Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer.

BACKGROUND

Platelet-derived microparticles (MPs) are believed to play an important role in coagulation and inflammatory disorders. Unfortunately, MP size renders them difficult to study and analyze by conventional flow cytometry.

METHODS

We analyzed and characterized platelet-derived MPs, using antibodies against the major surface glycoproteins (GP), the platelet activation antigen P-selectin (CD62P), and a marker of procoagulant activity (phosphatidylserine exposure). MPs were generated by exposure of platelets to thrombin receptor activating peptide (TRAP) or ionophore. Both agonists induced significant microvesiculation of platelets, and the resulting MPs were analyzed by a new digital flow cytometer: Becton-Dickinson FACSAria.

RESULTS

Membrane GPs were equally well represented in MPs generated by either reagent. In contrast, P-selectin was more intensely expressed in TRAP-MPs, while phosphatidylserine (PS) expression was markedly increased in ionophore-MPs. Two distinct populations of TRAP-MPs (one PS-positive and another PS-negative) were apparent. The latter characteristic facilitated sorting of MPs according to their PS exposure.

CONCLUSIONS

The data presented herein show a significant improvement in the methodology applied until now to the characterization of MPs. The ability to characterize and sort MP subpopulations may help to resolve their contributions to normal and pathological functions.