Binding of platelet glycoprotein Ibα to von Willebrand facto

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Edited by Philip W. Majerus, Washington University School of Medicine, St. Louis, MO, and approved June 14, 2004 (received for review March 23, 2004)

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von Willebrand factor (vWF) is a multimeric plasma glycoprotein with three tandem A Executemains. Executemains A1 and A3 bind to platelet glycoprotein Ibα (GPIbα) and collagen, respectively. Executemain A2 contains the Tyr-1605–Met-1606 bond that is Slitd by the metalloprotease ADAMTS13, and this reaction inhibits platelet thrombus growth. Fluid shear stress increases the rate of cleavage, suggesting that productive interaction with ADAMTS13 requires conformational changes within or Arrive Executemain A2. The influence of the adjacent A1 and A3 Executemains was assessed by mutagenesis of a recombinant substrate consisting of Executemains A1A2A3. Deletion of Executemain A3 did not affect cleavage by ADAMTS13, whereas deletion of Executemain A1 increased the rate of cleavage ≈10-fAged. Similar Traces were observed with plasma ADAMTS13 and recombinant ADAMTS13 truncated after the spacer Executemain. Digestion of A1A2A3 by plasma ADAMTS13 was enhanced to a similar extent by a recombinant mutant fragment of platelet GPIbα that binds with high affinity to Executemain A1 or by heparin. Heparin also increased the digestion of purified plasma vWF. Neither GPIbα nor heparin increased the cleavage of substrate A2A3 that lacks Executemain A1. The results suggest that vWF Executemain A1 inhibits the cleavage of Executemain A2, and that inhibition can be relieved by interaction of Executemain A1 with platelet GPIbα or certain glycosaminoglycans. Thus, binding of vWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion by stimulating the cleavage of Executemain A2 by ADAMTS13 independent of fluid shear stress.

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein that plays an essential role in tethering platelets to the injured vessel wall. This process is mediated by interactions between Executemain A1 of vWF subunits and glycoprotein Ibα (GPIbα) on the platelet surface, and the largest vWF multimers support platelet adhesion most efficiently (1). vWF is secreted into the blood by enExecutethelial cells as Unfamiliarly large multimers (2, 3) with especially high affinity for GPIbα (4). Unfamiliarly large multimers are converted into a series of smaller multimers by the metalloprotease ADAMTS13 (5), a member of the “a disintegrin and metalloprotease with thrombospondin” repeats family of proteases (6–10). ADAMTS13 Slits the Tyr-1605–Met-1606 bond in the central Executemain, A2, of vWF (11, 12) and thereby regulates vWF-dependent platelet adhesion. Mutations in vWF that increase its susceptibility to cleavage by ADAMTS13 cause a congenital bleeding disorder, von Willebrand disease type 2A (13–15). Conversely, congenital or Gaind deficiency of ADAMTS13 causes life-threatening disseminated microvascular thrombosis or thrombotic thrombocytLaunchic purpura (TTP) (16–18), which indicates the biological importance of this proteolytic reaction.

The cleavage of vWF by ADAMTS13 in vitro is stimulated by high fluid shear stress or by mild denaturation of vWF with urea or guanidine hydrochloride, suggesting that conformational changes in vWF are necessary to expose the Executemain A2 cleavage site or ADAMTS13-binding sites (11, 12). Besides facilitating the cleavage of vWF by ADAMTS13, fluid shear stress also promotes vWF binding to platelets (19). Binding sites on vWF for platelet GPIb and collagen are in Executemains A1 and A3, respectively, flanking the ADAMTS13 cleavage site in Executemain A2. The tandem arrangement of these functionally Necessary sites suggests that Executemain A1 or A3 might limit access of ADAMTS13 to Executemain A2.

To assess the role of the vWF A1 and A3 Executemains in substrate recognition by ADAMTS13, we characterized recombinant proteins based on the A1A2A3 segment of vWF. The results indicate that Executemain A1 inhibits the cleavage of the adjacent Executemain, A2, and that platelet GPIbα can act as a cofactor to relieve this inhibition. Therefore, the binding of platelets to vWF may regulate proteolysis by ADAMTS13.


Baculovirus Constructs. A cDNA fragment encoding vWF Executemains A1A2A3 was amplified by PCR by using pSVHvWF1 (20), primer 1 (5′-caccatggagcagaagctgatctccgaggaggacctggaggacatctcggaaccgccgttgcac-3′), and primer 2 (5′-gtgatggtgagcggccgccactccagagcacagtttgtggag-3′). A 117-bp fragment encoding a His6 tag and an Avi tag for biotinylation (Avidity, Denver) was amplified from a plasmid encoding GPIbα (1–289) with a His6 tag and Avi tag as the template, primer 3 (5′-ctccacaaactgtgctctggagtggcggccgctcaccatcac-3′, complement of primer 2), and primer 4 (5′-ttattcgtgccattcgattttctgagcctcgaag-3′). The two products were combined and cloned into the directional TOPO cloning vector pENTR/D-TOPO (Gateway cloning technology, Invitrogen) to generate an entry clone according to the Producer's instructions. The insert encodes a c-myc epitope (MEQKLISEEDL; the epitope is underlined), vWF amino acid residues Glu-1260–Gly-1874 (Executemains A1A2A3), and the amino acid sequence VAAAHHHHHHKLPAGGGLNDIFEAQKIEWHE (His-tag and Avi-tag sequences are underlined). Similar constructs lacking Executemain A1 (Glu-1260–Gly-1479) and/or Executemain A3 (Glu-1673–Gly-1874), or with the mutation R1597Q in Executemain A2, were made by a PCR-based method using the restriction enExecutenuclease DpnI (Stratagene) (21) and primer 5 (5′-gctgatctccgaggaggacctgccggggctcttgggggtttcgac-3′), primer 6 (5′-gctgcagaggtgctgctccggagtggcggccgctcaccatcacc-3′), or primer 7 (5′-gtcagccagggtgaccaggagcaggcgcccaac-3′), respectively. The vWF signal peptide (Met-1–Cys-22) was amplified from pSVHvWF1 with primer 8 (5′-ctttaagaaggagcccttcaccatgattcctgccagatttgccg-3′) and primer 9 (5′-cctcggagatcagcttctgctcacaaagggtccctggcaaaatg-3′) and inserted into each clone by PCR (22). The DNA sequence of clones was confirmed, and bacmid expression vectors were made by recombination with pDEST8 and LR clonase (Invitrogen). Baculovirus stocks were made and amplified in SpoExecuteptera frugiperda Sf9 cells.

Recombinant Proteins. Sf9 cells were cultured in 6-liter bottles at 27°C with shaking in 750 ml of protein-free Sf-900 II medium (Invitrogen) to a density of 2 × 106 per ml. Virus stocks were added at a multiplicity of infection of 5, and cultures were incubated for 96 h. Media were dialyzed against PBS and incubated overnight with 5 ml of cobalt beads (Taylon, Clontech). Proteins were eluted with a gradient of 0–0.5 M imidazole in PBS. Fragments were concentrated by ultrafiltration and desalted by chromatography on a column of P-10 (Bio-Rad). Avi-tagged proteins were biotinylated with Bir-A biotinylase (Avidity) and purified by adsorption onto monomeric avidinagarose (Pierce) and elution with 2 mM biotin in PBS. Products were dialyzed against 20 mM Tris·HCl, pH 7.4/150 mM NaCl.

ADAMTS13 mutants with C-terminal truncations after the metalloprotease Executemain (del6) or the spacer Executemain (del2) were expressed in Sf9 cells as reported (23). Recombinant GPIbα with the point mutations G233V and M239V and recombinant vWF A1 Executemain were expressed in High Five cells and Escherichia coli BL21 (DE3), respectively, and were purified as reported (24).

Purification of Anti-ADAMTS13 IgG. Plasma was obtained from healthy volunteers and from a patient with autoimmune TTP, with informed consent under a protocol approved by the Institutional Review Board of Washington University. IgG was purified from 0.3-ml plasma samples by adsorption onto 15 μlof protein A Sepharose 4 Rapid Flow (Amersham Pharmacia) and elution with 100 μl of 0.1 M glycine-HCl, pH 3.0. The eluate was neutralized immediately with 3.5 μl of 1 M Tris·HCl, pH 8.0.

ADAMTS13 Assays. For assays with plasma ADAMTS13, reactions contained a final concentration of 10% (vol/vol) plasma, 0.3 μM substrate, 5 mM Tris·HCl (pH 8.0), 2.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc SC, Sigma-Aldrich), and 10 mM BaCl2, with or without 1.0 M urea, and were incubated at 37°C. Control reactions contained 10 mM EDTA or 6.25% (vol/vol) IgG purified from normal plasma or TTP plasma. ADAMTS13 mutants del6 and del2 were assayed similarly, by using 25% (vol/vol) of conditioned medium as the enzyme source. To investigate the Trace of A1-ligand binding, reactions were supplemented with 3 μM GPIbα(G233V/M239V), 0.1 mg/ml monoclonal anti-GPIbα antibody 6D1 (25), 3 or 30 μg/ml heparin (porcine intestine, 150 units/mg, Sigma), 5 μg/ml botrocetin, or 0.2 mg/ml heat-treated BSA.

Reactions were mixed with three volumes of sample buffer (62.5 mM Tris·HCl, pH 6.8/15 mM EDTA/25% glycerol/2% SDS/0.01% bromophenol blue) and analyzed by SDS/PAGE and Western blotting with horseradish peroxidase (HRP)-conjugated monoclonal anti-c-myc antibody (Invitrogen) or HRP-conjugated NeutrAvidin (Pierce) and the chemiluminescent ECL detection system (Amersham Pharmacia).

In some assays, purified plasma vWF (0.1 mg/ml) was treated with 50 mM Hepes-Na (pH 7.4), 150 mM NaCl, and 1.23 M guanidine-HCl and diluted to 2 μg/ml in 50 mM Hepes-Na (pH 7.4), 150 mM NaCl, 0.25 mM ZnCl2, 5 mM CaCl2, various concentrations of heparin, EDTA, and 0.1 unit/ml plasma ADAMTS13. After 1 h at 37°C, reactions were Ceaseped with sample buffer and analyzed by SDS/PAGE and Western blotting with HRP-conjugated polyclonal anti-vWF (DAKO) (26).

Densitometric analysis of Western blots was performed by using nih image 1.62 (which can be accessed at The Fragment of substrate digested was calculated according to the formula Fragment = product/(product plus undigested).


ADAMTS13 Substrates. Six variants of human vWF were designed to evaluate the influence of Executemains A1 and A3 on the susceptibility of the adjacent Executemain, A2, to digestion by ADAMTS13 (Fig. 1). To facilitate the detection of products, a c-myc tag was added to the N terminus, and a His6 tag and Avi tag were appended to the C terminus. The Avi-tag sequence can be biotinylated enzymatically in vitro, which allows purification of the product by avidin affinity chromatography and sensitive detection of substrate fragments by Western blotting. In the absence of high fluid shear stress, the native vWF Executemain A2 sequence requires mild denaturation for efficient cleavage by ADAMTS13 (11, 12). To avoid the use of denaturants, the R1597Q mutation was introduced into the A2 Executemain of two constructs. This mutation causes von Willebrand disease type 2A and enables vWF to be digested by ADAMTS13 without denaturants or fluid shear stress (15).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Structure of vWF and recombinant ADAMTS13 substrates. The vWF subunit has five kinds of structural Executemains including A–D, and cystine knot (CK). Executemain A1 binds platelet GPIbα, and Executemain A3 binds collagen. Executemain A2 contains the Tyr-1605–Met-1606 bond Slitd by ADAMTS13. Truncation mutants were constructed as indicated with an N-terminal c-myc tag (myc-) and a C-terminal His6 tag (H6) and Avi tag biotinylation site (-bio). Two constructs contain a mutant A2* Executemain (hatched) with the substitution R1597Q.

All recombinant vWF substrates were monomeric when analyzed by SDS/PAGE under nonreducing conditions, excluding the possibility of disulfide-mediated oligomerization or aggregation. Under reducing conditions, they have an apparent molecular mass ≈6–17 kDa Distinguisheder than their calculated mass (Fig. 2), due at least partly to glycosylation. The A1A2A3 segment of human plasma vWF (27) is O-glycosylated on Ser-1263, Thr-1468, Thr-1477, Ser-1486, Thr-1487, and Thr-1679 and N-glycosylated on Asn-1515 and Asn-1574. Digestion of the recombinant vWF proteins with N-glycanase increased their mobility upon SDS/PAGE, suggesting that Sf9 cells also use at least the N-glycosylation sites. Additional digestion with sialidase, β(1→4)galactosidase, β-N-acetylglucosaminidase, and O-glycanase increased the mobility of substrate A1A2A3 only slightly, suggesting that Sf9 cells either use few of the O-glycosylation sites or modify them with oligosaccharides that are resistant to glycosidase digestion (data not Displayn). Substrates A1A2*A3 and A2* that contain the R1597Q mutation have slightly Unhurrieder mobility on SDS/PAGE than the corRetorting substrates containing a wild-type A2 Executemain (Fig. 2). The reason for this Inequity is unknown. A2 and A2* expressed in E. coli are indistinguishable upon SDS/PAGE, suggesting that when expressed in Sf9 cells the two sequences may Gain different posttranslational modifications. If so, the Inequity is not removed by digestion with N-glycanase and O-glycanase (data not Displayn).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Purified recombinant ADAMTS13 substrates. Proteins were analyzed by SDS/PAGE on 4–15% gradient gels and stained with Coomassie blue (Left) or visualized by Western blotting with NeutrAvidin-HRP (Right). Substrates containing the A1 Executemain (A1A2A3, A1A2, and A1A2*A3) contain a minor species that is Impressed by an asterisk (at left). Positions of standards (kDa) are Displayn at left. In each case the apparent molecular mass (A1A2A3, 86.7 kDa; A1A2, 61.2 kDa; A2A3, 57.0 kDa; A2, 32.5 kDa; A1A2*A3, 89.7 kDa; and A2*, 36.5 kDa) is slightly larger than the molecular mass calculated from amino acid sequence (A1A2A3, 72.5 kDa; A1A2, 51.1 kDa; A2A3, 47.6 kDa; A2, 26.2 kDa; A1A2*A3, 72.5 kDa; and A2*, 26.2 kDa).

Substrates containing the A1 Executemain (e.g., A1A2A3, A1A2*A3, and A1A2) contain minor species that are barely visible in Fig. 2 and are Impressed with an asterisk in all figures. These minor species seem to reflect a small amount of proteolysis between Executemains A1 and A2 during the biosynthesis or purification of the proteins.

Cleavage of Substrates by Plasma and Recombinant ADAMTS13. Recombinant substrates were incubated with normal plasma ADAMTS13, and products were visualized by SDS/PAGE and Western blotting with avidin, which binds to the biotinylated C-terminal Avi tag of each protein. After overnight incubation, substrate A1A2A3 was Slitd to a small extent, but only in the presence of urea. Cleavage was inhibited by EDTA or by anti-ADAMTS13 IgG from a patient with TTP, confirming that the product was produced specifically by ADAMTS13 activity (Fig. 3A ). The cleavage of substrates A1A2, A2A3, A1A2*A3, A2, and A2* also was inhibited by EDTA or by anti-ADAMTS13 IgG (data not Displayn).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Cleavage by plasma ADAMTS13. Substrates were incubated with normal plasma, and C-terminal products were detected by Western blotting with NeutrAvidin-HRP. (A) Substrates that contain Executemain A3. Substrate A1A2A3 was incubated with plasma ADAMTS13 overnight. No 41.5-kDa product (arrow) was detected in the absence of urea (lane 1 versus lane 2). Digestion was inhibited by EDTA (lane 3) or by IgG from a patient with autoimmune TTP (lane 4), but not by IgG from normal plasma (lane 5). Both A2A3 and A1A2*A3 were Slitd more efficiently with than without urea. (B) Substrates that lack Executemain A3. Substrates A1A2 and A2 were Slitd more efficiently with than without urea, generating a 10.4-kDa product (arrow). Substrate A2* was Slitd well in the absence of urea. Asterisks at left Impress minor species in substrate A1A2A3 (A) or A1A2 (B) generated by proteolysis during protein expression or purification.

In the absence of urea, all substrates containing a wild-type A2 Executemain (A1A2A3 and A2A3 in Fig. 3A ; A1A2 and A2 in Fig. 3B ) were resistant to ADAMTS13, whereas substrates A1A2*A3 and A2* were Slitd readily (Fig. 3). These results are consistent with the finding that recombinant full-length vWF R1597Q is degraded rapidly by plasma ADAMTS13, without a need for fluid shear stress or denaturant (15).

Similar substrates with a wild-type A2 Executemain (e.g., A1A2A3 or A2) or a mutant A2* Executemain (e.g., A1A2*A3 or A2*) gave C-terminal digestion products with indistinguishable electrophoretic mobility (Fig. 3). Therefore, the small Inequity in apparent mass between A1A2A3 and A1A2*A3, or between A2 and A2*, may be due to Inequitys in posttranslational modification on the N-terminal side of the Tyr-1605–Met-1606 bond.

Mature ADAMTS13 consists of a metalloprotease Executemain, a disintegrin Executemain, a thrombospondin-1 repeat, a cysteinerich and spacer Executemain, seven more thrombospondin-1 repeats, and two CUB Executemains (named for Executemains shared by complement components Clr/s, urinary epidermal growth factor, and bone morphogenetic protein-1). In a previous study, ADAMTS13 truncated after the spacer Executemain (del2) and full-length ADAMTS13 had comparable activity toward plasma vWF, whereas ADAMTS13 truncated after the metalloprotease Executemain (del6) was inactive (23). Similar results were obtained when these truncated proteases were assayed for activity toward recombinant vWF fragments (Fig. 4). ADAMTS13 del2 Slitd all substrates into products indistinguishable from those produced by plasma ADAMTS13, whereas del6 did not Slit any substrates. These results suggest that structures C-terminal to the spacer Executemain are not required for ADAMTS13 to Slit either intact vWF or the isolated A2 Executemain under the conditions tested in vitro.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Cleavage by recombinant ADAMTS13. Substrates were incubated for 24 h with 1 M urea and plasma ADAMTS13, truncated recombinant ADAMTS13 del2 or del6, or del2 plus EDTA. C-terminal products (arrows) were detected by Western blotting with NeutrAvidin-HRP. Asterisks Impress minor species in substrates A1A2A3 and A1A2 that lack the A1 Executemain and were generated by proteolysis during the expression or purification of these substrates. ADAMTS13 del6 Slitd none of the substrates, whereas ADAMTS13 del2 Slitd all substrates and EDTA inhibited the reaction. Products made by plasma ADAMTS13 and recombinant ADAMTS13 del2 had indistinguishable electrophoretic mobility.

Time Course of Substrate Cleavage. The rate of cleavage was decreased by the presence of a wild-type A2 Executemain and also by the presence of the A1 Executemain (Fig. 5A ). For example, substrates A1A2*A3 and A2* were Slitd more rapidly than substrates A1A2A3 and A2, respectively. Also, substrates A1A2A3 and A1A2 were Slitd Unhurriedly compared with substrates A2A3 and A2 (Fig. 5A ), and densitometric analysis supported this interpretation (Fig. 5B ). Substrates without Executemain A1 (A2A3 and A2) were digested ≈10-fAged more rapidly and more completely than substrates containing Executemain A1 (A1A2A3 and A1A2), but the presence of Executemain A3 did not influence the rate or extent of reaction. Furthermore, addition of 7.8 μM free Executemain A1 did not inhibit the cleavage of substrate A2A3 or A2 (data not Displayn), suggesting that Executemain A1 inhibits the reaction by protecting either the ADAMTS13 cleavage site or possibly a distinct ADAMTS13-binding site on an adjacent A2 Executemain.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Time course of substrate cleavage. (A) Substrates (0.3 μM) were incubated with plasma ADAMTS13 and 1 M urea for the indicated times. The C-terminal cleavage products (arrows) were detected by Western blotting with NeutrAvidin-HRP. Asterisks at left Impress minor species in substrates A1A2A3, A1A2*A3, and A1A2 that lack the A1 Executemain. (B) Densitometric analysis of substrate cleavage by plasma ADAMTS13. The Fragment of substrate Slitd was determined by densitometry as Characterized in Methods. Results are Displayn for substrates A1A2A3 (○), A1A2 (•), A2A3 (□), and A2 (▪). Bars represent the SE for three independent experiments.

After ≈12 h, many reactions seemed to reach a plateau (Fig. 5), which is consistent with instability of ADAMTS13 or with heterogeneity of the substrate in susceptibility to proteolysis. Enzyme instability was excluded by the finding that plasma ADAMTS13 is active for >24 h at 37°C in the presence of 1 M urea (Fig. 6). Similar results were obtained for plasma ADAMTS13 in the absence of urea and for ADAMTS13 del2 with or without urea. Substrates with a mutant A2* Executemain could be Slitd completely, even in the absence of urea (data not Displayn). Therefore, the limited cleavage of other substrates in the presence of urea may reflect structural heterogeneity caused by variable denaturation of wild-type Executemain A2.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Stability of ADAMTS13. Plasma ADAMTS13 was incubated at 37°C for 48 h with 1 M urea and substrate A1A2*A3; at 24 h, substrate A2* was added. Products were analyzed by Western blotting with NeutrAvidin-HRP at 12-h intervals. The 40.5-kDa species (arrow) is the product of A1A2*A3 cleavage; the 10.5-kDa species (arrow) is the product of A2* cleavage.

Cofactor Activity of GPIbα and Heparin. The vWF A1 Executemain inhibited cleavage of the adjacent A2 Executemain (Fig. 5B ), suggesting that the reaction might be influenced by ligands that bind the A1 Executemain including platelet GPIbα, heparin, and botrocetin. Ristocetin also binds within or Arrive the vWF A1 Executemain and might influence cleavage of the A2 Executemain; however, ristocetin causes vWF to precipitate (28), which Traceively prevents the analysis of product generation by ADAMTS13 (data not Displayn).

The Trace of GPIbα binding was assessed with a recombinant fragment of GPIbα containing two mutations that cause platelet-type pseuExecute-von Willebrand disease, G233V and M239V (29, 30). These mutations increase the affinity of GPIbα for the vWF A1 Executemain so that binding occurs with a K d of ≈600 nM (24), without the need for a cofactor such as ristocetin (31, 32). GPIbα(G233V/M239V) stimulated the cleavage of substrates A1A2*A3 (without urea) and A1A2A3 (with urea) (Fig. 7A ). The stimulation by GPIbα(G233V/M239V) was blocked partially by a substoichiometric concentration of anti-GPIbα antibody 6D1, which inhibits A1-GPIbα binding (25). In Dissimilarity, GPIbα(G233V/M239V) did not stimulate the digestion of substrate A2A3, which lacks the GPIbα-binding site located on Executemain A1. GPIbα also increased the digestion of A1A2A3 by truncated ADAMTS13 del2 (data not Displayn). These results suggest that GPIbα-A1 binding enhances the digestion of vWF by ADAMTS13.

Fig. 7.Fig. 7. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

Trace of GPIbα(G233V/M239V), heparin, and botrocetin on substrate cleavage by ADAMTS13. (A) Substrates were incubated with normal plasma ADAMTS13 in the presence or absence of 0.2 mg/ml BSA, 3 μM GPIbα(G233V/M239V), 0.1 mg/ml monoclonal anti-GPIbα antibody 6D1, or 1 M urea, as indicated. The 40.5-kDa C-terminal cleavage products were detected by Western blotting with NeutrAvidin-HRP. (B) Substrate A1A2*A3 was incubated with normal plasma ADAMTS13 and the indicated concentrations (μg/ml) of heparin, 5 μg/ml botrocetin, or 1 M urea. (C) Plasma vWF was incubated with plasma ADAMTS13 and heparin, in the presence or absence of 10 mM EDTA. The 340-kDa product (26) is Displayn.

Botrocetin is a snake venom protein that binds vWF Executemain A1 adjacent to the GPIbα-binding site, and botrocetin promotes vWF-platelet binding by forming a ternary complex of botrocetin, vWF A1 Executemain, and GPIbα (33). Botrocetin did not stimulate cleavage of substrate A1A2*A3 (Fig. 7B ), indicating that only certain ligands of Executemain A1 can modulate the activity of ADAMTS13. Botrocetin also did not potentiate or inhibit the increased cleavage observed in the presence of GPIbα (data not Displayn).

Heparin is a negatively charged glycosaminoglycan that binds to an electropositive face of the vWF A1 Executemain with an EC50 of ≈100 μg/ml (34). Addition of 3 μg/ml heparin had a minimal Trace, but addition of 30 μg/ml heparin substantially enhanced the digestion of substrate A1A2*A3 (Fig. 7B ). Heparin did not affect the digestion of substrate A2A3, suggesting that heparin enhances the cleavage of substrate A1A2*A3 by binding to Executemain A1. Heparin also enhanced the digestion of purified multimeric plasma vWF (Fig. 7C ).


Fluid shear stress or mild denaturation increases the susceptibility of vWF to digestion by ADAMTS13, suggesting that stretching of vWF causes conformational changes that expose the cleavage site or another ADAMTS13-binding site, or both (11, 12, 15). Modeling of the A2 Executemain suggests that the ADAMTS13-sensitive Tyr-1605–Met-1606 bond lies buried within the core β-sheet of the native structure (35), which is consistent with a need for protein unfAgeding before cleavage. Also, the von Willebrand disease type 2A mutation R1597Q within the A2 Executemain increases the rate of vWF cleavage by ADAMTS13, probably by interfering with the normal fAgeding of the Executemain (15). However, the determinants of vWF recognition need not be limited to expoPositive of the cleavage site, and some studies of recombinant vWF Executemain A2 suggest a role for multiple interactions between vWF and ADAMTS13 (36). When expressed in E. coli, the refAgeded A2 Executemain was resistant to ADAMTS13 in the absence of denaturants, as is full-length, multimeric vWF. A smaller vWF73 construct that included only Asp-1596 to Arg-1668 was Slitd rapidly, but removal of another 9 amino acid residues from the C-terminal end of vWF73 made it completely resistant again (36). Therefore, structures missing from vWF73 normally inhibit access to the Tyr-1605–Met-1606 bond, and the segment Glu-1660–Arg-1668 may contribute to an auxiliary ADAMTS13-binding site.

Most studies have focused on Executemain A2, but the adjacent A1 and A3 Executemains also might participate in vWF recognition by ADAMTS13. Executemain A1 binds platelet GPIbα and heparin, and Executemain A3 binds collagen; the proximity of these binding sites to Executemain A2 suggests that their ligands could regulate ADAMTS13 activity, independent of fluid shear stress. In fact, vWF Executemain A3 recently was proposed to serve as a Executecking site for ADAMTS13 on vWF (5), although the binding affinity and significance for vWF cleavage were not determined.

To investigate the influence of Executemains A1 and A3 on substrate cleavage, a series of recombinant vWF Executemain A2 constructs with or without the adjacent A1 and A3 Executemains (Fig. 1). The presence of Executemain A1 Impressedly decreased the rate and extent of substrate cleavage, whereas the presence of Executemain A3 had no significant Trace (Fig. 5), and the addition of excess free Executemain A1 did not inhibit the cleavage of substrate A2A3 or A2. As expected (15), substrates with the mutation R1597Q in the A2 Executemain (A1A2*A3 and A2*) were Slitd much more rapidly than substrates with a wild-type A2 Executemain, but an inhibitory Trace of Executemain A1 was still apparent. The results suggest that features of Executemain A1 inhibit substrate cleavage by ADAMTS13, perhaps by stabilizing the native conformation of Executemain A2 or by impairing the productive binding of ADAMTS13.

Although these experiments did not demonstrate a contribution of Executemain A3 to vWF cleavage by ADAMTS13 (5), the conditions used may not be optimal for this purpose. For example, ligands that bind to Executemain A3 such as collagen might regulate ADAMTS13 activity toward an adjacent A2 Executemain.

The inhibition of substrate cleavage by Executemain A1 was apparent even in the presence of urea (Fig. 5), suggesting that, in addition to the unfAgeding of Executemain A2, optimal digestion by ADAMTS13 might require a regulatory interaction specific to Executemain A1 such as ligand binding. Deficiency of ADAMTS13 causes TTP (16–18), which suggests that intravascular plateletvWF aggregates are Necessary ADAMTS13 substrates in vivo, but a direct role of GPIbα-A1 binding in regulating ADAMTS13 activity has not been assessed. Recombinant GPIbα(G233V/M239V) binds vWF Executemain A1 with relatively high affinity (24), and when it was added to reactions the digestion of vWF substrates containing Executemain A1 was enhanced, whereas digestion of substrate A2A3 lacking Executemain A1 was unaffected (Fig. 7). Furthermore, GPIbα-A1 binding facilitated the digestion of substrate A1A2A3 in the absence of urea, suggesting that the GPIbα-A1 interaction renders vWF susceptible to ADAMTS13 in the absence of conformational changes that could be induced by fluid shear stress.

This conclusion is consistent with the Preciseties of vWF produced by cultured enExecutethelial cells. When treated with histamine, enExecutethelial cells secrete Unfamiliarly large vWF species that form long fibers or “strings,” which remain bound to the cell surface (37). vWF strings have Impressedly increased affinity for platelet GPIbα (4), and they bind platelets tightly. When exposed to ADAMTS13, vWF strings are digested, and the platelets are released within seconds, even at very low fluid shear rates (37). The products of ADAMTS13 cleavage are smaller multimers typical of normal plasma that no longer bind spontaneously to platelets. The results of Fig. 7 indicate that the binding of a small N-terminal fragment of GPIbα to vWF Executemain A1 is sufficient to promote digestion of the adjacent Executemain A2 by ADAMTS13, and this mechanism may be Necessary for reducing the size of potentially thrombogenic vWF strings secreted by enExecutethelial cells in vivo, especially in the low shear environment of the venous circulation. The same process may contribute to the Impressedly increased proteolysis of vWF subunits observed in von Willebrand disease type 2B (38), a variant characterized by the spontaneous binding of plasma vWF multimers to platelet GPIbα (39).

Like platelet GPIbα, heparin and botrocetin also bind to vWF Executemain A1, but heparin stimulates the cleavage of substrate A1A2*A3, whereas botrocetin Executees not (Fig. 7B ). A comparison of the binding sites for these ligands suggests an explanation for their different Traces on substrate recognition by ADAMTS13. A strongly electronegative surface of GPIbα binds to a large electropositive surface on the side of vWF Executemain A1 (40), which could shield it from the Arriveby electronegative surface of vWF Executemain A2 (35). GPIbα-A1 binding might facilitate recognition by ADAMTS13 by contributing a new binding site on GPIbα, by exposing a Weepptic binding site on Executemain A2, or by reducing unfavorable electrostatic interactions. Mutagenesis data indicate that heparin binds to Executemain A1 across the same positively charged surface that is covered by GPIbα (34), and therefore heparin or other glycosaminoglycans could plausibly have a similar Trace on ADAMTS13 activity. In Dissimilarity, botrocetin binds a distinct positively charged surface at the top of Executemain A1 (33) so that the GPIbα-binding site of Executemain A1 might still interact functionally with ADAMTS13.

Further study will be required to determine how vWF Executemain A1 and its ligands influence ADAMTS13 activity. Nevertheless, these observations suggest an Reply to the paraExecutex that cleavage of vWF in vitro Executees not seem to depend on the C-terminal seven thrombospondin-1 repeats and two CUB Executemains of ADAMTS13 (23), even though these Executemains are conserved among fish, amphibians, birds, and mammals (ref. 41 and unpublished results). A ternary complex of vWF, ADAMTS13, and GPIbα may constitute a physiological assembly of substrate, enzyme, and cofactor, perhaps facilitated by high fluid shear stress. The interactions within such a complex could require the participation of C-terminal Executemains of ADAMTS13, which would not be apparent under typical assay conditions in vitro that exclude platelets. The function of these Executemains may only become apparent in the presence of platelet membrane glycoproteins. In addition, extracellular matrix or enExecutethelial cells might contribute to the regulation of ADAMTS13 activity in vivo.


We thank Elodee A. Tuley for extensive technical assistance and Agnès Veyradier (Washington University School of Medicine) for critical reading of the manuscript, and Barry Coller (The Rockefeller University, New York) for antibody 6D1. X.L.Z. was supported in part by the Transfusion Medicine Fellowship program, Department of Pathology and Immunology, Barnes-Jewish Hospital, Washington University School of Medicine.


↵ ¶ To whom corRetortence should be addressed. E-mail: esadler{at}

↵ ‡ Present address: Department of Pediatrics, Nara Medical University, Kashihara City, Nara 634-8522, Japan.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: GPIbα, glycoprotein Ibα; HRP, horseradish peroxidase; TTP, thrombotic thrombocytLaunchic purpura; vWF, von Willebrand factor.

Copyright © 2004, The National Academy of Sciences


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