A Single Cleavage Reaction in the Proteolytic Activation of Factor V to the Cofactor Is Rate-Limiting in a Step That Is Selectively Dependent on Anion Binding Exosite 2 of Thrombin

Human factor V (hFV) is a procofactor with a domain organization of A1-A2-B-A3-C1-C2. It requires proteolytic activation before it can assemble within the prothrombinase complex and function as a cofactor. Activation by thrombin results from cleavages following Arg⁷⁰⁹, Arg¹⁰¹⁸ and Arg¹⁵⁴⁵ to release the B domain in two fragments and form heterodimeric factor Va (FVa, A1-A2/A3-C1-C2). While A1-A2/A3-C1-C2 is the prototypic active cofactor, variable cofactor function is attributed to partially cleaved species. Several studies have proposed that meizothrombin (mIIa), an intermediate produced during prothrombin activation, is also an efficient activator of membrane-bound hFV and possibly responsible for FVa formation early in the clotting process. However, this is inconsistent with the proposed importance of anion binding exosite 2 (ABE2) for hFV activation by thrombin which is occluded by the covalently attached propiece in mIIa. We have studied the kinetics of hFV cleavage using a quantitative two-color immunoblotting approach to simultaneously and unambiguously identify intermediates and products containing A1-A2 and/or A3-C1-C2. We have also compared hFV cleavage by thrombin by cleavage with a stable mIIa variant and by mIIa lacking both the 4-carboxyglutamic acid and kringle 1 domains (mIIa-desF1) in the presence of membranes. Addition of 0.1 nM thrombin yielded quantitative cleavage of hFV (50 nM) in ~6 minutes, producing products consistent with cleavage at Arg⁷⁰⁹ and/or Arg¹⁰¹⁸. However, the C-terminal intermediate beginning at 1019 but uncleaved at Arg¹⁵⁴⁵ persisted for more than 90 minutes. A3-C1-C2, arising from cleavage at Arg¹⁵⁴⁵, appeared at a ~20-fold slower rate than the A1-A2 species. Thus, for the reaction with thrombin, cleavage at Arg¹⁵⁴⁵ represents the rate limiting step in the conversion of hFV to heterodimeric FVa. Similar rates of hFV consumption and appearance of species cleaved at Arg⁷⁰⁹ and/or Arg¹⁰¹⁸ were seen with mIIa. However, cleavage at Arg¹⁵⁴⁵ to produce A3-C1-C2 was substantially slower, by a factor of ~100, than A1-A2 formation. Equivalently large differences in the rates of A1-A2 and A3-C1-C2 formation were evident in the absence of membranes except the rate of all cleavages by mIIa was further reduced. Because the results obtained with mIIa were replicated with mIIa-desF1, the data imply that it is covalent occlusion of ABE2 in these mIIa variants that underlies their selectively impaired ability to cleave at Arg¹⁵⁴⁵ and produce heterodimeric Va but does not affect cleavage at Arg⁷⁰⁹ or Arg¹⁰¹⁸. The binding of Xa to activated FV, essential for membrane-dependent prothrombinase assembly, is a prerequisite for cofactor function. We correlated hFV cleavage with the development of cofactor function by binding measurements using Xa inactivated with a fluorescent probe covalently tethered to the active site with a peptidyl chloromethylketone. For hFV cleavage by thrombin, Xa binding and the assembly of prothrombinase proceeded unexpectedly slowly and correlated with cleavage at Arg¹⁵⁴⁵, widely separated in time from rapid cleavage at Arg⁷⁰⁹ and/or Arg¹⁰¹⁸. Therefore, slow cleavage at Arg¹⁵⁴⁵ dictates both the formation of heterodimeric FVa and the ability of the product to bind Xa with high affinity to form prothrombinase. Only a small fraction of Xa binding developed within an hour in hFV cleavage reactions initiated with mIIa in comparison to the same concentration of thrombin. Our findings indicate that ABE2 in thrombin variants plays an essential role in the rate-limiting cleavage at Arg¹⁵⁴⁵ required for the formation of heterodimeric FVa and for the assembly of prothrombinase. Occlusion of ABE2 by the covalently linked propiece in mIIa variants greatly impairs their ability to activate hFV to the active cofactor by selectively decreasing the rate of cleavage at Arg¹⁵⁴⁵. The surprising and near absolute need for cleavage at Arg¹⁵⁴⁵ to produce cofactor is consistent with new biochemical and structural evidence implicating an essential role played by an acidic sequence immediately N-terminal to Arg¹⁵⁴⁵ in concert with a basic sequence C-terminal to Arg¹⁰¹⁸ within the B domain in restricting the binding of Xa to the incompletely cleaved procofactor and preventing cofactor function.