Structure, stability, and interaction of fibrin αC-domain polymers.

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa.

Noncovalent interaction of alpha(2)-antiplasmin with fibrin(ogen): localization of alpha(2)-antiplasmin-binding sites.

Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance.

Structure, stability, and interaction of the fibrin(ogen) alphaC-domains.

Our recent study established the NMR structure of the recombinant bAalpha406-483 fragment corresponding to the NH(2)-terminal half of the bovine fibrinogen alphaC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen alphaC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAalpha425-503, homologous to bovine bAalpha406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAalpha406-483.

Direct evidence for specific interactions of the fibrinogen alphaC-domains with the central E region and with each other.

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy.

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions.

Interaction of fibrin(ogen) with apolipoprotein(a): further characterization and identification of a novel lysine-dependent apolipoprotein(a)-binding site within the gamma chain 287-411 region.

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear.

Identification of an ordered compact structure within the recombinant bovine fibrinogen alphaC-domain fragment by NMR.

The NMR solution structure of the bovine fibrinogen alphaC-domain fragment, including residues Aalpha374-538, reveals a type-I' beta-hairpin, restricted at the base by a C423-C453 disulfide linkage and a short turn preceding C423. Although both faces of the hairpin are formed mainly by hydrophilic residues, one of them is uncharged while the other has a characteristic pattern of charged residues which are highly conserved among vertebrate species. Chemical shift indexing and relaxation data indicate the presence of a collapsed hydrophobic region next to the hairpin that includes approximately 30 residues with slower concerted motion and higher content of nonpolar residues and, according to a previous study (Tsurupa, G.

Transglutaminase-mediated oligomerization of the fibrin(ogen) alphaC domains promotes integrin-dependent cell adhesion and signaling.

Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen alphaC domains with factor XIIIa generates ordered alphaC oligomers mimicking polymeric arrangement of the alphaC domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the alphaC domain-mediated interaction of fibrin with endothelial cells.

Do the isolated fibrinogen alphaC-domains form ordered oligomers?

Previous electron microscopy (EM) studies revealed that the proteolytically prepared, truncated, bovine fibrinogen alphaC-domain (Aalpha223-539 fragment) upon transfer from acidic to neutral pH formed ordered oligomers which could mimic alpha polymers of cross-linked fibrin. In this study, we demonstrated that although its recombinant analog, bAalpha224-538, as well as the full-length version of the alphaC-domain (bAalpha224-568), upon similar treatment also formed oligomers with ordered structure, both were monomeric when kept in neutral pH buffer. To search further for conditions for their oligomerization, we treated bAalpha224-568 with factor XIIIa, purified the cross-linked soluble fraction, and confirmed that it consisted of oligomers.

Identification and characterization of novel lysine-independent apolipoprotein(a)-binding sites in fibrin(ogen) alphaC-domains.

Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a).

Unisex pheromone detectors and pheromone-binding proteins in scarab beetles.

Olfaction was studied in two species of scarab beetle, Anomala octiescostata and Anomala cuprea (Coleoptera: Scarabaeidae: Rutelinae), which are temporarily isolated and use the same sex pheromone compounds, (R)-buibuilactone and (R)-japonilure. Single sensillum recordings in A. octiescostata revealed highly sensitive olfactory receptor neurons (ORNs) (threshold <1 pg) that were tuned to the detection of the green leaf volatile compound (Z)-3-hexenyl acetate.

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site.

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments.

Structural organization of the fibrin(ogen) alpha C-domain.

We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure [Weisel and Medved (2001) Ann. N.Y.