Single Particle Tracking of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type-1 Repeats) Molecules on Endothelial von Willebrand Factor Strings

von Willebrand factor (VWF) strings are removed from the endothelial surface by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats)-mediated proteolysis. To visualize how single ADAMTS13 molecules bind to these long strings, we built a customized single molecule fluorescence microscope and developed single particle tracking software. Extensive analysis of over 6,000 single inactive ADAMTS13^E225Q enzymes demonstrated that 20% of these molecules could be detected in at least two consecutive 60-ms frames and followed two types of trajectories. ADAMTS13^E225Q molecules either decelerated in the vicinity of VWF strings, whereas sometimes making brief contact with the VWF string before disappearing again, or readily bound to the VWF strings and this for 120 ms or longer. These interactions were observed at several sites along the strings. Control experiments using an IgG protein revealed that only the second type of trajectory reflected a specific interaction of ADAMTS13 with the VWF string. In conclusion, we developed a dedicated single molecule fluorescence microscope for detecting single ADAMTS13 molecules (nm scale) on their long, flow-stretched VWF substrates (μm scale) anchored on living cells. Comprehensive analysis of all detected enzymes showed a random interaction mechanism for ADAMTS13 with many available binding sites on the VWF strings.     

A conformation-sensitive monoclonal antibody against the A2 domain of von Willebrand factor reduces its proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13.     

Probing ADAMTS13 Substrate Specificity using Phage Display

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.     

ADAMTS13 substrate recognition of von Willebrand factor A2 domain

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593-1668), VWF115-(1554-1668), VWFA2-(1473-1668)) and one A2-A3 (VWF115-A3-(1554-1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 microM; kcat 0.14 s(-1)), from which the specificity constant kcat/Km was calculated, 8.70 x 10(4) m(-1) s(-1). Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved approximately 8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614 on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to approximately 1.3 microM), compared with VWF115 (KD 20 nM). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.     

Role of thrombospondin-1 in control of von Willebrand factor multimer size in mice

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr(842)-Met(843) peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet alpha-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets.     

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ～10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.     

Zinc and calcium ions cooperatively modulate ADAMTS13 activity

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.     

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.     

ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor

Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm2) and arterial (30 dynes/cm2) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.     

ADAMTS13 Bound to Endothelial Cells Exhibits Enhanced Cleavage of von Willebrand Factor

ADAMTS13 is a plasma metalloprotease that cleaves ultralarge von Willebrand factor multimers to generate less thrombogenic fragments. Although this cleavage can occur at the surface of endothelial cells, it is currently unknown whether this process involves binding of the ADAMTS13 to the endothelial cell plasma membrane. Using different assay systems, we present evidence that ADAMTS13 binds to endothelial cells in a specific, reversible, and time-dependent manner with a K_d of 58 nm. This binding requires the COOH-terminal thrombospondin type 1 repeats of the protease. Binding is inhibited in the presence of heparin and by trypsin treatment of the cells. ADAMTS13 that was prebound to endothelial cells exhibited increased proteolysis of VWF as compared with ADAMTS13 present only in solution. These data support the notion that cleavage of VWF occurs mainly at the endothelial cell surface.     

Cleavage of von Willebrand factor by granzyme M destroys its factor VIII binding capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.     

Degradation of circulating von Willebrand factor and its regulator ADAMTS13 implicates secreted Bacillus anthracis metalloproteases in anthrax consumptive coagulopathy

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.     

Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force–dependent cleavage at a single Tyr–Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13–VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.     

Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/F?rster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.     

Factor VIII and Platelets Synergistically Accelerate Cleavage of von Willebrand Factor by ADAMTS13 under Fluid Shear Stress

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2–3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of ∼8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 × 10³/μl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by ∼10-fold (to ∼0.3 nm) compared with that in the absence of platelets (∼3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1bα IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1bα-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.     

Binding of von Willebrand factor cleaving protease ADAMTS13 to Lys-plasmin(ogen)

The metalloprotease ADAMTS13 affects platelet adhesion and aggregation through depolymerization of von Willebrand factor (VWF) multimers. Identification of ADAMTS13-binding proteins would reveal the hitherto unrecognized mechanisms underlying microvascular thrombus. To identify ADAMTS13-binding proteins, we performed a yeast two-hybrid screen using the Cys-rich and spacer domains of ADAMTS13, the critical regions for the binding and cleavage of VWF, as a bait region. We identified Lys-plasminogen, an amino-terminal truncated form of plasminogen, as the binding protein to ADAMTS13. Intact Glu-plasminogen did not bind to ADAMTS13. Active-site blocked Lys-plasmin bound to ADAMTS13. Domain truncation of ADAMTS13 and elastase digest of plasminogen indicated that the Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen served as the main binding sites. Biacore measurements revealed that Lys-plasminogen bound to ADAMTS13 with a K(d) of 1.9 ± 0.1 × 10(-7) M and Glu-plasminogen exhibited a significantly lower affinity to ADAMTS13. Specific activity measurements revealed that ADAMTS13 and Lys-plasmin were still active even after the binary complex was formed. The binding of ADAMTS13 to Lys-plasminogen may play an important role to localize these two proteases at sites of thrombus formation or vascular injury where the fibrinolytic system is activated.     

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K_D, ∼15 nm). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII^−/− mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.     

ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.     

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu^373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser^341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu^2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.