Key Points• Tolerance of rituximab is acceptable in ITP, and with its benefit/risk ratio may remain a valid option for treating ITP in adults.We conducted a prospective multicenter registry of 248 adult patients with immune thrombocytopenia (ITP) treated with rituximab to assess safety. We also assessed response and predictive factors of sustained response. In total, 173 patients received 4 infusions of 375 mg/m 2 and 72 received 2 fixed 1-g infusions 2 weeks apart. The choice of the rituximab regimen was based on the physician's preference and not patient characteristics. Overall, 38 patients showed minor intolerance to rituximab infusions; infusions had to be stopped for only 3 patients. Seven showed infection (n 5 11 cases), with an incidence of 2.3 infections/100 patient-years. Three patients died of infection 12 to 14 months after rituximab infusions, but the role of rituximab was questionable. In total, 152 patients (61%) showed an overall initial response (platelet count ‡30 3 10 9 /L and ‡2 baseline value). At a median follow-up of 24 months, 96 patients (39%) showed a lasting response. On multivariate analysis, the probability of sustained response at 1 year was significantly associated with ITP duration <1 year (P 5 .02) and previous transient complete response to corticosteroids (P 5 .05). The pattern of response was similar with the 2 rituximab regimens. With its benefit/risk ratio, rituximab used off-label may remain a valid option for treating persistent or chronic ITP in adults. This trial was registered at www.clinicaltrials.gov as #NC1101295. (Blood. 2014;124(22):3228-3236)

(CUB 2 ), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L-and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB 1 and CUB 2 , located in close vicinity of their Ca 2؉ -binding sites and stabilized by the Ca 2؉ ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca 2؉ ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL⅐MASP complex is proposed.The lectin pathway of complement is increasingly recognized as an important component of innate immunity against pathogens. This pathway is triggered by oligomeric lectins that recognize patterns of neutral and acetylated carbohydrates on the surface of pathogens and share the ability to associate with and trigger activation of modular proteases termed mannanbinding lectin-associated serine proteases (MASPs) 3 (1, 2). Four such oligomeric lectins have been described: mannanbinding lectin (MBL) and ficolins L, H, and M (3-7). These proteins all assemble as oligomers of homotrimeric subunits, each comprising N-terminal collagen-like triple helices prolonged by recognition domains endowed with lectin-like binding activities. There are three different MASPs (MASP-1, -2, and -3) (4,8,9), and these feature modular structures homologous to those of C1r and C1s, the proteases of the C1 complex of complement, with an N-terminal CUB module (10), an epidermal growth factor (EGF)-like module belonging to the Ca 2ϩ -binding subset (11), a second CUB module, two complement control protein (CCP) modules (12), and a chymotrypsin-like serine protease domain. MASP-1 and MASP-3 are alternative splicing products of the MASP1/3 gene and include different serine protease domains but share identical CUB 1 -EGF-CUB 2 -CCP 1 -CCP 2 segments (9). Likewise, alternative splicing of the MASP2 gene generates MBL-associated protein 19 (MAp19), a truncated protein comprising the N-terminal CUB 1 -EGF segment of MASP-2 prolonged by four residues specific to MAp19 (13,14). From a functional standpoint, the ability of MASP-2 to trigger the lectin pathway of complement is clearly established (8). In contrast, whether MASP-1 is involved in this pathway is still a controversial issue (15,16), and the function of MASP-3 remains elusive.Studies on human (17-20) and rat (21, 22) proteins have established that the MASPs and MAp19 each associate as homodimers through their CUB 1 -EGF segment. In turn, the MASPs and MAp19 each form individual Ca 2ϩ -dependent complexes with MBL and the ficolins. The interaction involves a major site located in the CUB 1 -EGF moiety of each protein but is strengthened by module CUB 2 (18,19,22,23). Resolution of the x-ray structure of human MAp19 has allowed identification of a Ca 2ϩ...

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 ± 170 Da (MBL-I) and 304,899 ± 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 ± 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable KD values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.

CUB domains are 110-residue protein motifs exhibiting a β-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.