What It Takes To Be a Platelet: Evolving Concepts in Platelet Production.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.

Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

The bone marrow is the primary site of thrombopoiesis.

ABSTRACT: Megakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and light-sheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

A thrombus is formed by a gradient of platelet activation and procoagulant endothelium.

Background: The contribution of platelets in thrombosis within microcirculation has been extensively documented in the literature. We previously showed, in vivo, that platelet activation revealed by intracellular calcium mobilization was a crucial step in the growth of thrombi following laser-induced injury, a model of thromboinflammation. Objective: Our goal was to investigate the extent of platelet activation and the spatial distribution of platelets throughout a growing thrombus. Methods: We employed a multimodal, correlative microscopy approach and computational biology to study the state of platelets on a growing thrombus obtained after a laser injury. Results: We observed a reversible intracellular platelet calcium mobilization that correlates with the time a platelet resides during thrombus growth. Our bioinformatics analysis displayed the following 3 distinct platelet subpopulations resident within a thrombus: (1) resting, (2) partially activated, and (3) "fully" activated platelets. The spatial distribution of the platelet subpopulations in the thrombus creates a double gradient in both the transversal and longitudinal axis, with the maximal percentage of fully activated platelets close to the site of injury. However, these activated platelets did not express negative phospholipids. The injured endothelium was identified to play a vital role in activating the blood coagulation cascade in this model of thrombosis. Conclusion: Following a laser-induced injury, thrombi are formed by a gradient of activated platelets from the injury site to the periphery of the thrombus. These different activation states of platelets throughout the thrombi regulate the biomechanics of the thrombus. The injured endothelium, rather than platelets, was identified to play a key role in the activation of the blood coagulation cascade in this model of thromboinflammation.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts.

Role of Neutrophils and NETs in Animal Models of Thrombosis.

Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as "neutrophil extracellular traps" (NETs). Initially, described as "dead sacrifices for a good cause" that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research.

DNAse-dependent, NET-independent pathway of thrombus formation in vivo.

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)-activated neutrophils play a key role in initiating the tissue factor-dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis.

Platelets promote metastasis, however, their role in tumor growth remains controversial. Here, we investigated the effect of platelet interactions with colorectal tumor cells. Platelets extravasated into the tumor microenvironment and interacted with tumor cells in a cadherin-6-dependent manner. The interaction induced platelet spreading, release of their granule content, and the generation of three types of microparticles (iMP) that expressed platelet markers, tumor markers, or both. The presence of iMPs was confirmed in colorectal cancer tissue specimens. Platelets significantly reduced tumor growth and increased intratumoral macrophages. This was mediated by iMP recruitment of macrophages via the chemoattractants RANTES, MIF, CCL2, and CXCL12 and activation of their tumor cell killing capacity through IFNγ and IL4, which led to cell-cycle arrest of tumor cells in a p21-dependent manner. In contrast, in the bloodstream, iMPs activated endothelial cells and platelets and induced epithelial-to-mesenchymal transition of tumor cells, promoting metastasis. Altogether, these results indicate that depending on the environment, local or bloodstream, the consequences of the interactions between platelets and a tumor may promote or prevent cancer progression. SIGNIFICANCE: Tumor cell interaction with platelets produces chimeric extracellular vesicles that suppress primary tumor growth by activating tumor-eliminating macrophages, while promoting metastasis through EMT and endothelial activation.