Identification of Megakaryocytes As a Functional Component of Circulation Blood

Peripheral blood (PB), a central component of the circulation system, has well defined cellular contents essential for overall immunity and body homeostasis. While the conventional platelet-generating megakaryocytes (MKs) are recently emerging as a unique type of immune cells, they primarily reside in bone marrow (BM) as well as in lung and spleen, and are not considered as a regular constitute of PB. MKs lack of cytoplasm (naked MK) can be observed inpulmonary venous blood after releasing platelets into the pulmonary circulation. Intriguingly, suspected MKs population are detected in PB by single cell transcriptomic analysis in various pathological states including COVID-19 infection and autoimmune disorders. However, the true identity of those PB-MKs remains elusive. It remains unclear whether functional megakaryocytes are naturally present in circulation blood.

Here, by employing flow cytometry sorting of CD41⁺ cells with immunofluorescence and electron microscopy assays, we unexpectedly identified MKs with intact and classical MK morphology in PB nucleated cells (PBNCs). MKs comprise approximately 0.05% of PBNCs of healthy humans and mice. These cells express MK surface markers CD42 and CD61 and exhibit classical MK functions, such as proliferative response and proplatelet formation upon thrombopoietin (TPO) stimulation, and initiation of thrombin-induced clotting in vitro. However, in contrast to BM-derived MKs (BM-MKs), which are primarily composed of polyploid (4N to 64N) cells with diameters ranging from 9.2μm to 40.1μm, PB-MKs were predominantly 2N and smaller in size with an average diameter of 15μm.

To investigate the cellular and molecular characteristics of PB-MKs, we performed high-quality scRNA sequencing and analysis of manually picked CD41⁺ cells from PB. Notably, we identified two distinct subpopulations within PB-MKs: platelet-generating MKs (pMKs) (~15%) and immune MKs (iMKs) (~85%), with no detectable polyploidization and hematopoietic stem cell (HSC)-niche MKs typically found in bone marrow. The transcriptomic profile of hypoploid PB-pMKs (2N-4N) exhibited an unexpectedly high-level expression of platelet production signature compared to BM-MKs of the same ploidy. Developmental trajectory and regulatory gene network analysis revealed that the PB-pMKswere in a more mature state than their BM counterparts (BM-pMKs). Additionally, PB-MKs showed higher expression of genes associated with initiation of platelet generation (NFE2, STAT3, and ESAM), while BM-pMKs expressed genes involved in cytoskeleton extension (RUNX1, TUBB1, and MYL9). Real-time monitoring of in vitro MK culture demonstrated that proplatelet formation in PB-MKs occurs significantly earlier than that in BM-MKs (7.45±3.38 hours vs. 30.47±13.66 hours), indicating their potential in emergency response to bleeding.

The predominant component of PB-MKs is the immune MKs (iMK) subpopulation, which can be further subdivided into three distinct sub-clusters by unsupervised clustering. The antigen-presentation iMK-1 and phagocytosis iMK-2 sub-clusters in PB are also present in BM and lung. Notably, a CD3⁺ T-like iMK-3 sub-cluster was uniquely identified in PB but not in BM and lung. The iMK-3 exhibit gene expression signatures associated with cytotoxic T-cell activity and an enhanced interferon response. Examination of isolated single CD41⁺CD3⁺ cells from PB revealed an oval or multilobulated "rose petal" morphology, similar to classical MKs (CD41⁺CD3^-), yet distinct from the round shape of T cells. Furthermore, these cells upregulated CD62P upon thrombin stimulation, a functional characteristic of typical MK. To explore their pathological role, we re-analyzed several sc-RNA public datasets derived from human cohorts with viral (COVID-19, HIV and SFTS) or bacterial (sepsis) infection. Remarkably, PB in these patients contained a significantly expanded CD3⁺ T-like iMKs (>10-fold) compared to that in healthy donors. These PB-iMKs also exhibited increased expression in interferon signaling and cytokine secretion, pointing to a functional role in anti-infection responses.

In summary, our study uncovered MKs as a previously unappreciated component of circulation system. These MKs are biologically and functionally distinct from these in BM and known extramedullary tissues, and may play important roles in emergency response of bleeding and self-defense against pathogen.