The molecular basis of factor V and VIII procofactor activation

Abstract

Activation of precursor proteins by specific and limited proteolysis is a hallmark of the hemostatic process. The homologous coagulation factors (F)V and FVIII circulate in an inactive, quiescent state in blood. In this so-called procofactor state, these proteins have little, if any procoagulant activity and do not participate to any significant degree in their respective macromolecular enzymatic complexes. Thrombin is considered a key physiological activator, cleaving select peptide bonds in FV and FVIII which ultimately leads to appropriate structural changes that impart cofactor function. As the active cofactors (FVa and FVIIIa) have an enormous impact on thrombin and FXa generation, maintaining FV and FVIII as inactive procofactors undoubtedly plays an important regulatory role that has likely evolved to maintain normal hemostasis. Over the past three decades there has been widespread interest in studying the proteolytic events that lead to the activation of these proteins. While a great deal has been learned, mechanistic explanations as to how bond cleavage facilitates conversion to the active cofactor species remain incompletely understood. However, recent advances have been made detailing how thrombin recognizes FV and FVIII and also how the FV B-domain plays a dominant role in maintaining the procofactor state. Here we review our current understanding of the molecular process of procofactor activation with a particular emphasis on FV.

Fig. 1

Schematic representation of factor (F)VIII, FVIIIa and FVIII-SQ. Boundaries of the acidic regions denoted by a1, a2, and a3 are indicated. ‘Activation’ represents thrombin-mediated proteolysis of FVIII and cleavage sites are indicated as well as the molecular weight of the various fragments. The ‘SQ-linker’ in rFVIII-SQ is given above the schematic.

Fig. 2

Schematic representation of factor (F)V, FVa and FV-810. ‘Activation’ represents thrombin-mediated proteolysis of FV and cleavage sites are indicated as well as the molecular weight of the various fragments. The sequence above FV-810 indicates which B-domain elements have been deleted.

Fig. 3

Schematic representation of the factor (F)V B-domain. The B-domain is defined by residues 710–1545 which are liberated after thrombin-mediated proteolysis. An expanded view of the B-domain is indicated along with the sequence of the basic region (963–1008) implicated in preserving the FV procofactor state. Yellow circles, potential N-linked glycosylation sites; green box, 31X-9 amino acid tandem repeat region; red box, 2X-17 amino acid repeat region; blue box residues 963–1008.

Fig. 4

Alignment of factor (F)V B-domain sequences from the highly basic region. The conserved basic region (residues 963–1008) derived from the human FV sequence was aligned using a modified CLustal W algorithm (AlignX Module; Invitrogen Corporation town, state (if applicable), and country.) to corresponding regions from several select vertebrates (see Table 1 for common names).