Catabolism of coagulation factor VIII (fVIII) is mediated by the hepatic multiligand receptor low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CRs) organized in four clusters, among which clusters II and IV bind most of LRP ligands. In turn, fVIII contains two major LRP-binding sites, located in A2 and A3 domains (Saenko et al, JBC 1999; Bovenschen et al, JBC 2003). In present work, we characterized binding sites in LRP for A2 domain (A2) and heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. Using a baculovirus expression system, we generated CR clusters II, III and IV, along with eight overlapping CR triplets encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to clusters II and IV, and to the same sets of their CR triplets with similar affinities (KDs 25–50 nM). The same kinetic parameters of interaction of both A2 and HD were observed for several CR doublets from cluster II, shown previously to be minimal binding sites for a classical ligand of LRP, receptor associated protein (RAP) (Andersen et al, JBC 2000). The specificity of A2 and HD interactions with all tested fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit binding of 125I-A2 and 125I-HD to immobilized LRP in a solid-phase assay, and LRP-mediated catabolism of 125I-A2 and 125I-HD in cell culture. Notably, some mutations of the LRP-binding site in A2 resulted in significant reduction or abolishment of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. In summary, we demonstrated that i) A2 and HD interact with LRP via its multiple binding sites spanning CRs 3–8 in cluster II and CRs 24–29 in cluster IV, and ii) the elementary binding unit of LRP is formed by at least two adjacent CRs, similar to that shown for RAP. The above data also suggest that besides regulating fVIII levels, LRP also plays a role in clearance of the products of dissociation of activated fVIII.

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Clearance of coagulation factor VIII (fVIII) is mediated by a hepatic receptor low-density lipoprotein receptor-related protein (LRP), a member of low-density lipoprotein receptor (LDLR) family. It has been recently discovered that LDLR acts in concert with LRP in regulating fVIII level. FVIII has the domain structure A1-A2-B-A3-C1-C2, and to identify the portions providing the interaction with LDLR, in surface plasmon resonance-based assay we studied the binding of fVIII and its fragments to immobilized recombinant ligand-binding domain of LDLR (residues 1-292). The affinity values were determined from the families of binding signals obtained for five concentrations (10–150 nM) of each analyte. The binding signals for full-length fVIII, and its portions A3-C1–C2 (or light chain, LCh) and A1/A3-C1–C2 heterodimer (derived from activated fVIII) were best fitted to a two-site model, with equilibrium dissociation constants KD(1) ~1, 4, 14 nM and KD(2) ~14, 45 and 37 nM, respectively. Noteworthy, we did not observe any significant binding for the isolated C2 domain (at 300 nM). This suggests that the LDLR-binding site within LCh is likely located within the A3 domain, similar to that found previously for LCh-LRP interaction. The binding signals for A1-A2-B (heavy chain, HCh) were best fitted to a one-site model, with KD ~60 nM. We registered a dose-dependent, high-affinity binding of the isolated A2 domain to LDLR, with KD ~14 nM whereas the A1 domain showed no appreciable binding. This suggests that within HCh, A2 domain bears the LDLR-binding site. Von Willebrand factor did not significantly block the binding of fVIII to LDLR as compared to a 3-fold inhibition of fVIII binding to LRP. This indicates that within fVIII/vWf complex, the A2 binding site for LDLR is more available than that for LRP. Anti-A2 monoclonal antibody 413 (epitope 484–509) inhibited the A2 binding to LDLR in a dose-dependent manner, similarly to that demonstrated for fVIII-LRP interaction. A number of A2 point mutants with substitutions of the residues critical for A2 binding to LRP, megalin and VLDL were found to have significantly reduced affinity also for LDLR. The obtained data indicate that fVIII interacts with LDLR preferentially via the binding sites located within the A2 domain of HCh and within the A3 domain of LCh, and that the A2 site is likely to be universal for the interactions with four tested members of LDL receptor family.

SummaryCatabolism of coagulation factorVIII (FVIII) is mediated by lowdensity lipoprotein receptor-related protein (LRP). The ligandbinding 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 complementtype 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.

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B domain of coagulation factor VIII (fVIII) was previously considered to be dispensable for fVIII function. Recently, it was found that the B domain is important for fVIII intracellular interaction with its chaperon and likely involved in fVIII clearance via asialoglycoprotein receptor. At the same time, the major clearance mechanism of fVIII involves initial interaction with heparan sulfate proteoglycans (HSPGs) followed by internalization via low-density lipoprotein receptor-related protein (LRP), member of low-density lipoprotein receptor (LDLR) family (Saenko et al, 1999; Sarafanov et al, 2001). It is possible that recently discovered clearance of fVIII via LDLR (Bovenschen et al, 2005) occurs in the same way. Since it was previously shown that fVIII binding sites for LRP are not located within B domain, we investigated if the latter regulates fVIII interaction with HSPGs. To explore this role of B domain, we compared the binding of plasma-derived fVIII (pd-fVIII) and recombinant B domain-deleted fVIII (BDD-fVIII) to immobilized LRP and heparin (a model of HSPGs) in surface plasmon resonance-based assay. The corresponding affinities were assessed by processing the binding signals obtained for five different concentrations of each analyte. Both pd-fVIII and BDD-fVIII showed similar affinities for LRP (KD 42–60 nM). The LRP-binding site of BDD-fVIII was partially blocked by pre-incubation with its carrier protein von Willebrand factor (vWf) indicating that it is only partially accessible within fVIII/vWf complex. This was further confirmed by the finding that monoclonal antibody 413, which recognizes a high-affinity LRP-binding site within the fVIII A2 domain, interacted with ~25% of BDD-fVIII molecules bound to immobilized vWf. The affinities of pd-fVIII and recombinant BDD-fVIII for immobilized heparin were similar (KD ~20 nM) and 2-fold higher than that for purified A2 domain (KD ~46 nM). Noteworthy, the maximal binding level (Rmax) proved to be 10-fold lower for pd-fVIII in comparison with BDD-fVIII indicating that in the circulation intact fVIII may have limited interaction with HSPGs. Importantly, pre-incubation with vWf did not interfere with the interaction between BDD-fVIII and heparin (KD ~ 19.5 and 21.8 nM, Rmax ~ 194 and 354 RU for BDD-fVIII and BDD-fVIII/vWf, respectively) thus revealing that heparin-binding site of fVIII is completely exposed in BDD-fVIII/vWf complex. These findings suggest that the presence of B domain in circulating fVIII/vWf complex may regulate fVIII clearance by preventing its interaction with HSPGs. The absence of B domain leads to exposure of heparin-binding site within fVIII and binding of fVIII/vWf complex to HSPGs. This binding may be a driving force in fVIII clearance which involves subsequent exposure of LRP (LDLR)-binding site(s) and internalization of fVIII from its complex with vWf via these receptors.

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Методом трансмиссионной электронной микроскопии негативно контрастированных образцов исследована структура сополимеров фибриногена (F) с N-концевым дисульфидным узлом дез-А ABB-фибрина (tN-ДСУ). Показано, что первичным продуктом сополимеризации является нить из молекул фибриногена, расположенных по типу конец-к-концу. Молекулы tN-ДСУ, связывающие между собой молекулы фибриногена, на электронное рам мах не просматриваются ввиду, как мы полагаем, их малого размера и отсутствия глобулярной структуры. Образующиеся однонитчатые сополимеры латерально агрегируют в фибриллы различной толщины, создающие трехмерную фибриллярную сеть. Полученные данные подтверждают ранее предложенную гипотезу о струк турной основе F-tN-ДСУ сополимера как нити расположенных конец-к-концу молекул фибриногена, соседние Д-домены которых связаны молекулами tN-ДСУ по центрам полимеризации фибрина а-А и Ь-В. Показанный нами многостадийный процесс сборки сополимера подобен таковому полимери зации фибрина, что свидетельствует о лежащем в их основе едином механизме самосборки путем связывания друг с другом комплементарных центров полимеризации фибрина.