Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.

Catabolism of coagulation factor VIII (FVIII) is mediated by low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complement-type repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25-90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3-4, 4-5, 5-6, 6-7 and 7-8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay, LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3-8 in cluster II and CR 23-29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

Interaction of the fibronectin COOH-terminal Fib-2 regions with fibrin: further characterization and localization of the Fib-2-binding sites.

Incorporation of fibronectin into fibrin clots is important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. Previous studies revealed that this incorporation occurs through noncovalent interaction between two NH2-terminal Fib-1 regions of fibronectin (one on each chain) and the alphaC-regions of fibrin, and is further reinforced by factor XIIIa-mediated covalent cross-linking of fibronectin to the fibrin matrix. To clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin, we prepared by limited proteolysis a dimeric 140 kDa (Fib-2)2 fragment containing both Fib-2 regions and tested its interaction with recombinant fragments corresponding to the alphaC regions of fibrin(ogen). In both ELISA and surface plasmon resonance (SPR) experiments 140 kDa (Fib-2)2 bound to the immobilized Aalpha221-610 alphaC-fragment. However, the affinity of binding was substantially lower than that for Fib-1. Ligand blotting and ELISA established that the Fib-2 binding site is located in the connector part of the alphaC region including residues Aalpha221-391. Analysis of the SPR-detected binding of fibronectin to the immobilized Aalpha221-610 alphaC-fragment revealed two types of fibronectin-binding sites, one with high affinity and another one with much lower affinity. Competition experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment suggesting partial overlap of the two sets of binding sites. Based on these results and our previous studies we propose a mechanism of interaction of fibronectin with fibrin in which both Fib-1 and Fib-2 play a role.

Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

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. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.

Factor XIIIa incorporates thymosin beta4 preferentially into the fibrin(ogen) alphaC-domains.

It was shown recently that tissue transglutaminase and presumably plasma transglutaminase, factor XIIIa, can covalently incorporate into fibrin(ogen) a physiologically active peptide, thymosin beta(4) [(Huff et al. (2002) FASEB J. 16, 691-696]. To clarify the mechanism of this incorporation, we studied the interaction of thymosin beta(4) with fibrinogen, fibrin, and their recombinant fragments, the gamma-module (gamma-chain residues 148-411), and the alphaC-domain (Aalpha-chain residues 221-610) and its truncated variants by immunoblot and ELISA. No significant noncovalent interaction between them was detected in the absence of activated factor XIII, while in its presence thymosin beta(4) was effectively incorporated into fibrin and to a lesser extent into fibrinogen. The incorporation at physiological concentrations of fibrin(ogen) and factor XIII was significant with molar incorporation ratios of thymosin beta(4) to fibrinogen and fibrin of 0.2 and 0.4, respectively. Further experiments revealed that although activated factor XIII incorporates thymosin beta(4) into the isolated gamma-module and alphaC-domain, in fibrin the latter serves as the major incorporation site. This site was further localized to the COOH-terminal portion of the alphaC-domain including residues 392-610.