Inhibiting normal apoptosis leads to extended platelet survival, but these longer-living or “exhausted” platelets may have compromised hemostatic efficacy, according to research presented by Irina Pleines, PhD, at the 2015 ASH Annual Meeting. The results demonstrate that efforts to extend platelet survival times in blood bank storage serve no real purpose.
Platelet lifespan is limited to 10 days in humans and five days in mice and is regulated by the intrinsic apoptosis pathway, in which the pro-survival protein Bcl-xL restrains the essential death mediators Bak and Bax. “Hence, platelet lifespan and platelet counts in mice are increased in the absence of Bak and Bak/Bax,” Dr. Pleines, from the Walter & Eliza Hall Institute of Medical Research in Parkville, Australia, and co-authors explained. “However, the function of these long-lived platelets has not been investigated.”
To examine the functional outcomes of extending platelet survival, the researchers first washed platelets from mice with a constitutive deletion of Bak and a platelet-specific deletion of Bax, confirming the hypothesis that removing Bak and Bax leaves platelets fully resistant to apoptosis. However, on electrolytic thrombosis models, the thrombi formed in mice with these washed platelets were unstable – despite having normal time to arterial occlusion.
Next, to investigate potential defects in platelet signaling pathways in the absence of Bax and/or Bak, Dr. Pleines and colleagues performed in vitro platelet activation assays. Flow cytometric measurements revealed that, in the absence of Bak and Bak/Bax, activation of the protease-activated receptor (PAR4) led to reduced platelet degranulation, a process essential to normal platelet function and thrombus formation. Loss of Bax alone, however, had no effect.
“In contrast, the response to activation with the platelet agonist adenosine diphosphate (ADP), which does not induce granule release, was similar in platelets from all genotypes,” the researchers reported. “Similarly, platelet aggregation in response to intermediate concentrations of PAR4 was severely reduced in the absence of Bak and Bak/Bax, but normal in response to ADP.”
Dr. Pleines and colleagues then investigated whether platelet age was a factor behind the observed functional differences by synchronizing platelet age to three days in both Bak and Bak/Bax-deficient mice and wild-type controls. Platelets were then depleted in vivo by injection of anti-platelet serum, and newly generated platelets were collected at 72 hours post-injection, when platelet counts had returned to normal levels. They found that synchronized platelet age normalized functional outcomes in the absence of Bak and Bak/Bax to control levels; platelet aggregation and release defects were also rescued. “Strikingly, synchronization of platelet age to three days rescued the hemostatic defect in Bak– and Bak/Bax-deficient mice,” Dr. Pleines and co-authors added, suggesting that increased platelet age was responsible for the decrease in functional outcomes in this mouse model.
“We conclude that extended platelet survival leads to platelet exhaustion, with reduced ability to mobilize granular release,” the researchers wrote. “Our studies suggest that, in the context of blood bank storage, extending platelet survival times by pharmacologically inhibiting apoptosis may result in a hemostatically compromised product.”
Pleines I, Lebois M, Au A, et al. Extended platelet in vivo survival results in exhausted platelets. Abstract #416. Presented at the 2015 ASH Annual Meeting, December 7, 2015; Orlando, Florida.