Apical sorting of ADAMTS13 in vascular endothelial cells and Madin-Darby canine kidney cells depends on the CUB domains and their association with lipid rafts

ADAMTS13 biosynthesis appeared to occur mainly in hepatic stellate cells, but detection of ADAMTS13 mRNA in many other tissues suggests that vascular endothelium may also produce ADAMTS13. We showed that ADAMTS13 mRNA and protein were detectable in human umbilical vein endothelial cells, aortic endothelial cells, and endothelium-derived cell line (ECV304). ADAMTS13 in cell lysate or serum-free conditioned medium cleaved von Willebrand factor (VWF) specifically. ADAMTS13 and VWF were localized to the distinct compartments of endothelial cells. Moreover, ADAMTS13 was preferentially sorted into apical domain of ECV304 and Madin-Darby canine kidney (MDCK) cells. Apical sorting of ADAMTS13 depended on the CUB domains and their association with lipid rafts. A mutation in the second CUB domain of ADAMTS13 (4143-4144insA), naturally occurring in patients with inherited thrombotic thrombocytopenic purpura, resulted in a significant reduction of ADAMTS13 secretion and a reversal of its polarity in MDCK cells. These data demonstrated that ADAMTS13 is synthesized and secreted from endothelial cells; the apically secreted ADAMTS13 from endothelial cells may contribute significantly to plasma ADAMTS13 proteases. The data also suggest a critical role of the CUB domains and a novel cargo-selective mechanism for apical sorting of a soluble ADAMTS protease in polarized cells.     

Platelet hyperaggregability is associated with decreased ADAMTS13 activity and enhanced endotoxemia in patients with acute cholangitis

Aim

Insufficient ADAMTS13 activity (ADAMTS13:AC) leads to increased levels of unusually large von Willebrand factor (VWF) multimers and causes microcirculatory disturbance and multiple organ failure (MOF). Endotoxin (Et) triggers the activation of coagulation and cytokine cascades, leading to MOF in severe inflammatory response syndrome. Here, we investigated the potential role of endotoxemia‐related ADAMTS13 in acute cholangitis.

Methods

Twenty‐four patients with acute cholangitis, including 7 with severe acute cholangitis, were recruited in this study. The levels of ADAMTS13:AC, VWF antigen (VWF:Ag), interleukin (IL)‐6, IL‐8, and tumor necrosis factor (TNF)‐α in each patient were determined by enzyme‐linked immunosorbent assay, whereas Et levels were determined by Et activity assay (EAA) analysis.

Results

The ADAMTS13:AC and VWF:Ag levels were significantly lower and higher, respectively, in patients with acute cholangitis than in controls. The EAA levels were higher in patients with acute cholangitis than in controls, and were inversely correlated with that of ADAMTS13:AC. Patients with severe acute cholangitis had significantly lower ADAMTS13:AC and higher VWF:Ag levels than those with mild to moderate cholangitis. Notably, ADMTS13:AC was directly correlated with platelet counts and inversely correlated with IL‐6 levels, and the VWF:Ag/ADAMTS13:AC ratio was directly correlated with IL‐8 and TNF‐α levels.

Conclusions

Imbalance of ADAMTS13:AC and VWF:Ag levels might be associated with severe acute cholangitis, reflecting platelet hyperaggregability. Severe acute cholangitis has severe pathophysiological features and is complicated by endotoxemia and MOF. Notably, this is the first report indicating an association between the levels of ADAMTS13:AC and VWF:Ag and those of EAA and cytokines in acute cholangitis.     

Changes in von Willebrand factor-cleaving protease (ADAMTS13) activity after infusion of desmopressin

von Willebrand factor-cleaving protease (ADAMTS13) cleaves von Willebrand factor (VWF) and regulates its physiologic function. To investigate the relation between ADAMTS13 activity and VWF, we compared ADAMTS13 activity with the VWF-related parameters VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CBA), VWF-propeptide, proVWF, and VWF multimeric composition in 10 healthy volunteers and 3 patients with type 1 von Willebrand disease before and after infusing 0.3 microg/kg desmopressin. The VWF-related parameters in the volunteers increased 60 minutes after start of infusion by 3.7-fold for VWF:Ag, 7.2-fold for propeptide, and 2.2-fold for VWF:CBA. Unusually large VWF multimers and traces of proVWF appeared. The ADAMTS13 activity decreased to about half the initial value. After 24 hours values returned to baseline. Patients with type 1 von Willebrand disease showed similar results. We conclude that the inverse correlation between ADAMTS13 and VWF-related parameters suggests a consumption of ADAMTS13 after the desmopressin-induced release of higher multimers of VWF.     

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage

Type 2A VWD is characterized by the absence of large VWF multimers and decreased platelet-binding function. Historically, type 2A variants are subdivided into group 1, which have impaired assembly and secretion of VWF multimers, or group 2, which have normal secretion of VWF multimers and increased ADAMTS13 proteolysis. Type 2A VWD patients recruited through the T. S. Zimmerman Program for the Molecular and Clinical Biology of VWD study were characterized phenotypically and potential mutations identified in the VWF D2, D3, A1, and A2 domains. We examined type 2A variants and their interaction with WT-VWF through expression studies. We assessed secretion/intracellular retention, multimerization, regulated storage, and ADAMTS13 proteolysis. Whereas some variants fit into the traditional group 1 or 2 categories, others did not fall clearly into either category. We determined that loss of Weibel-Palade body formation is associated with markedly reduced secretion. Mutations involving cysteines were likely to cause abnormalities in multimer structure but not necessarily secretion. When coexpressed with wild-type VWF, type 2A variants negatively affected one or more mechanisms important for normal VWF processing. Type 2A VWD appears to result from a complex intersection of mechanisms that include: (1) intracellular retention or degradation of VWF, (2) defective multimerization, (3) loss of regulated storage, and (4) increased proteolysis by ADAMTS13.     

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.     

H1N1 influenza (swine flu)-associated thrombotic microangiopathy with a markedly high plasma ratio of von Willebrand factor to ADAMTS13

We describe an 18-year-old woman infected with H1N1 influenza followed by thrombotic microangiopathy. During the acute phase, her plasma levels of von Willebrand factor (VWF) were remarkably elevated, whereas those of ADAMTS13 were reduced without its inhibitors, generating a markedly high ratio of VWF to ADAMTS13 in circulation. A retrospective analysis established the following hypothesis: an influenza-mediated cytokine storm induced an enhanced release of unusually large VWF multimers (UL-VWFM) from vascular endothelial cells, generating platelet thrombi in microcirculatures under high shear stress. Plasma exchange removed UL-VWFM and cytokines, and rescued her life. This report sheds a light on a hitherto unrecognized influenza complication.     

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.     

ADAMTS13 promotes angiogenesis and modulates VEGF-induced angiogenesis

Plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 and its interactions with VEGF-mediated angiogenesis, the effects of ADAMTS13 on human umbilical vein endothelial cell (HUVEC) were studied in Matrigel tube formation, proliferation, cell migration, and scratch wound assays. Treatment of endothelial cells with exogenous recombinant full-length ADAMTS13 alone promoted angiogenesis in a dose-dependent manner. HUVEC incubated with 200 ng/mL ADAMTS13 (1.4 nM) resulted in a 65% increase in cell tube formation when compared to the EBM-2 control. HUVEC treated with 30 ng/mL ADAMTS13 (204.1 pM) resulted in an 83% increase in proliferation in a visual counting assay, whereas HUVEC treated with 10 ng/mL ADAMTS13 (68.0 pM) yielded a 295% increase in EC migration in a Boyden chamber assay. In contrast, ADAMTS13 inhibited VEGF-induced angiogenesis in a dose-dependent manner, with 200ng/mL inhibiting tube formation by 35%. HUVEC co-incubated with ADAMTS13 and an antibody to the ADAMTS13 thrombospondin domains 5-7 reversed the inhibition of tube formation. HUVEC treated with 30 ng/mL ADAMTS13 and 6.2 ng/mL (323.0 pM) VEGF proliferated 40% slower than the VEGF control after 24 h of incubation as measured by visual counting assay. Treatment of HUVEC with 6.2 ng/mL VEGF and 10 ng/mL ADAMTS13 inhibited cell migration by 48%, compared to the VEGF control. Substitution of ADAMTS13 with truncated ADAMTS13 (deletion of C-terminal TSP1 domain) did not significantly increase angiogenesis or suppress VEGF-induced angiogenesis, suggesting that the TSP1 domain is involved in ADAMTS13 angiogenic activities. Co-immunoprecipitation experiments provided further evidence that ADAMTS13 binds to VEGF via its TSP1 domain.     

Association between ADAMTS13 activity–VWF antigen imbalance and the therapeutic effect of HAIC in patients with hepatocellular carcinoma

BACKGROUNDPrediction of HAIC treatment response is important for improving the prognosis in patients with hepatocellular carcinoma (HCC). The progression of HCC is related to hypercoagulability and angiogenesis. It is known that ADAMTS13 and von Willebrand factor (VWF) are related to hypercoagulability. In addition, previous study reported that the association between ADAMTS13 and VWF, and angiogenesis via vascular endothelial growth factor (VEGF). Recently, ADAMTS13 and VWF have been associated with the prognosis in patients with various kinds of cancer undergoing chemotherapy.AIMTo investigate whether ADAMTS13 and VWF become useful biomarkers of treatment response in HCC patients before the initiation of HAIC treatment.METHODSSeventy-two patients were enrolled in this study. ADAMTS13 activity (ADAMTS13:AC), VWF antigen (VWF:Ag) and VEGF levels were determined via enzyme-linked immunosorbent assay. Univariable and multivariable analyses were performed to determine the predictive factors of treatment response in patients with HCC undergoing HAIC treatment.RESULTSADAMTS13:AC levels in HCC patients with stable disease (SD) + partial response (PR) of HAIC treatment were significantly higher than those with progressive disease (PD) (P < 0.05). In contrast, VWF:Ag/ADAMTS13:AC ratio and VEGF levels in HCC patients with SD + PR were significantly lower than those with PD (both P < 0.05). Patients with high VWF:Ag/ADAMTS13:AC ratio (> 2.7) had higher VEGF levels than those with low ratio (≤ 2.7). Multivariable analysis revealed that VWF:Ag/ADAMTS13:AC ratio was a predictive factor of HAIC treatment response.CONCLUSIONVWF:Ag/ADAMTS13:AC ratio may become a useful biomarker of treatment response in HCC patients before the initiation of HAIC treatment.     

Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.     

Size regulation of von Willebrand factor-mediated platelet thrombi by ADAMTS13 in flowing blood

The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s(-1). At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.     

von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome

Whether measurement of ADAMTS13 activity may enable physicians to distinguish thrombotic thrombocytopenic purpura (TTP) from hemolytic uremic syndrome (HUS) is still a controversial issue. Our aim was to clarify whether patients with normal or deficient ADAMTS13 activity could be distinguished in terms of disease manifestations and multimeric patterns of plasma von Willebrand factor (VWF). ADAMTS13 activity, VWF antigen, and multimeric pattern were evaluated in patients with recurrent and familial TTP (n = 20) and HUS (n = 29). Results of the collagen-binding assay of ADAMTS13 activity were confirmed in selected samples by testing the capacity of plasma to cleave recombinant VWF A1-A2-A3. Most patients with TTP had complete or partial deficiency of ADAMTS13 activity during the acute phase, and in some the defect persisted at remission. However, complete ADAMTS13 deficiency was also found in 5 of 9 patients with HUS during the acute phase and in 5 patients during remission. HUS patients with ADAMTS13 deficiency could not be distinguished clinically from those with normal ADAMTS13. In a subgroup of patients with TTP or HUS, the ADAMTS13 defect was inherited, as documented by half-normal levels of ADAMTS13 in their asymptomatic parents, consistent with the heterozygous carrier state. In patients with TTP and HUS there was indirect evidence of increased VWF fragmentation, and this occurred also in patients with ADAMTS13 deficiency. In conclusion, deficient ADAMTS13 activity does not distinguish TTP from HUS, at least in the recurrent and familial forms, and it is not the only determinant of VWF abnormalities in these conditions.     

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.     

Association between thrombotic microangiopathy and reduced ADAMTS13 activity in malignant hypertension

The thrombotic microangiopathy observed in malignant hypertension is similar to that of thrombotic thrombocytopenic purpura, which is associated with a deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving protease that cleaves large prothrombogenic multimers. We hypothesized that ADAMTS13 is deficient in malignant hypertension and that the severity of thrombotic microangiopathy is associated with decreased ADAMTS13 activity. We included 20 patients with malignant and 20 patients with severe hypertension, and 20 matched normotensive individuals served as control subjects. VWF, active VWF, and free hemoglobin were assessed to explore predictors of ADAMTS13 activity. Patients with malignant hypertension had lower ADAMTS13 activity (80%; interquartile range: 53% to 130%) compared with control subjects (99% interquartile range: 82% to 129%; P<0.01) but not compared with patients with severe hypertension (P=0.14). ADAMTS13 activity negatively correlated with lactic dehydrogenase levels after logarithmic transformation (r=-0.65; P<0.001) and was associated with platelet count (r=0.34; P=0.04) and the presence of schistocytes (r=-0.37; P=0.02). Apart from the association with thrombotic microangiopathy, ADAMTS13 was inversely associated with creatinine (r=-0.42; P=0.008). Increasing levels of VWF were associated with a decrease in ADAMTS13 activity (r=-0.34; P=0.03). There was no significant association between ADAMTS13 activity and other parameters, including blood pressure. In conclusion, ADAMTS13 is decreased in malignant hypertension and associated with the severity of thrombotic microangiopathy, likely because of the release of VWF after endothelium stimulation. A severe deficiency could not be demonstrated. More studies are needed to identify the role of ADAMTS13 in the thrombotic microangiopathy and ischemic complications of malignant hypertension.     

An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers

Severe deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated into a high and a low group that differed by approximately 10 fold. Low ADAMTS13 activity was detected in mice containing 2 alleles of intracisternal A-type particle (IAP) retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified 2 truncated variants (IAP-a and IAP-b) in the low-activity mice. Both of the IAP variants lacked the 2 carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared with full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.     

Phytoestrogen alpha-zearalanol antagonizes oxidized LDL-induced inhibition of nitric oxide production and stimulation of endothelin-1 release in human umbilical vein endothelial cells

Oxidative modification of low-density lipoprotein (LDL) leads to formation of the atherogenic molecule oxidized LDL (oxLDL), which is considered to be an important mediator for vascular endothelial dysfunction and atherosclerosis. It is speculated that reduced nitric oxide (NO) release/bioavailability and enhanced release of endothelin-1 (ET-1) may contribute to oxLDL-induced endothelial dysfunction. Estrogen may improve lipid profile and inhibit oxLDL-induced endothelial damage. However, estrogen replacement therapy has been suspended due to uncertainty in benefits versus risk (such as cancer progression) in postmenopausal women. This study was designed to evaluate the effect of a novel phytoestrogen, α-zearalanol (α-ZAL), on oxLDL-induced effect on NO and ET-1 production in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with oxLDL (50 μg/mL) for 24 h in the absence or presence of α-ZAL (0–1000 nM), 17β-estradiol (E₂, 10 nM), or the E₂ receptor antagonist ICI 182780 (1 μM). Levels of NO and ET-1 were measured by spectrophotometry and enzymatic immunoassay, respectively. NOS activity was evaluated by conversion of ³H-arginine to ³H-citrulline. Protein and mRNA expression of NOS and ET-1 were measured by Western blot and RT-PCR. Our results indicated that oxLDL significantly reduced NO release and NOS activity, and enhanced ET-1 production associated with reduced NOS3 (but not NOS2) expression and enhanced ET-1 mRNA expression. All these oxLDL-induced alterations were significantly attenuated or abolished by co-incubation with α-ZAL or E₂, both through an E₂ receptor-dependent mechanism. α-ZAL, E₂, and ICI182780 had no effect on NO/ET-1 release, NOS activity, or expression of NOS and ET-1. These data suggested that the phytoestrogen α-ZAL, like E₂, may effectively antagonize oxLDL-induced decrease in NO and increase in ET-1, which may be protective for endothelial function.     

ADAMTS13 content in plasma‐derived factor VIII/von Willebrand factor concentrates

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet‐rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti‐ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood‐borne infectious agents. A number of recent reports indicate that certain plasma‐derived VWF‐factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma‐derived VWF‐FVIII concentrates, showing that Koate^®‐DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate^® (Grifols) was the closest other product in terms of protease content. Koate^®‐DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF‐FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed. Am. J. Hematol. 88:895–898, 2013. © 2013 Wiley Periodicals, Inc.     

Levels and activities of von Willebrand factor and metalloproteinase with thrombospondin type-1 motif,number 13 in inflammatory bowel diseases

AIM To evaluate the levels of von Willebrand factor(VWF) and metalloproteinase with thrombospondin type-1 motif, number 13(ADAMTS13) in inflammatory bowel disease(IBD) and correlate them with the disease activity.METHODS Consecutive patients with IBD aged 18 years or older were enrolled in the study. Forty-seven patients with ulcerative colitis(UC), 38 with Crohn's disease(CD), and 50 healthy controls were included. The white blood cell count, haematocrit, platelet count, fibrinogen, partial activated thromboplastin time, C-reactive protein, albumin, VWF antigen level(VWF:Ag), VWF ristocetin cofactor activity(VWF:RCo), VWF collagen-binding activity(VWF:CB), and ADAMTS13 antigen level(ADAMTS13:Ag) and activity(ADAMTS13act) were measured. The following ratios were assessed: V W F : R C o/V W F : A g, V W F : C B/V W F : A g, V W F : A g/ADAMTS13 act, and ADAMTS13act/ADAMTS13:Ag. RESULTS Compared to controls, the odds ratio(OR) of an elevated VWF: Ag 150% was 8.7(95%CI: 2.7-28.1) in the UC group and 16.2(95%CI: 4.8-54.0) in the CD group. VWF:CB was lower in UC patients, and active CD was associated with a higher VWF: RCo(+38%). The ORs of VWF:CB/VWF:Ag 0.7(a marker of acquired von Willebrand syndrome) in the UC and CD groups were 11.9(95%CI: 4.4-32.4) and 13.3(95%CI: 4.6-38.1), respectively. Active UC was associated with lower ADAMTS13:Ag(-23%) and ADAMTS13act(-20%) compared to UC in remission. Patients with active CD had a 15% lower ADAMTS13 act than controls. The activity of UC, but not that of CD, was inversely correlated with ADAMTS13:Ag(r =-0.76) and ADAMTS13act(r =-0.81). CONCLUSION Complex VWF-ADAMTS13-mediated mechanisms disturb haemostasis in IBD. A reduced WVF:CB is a risk factor for bleeding, while a lower ADAMTS13 level combined with an elevated VWF:Ag could predispose one to thrombosis.     

Prospective study on the behaviour of the metalloprotease ADAMTS13 and of von Willebrand factor after bone marrow transplantation

Thrombotic microangiopathies (TMAs) are rare but serious complications of bone marrow transplantation (BMT). Clinical manifestations are similar to those of thrombotic thrombocytopenic purpura (TTP), but prognosis is generally poorer despite plasma exchange. The enzymatic activity of the plasma metalloprotease ADAMTS13, which cleaves ultralarge thrombogenic multimers of von Willebrand factor (VWF) derived from activated endothelial cells, is very low or undetectable in patients with classic TTP, and protease deficiency is thought to play a mechanistic role in the formation of platelet thrombi in the microcirculation. This is the first prospective study to evaluate the incidence of TMA in 46 consecutively recruited patients undergoing autologous or allogeneic BMT and explore in parallel the behaviour of ADAMTS13, VWF antigen and VWF multimer size. The incidence of post-BMT TMA was 6% (three of 46); all cases occurred after allogeneic BMT. Compared with baseline values plasma ADAMTS13 activity was significantly reduced in patients undergoing BMT, particularly after the conditioning regimen (mean values: 50 +/- 22 vs. 77 +/- 32%; P < 0.0001). In the three patients who developed TMA, ADAMTS13 decreased after conditioning, but was very low in one case only (8%). VWF antigen levels progressively increased after the conditioning regimen (228 +/- 75 vs. 178 +/- 76% at baseline, P = 0.002). The mean proportion of high-molecular weight VWF multimers did not change in the various stages of BMT, even though ultralarge multimers were transiently found in same cases with and without TMA. Hence, the measurements evaluated in this study are not clinically useful to predict the occurrence of post-BMT TMA.     

The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow

ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr(1605) and Met(1606) residues at the central A2 subunit. The amino-terminus of ADAMTS13 protease appears to be sufficient to bind and cleave VWF under static and denatured condition. However, the role of the carboxyl-terminus of ADAMTS13 in substrate recognition remains controversial. Present study demonstrates that ADAMTS13 cleaves VWF in a rotation speed- and protease concentration-dependent manner on a mini vortexer. Removal of the CUB domains (delCUB) or truncation after the spacer domain (MDTCS) significantly impairs its ability to cleave VWF under the same condition. ADAMTS13 and delCUB (but not MDTCS) bind VWF under flow with dissociation constants (K(D)) of about 50 nM and about 274 nM, respectively. The isolated CUB domains are neither sufficient to bind VWF detectably nor capable of inhibiting proteolytic cleavage of VWF by ADAMTS13 under flow. Addition of the TSP1 5-8 (T5-8CUB) or TSP1 2-8 repeats (T2-8CUB) to the CUB domains restores the binding affinity toward VWF and the inhibitory effect on cleavage of VWF by ADAMTS13 under flow. These data demonstrate directly and quantitatively that the cooperative activity between the middle carboxyl-terminal TSP1 repeats and the distal carboxyl-terminal CUB domains may be crucial for recognition and cleavage of VWF under flow.