Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species

Abstract

Background Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. Methods The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field imaging were used to gather platelet images and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Results Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Conclusion Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to more granules assisting in spreading. General Significance Highlights fundamental membrane differences and their effects on platelet secretion

Variations in Fusion Pore Formation in Cholesterol-Treated Platelets

“…2 B). Normal levels of foot events reported with CFMA are traditionally around 10-30% (2,13,14), which is nominally lower than what we report; this discrepancy is likely due to our inclusion of nontraditional type events and postfoot-containing events when determining the percentage of events with features of interest. When only events with prefoot characteristics are taken into account, the percentage of events falls in previously described ranges with 15.2 5 1.8%, 21.9 5 2.0%, and 21.8 5 2.3% for increasing cholesterol concentrations, respectively (Fig.…”

“…Even though the fusion pore dynamics of human platelets have not been studied directly with CFMA, it has been shown that platelets from other species, particularly the cow, which has comparatively increased levels of cholesterol, demonstrate similar fusion pore dynamics as the rabbit platelets incubated with cholesterol. These included increased T rise and T 1/2 times compared to rabbit platelets without cholesterol modification, which are often associated with NTEs (2,13). In addition, because platelets are anucleate, the up-and down-regulation of proteins and receptors is limited, suggesting that the only difference between the conditions was the concentration of cholesterol.…”

“…To set an upper limit on the release that could be attributed to kissand-run, we averaged the peaks in the control platelets considered to be kiss-and-run and found the highest value within one standard deviation of the mean, and then made this our maximum release value. Previous data obtained by high-performance liquidity chromatography indicated that a 5-min ionomycin stimulation on rabbit platelets resulted in the average release of 52% of the total serotonin content (13). The average area (250 fC per granule in control platelets) was thus assumed to be 52% content release.…”

Effect of Clostridium perfringens β-Toxin on Platelets

“…Possible reasons are species-specific characteristics of platelets. It is well known that differences in receptor expression exist together with differences in granule content [ 22 , 23 , 24 ]. Composition and quantity of coagulation factors are also influenced by the species, which plays an important role in the hemostatic process [ 22 , 25 ].…”

The role of cholesterol in membrane fusion

“…Cholesterol’s direct effects on the dynamics of forming fusion pores has been measured in cells using amperometery (Ge et al, 2010; Wang et al, 2010; Koseoglu et al, 2011; Gruba et al, 2015; Finkenstaedt-Quinn et al, 2016). Recapituating these effects observed in cells, reconstitution of SNARE-mediated fusion in different biochemical settings also shows more efficient fusion when the cholesterol content is increased in the membrane (Tong et al, 2009; Kreutzberger et al, 2015; Stratton et al, 2016).…”