S. F. De Meyer

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13).

Summary.  Background: Over the last 4 years ADAMTS‐13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS‐13‐deficient plasma (arbitrarily set at 0%) into one normal‐pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed‐vs.‐expected ADAMTS‐13 levels assessed as r2 ranged from 0.931 to 0.998. Between‐run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F‐values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS‐13 levels (the higher the F‐value, the better the capacity) ranged from 3965 to 137. Between‐method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS‐13 offer the best performance characteristics. Conclusions: New assays for ADAMTS‐13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo

Summary.  Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C‐terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13−/− mouse. Results: Murine MDTCS (devoid of T2‐8 and CUB domains) retained full enzyme activity in vitro towards FRETS‐VWF73 and the C‐terminal T6‐8 (del(T6‐CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet‐decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6‐CUB)) or complete (delCUB) addition of the T2‐8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2‐8 domains influence proteolysis of platelet‐decorated VWF strings in vivo.

Development of Monoclonal Antibodies that Inhibit Platelet Adhesion or Aggregation as Potential Anti-Thrombotic Drugs

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13

Recently, conformational activation of ADAMTS‐13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2‐CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).

The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice

Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin‐like And Metalloprotease domain with ThromboSpondin type‐1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP.

In vivo von Willebrand factor size heterogeneity in spite of the clinical deficiency of ADAMTS‐13

rates following IVF treatment, using two recombinant FSH preparations for ovarian stimulation. Hum Reprod 2002; 17: 304–9. 3 GoswamyRK,Williams G, Steptoe PC. Decreased uterine perfusion – a cause of infertility. Hum Reprod 1988; 3: 955–9. 4 Groeneveld E, Broeze KA, Lambers MJ, Haapsamo M, Dirckx K, Schoot BC, Salle B, Duvan CI, Schats R, Mol BW, Hompes PG, for the IPD MARIA study group. Is aspirin effective in women undergoing in vitro fertilization (IVF)? Results from an individual patient data meta-analysis (IPD MA). Hum Reprod Update 2011; 17: 501–9. 5 Nelson SM, Greer IA. The potential role of heparin in assisted conception. Hum Reprod Update 2008; 14: 623–45. 6 McGinn T, Wyer PC, Newman TB, Keitz S, Leipzig R, For GG. Evidence-based Medicine Teaching Tips Working Group. Tips for learners of evidence-based medicine: 3. Measures of observer variability (kappa statistic). CMAJ 2004; 171: 1369–73. 7 Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. BMJ 2003; 327: 557–60. 8 Di Micco P, D Uva M, Lodigiani C, Rota LL. Thrombophilia and repeated in vitro fertilisation and embryo transfer failure: an open issue. Thromb Haemost 2010; 103: 472–3. 9 Berker B, Taskin S, Kahraman K, Taskin EA, Atabekoğlu C, Sönmezer M. The role of low-molecular-weight heparin in recurrent implantation failure: a prospective, quasi-randomized, controlled study. Fertil Steril 2011; 95: 2499–502. 10 Noci I, Milanini MN, Ruggiero M, Papini F, Fuzzi B, Artini PG. Effect of dalteparin sodium administration on IVF outcome in nonthrombophilic young women: a pilot study. Reprod Biomed Online 2011; 22: 615–20. 11 Urman B, Ata B, Yakin K, Alatas C, Aksoy S, Mercan R, Balaban B. Luteal phase empirical lowmolecular weight heparin administration in patients with failed ICSI embryo transfer cycles: a randomized openlabeled pilot trial. Hum Reprod 2009; 24: 1640–7. 12 Qublan H, Amarin Z, DabbasM, Farraj AE, Beni-Merei Z, Al-Akash H, Bdoor AN, Nawasreh M, Malkawi S, Diab F, Al-Ahmad N, Balawneh M, Abu-Salim A. Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial.HumFertil (Camb) 2008; 11: 246–53. 13 Stern C, Chamley L, Norris H, Hale L, Baker HW. A randomized, double-blind, placebo-controlled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies. Fertil Steril 2003; 80: 376–83. 14 Sher G, Zouves C, FeinmanM, Maassarani G, Matzner W, Chong P, ChingW. A rational basis for the use of combined heparin/aspirin and IVIG immunotherapy in the treatment of recurrent IVF failure associated with antiphospholipid antibodies. Am J Reprod Immunol 1998; 39: 391–4. 15 Kutteh WH, Yetman DL, Chantilis SJ, Crain J. Effect of antiphospholipid antibodies in women undergoing in-vitro fertilization: role of heparin and aspirin. Hum Reprod 1997; 12: 1171–5. 16 Lacey H, Haigh T, Westwood M, Aplin JD. Mesenchymally-derived insulin-like growth factor 1 provides a paracrine stimulus for trophoblast migration. BMC Dev Biol 2002; 24: 2. 17 Lodigiani C, Di Micco P, Ferrazzi P, Librè L, Arfuso V, Polatti F, Michela B, Rossini R, Morenghi E, Rota L, Brenner B, Paolo EL. Low-molecular-weight heparin in women with repeated implantation failure. Womens Health (Lond Engl) 2011; 7: 425–31. 18 Grandone E, Colaizzo D, Lo Bue A, Checola MG, Cittadini E, Margaglione M. Inherited thrombophilia and in vitro fertilization implantation failure. Fertil Steril 2001; 76: 201–2. 19 Di Nisio M, Rutjes AW, Ferrante N, Tiboni GM, Cuccurullo F, Porreca E. Thrombophilia and outcomes of assisted reproduction technologies: a systematic review and meta-analysis. Blood 2011; 118: 2670–8.

An open conformation of ADAMTS‐13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura

Essentials Conformational changes in ADAMTS‐13 are part of its mode‐of‐action. The murine anti‐ADAMTS‐13 antibody 1C4 discriminates between folded and open ADAMTS‐13. ADAMTS‐13 conformation is open in acute acquired thrombotic thrombocytopenic purpura (TTP). Our study forms an important basis to fully elucidate the pathophysiology of TTP.

On the impact of indium and boron on the Reversed Narrow-Channel Effect (RNCE) in BULK and SOI MOSFETs

For the first time, experimental results are presented on the Reverse Narrow-Channel Effect (RNCE) in SOI and bulk MOSFETs using indium as a channel dopant. The presented results show that devices with an indium channel exhibit the same RNCE as devices with a boron channel, which refers to the same diffusion mechanisms in deep submicron devices.

High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease.

Essentials von Willebrand disease (VWD) is the most common inherited bleeding disorder. Gene therapy for VWD offers long‐term therapy for VWD patients. Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice. Liver‐directed transposons support sustained VWF expression with suboptimal multimerization.

Enhanced activity of an ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischemic stroke

Essentials ADAMTS13 requires a substrate‐induced conformational change to attain full activity in vitro. The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre‐activation. A pre‐activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates. This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13.