Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood

Introduction The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. By now, there are no existing tools for diagnosis of platelet function disorders in small blood volumes. AKT signaling plays a key role in platelet activation during hemostasis and can be visualized by flow cytometry in small volumes of blood.

Methods Platelet-rich plasma obtained by the centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, TRAP-6, collagen, ADP, CRP, and epinephrine. Briefly after activation of platelet-rich plasma, samples were fixed for 15 minutes and then permeabilized for 15 minutes. The phosphorylation of Akt was assessed by flow cytometer on a Navios cytometer.

Results Healthy volunteers showed a reproducible phosphorylation of AKT. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP and CRP cause considerable increases in percentage of pAKT expression throughout all the tested healthy volunteers (Table 1).

Conclusion An activation of the AKT signal pathway by different agonists can clearly be detected on the flow cytometer. Thus, visualization of signaling in platelets by flow cytometry might be an alternative for light transmission aggregometry in young children.

Table 1

	MV (FI)	Min (FI)	Max (FI)
Arachidonic acid	13.88	3.96	18.14
Collagen	10.40	1.78	20.12
Epinephrine	3.21	1.10	6.42
ADP	24.96	3.62	56.55
TRAP	33.00	13.41	51.62
CRP (collagen-related peptide)	21.44	13.11	35.68

Publication History

Article published online:
13 November 2020