It has long been known that cells often have a very large pool of unpolymerized actin, sometimes constituting as much as half the actin in the cell. Since cellular actin levels can be as high as 100–400 µM, this means that there can be as much as 50–200 µM unpolymerized actin in cells. Since the critical concentration in vitro is about 0.2 µM, why doesn’t all this actin polymerize? The answer lies, at least in part, in the presence of actin monomer sequestering proteins. One of these proteins is thymosin-β₄, a small protein that binds to ATP–G-actin in such a way that it inhibits addition of the actin subunit to either end of the filament. Thymosin-β₄ can be very plentiful, for example, in human blood platelets. These disk-shaped cell fragments are very abundant in the blood, and when they are activated during blood clotting, they undergo a burst of actin assembly. Platelets are rich in actin: they are estimated to have a total concentration of about 550 µM actin, of which about 220 µM is in the unpolymerized form. They also contain about 500 µM thymosin-β₄, which sequesters much of the free actin. However, as in any protein-protein interaction, free actin and free thymosin-β₄ are in a dynamic equilibrium with actin–thymosin-β₄. If some of the free actin is used up for polymerization, more actin–thymosin-β₄ will dissociate, providing more free actin for polymerization (see Figure 17-11). Thus thymosin-β₄ behaves as a buffer of unpolymerized actin, making it available when it is needed.