Function of Large and Small Platelets Differs, Depending on Extracellular Calcium Availability and Type of Inductor

It is widely anticipated that large platelets are more reactive than small platelets. This was mainly shown in Ca²⁺-poor media albeit extracellular Ca²⁺ is utilized by platelets for activation. We determined the impact of extracellular Ca²⁺ on functional differences between large and small platelets in response to thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP), and epinephrine. In Ca²⁺-poor buffer, large platelets responded stronger to TRAP-6 which equalized in Ca²⁺ containing buffer. Large platelets contained and mobilized more Ca²⁺ from their intracellular stores upon TRAP-6 stimulation explaining their better reactivity in Ca²⁺-poor media. Stronger aggregation of large platelets in response to ADP also equalized in presence of Ca²⁺, whereas large platelets responded weaker to ADP in flow cytometry (CD62P-expression: 9.7 mean fluorescence intensity [MFI] [4.4-17.9] vs. 17.5 MFI [6.1-45.6], p = 0.0234) and PAC-1 binding (11.1 MFI [5.7-19.6] vs. 20.5 MFI [14.4-35.0], p = 0.0078). Epinephrine response was stronger in large platelets (CD62P-expression: 11.8 MFI [6.8-33.0] vs. 6.8 MFI [2.5-15.2], p = 0.0078; PAC-1 binding 18.9 MFI [13.6-38.4] vs. 13.0 MFI [6.8-22.4], p = 0.0234; max. aggregation 82.9% [58.7-94.8] vs. 77.2% [19.8-88.8], p = 0.0313), which expressed more α2A receptors. Epinephrine further increased phosphatidylserine (PS) exposure especially in large platelets. PS-positive platelets progressively divided into two subpopulations with high or basic intracellular Ca²⁺ dependent on extracellular Ca²⁺. Thrombin generation was faster with small, but accelerated by PS exposure and epinephrine-coactivated large platelets. We show that responses of large and small platelets differ depending on extracellular Ca²⁺ availability and the inductor. Careful control of extracellular Ca²⁺ is necessary in functional studies with large and small platelets.