Deletion of GARP on mouse regulatory T cells is not sufficient to inhibit the growth of transplanted tumors.

GARP is a transmembrane protein that presents latent TGF-ß1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-ß1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.

Virology analyses of HCV genotype 4 isolates from patients treated with simeprevir and peginterferon/ribavirin in the Phase III RESTORE study.

Simeprevir is a hepatitis C virus NS3/4A protease inhibitor. Hepatitis C virus baseline NS3/4A polymorphisms and emerging mutations were characterized in treatment-naїve and treatment-experienced genotype 4-infected patients treated with simeprevir+peginterferon/ribavirin in the RESTORE study. Population sequencing of the NS3/4A region was performed and in vitro simeprevir activity against site-directed mutants or chimeric replicons with patient-derived NS3 protease sequences was assessed in a transient replicon assay. Simeprevir remained active against most (83/91 [91%]) baseline isolates tested in the chimeric replicon assay. Eight baseline isolates reduced simeprevir activity; these carried I132L or D168E substitutions reducing simeprevir median activity by 4.6- and 39-fold, respectively. Six of these eight isolates were from patients achieving sustained virologic response. Baseline NS3 Q80K polymorphism was not observed in the genotype 4-infected patients. Of the 107 simeprevir-treated patients, 37 did not achieve sustained virologic response for any reason. Of the 32 patients who failed treatment and had sequencing information, 28 (88%) had emerging mutations at NS3 positions 80, 122, 155, 156 and/or 168 at time of failure, similar to those in genotype 1. Emerging mutations were mainly D168V and D168E alone or combined with mutations at position 80. In general, isolates obtained at time of failure displayed high-level in vitro resistance to simeprevir (fold change ≥50) in a chimeric replicon assay with a median simeprevir fold change value of 440, consistent with observed mutations. In conclusion, emerging mutations in genotype 4 patients failing simeprevir+peginterferon/ribavirin treatment were similar to those in genotype 1 and conferred high-level resistance to simeprevir.

Effect of laser-activated irrigation on biofilms in artificial root canals.

AIM: To evaluate the antimicrobial effect of laser-activated irrigation (LAI) on biofilms formed in simulated root canals. METHODOLOGY: A dual-species biofilm of Enterococcus faecalis and Streptococcus mutans was grown in a resin root canal model. Biofilms were formed over 48 h and subsequently subjected to the following treatments, all executed for 20 s: syringe irrigation (SI) with a 27G needle, ultrasonically activated irrigation (UAI) with a size 20 Irrisafe file, and LAI with a 2940 nm Er:YAG laser (20 Hz, 50 µs, 20 or 40 mJ, conical fibre tip at two positions). Tests were performed with both sterile saline as well as NaOCl (2.5%) as the irrigant. Surviving bacteria were harvested and the number of CFU was determined by plate counting and compared across groups (anova, P ≤ 0.05). RESULTS: Using saline as the irrigant, significant reductions in viable counts compared to untreated controls were observed for ultrasonically activated irrigation (0.52 log10 reduction) and for all laser-activated irrigation groups (>1 log10 reduction), but not for syringe irrigation (<0.25 log10 reduction). The reductions in the laser-activated irrigation groups were significantly greater than those of ultrasonically activated irrigation. With NaOCl as the irrigant, significant reductions (>2.2 log10 units) in the number of attached bacteria were observed for all treatment groups with no significant differences between laser-activated and ultrasonically activated irrigation. CONCLUSIONS: Within the limitations of this in vitro set-up, laser-activated irrigation removed more biofilm than ultrasonically activated irrigation when using saline as the irrigant, indicating greater physical biofilm removal. The use of NaOCl resulted in greater biofilm reduction with no significant differences between treatment groups.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor.

BACKGROUND: Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion. METHODS: GalTKO.hCD46 transgenic pig lungs were perfused with heparinized fresh human blood. Results from perfusions in which αGPIb Fab (6B4, 10 mg/l blood, n = 6), αGPIIb/IIIa Fab (ReoPro, 3.5 mg/l blood, n = 6), or both drugs (n = 4) were administered to the perfusate were compared to two additional groups in which the donor pig received 1-desamino-8-d-arginine vasopressin (DDAVP), 3 µg/kg (to pre-deplete von Willebrand Factor (pVWF), the main GPIb ligand), with or without αGPIb (n = 6 each). RESULTS: Platelet sequestration was significantly delayed in αGPIb, αGPIb+DDAVP, and αGPIb+αGPIIb/IIIa groups. Median lung "survival" was significantly longer (>240 vs. 162 min reference, p = 0.016), and platelet activation (as CD62P and ßTG) were significantly inhibited, when pigs were pre-treated with DDAVP, with or without αGPIb Fab treatment. Pulmonary vascular resistance rise was not significantly attenuated in any group, and was associated with residual thromboxane and histamine elaboration. CONCLUSIONS: The GPIb-VWF and GPIIb/IIIa axes play important roles in platelet sequestration and coagulation cascade activation during GalTKO.hCD46 lung xenograft injury. GPIb blockade significantly reduces platelet activation and delays platelet sequestration in this xenolung rejection model, an effect amplified by adding αGPIIb/IIIa blockade or depletion of VWF from pig lung.

Incidence of virological failure and emergence of resistance with twice-daily vs every 8-h administration of telaprevir in the OPTIMIZE study.

The OPTIMIZE study demonstrated noninferior efficacy between telaprevir (TVR) twice daily (bid) vs every 8-h (q8h) administration. This analysis compared the selective pressure of both dosing regimens by characterisation of the hepatitis C virus (HCV) variants emerging in genotype 1 (G1) HCV-infected patients who did not achieve sustained virological response (SVR). HCV NS3•4A population sequencing was performed at baseline and time of failure (viral breakthrough, stopping rule or relapse). TVR-resistant variants were classified by fold change in inhibitory concentration (IC50 ). Baseline TVR-resistance was low (<5%) and did not preclude achieving SVR in either arm. The proportion of patients with TVR-resistant variants at time of failure was similar in the bid (15%) and q8h (17%) dosing arms. The majority of variants and virological failures occurred in G1a patients, and mutations V36M, R155K and R155T (G1a), and V36A, T54A and A156S (G1b) were significantly enriched in both treatment arms. The number and type of emerging TVR-resistant variants in non-SVR patients were comparable between treatment arms and were consistent with previous observations. No differences in viral resistance profiles were observed between TVR-based treatment arms in non-SVR patients, indicating a similar selective pressure of TVR bid and q8h dosing.

Analysis of genotype 2 and 3 hepatitis C virus variants in patients treated with telaprevir demonstrates a consistent resistance profile across genotypes.

Study C209 evaluated the activity of telaprevir in treatment-naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir-based treatment of G2/G3 HCV-infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa-2a and ribavirin (triple therapy), or placebo plus peginterferon alfa-2a and ribavirin (control), followed by 22-24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty-three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2-week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3-specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

Histological stroke clot analysis after thrombectomy: Technical aspects and recommendations.

The recent advent of endovascular procedures has created the unique opportunity to collect and analyze thrombi removed from cerebral arteries, instigating a novel subfield in stroke research. Insights into thrombus characteristics and composition could play an important role in ongoing efforts to improve acute ischemic stroke therapy. An increasing number of centers are collecting stroke thrombi. This paper aims at providing guiding information on thrombus handling, procedures, and analysis in order to facilitate and standardize this emerging research field.

Measuring gender norms about relationships in early adolescence: Results from the global early adolescent study.

INTRODUCTION: Gender norms are increasingly recognized as drivers of health and wellbeing. While early adolescence constitutes a critical window of development, there is limited understanding about how adolescents perceive gender relations across different cultural settings. This study used a mixed-method approach, grounded in the voices of young people around the world, to construct and test a cross-cultural scale assessing the perceptions of gender norms regulating romantic relationships between boys and girls in early adolescence. METHODS: The study draws on the Global Early Adolescent study (GEAS), a study focusing on gender norms and health related outcomes over the course of adolescence in urban poor settings worldwide. In-depth interviews were first conducted among approximately 200 adolescents between 10-14 years in seven sites across 4 continents to identify common scripts guiding romantic relations in early adolescence. These scripts were then transformed into a multidimensional scale. The scale was tested among 120 adolescents in each of 14 GEAS sites, followed by a second pilot among 75 adolescents in six sites. We evaluated the psychometric criteria of each sub-scale using principal component analysis, and parallel analysis, followed by exploratory factor analysis to guide the selection of a more parsimonious set of items. RESULTS: Results suggested a two-factor structure, consisting of an "adolescent romantic expectations" subscale and a "Sexual Double Standard" subscale. Both subscales yielded high internal validity in each site, with polychoric Cronbach alpha values above 0.70 with the exception of Kinshasa for the adolescent romantic expectations scale (0.64) and Hanoi for the sexual double standard scale (0.61). CONCLUSION: This study reveals common perceptions of gendered norms about romantic engagement in early adolescence, normative for both sexes, but socially valued for boys while devaluated for girls. The findings illustrate that social hierarchies of power in romantic relationships form early in adolescence, regardless of cultural setting.

Macroscopic x-ray powder diffraction imaging reveals Vermeer's discriminating use of lead white pigments in Girl with a Pearl Earring.

Until the 19th century, lead white was the most important white pigment used in oil paintings. Lead white is typically composed of two crystalline lead carbonates: hydrocerussite [2PbCO3·Pb(OH)2] and cerussite (PbCO3). Depending on the ratio between hydrocerussite and cerussite, lead white can be classified into different subtypes, each with different optical properties. Current methods to investigate and differentiate between lead white subtypes involve invasive sampling on a microscopic scale, introducing problems of paint damage and representativeness. In this study, a 17th century painting Girl with a Pearl Earring (by Johannes Vermeer, c. 1665, collection of the Mauritshuis, NL) was analyzed with a recently developed mobile and noninvasive macroscopic x-ray powder diffraction (MA-XRPD) scanner within the project Girl in the Spotlight. Four different subtypes of lead white were identified using XRPD imaging at the macroscopic and microscopic scale, implying that Vermeer was highly discriminatory in his use of lead white.

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. SUMMARY: Background ADAMTS-13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate-induced conformational change. A gain-of-function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated. Objectives To establish how the hyperactivity of GoF ADAMTS-13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke. Methods The ability of GoF ADAMTS-13 to dissolve VWF-platelet agglutinates was examined with an assay of ristocetin-induced platelet agglutination and in parallel-flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS-13. Results Wild-type (WT) ADAMTS-13 required conformational activation to attain full activity against VWF-mediated platelet capture under flow. In this assay, GoF ADAMTS-13 had an EC50 value more than five-fold lower than that of WT ADAMTS-13 (0.73 ± 0.21 nm and 3.81 ± 0.97 nm, respectively). The proteolytic activity of GoF ADAMTS-13 against preformed platelet agglutinates under flow was enhanced more than four-fold as compared with WT ADAMTS-13 (EC50 values of 2.5 ± 1.1 nm and 10.2 ± 5.6 nm, respectively). In a murine stroke model, GoF ADAMTS-13 restored cerebral blood flow at a lower dose than WT ADAMTS-13, and partially retained the ability to recanalize vessels when administration was delayed by 1 h. Conclusions The limited proteolytic activity of WT ADAMTS-13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS-13 variant translates to a more pronounced protective effect in experimental stroke.

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. SUMMARY: Background Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. Objectives To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. Methods We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf-/- mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery. Results The sandwich SB transposon was suitable to deliver the full-length mVWF cDNA (8.4 kb) and supported supra-physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride-induced liver regeneration. Analysis of integration sites by high-throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long-term expression of supra-physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long-term expression of VWF by hepatocytes resulted in relatively reduced amounts of high-molecular-weight multimers, potentially limiting its hemostatic efficacy. Conclusions Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type-specific targeting is yet to be achieved.

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. SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies. Recently, it was shown that ADAMTS-13 adopts a folded or an open conformation. Objectives As conformational changes in self-antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS-13 changes during acute aTTP. Methods Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS-13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic-uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA. Results The antibody 1C4 recognizes a cryptic epitope in ADAMTS-13. Therefore, we were able to discriminate between a folded and an open ADAMTS-13 conformation. We showed that ADAMTS-13 in HDs does not bind to 1C4, indicating that ADAMTS-13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS-13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS-13 is open during an acute aTTP episode. Conclusions Our study shows that, besides absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies, an open ADAMTS-13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS-13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.

Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

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

BACKGROUND: 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). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

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

BACKGROUND: 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. OBJECTIVES: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. METHODS: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. RESULTS: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. CONCLUSIONS: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.

New approaches for antithrombotic antiplatelet therapies.

Cardiovascular diseases are one of the major causes of mortality in the western world. As platelet dependent thrombosis is of central importance in their pathophysiology, several successful strategies, targeting a specific platelet function or interaction, have been developed to prevent or treat these disorders. However, as the current antiplatelet strategies are limited in efficacy and safety, and often influence normal haemostatic functions, new compounds are being developed with improved characteristics. This review deals with the development of novel antiplatelet compounds for which evidence is available on their antithrombotic action in vivo. In a first part, these compounds, their targets and their potential applicability are discussed. The second part of this review focuses on BT tests and bleeding models and their usefulness for determination and/or prediction of the safety of novel antiplatelet compounds.

Animal models for thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a puzzling disorder in many ways. The disease is difficult to diagnose as analogous symptoms are also found in other microangiopathic disorders. Although ADAMTS13 deficiency is generally required to develop TTP, only some patients with severe ADAMTS13 deficiency do spontaneously develop this disease. It is therefore assumed that environmental and/or genetic factors are needed to cause acute TTP. Nevertheless, acute TTP-like symptoms have also been observed in patients with moderate or normal levels of ADAMTS13. The development of animal models for TTP has allowed a closer look at the specific need for ADAMTS13 deficiency and the necessity for additional triggers in the pathophysiology of TTP. Mouse models for congenital TTP and a baboon model for acquired TTP have been generated. These animal models have also proven to be extremely valuable in developing new treatment strategies for TTP. In the current review, we discuss current animal models for TTP, what we have learned from them and how they were used to test new treatment strategies.

Neutrophil extracellular traps promote deep vein thrombosis in mice.

BACKGROUND: Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be prothrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). OBJECTIVE: To explore the source and role of extracellular chromatin in DVT. METHODS: We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). RESULTS: We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared with sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. CONCLUSIONS: Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development.

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

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.

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

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.