The history and origin of platelets

The discovery of platelets in the early 19^th century can be credited to many scientists who all described the existence of the 'blood plates/plaques' in some way or the other. Notable amongst them were, Max Schultze who was one of the first anatomists to accurately describe these cells after looking at them under a microscope in the year 1865. At that time he referred to them as 'granules' which he saw in his own as well as other people's blood and described them as a normal constituent of the blood which tended to form irregular clumps of various sizes. He further 'enthusiastically recommended' the study of these objects 'to those who are concerned with the in-depth study of the blood of humans' [16]. In the later years, Giulio Bizzozero (1882) identified these cells when he used 'intravital microscopy of mesenteric venules of guinea pigs' [94] and observed them to be 'disk-shaped and circulating in isolation' in blood [94]. He also quoted that what he was studying were the same 'granules' that Schultze

7 had previously described. He called them blutplättchen in German and 'blood plates' in English which is most likely the source of the current use of the word 'platelets' [16, 33].

Bizzozero's contemporary William Oslar (1881-1186), built on his observations and established a clear role for these cells in thrombosis and termed these cells as blood 'plaques' [94]. He noted that these blood plaques ''are the elements which first settle on the edges of a wounded vessel and form the basis for thrombosis'' [33]. At this time, the origin of blood platelet production was unknown. Although Bizzozero had also identified bone marrow megakaryocytes (MKs), he was unaware that they were the precursors of blood platelets [94].

Then in 1906, Homer Wright using his self-made polychrome staining solution (Wright's stain) noted that the red to violet granules present in both platelets and MKs were similar in shape and color and thus clarified that platelets originated from the MKs in the bone marrow [33, 94].

There are several models proposed in the literature to suggest how platelets form from their progenitor MKs [94]. The most promising model is that of the production of platelets via proplatelets that extend from MKs because proplatelets have been found in blood [153] and their extension from MKs into blood vessels has been shown [94]. The production of platelets based on this model is briefly summarized here and a schematic is shown in Figure 2.2 [88].

In response to the hormone thrombopoietin (TPO), hematopoietic stem cells (HSCs) which in adults are present mainly in the bone marrow, differentiate into immature MKs (Figure 2.2- step 1). The nucleated MKs then undergo maturation and become polyploid by endomitosis i.e. several cycles of DNA replication which takes place without cell division. During this time the mature MKs migrate to the vascular niche and both the endomitosis and migration are regulated by the acto-myosin cytoskeleton [88, 115]. During maturation, the MKs increase in size (50-100 µm diameter) thus allowing them to accumulate protein, mRNA, organelles, platelet-specific granules and internal membrane pools that are later distributed in the platelets [115]. They also develop an invaginated membrane system (IMS) that serves as a reservoir for the next step of proplatelet formation (Figure 2.2-steps 2 and 3). At the vascular niche, the MKs extend protrusions called proplatelets which appear as barbell-shaped structures (Figure 2.2- step 4) that are released in the vascular sinusoids. The MTs have a major role during this process when they slide past each other and provide the driving force to the proplatelet extension via the MT motor protein dynein. Bipolar MTs line the length of these proplatelets and serve as a cargo route on which the organelles and granules that the MKs have accumulated during maturation are transported towards the proplatelet tips driven by the MT motor protein kinesin [88, 115]. Once the proplatelets are formed, the terminal formation of

8 platelets from them can occur in the blood stream [88]. Recent studies have speculated that MKs release a heterogeneous mixture of nascent platelets in the blood stream which then finally mature into platelets in circulation. These speculations come from the identification of proplatelets in blood [88, 153]. A recent in vitro study has shown that proplatelets are capable of generating progeny [133] and another study has identified intermediate forms between proplatelets and platelets, termed as preplatelets [154]. These preplatelets (discoid, 2-10 µm diameter) can twist their MT cytoskeleton in the center [154] and reversibly convert into the barbell-shaped proplatelets (Figure 2.2-step 5). The nuclei from the MKs, which are converted to the pre/proplatelets, are extruded and eventually phagocytosed [88]. In the final step, the barbell-shaped undergo a fission event to divide into two individual anucleate platelets that are released into circulation [88, 115]. These proplatelets have MT coils (~ 2 µm diameter) at each end and these MT coils are further retained in the individual platelets. The conversion of preplatelets to proplatelets is driven by MT forces as discussed above and formation of these proplatelets is determined by the marginal MT coil diameter and thickness [88, 153]. These cortical forces thus regulate the final sizes of platelets [153]. The actin and MT cytoskeleton hence play a major role in the production and final sizes of the platelets.

9 Figure 2.2: Scheme of platelet production in the bone marrow

1) In the bone marrow, HSCs differentiate into immature MKs in reponse to the hormone TPO.

2 & 3) Then these MKs mature during which they become polyploid by undergoing endomitosis, increase in size, accumulate mRNA, protein, organelles etc. and develop an IMS. 4) The MKs then migrate to the vascular niche and extend proplatelets which can also interconvert to 5) preplatelets.

The nuclei from these pre/proplatelets are extruded and phagocytosed and in the final step 6) in an MT-dependent process, the proplatelets undergo a fission event and form two individual platelets.

The figure caption information is adapted and the figure is reproduced with permission from Machlus KR and Italiano JE., 2013 originally published in The Journal of Cell Biology http://dx.doi.org/10.1083/jcb.201304054 [88]. © 1979 Rockefeller University Press, License number- 4003210614697, Licensed content publisher- Rockefeller University Press.