LRP1 was the first found FVIII clearance receptor, described independently by research groups of Saenko and Lenting in 1999 [15,28]. LRP1 is the major clearance receptor for both FVIII and VWF, based on many in vitro, animal, and genetic studies [14]. In circulation, LRP1 is expressed on hepatic cells where it internalizes ~15 ligands, such as alpha-2 macroglobulin, proteases, coagulation factors, and lipoproteins, as well as a large variety of ligands in other tissues [29,30]. LRP1, similar to other related receptors (see below), interacts with its ligands via a flexible string of adjacent complement-type repeat domains (CRs), and lysine residues on ligands are the major determinants for the interactions [31].

On FVIII, such lysine residues are located on the LCh throughout its A3, C1, and C2 domains [28,32,33]. The FVIII-LRP1 interaction (K_D ~20 nM) occurs via formation of multiple alternative binding combinations in a dynamic mode where a real-time interactive site involves a string with three or more CR domains of the receptor and multiple lysines of FVIII, as found by us recently [33]. 

Upon FVIII activation, another LRP1-binding site is exposed on the A2 domain. The functional relevance of that may reflect a participation of LRP1 in plasma clearance of inactivated FVIIIa remnants (A2 domain and A1/A3′-C1-C2 heterodimer). Indeed, each of these fragments is cleared from mouse plasma with the involvement of LRP1 [34,35].

On VWF, the LRP1-binding site is located within its A1 domain [36]; thus, all VWF monomers carry such sites. In contrast to VWF sites for binding FVIII, its LRP1-binding sites are not exposed under static fluid conditions in vitro due to folded state of molecule and become exposed only when it unfolds under fluid shear force in blood vessels [37].

LDLR was identified as the second FVIII plasma clearance receptor in 2005 [16]. LDLR belongs to a large family of endocytic receptors named after LDLR itself and involving seven members in mammals, including LRP1 [38]. The family members are expressed in many tissues where they interact with numerous ligands and regulate many processes, including those with medical importance [29,30,39,40].

Expressed in the liver, LDLR catabolizes a smaller number of ligands, mainly low-density lipoprotein (LDL), relevant to cholesterol metabolism [41]. The role of LDLR in FVIII clearance was supported in a mouse model, where LDLR deficiency resulted in ~1.5-fold prolongation of FVIII half-life, similarly to LRP1. In turn, combined deficiency in both LRP1 and LDLR resulted in ~5-fold prolongation of FVIII plasma half-life with an increase in its plasma level. The authors concluded that LRP1 and LDLR cooperate in FVIII plasma clearance [16], which was supported in further genetic studies [42,43].

Compared to LRP1, the affinity of LDLR for FVIII is lower (K_D 30–60 nM), which may explain its apparently smaller contribution to FVIII plasma clearance. As we found previously, the organization of FVIII binding sites for both receptors is similar as they are located on the FVIII LCh with the same significance of the C1 domain for LDLR binding [44] and due to presence of the binding site also for LDLR in the A2 domain in FVIIIa [45,46]. Reanalysis of these data [44], considering the FVIII-LRP1 interaction mode [33], indicates that the mechanism of FVIII-LDLR interaction is also based on formation of alternative binding combinations.

In addition to LDLR and LRP1, other members of the LDLR family exposed to circulation are very low-density lipoprotein receptor (vLDLR) and megalin, expressed on endothelial and kidney cells, respectively. Both can interact with FVIII and FVIIIa in vitro similarly to LRP1 and LDLR, however, with unknown functional relevance [45,46]. In particular, the role of vLDLR in FVIII clearance was not supported in the mouse model [47]. 

HSPGs have covalent attachment of several negatively charged sulfated polysaccharide chains and are localized on cell membranes. There is a large variety of HSPGs which interact with numerous protein ligands via their basic residues [48,49]. HSPGs serve as receptors or coreceptors, in particular of LRP1, facilitating its ligand recognition. In plasma, such ligands involve lipoprotein lipase [50], apo E-containing lipoproteins [51,52], thrombospondin [53], thrombin-protease nexin 1 complex [54], tissue factor pathway inhibitor [55], thrombin, antithrombin III, and FX [49], and FVIII [17].

In these processes, HSPGs preconcentrate the ligands to facilitate their further interactions with other receptors. In particular, FVIII has extremely low plasma concentration (≤1 nM) [56], insufficient for its effective interaction with LRP1 (K_D ~20 nM). The involvement of HSPGs, abundantly expressed on cells [17], altogether with the cell membrane interaction of FVIII [57], is believed to increase its local concentration and facilitate LRP1 binding to LRP1. Similarly, HSPGs may facilitate FVIII interactions with other clearance receptors.

Other clearance receptors of FVIII and VWF (lectins) recognize their carbohydrates. Activated form of FVIII contains four N-linked glycans, whereas the dissociated B-domain contains ~14 N- and ~7 O-linked glycans [4,58]. VWF contains 23 glycans (N- or O-linked) per monomer [14,59], with an estimated number of >1000 on the molecule. In both proteins, the N-glycans contain bi-, tri-, and tetra-antennary structures with high mannose portions, ABO(H)-determinants; and the O-glycans contain mainly T-antigen. These glycans (as well as in other proteins) are terminally capped by sialic acid sugars with ≥80% occupancy [58,59,60,61,62,63]. Notably, the N-glycosylation of rFVIII differs from pdFVIII mainly in sialic acids, ABO(H) blood group, etc. The greatest difference was found in rFVIII expressed in human embryonic kidney (HEK) cells, whereas the use of baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells also resulted in such a difference [58]. The half-life of VWF is dependent on its ABO(H) group glycans, where the O-type glycan-bearing VWF variants have significantly shorter plasma half-life, associated with FVIII level as noted above [11].

ASGPR (Ashwell receptor), a member of the C-type family of lectins recognizing terminal asialic sugars [64], was found to be a clearance receptor for both FVIII and VWF. ASGPR binds with high affinity to FVIII asialylated N-glycans (K_D ~2 nM), where the major binding determinant is the abundantly glycosylated B-domain. However, removal of the B-domain does not significantly affect FVIII plasma half-time in various species including humans. Therefore, the authors concluded that ASGPR likely plays a role only in quality control of FVIII biosynthesis by eliminating incompletely glycosylated protein [18]. Other causes of proteins desialylation are proteins “aging” [65] and infection with pathogens [19,66]; both processes likely engage ASGPR in the clearance of such proteins.

Regarding VWF, a deficiency in ASGPR resulted in ~1.5-fold prolongation of its plasma half-life in a mouse model; in parallel, FVIII level was increased [19]. Other plasma ligands of ASGPR include chylomicron remnants, fibronectin, lactoferrin, immunoglobulin A, members of the prolactin/growth hormone family, lipoprotein(a), urokinase-type plasminogen activator [18], and platelets [19].

In humans, there are 14 members of SIGLECs. These receptors are expressed on cells of hematopoietic origin, whereas each member is found on specific cell type(s). SIGLEC5 recognizes the two most common sialic acid sugars, N-acetylneuraminic acid and N-glycolylneuraminic acid, and is expressed on macrophages. SIGLEC5 binds to both FVIII and VWF with high affinity (K_Ds ~8 and ~14 nM, respectively). In cell culture, SIGLEC5 mediated internalization of both proteins; and in mice, overexpression of SIGLEC5 resulted in significant decrease in FVIII and VWF levels [20].

Within the FVIII/VWF complex, FVIII does not interact with SIGLEC5. In contrast to VWF, desialylation of FVIII does not affect its binding to SIGLEC5, indicating that FVIII interacts with receptors via protein moiety. Indeed, each of full-length (FL) FVIII and a B-domain deleted (BDD) FVIII, lacking ~85% of FL-FVIII glycans, had similar binding to SIGLEC5. The authors concluded that SIGLEC5 may regulate plasma level of the FVIII/VWF complex [20].

CLEC4M is a mannose-specific endocytic receptor expressed on endothelium of liver, lymph nodes, and placenta. CLEC4M interacts with both FVIII and VWF via their N-glycans and internalizes both proteins in cell culture; in the case of FVIII, the interaction occurred in either a VWF-dependent or independent manner. Overexpression of CLEC4M in mice resulted in decreased plasma levels of both FVIII and VWF. The authors suggested that CLEC4M is a clearance receptor of both FVIII and VWF [21,22].

This role of CLEC4M was further supported by finding association of polymorphism in the receptor gene with FVIII and VWF plasma levels (see below). Other known ligands of CLEC4M include pathogens, such as Mycobacterium tuberculosis, HIV-1, influenza A, SARS-CoV, Ebola, hepatitis C, and West Nile viruses [67], whereas no endogenous glycoprotein ligands besides FVIII and VWF were reported.

STAB2 is a class H endocytic receptor, recognizing glycosaminoglycans (mucopolysaccharides), and is expressed on endothelial cells in liver, spleen, and lymph nodes. Studies demonstrated association between STAB2 genetic polymorphism and plasma levels of both FVIII and VWF [68,69,70,71]. STAB2 binds and internalizes VWF, and binds FVIII in a VWF-independent mode. In STAB2-deficient mice, the half-lives of VWF and FVIII were prolonged ~1.5-fold upon injection of VWF, whereas FVIII itself interacted weakly with STAB2. The authors concluded that STAB2 is a clearance receptor of the VWF and FVIII/VWF complex. Other ligands of STAB2 include hyaluronic acid, heparins, chondroitin sulfates, collagen, and advanced glycation end products [72].

SCARA5 (SR-A5) is expressed on endothelial cells in the spleen and kidneys, where it recognizes lipopolysaccharides, ferritin, polyanions, and bacteria [24], indicating that recognized determinants are carbohydrates. SCARA5 binds VWF with very high affinity (K_D ~0.5 nM), whereas it does not bind FVIII. SCARA5-expressing human cells bound and internalized VWF, but not FVIII. Under the condition of SCARA5 deficiency in mice, the half-life of injected VWF was prolonged by ≤1.3 times. The authors concluded that SCARA5 is an endocytic receptor for VWF [24]. This was supported by the finding of association of SCARA5 gene polymorphism with VWF levels [69].

SCARA1 (SR-A1) is localized on macrophages where it recognizes polysaccharides, lipopolysaccharides, spectrin (a marker of apoptotic cells), bacteria, extracellular matrix, proteoglycans, etc. [73], most likely via carbohydrates. SCARA1 binds to VWF with high affinity (K_D ~14 nM) via multiple sites suggested to be on the D’D3, A1, and D4 domains via both protein and glycan determinants. VWF bound to SCARA1-expressing cells and had slower plasma clearance in SCARA1-defcient mice. Two VWF mutants, known for increased plasma clearance in humans, had significantly higher binding to SCARA5 in a purified system and on macrophages, and slower plasma clearance in SCARA1-defcient mice. The authors suggested that SCARA1 contributes to increased plasma clearance of certain VWF variants [25].

MGL is a C-type lectin, expressed on macrophages and recognizing terminal N-acetyl galactosamine or galactose sugars. FVIII binds to MGL via asialic O-linked glycans on the B-domain. In MGL-deficient mice, FVIII level was ~1.3-fold elevated, consistent with a role of MGL in clearance of both FVIII and VWF. In VWF-deficient mice, FVIII half-life was reduced ~8-fold, but prolonged 3–4-fold upon (i) inhibition of MGL with antibodies and (ii) depletion of macrophages by a specific reagent (clodronate). The authors concluded that MGL plays an important role in macrophage-mediated plasma clearance of both FVIII and the FVIII/VWF complex [26].

In an earlier study, dose-dependent binding of VWF to MGL was observed, as well as an enhanced clearance of hypo sialylated VWF in VWF/ASGPR-deficient mice, inhibited by coadministration of an MGL antibody. In MGL-deficient mice, VWF levels were significantly elevated [27]. Altogether, these studies demonstrated a role of MGL in plasma clearance of FVIII via both VWF-dependent and independent pathways.

In addition to receptors mentioned above, other factors were also indicated to participate in FVIII plasma clearance based on less-in-depth studies. Two lectins, Galectin-1 and -3 (Gal-1, Gal-3), expressed on endothelial cells and recognizing β-galactoside residues, bound FVIII with high affinity (K_Ds 0.1–0.5 nM) via N- and O-linked glycans, mostly located on the B-domain. Notably, a BHK-cell-derived rFVIII had enhanced affinity to both receptors compared to the CHO-cell-derived rFVIII [74], consistent with their difference in N-glycans mentioned above [58]. VWF also bound both galectins, mostly via N-glycans but with lower affinity (K_Ds 20–80 nM) [75]. The authors concluded that Gal-1, Gal-3 may influence FVIII procoagulant activity and VWF-mediated thrombus formation [74,75].

In several studies involving more than 50,000 subjects total, genetic polymorphism (a single nucleotide or amino acid change) in many genes was associated with FVIII and VWF plasma levels. The FVIII-linked genes were STAB2, VWF, F8, ABO, SCARA5, STXBP5, LBH, FAM46A, VAV2, ACCN1, KATNB1, LDLR, and LRP1; and the VWF-linked genes were STXBP5, SCARA5, ABO, STAB2, VWF, STX2, TC2N, CLEC4M, VPS8, EBP41L4A, KRT18P24, and SAFB2 [42,43,68,69,76]. A later study of Sabater-Lleal et al. identified additional 7 FVIII- and 11 VWF-linked genes/gene groups, supported by functional validation of results using cell culture where silencing of respective genes resulted in change of FVIII and VWF levels [77].

Recently, Swystun and Lillicrap described particular variants of LDLR, ASGPR, CLEC4M, TC2N, and ABO(H) affecting the plasma half-lives of FVIII and VWF in support of development of personalized treatment plans for patients with hemophilia A [11]. It should also be noted that elevated FVIII levels are associated with increased risk of thrombosis [78]. Overall, the genetic studies supported the role of FVIII and VWF clearance receptors reviewed above, whereas the roles of other identified genes remain to be explored.

Knowledge derived from understanding FVIII clearance mechanisms indicates that major FVIII determinants to clearance receptors are located on the LCh. This portion of FVIII carries extended binding sites for LRP1, LDLR, and VWF, for which interactions with FVIII significantly affect its plasma half-life. Therefore, blocking these sites on FVIII may potentially decrease or abolish these interactions and extend FVIII plasma half-life.