Interaction between complementary polymerization sites in the structural D and E domains of human fibrin

Plasmic degradation products of human fibrin, fragments DD, D, and E, bind to fibrin. It has been inferred from this observation that the binding occurs by attraction of complementary sites located in the NH₂-and COOH-terminal domains of the fibrin molecule. The interaction between fragments D₁ and E₁ has been investigated in this work since it represents the first step in the process of fibrin clot formation. Fragment D₁, that was initially as active as fragment DD, lost most of its anticoagulant activity after purification by cation-exchange chromatography. The lability of fragment D₁ function explained the previous unsuccessful attempts to form a complex between fragments D₁ and E₁. The loss of fragment D₁ anticoagulant activity was not associated with the cleavage of the γ63-85 chain segment, since fragments D_1A and D₁ identically inhibited the fibrin monomer polymerization rate. In order to demonstrate the formation of a complex between fragments D₁ and E₁, three lines of experiments were advanced. First, the anticoagulant activity of fragment D₁ was neutralized by fragment E₁ in a dose-dependent manner, demonstrating that the association between these fragments involved polymerization sites. Second, two products, D₁·E₁ and D₁·E₁·D₁, were stabilized in a reaction with bifunctional cross-linking reagents, proving the formation of D·E complexes in aqueous solution. Third, immobilized fragment D₁ bound fragments E₁ and E₂, but not fragment E₃, showing that fragments E₁ and E₂ attached via a polymerization site to the complementary one in fragment D₁, since this association was disrupted by fibrin polymerization inhibitory peptide GPRP. These results provided direct evidence for specific binding between the structural D and E domains of fibrin mediated through complementary polymerization sites. Thus, the initial formation of fibrin clot fibers appears to be driven by specific association of these sites.