates when blood vessels are damaged, stopping bleeding. To perform this function, platelets can implement several functional responses induced by various agonists and coordinated by a complex network of intracellular signaling triggered by a dozen of different receptors. This review, the second in a series, describes the known intracellular signaling pathways induced by platelet receptors in response to canonical and rare agonists. Particular focus will be on interaction points and “synergy” of platelet activation pathways and intermediate or “secondary” activation mediators that transmit a signal to functional manifestations.


Different degrees of the platelet activation in hemostasis. Upon weak stimulation, platelets pass into a weakly activated state, in which there is no clustering of platelet integrins and no significant change in the shape of platelets. This weak activation is reversible, and it corresponds to the state of platelets in the outer layers ("coat") of the thrombus. Upon stronger activation, platelet shape significantly changes. Platelets become irreversibly activated and aggregate. The secretion of platelet granules also occurs. At the maximum degree of activation, platelet mitochondria collapse, and platelets pass into a procoagulant state, exposing phosphatidylserine, which significantly accelerates blood plasma coagulation.
14 988
0
#platelets#intracellular signaling#physiology

Search by keyword
#intracellular signaling

Found 4 articles