Platelets, anucleated cells that play a critical role in blood clotting, store proteins and small molecules in alpha-granules and dense granules, respectively, for secretion. Alpha-granules contain several proteins including von Willebrand factor and fibrinogen and dense granules contain serotonin. Rab4, a marker for the early endosomes has been implicated in regulating alpha granule secretions (Sirakawa et al, 2010). Previous fluorescence microscopy mapping of alpha-granule protein distributions suggested that there are either two different alpha-granule types or subdomains within a single granule population (Storrie and Seghal, 2007; Italiano et al, 2008). More recent work based on electron tomography (Kamykowski et al, manuscript in preparation) indicates that human platelets are comprised of one alpha granule population. We hypothesized that there was a single population of alpha-granules in which all fibrinogen is similarly compartmentalized. Hence, fibrinogen endocytocized by guinea pig megakaryocytes and platelets in vivo at 4 h (short label) and 24 h (long label) would map to the same location.
We carried out several experiments to form a basis for future high-resolution (5 nm) electron tomography to establish packaging of HRP-conjugated fibrinogen or nanogold conjugated fibrinogen into platelet alpha-granules. (a) Using PD-10 columns, we prepared Cy3 conjugated fibrinogen. Using an in vivo guinea pig model to test the ability of guinea pig platelets to take up fluorescently labeled fibrinogen, we injected 10 mg/ml of Cy3 conjugated fibrinogen (short label, 4 h) and 10 mg/ml of commercially purchased AlexaFluor 488 conjugated fibrinogen (long label, 28 h) into guinea pigs. Platelets were then fixed, purified and confocal microscopy performed. (b) Using triple immunofluorescence, serotonin antibody was applied to fixed and purified resting state human and guinea pig platelets and immunofluorescence microscopy was performed to provide whole platelet information on the staining pattern of the dense granules in comparison to the alpha-granules and early endosomes. (c) Preliminary Electron Microscopy fixation conditions were also tested on guinea pig platelets.
For the uptake experiment, spinning-disk confocal microscopy was used to collect full platelet volume image stacks which were then deconvolved, pixel shift corrected for red and green channels and analyzed. Overlap of green and red fibrinogen conjugates was observed where the fluorescently tagged fibrinogens were taken up by structures presumed to be alpha-granules. For the triple labeling experiments, the distribution of serotonin, Rab4 and von Willebrand factor was observed in resting state platelets. Using spinning-disk confocal microscopy, full platelet volume image stacks were collected, deconvolved, pixel shift corrected for red, far red and green channels and analyzed. Serotonin antibody gave an abundant punctate staining pattern in both the triple-labeled human and guinea pig platelets. In both the human platelets and the guinea pig platelets, the serotonin positive punctate granules, presumed to be dense granules, had a more similar pattern to the von Willebrand factor positive punctate alpha granules, than to the Rab4 positive punctate granules, presumed to be the early endosomes. The triple label results were unexpected because previous electron microscopy studies have indicated that the dense granules in human platelets are fewer in number than the alpha-granules and fewer than the corresponding dense granules in guinea pig platelets. Results of the electron microscopy preparations are pending.
Our results indicate that the guinea pig model, while its platelets are a much smaller size than human platelets, is a good system for loading alpha-granules with labeled proteins for electron tomography. The serotonin distribution results together with previous electron tomography also raise the question as to whether dense granules could be a specialized form of the alpha-granules. A summary of this research will be presented at the Promoting Minorities in Hematology event during the 2010 ASH meeting.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.