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Anna M. Blom - One of the best experts on this subject based on the ideXlab platform.
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the novel complement inhibitor cmsd1 protein promotes factor i mediated degradation of c4b and c3b and inhibits mac assembly and c9 polymerisation
Molecular Immunology, 2013Co-Authors: Astrid Escuderoesparza, Nikolina Kalchishkova, E Kurbasic, Wen Guo Jiang, Anna M. BlomAbstract:Cub and Sushi multiple domains 1 (CSMD1) is a transmembrane protein formed from 14 N-terminal Cub domains separated by single complement control protein (CCP) domains and followed by a tandem repeat of 15 CCP domains which are characteristic for complement inhibitors. Therefore, shares homology with a number of proteins involved in many cellular processes such as growth, cell adhesion, cancer progression and in controlling the complement system. It was firstly identified as a candidate tumour suppressor gene of unknown function. By using a panel of healthy adult human samples, Q-PCR data revealed that CSMD1 is mainly found in testis, cerebellum, cerebral cortex and brain white matter. A high expression profile was also observed in fetal brain tissue. We expressed a soluble protein containing CCP domains 17–21 together with Fc-tag. The expression of this construct significantly decreased deposition of C4b and C3b on transfected mammalian cells and showed cofactor activity to FI. Furthermore, CSMD1 CCP 17–21 Fc inhibited MAC formation at the level of C7 and the polymerisation of C9, both when self-polymerisation of C9 was studied but also on the surface of erythrocytes in the presence of human serum. Moreover, reduction of expression in the T47 breast cancer cell line that expresses endogenous CSMD1, significantly increased C3b deposition on the surface. Considering that CSMD1 is frequently deleted in cancer, loss of CSMD1 and consequently loss of local complement inhibition may lead to inflammation, thus providing an ideal environment for cancer development.
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the novel complement inhibitor human Cub and sushi multiple domains 1 csmd1 protein promotes factor i mediated degradation of c4b and c3b and inhibits the membrane attack complex assembly
The FASEB Journal, 2013Co-Authors: Astrid Escuderoesparza, Nikolina Kalchishkova, E Kurbasic, Wen Guo Jiang, Anna M. BlomAbstract:Cub and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways.
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acquisition of complement inhibitor serine protease factor i and its cofactors c4b binding protein and factor h by prevotella intermedia
PLOS ONE, 2012Co-Authors: Sven Malm, Monika Jusko, Sigrun Eick, Jan Potempa, Kristian Riesbeck, Anna M. BlomAbstract:Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.
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Analysis of Binding Sites on Complement Factor I Using Artificial N-Linked Glycosylation
The Journal of biological chemistry, 2012Co-Authors: José I. Sánchez-gallego, Sara C. Nilsson, Bruno O. Villoutreix, Tom W.l. Groeneveld, Stefanie Krentz, Anna M. BlomAbstract:Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors, such as factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein. FI is composed of two chains linked by a disulfide bridge; the light chain comprises only the serine protease (SP) domain, whereas the heavy chain contains the FI membrane attack complex domain (FIMAC), CD5 domain, and low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based three-dimensional models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface-exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (Km) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished the ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespective of the cofactor used. On the other hand, only a few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI.
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Complement factor I in health and disease.
Molecular immunology, 2011Co-Authors: Sara C. Nilsson, Véronique Fremeaux-bacchi, Robert B. Sim, Susan M. Lea, Anna M. BlomAbstract:Factor I (FI) is a crucial inhibitor controlling all complement pathways due to its ability to degrade activated complement proteins C3b and C4b in the presence of cofactors such as factor H, C4b-binding protein, complement receptor 1 or CD46. Complete deficiency of FI, which is synthesized mainly in the liver is rare and leads to complement consumption resulting in recurrent severe infections, glomerulonephritis or autoimmune diseases. Incomplete FI deficiency is in turn associated with atypical haemolytic uremic syndrome, a severe disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure. Structurally, FI is a 88kDa heterodimer of a heavy chain consisting of one FI-membrane attack complex (FIMAC) domain, one CD5 domain and two low-density lipoprotein receptor domains (LDLr), and a light chain which is a serine protease domain (SP), linked to the heavy chain by a disulfide bond. FI cleaves its in vivo substrates C3b and C4b only in the presence of cofactors, it shows poor enzymatic activity towards synthetic substrates tested so far and it has no natural inhibitor.
Sara C. Nilsson - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Binding Sites on Complement Factor I Using Artificial N-Linked Glycosylation
The Journal of biological chemistry, 2012Co-Authors: José I. Sánchez-gallego, Sara C. Nilsson, Bruno O. Villoutreix, Tom W.l. Groeneveld, Stefanie Krentz, Anna M. BlomAbstract:Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors, such as factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein. FI is composed of two chains linked by a disulfide bridge; the light chain comprises only the serine protease (SP) domain, whereas the heavy chain contains the FI membrane attack complex domain (FIMAC), CD5 domain, and low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based three-dimensional models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface-exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (Km) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished the ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespective of the cofactor used. On the other hand, only a few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI.
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Complement factor I in health and disease.
Molecular immunology, 2011Co-Authors: Sara C. Nilsson, Véronique Fremeaux-bacchi, Robert B. Sim, Susan M. Lea, Anna M. BlomAbstract:Factor I (FI) is a crucial inhibitor controlling all complement pathways due to its ability to degrade activated complement proteins C3b and C4b in the presence of cofactors such as factor H, C4b-binding protein, complement receptor 1 or CD46. Complete deficiency of FI, which is synthesized mainly in the liver is rare and leads to complement consumption resulting in recurrent severe infections, glomerulonephritis or autoimmune diseases. Incomplete FI deficiency is in turn associated with atypical haemolytic uremic syndrome, a severe disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure. Structurally, FI is a 88kDa heterodimer of a heavy chain consisting of one FI-membrane attack complex (FIMAC) domain, one CD5 domain and two low-density lipoprotein receptor domains (LDLr), and a light chain which is a serine protease domain (SP), linked to the heavy chain by a disulfide bond. FI cleaves its in vivo substrates C3b and C4b only in the presence of cofactors, it shows poor enzymatic activity towards synthetic substrates tested so far and it has no natural inhibitor.
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Analysis of Binding Sites on Complement Factor I That Are Required for Its Activity
The Journal of biological chemistry, 2009Co-Authors: Sara C. Nilsson, Leendert A. Trouw, Bruno O. Villoutreix, Tom W.l. Groeneveld, Izabela Nita, Lisa Månsson, Anna M. BlomAbstract:The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1, and membrane cofactor protein. FI is a serine protease composed of two chains. The light chain comprises the serine protease domain, whereas the heavy chain contains several domains; that is, the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not altered, whereas some mutants showed increased maximum initial velocity (V(max)). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used, whereas only some mutations in the CD5 and LDLr1/2 domains had a similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.
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Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I.
European journal of immunology, 2009Co-Authors: Sara C. Nilsson, Véronique Fremeaux-bacchi, Leendert A. Trouw, Nikolina Kalchishkova, Bruno O. Villoutreix, Anna M. BlomAbstract:The complement system is regulated by inhibitors such as factor I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complement factor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. In conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS.
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a mutation in factor i that is associated with atypical hemolytic uremic syndrome does not affect the function of factor i in complement regulation
Molecular Immunology, 2007Co-Authors: Sara C. Nilsson, Leendert A. Trouw, Diana Karpman, Fariba Vazirisani, Anncharlotte Kristoffersson, Remi Salomon, Francois Provot, Veronique Fremeauxbacchi, Anna M. BlomAbstract:Factor I (FI) is the major complement inhibitor that degrades C3b and C4b in the presence of cofactors such as factor H (FH) and membrane cofactor protein (MCP). Recently, mutations and polymorphisms in complement regulator molecules FH and MCP but also in FI have been associated with atypical hemolytic uremic syndrome (aHUS). HUS is a disorder characterized by hemolytic anemia, thrombocytopenia and acute renal failure. In this study, we report three unrelated patients with an identical heterozygous mutation, G261D, in the FI heavy chain who developed severe aHUS at different time points in their lives. Two of the patients also have polymorphisms in FH previously associated with risk of developing aHUS. Testing in particular one patient and control serum samples we did not observe major differences in complement hemolytic activity, FI plasma levels or the capability to degrade C4b or C3b. A recombinant protein was produced in order to analyze the functional consequences of the mutation. Mutant FI had a slightly different migration pattern during electrophoresis under reducing conditions. An alteration due to alternative splicing or glycosylation was ruled out, thus the altered migration may be due to proximity of the mutation to a cysteine residue. The recombinant mutant FI degraded C3b and C4b in a manner comparable to wild-type protein. In conclusion, despite the association between the heterozygous mutation in FI and aHUS we did not observe any abnormalities in the function of FI regarding complement regulation.
Robert B. Sim - One of the best experts on this subject based on the ideXlab platform.
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Complement factor I in health and disease.
Molecular immunology, 2011Co-Authors: Sara C. Nilsson, Véronique Fremeaux-bacchi, Robert B. Sim, Susan M. Lea, Anna M. BlomAbstract:Factor I (FI) is a crucial inhibitor controlling all complement pathways due to its ability to degrade activated complement proteins C3b and C4b in the presence of cofactors such as factor H, C4b-binding protein, complement receptor 1 or CD46. Complete deficiency of FI, which is synthesized mainly in the liver is rare and leads to complement consumption resulting in recurrent severe infections, glomerulonephritis or autoimmune diseases. Incomplete FI deficiency is in turn associated with atypical haemolytic uremic syndrome, a severe disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure. Structurally, FI is a 88kDa heterodimer of a heavy chain consisting of one FI-membrane attack complex (FIMAC) domain, one CD5 domain and two low-density lipoprotein receptor domains (LDLr), and a light chain which is a serine protease domain (SP), linked to the heavy chain by a disulfide bond. FI cleaves its in vivo substrates C3b and C4b only in the presence of cofactors, it shows poor enzymatic activity towards synthetic substrates tested so far and it has no natural inhibitor.
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The catalytically active serine protease domain of human complement factor I.
Biochemistry, 2005Co-Authors: Stefanos A. Tsiftsoglou, Antony C. Willis, Xuehui Chen, Daniel A. Mitchell, Zihe Rao, Robert B. SimAbstract:Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by ...
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Complement factor I and cofactors in control of complement system convertase enzymes
Methods in enzymology, 1993Co-Authors: Robert B. Sim, Anthony J. Day, Beryl E. Moffatt, Monique FontaineAbstract:Publisher Summary This chapter describes the complement factor I and cofactors in control of complement system convertase enzymes. Complement factor I was first described and partially characterized in 1966–1968 as an enzyme involved in the physiological degradation of the major complement protein C3. It was previously named conglutinogen-activating factor (KAF), because its action on C3 exposed a binding site on a C3 fragment for the bovine protein conglutinin. Other former names include C3b inactivator, C3b INA, and C3b/C4b inactivator. The physiological substrates of factor I are produced only when the complement system is activated. Activation of the classical pathway of the complement system is generally mediated by binding of the C1 complex to a target, such as immune complexes or micro-organisms. The role of factor I is to control the activities of the convertase enzymes, C4b2a and C4b2a3b, by proteolytic cleavage of the C4b and C3b subunits of these enzymes. Before C3b or C4b can be cleaved by factor I, they must bind to one of several complement control proteins to form a non-covalent complex.
Tonya M Brubaker - One of the best experts on this subject based on the ideXlab platform.
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strontium isotope study of coal utilization by products interacting with environmental waters
Journal of Environmental Quality, 2012Co-Authors: Lev J Spivakbirndorf, Elizabeth C. Chapman, Karl T Schroeder, Rosemary C Capo, Brian W Stewart, Tonya M BrubakerAbstract:Sequential leaching experiments on coal utilization by-products (Cub) were coupled with chemical and strontium (Sr) isotopic analyses to better understand the influence of coal type and combustion processes on Cub properties and the release of elements during interaction with environmental waters during disposal. Class C fly ash tended to release the highest quantity of minor and trace elements—including alkaline earth elements, sodium, chromium, copper, manganese, lead, titanium, and zinc—during sequential extraction, with bottom ash yielding the lowest. Strontium isotope ratios ({sup 87}Sr/{sup 86}Sr) in bulk-Cub samples (total dissolution of Cub) are generally higher in class F ash than in class C ash. Bulk-Cub ratios appear to be controlled by the geologic source of the mineral matter in the feed coal, and by Sr added during desulfurization treatments. Leachates of the Cub generally have Sr isotope ratios that are different than the bulk value, demonstrating that Sr was not isotopically homogenized during combustion. Variations in the Sr isotopic composition of Cub leachates were correlated with mobility of several major and trace elements; the data suggest that arsenic and lead are held in phases that contain the more radiogenic (high-{sup 87}Sr/{sup 86}Sr) component. A changing Sr isotope ratio of Cub-interacting waters inmore » a disposal environment could forecast the release of certain strongly bound elements of environmental concern. This study lays the groundwork for the application of Sr isotopes as an environmental tracer for Cub–water interaction.« less
Jing Fei Dong - One of the best experts on this subject based on the ideXlab platform.
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Cysteine residues in Cub-1 domain are critical for ADAMTS13 secretion and stability
Thrombosis and haemostasis, 2010Co-Authors: Zhou Zhou, Hui Chun Yeh, Hua Jing, Christina Wang, Zhenyin Tao, Huiwan Choi, Khatira Aboulfatova, Jing Fei DongAbstract:Upon stimulation, endothelial cells release von Willebrand factor (VWF) enriched in ultra-large (UL) forms that are rapidly cleaved by ADAMTS13. The zinc metalloprotease fits in the consensus for members of the ADAMTS family, but also contains two unique C-terminal Cub domains. There are five and two cysteine residues in the Cub-1 and Cub-2 domains, respectively, instead of four as deducted from the consensus. In this study, we investigated the role of cysteine residues in the Cub-1 domain in ADAMTS13 synthesis and activity. Cub-1 and cysteine mutations were expressed in mammalian cell lines and examined for synthesis, secretion, stability, and VWF-cleaving activity. When expressed as an isolated domain, Cub-1, but not Cub-2, covalently aggregated. Converting any of the four cysteines that fit in the Cub consensus (C1192, C1213, C1236 and C1254) reduced the secretion of the mutants to the conditioned medium, but not to extracellular matrix. The mutations also resulted in a moderate increase in proteolytic degradation and decrease in cleaving VWF under static, but not flowing conditions. In contrast, replacing C1275, which was found to be in the thiol form, with a serine residue prevented covalent aggregation of Cub-1, but had no effect on secretion and VWF-cleaving activity. C1275S was also markedly resistant to proteolytic degradation. The data illustrate the importance of consensus cysteines in the secretion and proteolytic activity of ADAMTS13. They also identify an ADAMTS-13 mutant that is resistant to proteolytic degradation, while maintaining a normal VWF-cleaving activity.
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Effects of Naturally Occurring Mutations in Cub-1 Domain on ADAMTS-13 Synthesis, Stability and Activity.
Blood, 2007Co-Authors: Zhou Zhou, Hua Jing, Zhenyin Tao, Huiwan Choi, Khatira Aboulfatova, Jing Fei DongAbstract:Upon stimulation, vascular endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and prothrombotic forms. The hyperactive ULVWF multimers are rapidly cleaved by the metalloprotease ADAMTS-13 to smaller forms that are hemostatically active, but no longer prothrombotic. Lack of this VWF- cleaving activity results in accumulation of ULVWF, which agglutinates platelets, leading to microvascular thrombosis as demonstrated in patients with thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 deficiency seen in TTP results from either mutations in the ADAMTS13 gene in familial cases or autoantibodies against the metalloprotease in acquired cases. ADAMTS-13 differs from other members of the family metalloproteases by containing two C-terminal Cub domains, which contain the binding site for VWF and whose mutations are associated with TTP. However, recombinant ADAMTS-13 without Cub domains remains active in vitro . To study the functional role of Cub domains, three naturally occurring mutations in the Cub-1 domain (C1213Y, W1245del and K1256frameshift, the latter two removed Cub-2 and the C-terminal part of Cub-1 domain) were generated by site-directed mutagenesis and expressed in the mammalian Hela cells. The release, stability, and activity of the mutants were then examined. We found that the mutations significantly reduced the release of recombinant ADMATS-13 into the culture medium, but not to the extracellular matrix. The intracellular pools of the recombinants detected in cell lysates were comparable to that of wild-type, indicating that the mutations affected the targeted secretion of the metalloprotease. When measured during a course of up to one month at −80°C, C1213Y and K1256framshift significantly accelerated, whereas W1245del delayed, degradation of the recombinants, which was inhibited by 5 mM of EDTA. The observation demonstrates that Cub domains are also critical for the structural stability of the metalloprotease. In comparison, the three mutants remained active in cleaving (UL)VWF under static and flow conditions. Consistent with the activity measurements, all three mutants bound immobilized VWF with similar kinetics in ELISA assay, indicating that the N-terminal sequence (C1192-D1212) of Cub-1, which is not affected by the mutations, contains the binding site for VWF. The conclusion is supported by our previous data that the Cub-1 synthetic peptides derived from the N-terminal region (H1196-P1220) blocked VWF-ADAMTS-13 interaction and cleavage of ULVWF under flow. Together, these results demonstrate that the Cub domains (primarily Cub-2 and the C-terminal part of Cub-1) are crucial for regulating the targeted ADAMTS-13 secretion and the stability of the metalloprotease; the N-terminal sequence of Cub-1 domain contains the VWF binding site that remains intact after deleting the rest of Cub domains; and the primary cause of ADAMTS-13 deficiency found in patients carrying these mutations is likely due to defective secretion of the metalloprotease.
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Recombinant Cub-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions.
Blood, 2005Co-Authors: Zhenyin Tao, Yuandong Peng, Leticia Nolasco, Santiago Cal, Carlos López-otín, Joel L. Moake, José A. López, Jing Fei DongAbstract:The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal Cub domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the Cub region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the Cub domain in ADAMTS13 activity under flow conditions. We found that recombinant Cub-1 and Cub-1+2 polypeptides and synthetic peptides derived from Cub-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the Cub-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.