The power of large platelets: Comprehensive functionalcharacterizationoflarge and small platelets fromhealthy volunteers.

S. Handtke, R. Palankar, M. Ferrara, S. Cauhan, A. Greinacher, T. Thiele (Greifswald, Germany)

Platelets - Physiology and Disorders of platelet number and function
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: An increased mean platelet volume (MPV) is a known risk factor for poorer outcomes in cardiovascular diseases, which is attributed to a higher reactivity of large platelets. However, it is a major challenge to separate functional large and small platelets from the same individual. We developed a protocol that facilitates the separation of large and small human platelets and questioned whether the intensity and the velocity of the response of large and small platelets to different stimuli also differ in healthy volunteers.

Methods: Using sequential differential centrifugation steps in defined volumes, large and small platelet fractions were separated. MPV was determined by a cell counter. Generation of platelet derived microparticles and platelet aggregation was assessed after stimulation with collagen and epinephrine using flow cytometry and light transmission aggregometry, respectively. Platelet adhesion and spreading under flow was measured on collagen functionalized defined micropatterned arrays using live imaging and quantitative fluorescence microscopy. All experiments were performed at least n=6.

Results: After separation, large platelets had an average MPV of 12.03 fL ± 0.88 and small ones of 7.76 fL ± 0.53 (~1.6 fold difference, p<0.0001). Collagen induced aggregation was faster (lagtime 25s ± 21 vs 49s ± 28, p=0.0086) and the maximum epinephrine induced aggregation was higher (66% ± 23 vs 42% ± 30, p=0.0125) in large platelets. Large platelets secreted 2.5 to 3 times more microparticles compared to small platelets (collagen induced: 15300 ± 7109 vs 5192 ± 1728 microparticles per µl, p=0.0147; epinephrine induced: 13862 ± 8518 vs 5562 ± 2292 microparticles per µl, p=0.0285). On collagen functionalized defined micropatterned arrays, single large platelets covered an area being 1.4 fold higher than the area covered by small platelets (63.76 µm2 ± 21.13 in vs. 45.69 µm2 ± 17.9, p<0.0001).

Conclusion: In healthy volunteers, large platelets respond faster and stronger to different stimuli than small ones. Thus, an increase of platelet size increases the overall functional capacity of circulating platelets, which may be relevant in cardiovascular diseases. Our protocol facilitates the study of large and small human platelets in health and disease.