PMC

Kinetic constants measured for the individual half-reactions are listed. V/E denotes V_max/E for the indicated cleavage reactions and is more appropriate than referring to this term as kcat. Taken from Orcutt & Krishnaswamy ().

Initial cleavage of prothrombin following Arg³²⁰ yields a zymogen-like form (mIIa’) that interconverts slowly and reversibly with the proteinase-like form (mIIa). Because the proteinase-like conformation is required for further cleavage at Arg²⁷¹, it is only mIIa rather than mIIa’ that is processed to thrombin. The reversible conversion of mIIa’ to mIIa there represents a previously unanticipated rate-limiting step in thrombin formation.

The conversion of prothrombin to thrombin results from cleavages following Arg²⁷¹ and Arg³²⁰. Initial cleavage following Arg²⁷¹ yields the pathway on the left and produces the zymogen, prethrombin 2 (P2) and the propiece, fragment 1.2 (F12) as intermediates. P2 requires further processing at Arg³²⁰ to yield thrombin. The pathway on the right arises from initial cleavage following Arg³²⁰, which produces the proteinase meizothrombin (mIIa) as an intermediate. Further cleavage following Arg²⁷¹ is required to yield IIa and the propiece, F12. The Arg¹⁵⁵ site (Red Arrow) is susceptible to thrombin cleavage and separates the fragment 1 region from fragment 2 within F12.

Prothrombinase assembles through reversible interactions between the serine proteinase Xa and the protein cofactor Va on membranes containing phosphatidylserine. The enzyme complex cleaves the zymogen, prothrombin (II) at two sites to produce thrombin (IIa), which is composed of two chains in disulphide linkage and the release of the N-terminal propiece fragment 1.2 (F12).

Exosite-binding constrains substrate presentation such that when the substrate is the zymogen, Arg³²⁰ preferentially engages the active-site and is cleaved (Panel A). Conversely, the Arg²⁷¹ site is readily cleaved when the substrate is the proteinase (Panel B). Prior cleavage at Arg³²⁰ yields impaired subsequent cleavage at Arg²⁷¹ in a variant that remains zymogen-like and is defective in making the transition to proteinase (Panel C). Conversely conformational activation and stabilisation of uncleaved prothrombin in a proteinase-like state yields increased cleavage at Arg²⁷¹ at the expense of cleavage at Arg³²⁰ (Panel D).

Cleavage of the individual sites in intact prothrombin was assessed using prothrombin variants in which the two arginines were individually rendered uncleavable by mutation to Gln. The intermediates mIIa and P2/F12 were used to assess cleavage at the individual sites following cleavage at the first site. II_QQ denotes a prothrombin variant in which both Arg side chains were mutated to Gln to yield an uncleavable derivative. The products formed upon the limiting action of prothrombinase on these substrate variants are illustrated.

In the zymogen-like configuration, ABE1, the Na⁺ site and the active-site are not optimally configured while F12 binding to ABE2 is thermodynamically favoured. The reverse is true for the proteinase-like state in which ligands targeting ABE1, Na⁺ and the active-site bind more favourably while ABE2 is not optimally configured. Consequently, binding of F12 to ABE2 favours the zymogen-like form while substrates or inhibitors (S or I), Na⁺ or thrombomodulin (TM) favour the proteinase-like form. It remains to be established as to whether other ligands for ABE1 (i.e. proteinase activated receptor 1 (), fibrinogen ()) or ABE2 (i.e. heparin (), glycoprotein 1bα (,)) replicate the effects seen with F12 and TM at biologically relevant concentrations.

Kinetic scheme resolved for the action of prothrombinase on P2. The initial binding interaction between substrate (S) and prothrombinase (E) to form ES results from exosite-dependent interactions between S and E. Exosite binding is followed by a unimolecular binding step in which structures flanking the cleavage site engage the active-site of the enzyme before catalysis can occur. The product (P) is also bound to E by exosite interactions before it is released. The graphical legend highlights the important features of S and E. The composite nature of the steady state kinetic constants is illustrated by derivation employing the rapid equilibrium assumption. Ks* is defined as [E.S]/[E.S*].