Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

Abstract

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A␤ fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA (G14V). Analyses of fibrinopeptide release from A␤ fibrinogen showed that FpA release and FpA release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA release from A␤ fibrinogen was unaffected. Thus, with A␤ fibrinogen, fibrinopeptide release from the ␤ chain is similar to fibrinopeptide release from the ␣ chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A␤ fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A␤ fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the ␤ chain does not affect polymerization of fibrinogen.Fibrinogen is a 340-kDa plasma protein that is involved in the final phase of the coagulation cascade. Fibrinogen consists of two pairs of three polypeptide chains (A␣, B␤, and ␥) that fold to produce a trinodular protein with two distal (D) nodules connected to a central nodule (E) by coiled-coil regions. The central nodule of the molecule consists of the N termini of all six polypeptide chains, and the D nodules consist predominantly of the C termini of the ␤ and ␥ chains, each folded into a globular domain. To initiate polymerization, the serine protease thrombin cleaves four specific Arg-Gly bonds at the N termini of both the A␣ and B␤ chains, releasing fibrinopeptides A (FpA) 1 and B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the "A" site, which noncovalently interacts with the "a" site in the ␥ chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1). The release of FpB, a 14-residue peptide, exposes the "B" site (2-4), which presumably interacts with a "b" site in the ␤ chain of the D nodule of another molecule (5). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood. The final product of this polymerization is a complex, branching network of fibers.The interactions of thrombin with fibrinogen have been extensively studied (Refs. 6 and 7; for a review, see Ref. 8)....

Fibrinopeptides A and B release in the process of surface fibrin formation

“…22 At the same time, no delay of FpB release (lag phase) from side-on adsorbed fibrinogen was observed. Because such a delay was proposed to be connected with conformational changes accompanying FpA release 21 and protofibril formation, 22 such changes seem to have already occurred in adsorbed fibrinogen. This may also have been the reason for the absence of a lag phase in FpB release from the end-on adsorbed fibrinogen or fibrinogen attached to the primary fibrin layer (Figures 4A and 5).…”

“…on May 11, 2018. by guest www.bloodjournal.org From the N-terminal parts of the B␤ chains with the dimeric DD regions formed in protofibrils and fibers, bringing FpB into the vicinity of bound thrombin. [17][18][19][20][21][22] So far, fibrin(ogen) characteristics, such as the release of fibrinopeptides and fibrin-fibrinogen interactions, have been mostly studied in solution. 23,24 However, adsorbed fibrinogen acquires new properties important for biointeraction compared with fibrinogen in solution.…”

Genetic and Environmental Determinants of Fibrin Structure and Function

“…Release of fibrinopeptide A by thrombin is fast and exposes a polymerization site on the E region of fibrin. 4,5 This combines with a complementary binding site on the ␥ chain in the D region of an adjacent fibrin molecule to form a double-stranded twisted protofibril of fibrin. Cleavage of fibrinopeptide B by thrombin is slower but also exposes a binding site in the E region.…”

Directing thrombin

“…[37][38][39] Release of FPA is sufficient to induce fibrin polymerization, and, while the release of FPB follows closely that of FPA, it is determined by the relative specificity constants of thrombin for the A␣ and B␤ chains, and premature release of FPB does not affect polymerisation. 40 Exosite I is also involved in the binding of thrombin to the B␤ chain to cleave FPB (Table 1). Another early action of thrombin is activation of factor V, needed to promote further generation of thrombin.…”