Crystal structure of ADAMTS13 CUB domains reveals their role in global latency.

ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.

Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function.

Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.

Plasma ADAMTS-13 levels and the risk of myocardial infarction: an individual patient data meta-analysis.

BACKGROUND: Low ADAMTS-13 levels have been repeatedly associated with an increased risk of ischemic stroke, but results concerning the risk of myocardial infarction are inconclusive. OBJECTIVES: To perform an individual patient data meta-analysis from observational studies investigating the association between ADAMTS-13 levels and myocardial infarction. METHODS: A one-step meta-analytic approach with random treatment effects was used to estimate pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for confounding. Analyses were based on dichotomous exposures, with the 5th and 1st percentiles of ADAMTS-13 antigen levels as cut-off values. Quartile analyses, with the highest quartile as a reference category, were used to assess a graded association between levels and risk ('dose' relationship). Additionally, we assessed the risk of the combined presence of low ADAMTS-13 and high von Willebrand factor (VWF) levels. RESULTS: Five studies were included, yielding individual data on 1501 cases and 2258 controls (mean age of 49 years). Low ADAMTS-13 levels were associated with myocardial infarction risk, with an OR of 1.89 (95% CI 1.15-3.12) for values below the 5th percentile versus above, and an OR of 4.21 (95% CI 1.73-10.21) for values below the 1st percentile versus above. Risk appeared to be restricted to these extreme levels, as there was no graded association between ADAMTS-13 levels and myocardial infarction risk over quartiles. Finally, there was only a minor synergistic effect for the combination of low ADAMTS-13 and high VWF levels. CONCLUSIONS: Low ADAMTS-13 levels are associated with an increased risk of myocardial infarction.

Evidence that high von Willebrand factor and low ADAMTS-13 levels independently increase the risk of a non-fatal heart attack.

BACKGROUND: A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) may influence von Willebrand factor (VWF) levels and consequently the risk of myocardial infarction (MI). Moreover, ADAMTS-13 influences hemostatic plug formation in mouse models. We therefore studied their associations in the Glasgow MI Study (GLAMIS). METHODS AND RESULTS: We measured ADAMTS-13 and VWF antigen levels by ELISAs in stored plasma from a case-control study of 466 MI cases and 484 age- and sex-matched controls from the same north Glasgow population. There was no correlation between ADAMTS-13 and VWF levels in cases or controls. ADAMTS-13 levels correlated positively with serum cholesterol and triglycerides and body mass index, and negatively with high-density lipoprotein-cholesterol. VWF levels correlated with age, fibrinogen and C-reactive protein. In multivariable analyses including risk factors, VWF correlated positively with risk of MI, and ADAMTS-13 correlated negatively with risk of MI. These associations were independent of each other. The association of ADAMTS-13 with risk of MI was observed only in multivariable analysis. CONCLUSIONS: VWF and ADAMTS-13 levels were not associated in this study, and showed associations with MI risk in opposite directions but of similar strength. The association of ADAMTS-13 with MI is influenced by lipid levels, and consequently requires further investigation.

Prevalence of the ADAMTS-13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. OBJECTIVES: We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. PATIENTS/METHODS/RESULTS: We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66-126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity. CONCLUSION: The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.

The central role of thrombin in hemostasis.

Following vascular injury, blood loss is controlled by the mechanisms of hemostasis. During this process, the serine proteinase, thrombin, is generated both locally and rapidly at sites of vessel damage. It plays a pivotal role in clot promotion and inhibition, and cell signaling, as well as additional processes that influence fibrinolysis and inflammation. These functions involve numerous cleavage reactions, which must be tightly coordinated. Failure to do so can lead to either bleeding or thrombosis. The crystal structures of thrombin, in combination with biochemical analyses of thrombin mutants, have provided insight into the ways in which thrombin functions, and how its different activities are modulated. Many of the interactions of thrombin are facilitated by exosites on its surface that bind to its substrates and/or cofactors. The use of cofactors not only extends the range of thrombin specificity, but also enhances its catalytic efficiency for different substrates. This explains a paradox (i.e. thrombin is a specific proteinase, and yet one that has multiple, and sometimes opposing, substrate reactions). In this review, we describe the context in which thrombin acts during hemostasis and explain the roles that its exosites and cofactors play in directing thrombin function. Thereafter, we develop the concept of cofactor competition as a means by which the activities of thrombin are controlled.

Further characterization of ADAMTS-13 inactivation by thrombin.

BACKGROUND: The multimeric size and platelet-tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. OBJECTIVES: In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance. METHODS AND RESULTS: By N-terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS-13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS-13. Proteolysis of ADAMTS-13 by thrombin caused an 8-fold reduction in its affinity for VWF that contributed to its loss of VWF-cleaving function. Intriguingly, thrombin-cleaved ADAMTS-13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS-13, but not added ADAMTS-13, appeared resistant to coagulation-induced fragmentation. An estimation of the K(m) for ADAMTS-13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS-13. This was corroborated by the comparatively low affinity of ADAMTS-13 for thrombin (K(D) 95 nM). CONCLUSIONS: Together, our data suggest that ADAMTS-13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.