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.     

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.     

Conformational stability and domain unfolding of the Von Willebrand factor A domains

Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.     

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.     

A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-dependent manner

Reelin is a glycoprotein essential for brain development and functions. Reelin is subject to specific proteolysis at two distinct (N-t and C-t) sites, and these cleavages significantly diminish Reelin activity. The decrease of Reelin activity is detrimental for brain function, but the protease that catalyzes specific cleavage of Reelin remains elusive. Here we found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-specific manner. Among ADAMTS-4 isoforms, p50 cleaves the N-t site only, while p75 cleaves both sites. This is the first report identifying a protease that can specifically cleave Reelin.     

The Interaction between Factor H and Von Willebrand Factor

Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13.     

Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin

Human granzyme B (GrB) released from cytotoxic lymphocytes plays a key role in the induction of target cell apoptosis when internalized in the presence of perforin. Here we demonstrate that GrB also possesses a potent extracellular matrix remodeling activity. Both native and recombinant GrB caused detachment of immortalized and transformed cell lines, primary endothelial cells, and chondrocytes. Cell detachment by GrB induced endothelial cell death (anoikis). GrB also inhibited tumor cell spreading, migration, and invasion in vitro. Investigation into the underlying mechanism revealed that GrB efficiently cleaves three proteins involved in extracellular matrix structure and function: vitronectin, fibronectin, and laminin. In vitronectin, GrB cleaves after an Arg-Lys-Asp (RGD) motif, which is part of the integrin-binding site found in matrix proteins. We propose that targeting of the integrin-extracellular matrix interface by GrB may allow perforin-independent killing of target cells via anoikis, restrict motility of tumor cells, facilitate lymphocyte migration, or directly reduce virus infectivity. It may also contribute to tissue destruction in diseases in which extracellular GrB is evident, such as rheumatoid arthritis and atherosclerosis.     

ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan

A disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) is an extracellular matrix-anchored metalloproteinase. In this study we have demonstrated that ADAMTS-1 is able to cleave a major cartilage proteoglycan, aggrecan. N-terminal sequencing analysis of the cleavage product revealed that ADAMTS-1 cleaves the Glu(1871)-Leu(1872) bond within the chondroitin sulfate attachment domain of aggrecan. In addition, deletional analysis demonstrated that the C-terminal spacer region of ADAMTS-1 is necessary to degrade aggrecan. These results suggest that ADAMTS-1 may be involved in the turnover of aggrecan in vivo.     

Interaction of Shiga Toxin with the A-domains and Multimers of von Willebrand Factor

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.     

Role of chloride ions in modulation of the interaction between von Willebrand factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.     

Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.     

Severe dengue is associated with consumption of von Willebrand factor and its cleaving enzyme ADAMTS-13

Background

Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.

Methods and Principal Findings

In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease.

Conclusion

These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.     

Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products. GrB cleaves Mcl-1 after aspartic acid residues 117, 127, and 157, generating C-terminal fragments that all contain BH-1, BH-2, BH-3, and transmembrane domains. These fragments accumulate at an early apoptotic phase but are eliminated by further degradation during the apoptotic process. The major Mcl-1 C-terminal fragment generated by GrB (residues 118-350) was unable to induce or enhance apoptosis when transfected into tumor cells. Instead, this Mcl-1 C-terminal fragment maintained a partial protective capability against GrB-mediated apoptosis via its lower affinity to Bim. In comparison with ectopically expressed full-length Mcl-1, the stably transfected C-terminal fragments of Mcl-1 were less efficiently localized to the mitochondria. Knockdown of Mcl-1, as achieved by transfection with Mcl-1-specific short interfering RNA, resulted in a significant level of apoptosis in the absence of external apoptotic stimulation and, in addition, enhanced the susceptibility of breast carcinoma cells to GrB cytotoxicity. The significance of Bim in this GrB apoptotic cascade was indicated by the marked protection against GrB-mediated apoptosis endowed on these cells through Bim knockdown. Our studies suggest that the disruption of the Mcl-1.Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.     

Identification of a thrombin cleavage site and a short form of ADAMTS-18

We previously reported that C-terminal fragment of ADAMTS-18 induces platelet fragmentation through ROS release. We have shown that thrombin cleaves ADAMTS-18 and that a short form of ADAMTS-18 in in vitro translational assay. However, the exact thrombin cleavage site and whether a short form ADAMTS-18 presents in vivo are not clear. In this study, we first identified that the thrombin cleavage site is between Arg775 and Ser776 by thrombin cleavage of ADAMTS-18 peptide following mass spectrum assay. We then showed that a short form ADAMTS-18 presents in brain, kidney, lung, and testicle from C57BL/6 mouse embryo. Since alternative form of ADAMTS-18 could be a mechanism to regulate its activity, we then investigated the mechanism involves in the generation of ADAMTS-18 short form. However, neither protease inhibitors nor mutations in catalytic domain of ADAMTS-18 have any significant effect on the generation of ADAMTS-18 short form. Thus, our data demonstrate a thrombin cleavage site and confirm a short form of ADAMTS-18 presents in vivo.     

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.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.     

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.     

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.