Impact of sampling technique, anticoagulant, processing delay, and temperature on murine platelet function in whole blood

Abstract

Background: Platelets are highly sensitive to subtle changes in their microenvironment, making functional analyses challenging and prone to variation. Advances in understanding how experimental procedures influence human platelet activation have improved the accuracy and comparability of diagnostic and research data. However, despite the pivotal role of murine models, the effects of methodological variations on murine platelets remain incompletely understood. Objectives: To elucidate how blood draw techniques, anticoagulation, processing delay, and assay temperature affect murine platelets. Methods: Blood was obtained by retro-orbital, vena cava, or cardiac puncture and anticoagulated with heparin, citrate, or acid-citrate-dextrose ± recalcification. After 30 to 120 minutes, blood was stimulated at room temperature or 37 °C with adenosine diphosphate (ADP), protease-activated receptor 4-activating peptide (PAR4-AP), or cross-linked collagen-related peptide (CRP-XL), and platelets were analyzed by flow cytometry for CD62P, CD63, CD40L, and activated glycoprotein IIb/IIIa. Results: Blood sampling had minimal impact on ADP-induced platelet activation. However, platelets isolated via vena cava or cardiac puncture exhibited heightened responsiveness to PAR4-AP and CRP-XL, respectively, compared with retro-orbital sampling. Citrate and acid-citrate-dextrose significantly impaired PAR4-AP responses compared with heparin, whereas CRP-XL sensitivity was anticoagulant-independent. Processing delays as brief as 60 minutes significantly altered platelet reactivity to CRP-XL and PAR4-AP, with further delays producing minimal additional impact. Finally, ADP- and CRP-XL–induced platelet activation was significantly reduced at 37 °C compared with room temperature. Conclusion: Common variations in murine platelet handling influence in vitro responsiveness of platelets in an agonist-specific manner, highlighting the critical need for meticulous assay optimization to ensure experimental consistency and comparability.

Keywords

• animal experimentation
• anticoagulant
• blood specimen collection
• delayed processing
• method optimization
• platelet activation