Table 1. Controversies, challenges, and... | American Society of Hematology

Table 1.

Controversies, challenges, and definitions

The challenge	Description and solution
What defines a mature megakaryocyte?	A major determinant of in vitro platelet generation quality and quantity is the state of maturity of the precursor MK. A clear set of parameters, including the expression of surface markers, cell size, ploidy level, and internal membrane content, is needed to define standards for defining mature MKs for in vitro platelet generation.
What defines a functional platelet?	This seems to be an obvious point, but there is an urgent need for a clear definition of what constitutes the essence of a platelet so that we can gauge the effectiveness of in vitro platelet generation. Some major parameters that define a platelet and its normal functionality include the following: morphological and receptor characterization; the number and presence of organelles assessed using multiple, unbiased transmission electron micrographs, and key to this would be the presence of a microtubule ring that may be more easily assessed using immunofluorescent microscopy; bell-shaped size distribution of all produced platelet-like particles; expression levels of key surface receptors that are comparable with donor platelets (eg, CD41, CD42, and glycoprotein VI); functional capacity must be assessed; and minimal levels of pre-activation (low P-selectin and phosphatidyl serine surface expression before stimulation) should be ensured. The platelets should also respond to multiple agonists. This can be confirmed using flow cytometry (increased activated integrin αIIbβ3 and P-selectin expression) and in vitro thrombosis assays. Furthermore, cell viability should be assessed using an exclusion dye. The final steps should include in vivo survival and function of the platelets. The half-life and ability to incorporate into a thrombus could be assessed. In vivo assays may not always be easily accessible but should be performed once all other platelet criteria are met.
Proplatelet or large protrusion?	This is a major point, and clarity here in the use of the word proplatelet is required for unequivocal determination of the mechanisms of platelet generation. We would propose that proplatelets are restricted to elongated fine structures, derived from MKs, which may either still be attached to the MK cell body or detached from it. These structures should be driven by microtubule sliding, may include branches, and may include bulges at the tips and at intermediate points along the proplatelet (beads on a string²⁵), and there may be multiple proplatelet extensions from a single MK cell body. Large protrusions from MKs, in contrast, are likely to be driven by mechanisms that are different from those involved in microtubule sliding required for proplatelets, for example, our observation from correlative light electron microscopy that multiple membrane fusion sites at the tips may be responsible.²⁶ Large protrusions are less fine than proplatelets, are usually single structures from the cell body, and typically do not branch or contain bulges. Proplatelets are therefore distinct from large protrusions of MKs, and clarity is needed, particularly regarding defining extensions from MKs in vivo in which case the resolution of light microscopy is usually not sufficient to be able to distinguish these 2 structures. Further discussion of this point is made in “Location of platelet generation”.
Tissue space or within vasculature?	This is also a major point in which care over definition is critical for understanding cellular mechanism. It can be insufficient to state broadly that thrombopoiesis derives from a particular entire organ because the location of the MKs within that organ may be intravascular or tissue resident. If the MKs are intravascular when they are generating platelets, they may be derived from the organ in which they are found or they are likely, and more commonly, derived from a distant site (usually bone marrow) and the migrated into the vasculature. So, for example, it is possible that the majority of platelet-generating MKs derive from bone marrow and mature in that tissue space but that platelet generation from those MKs could take place in other regions of the body within the vasculature of other organs.
Megakaryopoiesis or thrombopoiesis?	Again, definitions should be self-evident, but accuracy is essential for understanding the process of platelet generation given that it is essentially a 2-step process of maturation of MKs (megakaryopoiesis), followed by the generation of platelets (thrombopoiesis). It is quite possible that, as described, these 2 events take place in different regions of the body so that the locations of high MK populations do not necessarily also correspond to sites of thrombopoiesis.
Do megakaryocyte subtypes produce platelet subtypes?	Although this is beyond the scope of this review, this is a key point of discussion within the field. A more developed understanding of this could drive tailored therapies.

The challenge	Description and solution
What defines a mature megakaryocyte?	A major determinant of in vitro platelet generation quality and quantity is the state of maturity of the precursor MK. A clear set of parameters, including the expression of surface markers, cell size, ploidy level, and internal membrane content, is needed to define standards for defining mature MKs for in vitro platelet generation.
What defines a functional platelet?	This seems to be an obvious point, but there is an urgent need for a clear definition of what constitutes the essence of a platelet so that we can gauge the effectiveness of in vitro platelet generation. Some major parameters that define a platelet and its normal functionality include the following: morphological and receptor characterization; the number and presence of organelles assessed using multiple, unbiased transmission electron micrographs, and key to this would be the presence of a microtubule ring that may be more easily assessed using immunofluorescent microscopy; bell-shaped size distribution of all produced platelet-like particles; expression levels of key surface receptors that are comparable with donor platelets (eg, CD41, CD42, and glycoprotein VI); functional capacity must be assessed; and minimal levels of pre-activation (low P-selectin and phosphatidyl serine surface expression before stimulation) should be ensured. The platelets should also respond to multiple agonists. This can be confirmed using flow cytometry (increased activated integrin αIIbβ3 and P-selectin expression) and in vitro thrombosis assays. Furthermore, cell viability should be assessed using an exclusion dye. The final steps should include in vivo survival and function of the platelets. The half-life and ability to incorporate into a thrombus could be assessed. In vivo assays may not always be easily accessible but should be performed once all other platelet criteria are met.
Proplatelet or large protrusion?	This is a major point, and clarity here in the use of the word proplatelet is required for unequivocal determination of the mechanisms of platelet generation. We would propose that proplatelets are restricted to elongated fine structures, derived from MKs, which may either still be attached to the MK cell body or detached from it. These structures should be driven by microtubule sliding, may include branches, and may include bulges at the tips and at intermediate points along the proplatelet (beads on a string²⁵), and there may be multiple proplatelet extensions from a single MK cell body. Large protrusions from MKs, in contrast, are likely to be driven by mechanisms that are different from those involved in microtubule sliding required for proplatelets, for example, our observation from correlative light electron microscopy that multiple membrane fusion sites at the tips may be responsible.²⁶ Large protrusions are less fine than proplatelets, are usually single structures from the cell body, and typically do not branch or contain bulges. Proplatelets are therefore distinct from large protrusions of MKs, and clarity is needed, particularly regarding defining extensions from MKs in vivo in which case the resolution of light microscopy is usually not sufficient to be able to distinguish these 2 structures. Further discussion of this point is made in “Location of platelet generation”.
Tissue space or within vasculature?	This is also a major point in which care over definition is critical for understanding cellular mechanism. It can be insufficient to state broadly that thrombopoiesis derives from a particular entire organ because the location of the MKs within that organ may be intravascular or tissue resident. If the MKs are intravascular when they are generating platelets, they may be derived from the organ in which they are found or they are likely, and more commonly, derived from a distant site (usually bone marrow) and the migrated into the vasculature. So, for example, it is possible that the majority of platelet-generating MKs derive from bone marrow and mature in that tissue space but that platelet generation from those MKs could take place in other regions of the body within the vasculature of other organs.
Megakaryopoiesis or thrombopoiesis?	Again, definitions should be self-evident, but accuracy is essential for understanding the process of platelet generation given that it is essentially a 2-step process of maturation of MKs (megakaryopoiesis), followed by the generation of platelets (thrombopoiesis). It is quite possible that, as described, these 2 events take place in different regions of the body so that the locations of high MK populations do not necessarily also correspond to sites of thrombopoiesis.
Do megakaryocyte subtypes produce platelet subtypes?	Although this is beyond the scope of this review, this is a key point of discussion within the field. A more developed understanding of this could drive tailored therapies.