Cytoskeleton of platelets in resting state

As has been discussed before, disc-shaped platelets that are released into the bloodstream from the ends of proplatelets circulate in humans for 7-10 days. The platelets can vary in size and in their granular contents. However, the structure of their internal cytoskeleton is very much consistent. This cytoskeleton helps to maintain the discoid shape of the platelets and also maintains their integrity, especially since they encounter high shear forces generated by blood flow [94]. The internal cytoskeleton of platelets in their resting state, in order of their occurrence from the plasma membrane towards to the inner side, is described in this section and a sketch of the cytoskeleton is shown in Figure 2.5.

2.5.1 Glycoprotein receptors

As has been discussed in 2.3, the plasma membrane of platelets has many glycoprotein-rich transmembrane receptors which mainly include the IIb3 integrins (indicated in Figure 2.5 by magenta color) which are the most abundant glycoproteins on the platelet surface and the GP-Ib-IX-V complex. Resting platelets contain about 80,000 copies of the IIb3 integrins on

18 their surfaces [135, 160]. In response to a stimulus for e.g. a vascular injury, these receptors help the platelets to adhere to the ECM at the site of injury. In resting platelets, the integrins are in a low-affinity state and have a bent conformation which prevents them from binding to extracellular ligands. However, once the platelets are activated by a stimulus, the integrins switch to the high-affinity state by unbending and change their conformation making it easier to bind to the ligands [94].

2.5.2 The spectrin cortex

The first structure beneath the platelet plasma membrane is a spectrin-based cytoskeleton (indicated in Figure 2.5 by amber color). The spectrin strands assemble in two-dimension and interconnect to each other at their ends by binding to actin filaments. There is an actin-binding site at each molecular end of the spectrin molecule. The RBCs also have a spectrin cytoskeleton which has been studied in great details. Under an electron microscope, the spectrin strands in RBCs are seen to be heterotetramers which are composed of head-to-head aggregates of  chains [94]. The subunit chains associate laterally to form heterodimers which in turn associate to form the heterotetramers. There is an actin-binding site at the amino terminus of the ß subunit of each heterodimer. Ankyrin helps to anchor the spectrin strands to the plasma membrane. In platelets, spectrin is highly expressed in MKs just before they start to make the proplatelets and it has been recently shown that the assembly of spectrin tetramers are essential for MK maturation, proplatelet extension and also in the maintenance of the 'barbell-shapes' of proplatelets [109].

2.5.3 The acto-myosin cortex and the cytoplasmic actin network

Underlying the spectrin is an actin cortex that too supports the platelet membrane skeleton and is both, directly and indirectly, connected to the spectrin cortex. Actin is the most abundant protein in platelets with a concentration of 0.55 mM, which approximately equals to 2 × 10⁶ copies per platelet. In the resting platelet, ~ 40% of the actin is in the form of filaments i.e. F-actin. About 800,000 actin molecules assemble into 2000-5000 actin filaments that span the cytoskeleton of the resting platelet [13, 94]. The rest of the actin (~ 60%) is in the form of monomeric globular actin i.e. G-actin [47]. Also, platelets express high concentrations of actin cross-linking proteins such as filamin and -actinin. Electron microscopy studies have described that the actin cytoskeleton of resting platelets resembles the structure of a spoked wheel [56]. At the rim of this wheel, is a two-dimensional spectrin-actin network where the

19 two spectrin strands interconnect with each other via long actin filaments. These filaments originate from a highly crosslinked three-dimensional actin filament core in the cytoplasm that forms the hub of this wheel (indicated in Figure 2.5 by red color). Filamin connections from the sides of actin filaments connect with the cytoplasmic tails of the GP-Ib-IX-V receptors and hold the membrane skeleton in compression in between the cytoplasmic actin and the plasma membrane. Apart from actin, platelets also have non-muscle myosin IIA. The actin filaments are polarized structures and are decorated with myosin heads whose stereospecific binding defines this polarity thus allowing the actin barbed ends and pointed ends to be distinguished [94]. It is believed that the presence of myosin at the membrane skeleton keeps it in a taut state by maintaining contractile tension on the actin filaments which are connected to the GP-Ib-IX-V receptors [56].

Apart from the physical constraints provided by the internal cytoskeleton to maintain the platelet discoid shape, there are also some biochemical constraints that prevent the platelets from polymerizing the G-actin and changing their shape. These include proteins like profilin and thymosin ß4 that sequester G-actin and lower free monomer concentrations. Furthermore monomer addition to the barbed ends of actin filaments is prevented by proteins like gelsolin and capZ which cap these ends. Additionally, the stable actin filaments are continuously undergoing treadmilling (as explained in section 2.4.1) which keeps the lengths of the filaments constant. The protein profilin, apart from sequestering monomers, helps in maintaining this treadmilling, as it catalyzes the exchange of ADP-G-actin to the ATP-G-actin, the form that can polymerize. However, to ensure that not all ADP-G-actin is exchanged, thymosin ß4 binds to ADP-G-actin and prevents the nucleotide exchange [25].

The F/actin filaments are further stabilized along their lengths by proteins like vasoactive-stimulated phosphoprotein (VASP), which prevents actin depolymerization from the () end.

2.5.4 The MT coil

As has been discussed before (see section 2.2), the platelets released from MKs contain MT.

In platelets, there exist several MTs which are organized in a circumferential ring structure called the marginal band (MB) which is located just beneath the actin cortex (indicated in Figure 2.5 by shades of blue). This 'MB' has also been referred to as circumferential MT coil [169] or microtubule (MT) coil [108] or even microtubular (MT) ring structure [36]. It has been recently shown that this MB is a bipolar array and consists of multiple MTs that coil several times and form a single long-lived, detyrosinated/acetylated (stable) MT and is

20 associated with 8-12 tyrosinated/deacetylated (dynamic) MTs which polymerize in both directions around the MB of the resting platelet. The dynamic MTs are required to efficiently and quickly change the MB size because platelets reduce in size as they age which in turn requires the shrinkage of the MT coil. Platelets do not have a MTOC but instead -tubulin within the MB nucleates the MTs [108]. Furthermore, an equilibrium between the MT (+) end directed motor protein kinesin and () end directed motor protein dynein keeps the platelet MBs in their resting state and the dynein motors anchor the MB to the acto-myosin cortex [122]. The MB of the platelets is important for maintaining their discoid shape (Figure 2.6A) as it has been observed that, mice that have 1-tubulin deficient MKs, are affected by platelet spherocytosis [64]. In this dissertation, the terms MT coil/ring refer to the MB structure described above.

Figure 2.5: Sketch of platelet cytoskeleton at rest

A simplified sketch of the structure of the platelet cytoskeleton at rest is shown. Platelets in the resting, quiescent state circulate in the bloodstream and have a discoid shape which is maintained due to their internal cytoskeleton. The discoid platelets have a plasma membrane (black) which has transmembrane receptors like IIb3 integrins (magenta). Below this is a spectrin cortex (orange) that forms the backbone of the cytoskeleton and which is connected to an acto-myosin cortex (short red filaments). Together actin and myosin provide tension and hence maintain the shape. Below this lies the circumferential MB (blue) that comprises stable and dynamic MTs. The cytoplasm is filled with a rigid network of F-actin filaments (long red filaments) that resemble the spokes of a wheel and maintain the platelet shape.

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