IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ zanardelli-2006-1 ADAMTS13 substrate recognition of von Willebrand factor A2 domain. in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation zanardelli-2006-2 ADAMTS13 substrate recognition of von Willebrand factor A2 domain. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation vwf_human von Willebrand factor VWF F8VWF protein protein human Homo sapiens Human MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK ats13_human A disintegrin and metalloproteinase with thrombospondin motifs 13 von Willebrand factor-cleaving protease ADAMTS13 C9orf8 UNQ6102/PRO20085 protein protein human Homo sapiens Human MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSPGAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPAPQPRRLLPGPQENSVQSSACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTLQSWVPEMQDPQSWKGKEGT adamts13-vwf 503086 N/A 503088 enzyme target enzyme target substrate substrate unspecified role unspecified role region VWF76 binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) N/A 503089 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. protein cleavage protein cleavage false false false kcat/Km 6.39 (plus or minus 1.18) E4 per M per s All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2A, 4A, 4C, Table 1 Cleavage product fractions were collected and analyzed by mass spectrometry. For all substrates, peak 1 corresponded to the tagged N-terminal cleavage product up to Tyr1605 and peak 2 to the C-terminal fragment, in each case starting at Met1606. This confirmed that the Tyr1605-Met1606 was cleaved. adamts13-vwf-8 503086 N/A 503089 enzyme enzyme unspecified role unspecified role n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. N/A 503088 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false P04275:p.Arg1583Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.[Asp1614Ala;Glu1615Ala;Lys1617Ala] asp1614ala, glu1615ala, lys1617ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P04275:p.Asp1587Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Asp1614Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Glu1615Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Lys1617Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Glu1638Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Glu1640Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false region VWF115 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false The scissile bond in VWF is predicted from a homology model to be located in the center of the folded A2 domain. For the ADAMTS13-dependent proteolysis of VWF115 (n equals 4), they derived a kcat of 0.14 plus or minus 0.06 per s. The Km for this reaction was 1.61 plus or minus 4.7 E-7 M and the specificity constant (catalytic efficiency, kcat/Km) was 8.70 E4 per M per s. From the gels in Fig. 4A, it seemed as if all of the mutants exhibited reduced rates of proteolysis. All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2B, 3D, 4, Table 1 The kcat_Km (E4 per M per s) are as follows: VWF115 7.83 plus or minus 1.18, VWF115-A3 6.70 plus or minus 2.78, VWF76 6.39 plus or minus 1.18, for VWF115 variants: arg1583ala 6.19, asp1640ala, 2.74, glu1638ala 2.74, lys1617ala 1.90, asp1614ala 1.36 plus or minus 0.26, glu1615ala 1.13 plus or minus 0.30, asp1587ala 0.64 plus or minus 0.10, asp1614ala_glu1615ala_lys1617ala 0.59. Mutation of Glu1638 and Glu1640 induced a mild (2�3-fold) reduction in catalytic efficiency. The most affected (over 12-fold reduction) single mutant was D1587A, the kcat/Km of which was 0.64 plus or minus 0.10x 10E4 per M per s. The kcat/Km for K1617A, D1614A, and E1615A, which are predicted to lie on the same surface-exposed loop, were 1.90, 1.36, and 1.13 E4 per M per s, respectively. The triple mutant DEK1614/5/7AAA containing all 3 residues substituted for alanine was generated. Whereas individually these mutations caused a 4�7-fold reduction in kcat/Km, in combination the catalytic efficiency was over 13-fold less than wild-type VWF115, implying that the amino acids in this charged loop may act in synergy. adamts13-vwf-9 503086 N/A 503089 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. N/A 503088 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region VWFA2 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P04275:p.Asp1614Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Glu1615Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Asp1587Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false 5 When samples containing 500 nM VWFA2 were analyzed by HPLC (Fig. 5C), it was clear that the cleavage reaction did not proceed toward completion (approx. 28% after 5 h). The D1587A, D1614A, and E1615A VWFA2 mutants were proteolyzed either at a similar or slower rate (Fig. 5, A and C), suggesting that these mutations do not alter the A2 domain structure markedly. Under denaturing conditions (1 M urea), the wild-type VWFA2 was proteolyzed faster, although it still did not reach completion (approx. 50% after 5 h) (Fig. 5, B and D). Although all the mutants were cleaved at a higher rate under denaturing conditions, they were cleaved appreciably slower than VWFA2, corroborating the direct influence of these mutations upon VWF115 proteolysis. All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. adamts13-vwf-1 503086 N/A 503088 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region VWF115-A3 sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503089 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. protein cleavage protein cleavage false false false kcat/Km 6.70 (plus or minus 2.78) E4 per M per s All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2 C, 4A and C, Table 1 Cleavage product fractions were collected and analyzed by mass spectrometry. For all substrates, peak 1 corresponded to the tagged N-terminal cleavage product up to Tyr1605 and peak 2 to the C-terminal fragment, in each case starting at Met1606. This confirmed that the Tyr1605-Met1606 was cleaved. adamts13-vwf-7 503087 N/A 503088 unspecified role unspecified role bait bait xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region VWF115 contains part of the A2 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false P04275:p.Asp1587Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Asp1614Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.Glu1615Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P04275:p.[Asp1614Ala;Glu1615Ala;Lys1617Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503089 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. direct interaction direct interaction false false false All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 6 The KD was 2.0 E-8 M for VWF115. The mutants has a reduced affinity. The estimate for the KD for the triple mutant was approx. 1.3 E-6 M but due to low binding for mutants KD values could not be derived. The results for the experiment using ADAMTS13 as the bait were not shown but the authors state that the results were similar.