Arterial and venous thromboses remain among the leading causes of mortality and morbidity worldwide, and their incidence rises significantly with age. While the causes of this are likely multifactorial, the role of platelets in age-related disease has come under increasing scrutiny. It is known that platelets from older individuals are more reactive, with enhanced aggregability and shorter bleeding times,1 indicating faster clot formation. Seminal work published approximately 10 years ago demonstrated that there was more than one cellular pathway by which megakaryocytes, and therefore platelets, are produced. In addition to the “canonical” route through which megakaryocyte differentiation from hematopoietic stem cells (HSCs) occurs via various multi-lineage progenitor stages, there also exists a “direct” route from a subset of HSCs that retain their multipotency but (if unperturbed) exclusively give rise directly to megakaryocytes.2-4 Subsequently, it was shown that platelet/megakaryocyte-biased HSCs expand with age, resulting in impaired lymphopoiesis and age-related impaired immunity.5-7 An attractive hypothesis has been that the megakaryocytes and platelets generated by these two pathways have distinct physiologies; however, this has not been fully validated functionally.
The Forsberg lab at the University of California, Santa Cruz, previously developed a sophisticated “FlkSwitch” mouse model that uses a Cre recombinase system, whereby expression of the tyrosine kinase receptor Flk2 causes permanent deletion of a red fluorescent reporter and replaces it with a green fluorescent protein (GFP). Flk2 expression occurs when HSCs transition to multipotent progenitor (MPP) stage. Therefore, cell progeny arising from HSCs via MPPs and the canonical route fluoresce green, while cells arising via pathways that bypass Flk2-positive MPPs retain their red fluorescence.
In the current study, the authors used this model to compare the HSC output (and, specifically, the platelet production pathways) of young mice (aged 3-4 months) and old mice (aged 20-24 months, equivalent to 60-70 years in humans). In the young mice, almost 100% of cells of all lineages were GFP-positive, i.e., arose via an MPP pathway. In contrast, the authors found a striking and continual decline in the percentage of GFP-positive cells with age exclusively in the megakaryocyte compartment, in line with the previously demonstrated expansion of megakaryocyte-biased HSCs.
To investigate whether this finding occurred due to changes in cell-intrinsic controls of cell fate or influences from the aged microenvironment, the authors then transplanted old HSCs into young mice and vice versa. Notably, old HSCs reverted to multilineage hematopoiesis in a young host; conversely, transplanting young HSCs into old mice did not trigger the shortcut pathway, indicating that the differentiation paths were not altered by the age of the recipient in a transplantation setting.
The authors next performed bulk and single-cell RNA sequencing, revealing that megakaryocyte progenitors (MkPs) produced via the shortcut route had a distinct molecular profile similar to that of old HSCs, with high expression of genes associated with HSC function. This supports the idea of a fast-track pathway direct from HSCs to MkPs, and also suggests that cells arising from these two routes might exhibit functional differences. To test this hypothesis, the authors transplanted MkPs into young wild-type recipient mice, demonstrating a higher contribution to platelets from the shortcut MkPs and higher and/or accelerated platelet production capacity. In line with this finding, platelet numbers were restored more quickly via the shortcut pathway in older mice following anti-glycoprotein Iba antibody-induced thrombocytopenia. Platelets from the canonical and shortcut pathway were found to have similar expression of surface proteins, as well as half-lives and lifespans. Also, the authors confirmed that the surplus of platelets seen in older mice is primarily comprised of platelets from the shortcut pathway.
Finally, the thrombotic propensity of platelets produced via direct and canonical routes was assessed using an intravital microscopy assay involving laser-induced lesions to arteriole walls. In young mice, the injury created only small thrombi made up of GFP-positive platelets from the canonical pathway. By contrast, old mice formed larger thrombi containing platelets from both direct and canonical pathways. Increased expression of platelet activation markers was found on old platelets compared to young platelets following stimulation with adenosine diphosphate and thrombin. It was also shown that it is the shortcut pathway platelets that contribute to this hyperreactivity, as they exhibit more rapid P-selectin expression and enhanced platelet spreading upon stimulation compared to canonical pathway-derived old platelets.
In Brief
Donna M. Poscablo, PhD, and colleagues demonstrated that while a shortcut pathway producing megakaryocytes/platelets expands with aging, similar direct differentiation “bypass pathways” do not occur for other blood lineages. This creates transcriptionally distinct, more expansive MkPs that show accelerated platelet production. Platelets from the shortcut pathway are hyperreactive and lead to thrombocytosis, contributing to larger, more stable thrombi in older mice. Concurrently published findings from an independent group using different methodology also identified two parallel platelet differentiation pathways, adding weight to these already significant findings.8 Additional work is required to confirm the relevance of these findings to humans in whom platelet counts decrease with age, in contrast to the increase in platelet counts seen in older mice. Further research should also consider to what extent the functional differences in HSC output and megakaryocyte/platelet function are due to changes in the intrinsic controls of cell fate versus environmental cues.5,9,10 Nevertheless, this study provides an attractive explanation for why thrombotic events are more common in the later decades of life.
Competing Interests
Dr. Lees indicated no relevant conflicts of interest. Dr. Psaila has reported consulting activity and research funding from Alethiomics, where she is a co-founder and major shareholder. She has also reported consulting/advisory board activity and paid speaking engagements for GSK, consulting activity for Blueprint medicines, advisory board activity and paid speaking engagements for Novartis, and research funding from Incyte.
