A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of γ chains by factor XIII _a

Abstract

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its ␣ chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIII a -catalyzed end-to-end ligation of the ␥ chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIII a reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of ␣ chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.The clotting of fibrinogen in human plasma is regulated by a remarkable series of biochemical controls (for a recent summary, see ref. 1). The key for unlocking the self-assembly potential of fibrinogen in its conversion to fibrin is the limited proteolytic attack by thrombin that, through removal of the N-terminal fibrinopeptide moieties (2, 3), generates a new set of Gly end groups (4, 5). The short sequence of amino acids including and following the newly exposed N-terminal Gly residues of the two ␣ chains of fibrin (6, 7) seems to be the necessary ligand for causing the noncovalent, reversible aggregation of fibrin into a clot (5,8,9). Gly-Pro-Arg-Pro, which resembles the natural Gly-Pro-Arg-Val sequences (knobs) in the fibrinopeptide A-denuded central E domain of fibrin, can prevent clot formation (10). The tetrapeptide blocks the polymerization pockets or holes located in the ␥ chains at the two D end domains of the protein (11-16). It is important to bear in mind that these polymerization pockets are present not only in fibrin but also in the parent fibrinogen molecule, i.e., thrombin action is not required for opening them for Gly-ProArg-Pro to bind. Thus, the two N-terminal knobs in the central E domain of the protein unmasked by thrombin seem to act as a bifunctional ligand for the end-to-end, noncovalent assembly of fibrin units into the well known, half-staggered, double array of protofibrils seen in the electron microscope (17, 18). This brings together each E domain with the D domains of two other molecules.The assembly process, as organized by the thrombinmodified E domains, imparts a major advantage for the next enzymatic phase of the coagulation cascade, the factor XIII acatalyzed covalent stabilization of fibrin filaments by N (␥-glutamyl)lysine crosslinks. Fibrinogen in solution is about an order of magnitude weaker substrate for factor XIII a than clotted fibrin (19,20), which, from the physiological point of view, is obviously an important feed-forward regulatory con...

Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms

“…In the presence of the tetrapeptide Gly-ProArg-Pro, which inhibits fibrin polymerization, the glutamine sites in the carboxy terminal tail of the ␥-chain are no longer aligned for rapid cross-linking by factor XIIIa. 50 Further evidence that end-toend alignment of the D-domains of fibrin plays an important role in the cross-linking reaction was provided by experiments performed by Lorand et al 51 The authors found that a double-headed ligand, produced from 2 Gly-Pro-Arg-Pro peptides linked to each end of a long polyethylene glycol molecule, could mimic the E-region in joining 2 D-regions and allow for ␥-chain cross-linking to occur. 51 In the absence of the double-headed ligand (or the E-region), no cross-linking takes place between 2 D-regions.…”

Modulation of fibrin matrix properties via knob:hole affinity interactions using peptide–PEG conjugates

“…A separate group also found that a bivalent GPRP-conjugated 900 Da PEG linker promotes the end-to-end crosslinking of fibrinogen γ chains by factor XIIIa, suggesting that this length of PEG spans the distance between two adjacent holes a within a fibrin network [13]. Previously, our group has shown that GPRP binds specifically to fibrin(ogen) even when presented in the context of a non-binding 20 kDa protein, implying that this short peptide sequence is sufficient for mediating knob:hole interactions with both fibrin and fibrinogen [14].…”

Synthetic Strategies for Engineering Intravenous Hemostats

“…79 Presence of free knob A peptide mimic in a mixture of thrombin and fibrinogen inhibits clot formation due to competition with fibrin monomers for hole a. 80 However, as demonstrated by Lorand et al, a bivalent construct terminated with knob A peptides is able to crosslink fibrin.…”

“…( B ) Bis(GlyProArgPro-amido)peg demonstrated biphasic behavior where lower “productive” polymer concentrations formed D-D crosslinks and higher “non-productive” polymer concentrations showed less efficient crosslinking due to competition of polymers for the finite number of hole a’s. ( A-B ) Reproduced with permission from 79 . Copyright © 1998 National Academy of Sciences, U.S.A. ( C ) Biphasic behavior was also observed in turbidity measurements for fibrin formed with 4-arm PEG-knob A conjugates of various molecular weights.…”