Preserved Expression of Mrna Coding Von Willebrand Factor–Cleaving Protease ADAMTS13 by Selenite and Activated Protein C

Preserved Expression of mRNA Coding von Willebrand Factor–Cleaving Protease ADAMTS13 by Selenite and Activated Protein C

Michael L Ekaney,1,2 Clemens L Bockmeyer,3 Maik Sossdorf,1,2 Philipp A Reuken,1,2 Florian Conradi,2 Tobias Schuerholz,4 Markus F Blaess,1,2 Scott L Friedman,5 Wolfgang Lösche,1,2 Michael Bauer,1,2 and Ralf A Claus1,2

1Center for Sepsis Control and Care, and 2Clinic for Anaesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany; 3Department of Nephropathology, and 4Department for Interdisciplinary Intensive Care, University Hospital Aachen, Aachen, Germany; and 5Mount Sinai School of Medicine, New York, New York, United States of America

In sepsis, the severity-dependent decrease of von Willebrand factor (VWF)–inactivating protease, a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), results in platelet aggregation and consumption, leading to sepsis- associated thrombotic microangiopathy (TMA) and organ failure. Previous reports assessing its functional deficiency have pin- pointed involvement of autoantibodies or mutations to propagate thrombotic thrombocytopenic purpura (TTP). However, mechanisms of acquired ADAMTS13 deficiency during host response remain unclear. To enhance understanding of ADAMTS13 deficiency in sepsis, we evaluated changes in expression of mRNA coding ADAMTS13 during septic conditions using primary cellular sources of the protease. We hypothesized that proinflammatory cytokines and constituents of serum from septic patients affect the transcriptional level of ADAMTS13 in vitro, and previously recommended therapeutic agents as adjunctive therapy for sepsis interact therewith. Cultured hepatic stellate cells (HSCs), endothelial cells (HMEC) and human precision-cut liver slices as an ex vivo model were stimulated with sepsis prototypic cytokines, bacterial endotoxin and pooled serum obtained from septic patients. Stimulation resulted in a significant decrease in ADAMTS13 mRNA between 10% and 80% of basal transcriptional rates. Cos- timulation of selenite or recombinant activated protein C (APC) with serum prevented ADAMTS13 decrease in HSCs and increased ADAMTS13 transcripts in HMEC. In archived clinical samples, the activity of ADAMTS13 in septic patients treated with APC (n = 5) increased with an accompanying decrease in VWF propeptide as surrogate for improved endothelial function. In conclusion, proinflammatory conditions of sepsis repress mRNA coding ADAMTS13 and the ameliorating effect by selenite and APC may support the concept for identification of beneficial mechanisms triggered by these drugs at a molecular level. Online address: http://www.molmed.org doi: 10.2119/molmed.2014.00202

INTRODUCTION ing from a systemic inflammatory re- Central to these responses are alter- Despite therapy with appropriate an- sponse to infection (5). The continuum ations in endothelial cell function and a tibiotics and intensive supportive care, of sepsis pathophysiology involves a shift to a proinflammatory and procoag- sepsis carries a persistently high mor- complex integrated response involving ulant surface, which amplifies the initial bidity and mortality (1–4). Sepsis is a immune cell activation, inflammatory signal intensity (6,7). Alterations in mi- progressive injurious process aggravat- mediators and the coagulation system. crocirculation and microvascular perme- ability precede early tissue injury and organ failure (8). However, pathogenic mechanisms and messengers involved Address correspondence to Ralf A Claus, Center for Sepsis Control and Care, and Clinic in processes resulting in remote organ for Anaesthesiology and Intensive Care Therapy, Jena University Hospital, Erlanger Allee injury require further investigation. 101, D-07747 Jena, Germany. Phone: +49-(0)-3641-9325860; Fax: +49-(0)-3641-9325862; During sepsis, von Willebrand factor E-mail: [email protected]. (VWF), a multimeric acute phase pro- Submitted October 9, 2014; Accepted for publication April 3, 2015; Published Online tein, is abundantly secreted into plasma (www.molmed.org) April 3, 2015. by activated endothelial cells and platelets (9,10). VWF in its native, ultralarge isoform (ulVWF) is only de- tectable in conditions of an activated

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endothelium (11) and functions as a de- induced mortality because the complete with 100 ng/mL of endotoxin as determinant of platelet activation and ag- absence of its regulating protease had scribed previously) (40). The stimulation gregation (12,13). ADAMTS13 is a only a minor impact in the murine sep- period was limited to a maximum of 24 plasma protease primarily synthesized sis model. h because ADAMTS13 mRNA tran- and secreted from hepatic stellate cells To gain insight into mechanisms of scripts remain stable during this period, (HSCs) and is the only enzyme reported ADAMTS13 deficiency in systemic in- therefore providing a threshold to iden- to inactivate ulVWF, thereby reducing flammation and sepsis, we investigated tify significant changes in ADAMTS13 its thrombogenicity (14,15). Congenital the effects of proinflammatory cytokines, transcripts. Longer stimulation times or immunologically induced deficiency endotoxins and constituents of serum ob- may provoke mRNA changes due to ar- of ADAMTS13 has been identified to re- tained from septic patients on tifacts resulting from cellular stress in- sult in thrombotic thrombocytopenic ADAMTS13 expression on a transcrip- duced by starvation and accumulation purpura (TTP) (16). In sepsis, the term tional level. Furthermore, we evaluated of cell debris and not the intended effect thrombotic microangiopathy (TMA) the contribution of selenite and APC as observed by the inflammatory stimuli. A was introduced to characterize throm- adjuvant sepsis therapeutics in regulat- concentration of 5% pooled serum from botic occlusion of arterioles less than ing inflammation-induced ADAMTS13 septic patients (n = 12) was used for 100 μm diameter independent of the changes in transcription. Finally, we stimulation where indicated. Serum underlying pathophysiology (17,18). compared data obtained from in vitro ex- samples were obtained within 24 h after Hence, thrombocytopenia is a risk fac- periments to an observational study co- the patients met the criteria for sepsis or tor for mortality in critically ill patients hort for consistency of our findings. severe sepsis (5). Pivotal parameters of (19) and is also an independent predic- the serum pool were determined as fol- tor of multiple organ failure and death MATERIALS AND METHODS lows (normal values are given in brack- (20–22). Institutional ethical approval by the ets): creatinine 121 μmol/L [44–106], Further evidence suggests ADAMTS13 local ethics committee of the medical fac- blood urea nitrogen 37.2 mg/dL deficiency in patients with systemic in- ulty of Friedrich-Schiller University Hos- [2.6–6.4], γ-glutamyltransferase 80 U/L flammation and severe sepsis (9,23–25). pital in Jena (A-1827-07/06 and A-1840- [6–28], glutamatoxalacetate-transami- This deficiency is associated with an in- 08/06) and written informed consent of nase 209 U/L [<18], lactate dehydroge- crease in ulVWF and is postulated to each patient were obtained. nase 463 U/L [135–223], ammonia cause TMA in patients (9,23,26). The 128 μg/dL [19–94], creatine-phosphoki- impact of ADAMTS13 deficiency in Cell Culture Experiments nase 640 U/L [<174], albumin 2.6 g/dL thrombocytopenia and organ failure is Using cell culture models, an immor- [3,5–5.5], glucose 6.9 mmol/L [3.9–5.6], supported by the observation that talized hepatic stellate cell line (LX-2) IL-6 2282.75 pg/mL, TNFα below detec- plasma exchange reversed organ dys- (37,38) and a human microvascular en- tion limit (10 pg/mL). function in thrombocytopenia-associ- dothelial cell line (HMEC) (39), both of Cells were also coincubated with one ated multiple organ failure in children human origin, were cultured under stan- of the following substances: noradrena- (27) and renal deposition of thrombi en- dard conditions (LX-2: 1% heat inacti- lin (NA), protein C–activated (Eli Lilly, riched from activated platelets and vated fetal calf serum [FCS] in Dul- Indianapolis, IN, USA), arginine vaso- VWF in a porcine sepsis model (28). becco’s Modified Eagle Medium pressin (AVP) or desmopressin

Also, an inverse relationship between (DMEM) media, 5% CO2; HMEC: 10% (DDAVP), sodium selenite (biosyn severity of inflammation and heat inactivated FCS in DMEM media, Arzneimittel GmbH, Fellbach, Germany) 6 ADAMTS13 activity is described in 5% CO2). Cells (1 × 10 ) were seeded in and tolvaptan (Sigma-Aldrich, St. Louis, clinical studies (29,30). However, the ac- media supplemented with pooled MO, USA). tual cause for ADAMTS13 deficiency is human AB-serum (1%), equilibrated for controversial, ranging from its con- 2 h and stimulated for 6 or 24 h with Human High Precision–Cut Liver Slices sumption (31), secretion defects (32) or human recombinant cytokines (tumor (HPLS) proteolytic degradation by enzymes cir- necrosis factor-α [TNFα], interferon γ As an established ex vivo model, HPLS culating in plasma of critically ill pa- [IFNγ], interleukin-1β [IL-1β], IL-2 or IL- from patients who underwent liver resec- tients (33,34). Furthermore, an unbal- 6; reconstituted in saline supplemented tion spatially distant to metastases con- anced expression of ADAMTS13 and with 0.1% human albumin) to final controlled by macroscopic inspection by two VWF was reported in mouse endotox- centration of 100 ng/mL, with endotoxin independent and experienced scientists emia (35) and in the cecal ligation and (200 ng/mL E. coli, serotype B4; saline were obtained. Specimens were precision- puncture model of sepsis (36). How- supplemented with 0.1% AB-serum), cut into 200-μm slices (5 mm diameter) ever, VWF secretion was found to func- and a mixture of cytokines (TNFα, IFNγ with a Krumdieck slicer (Alabama Re- tion as the major determinant of sepsis- and IL-1β [10 ng/mL] each combined search and Development, Munford, AL,

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USA). Slices were then incubated into primary cultures for 24 h before stimulation as described previously (41).

RNA Preparation and Determination of Transcript Level RNA was isolated with RNeasy kit (Qi- agen, Hilden, Germany), reverse tran- scribed (Bio-Rad, Berkeley, CA, USA) and ADAMTS13 transcripts were quantified using an iCycler. Data were normalized Figure 1. ADAMTS13 expression in inflammatory conditions. (A) ADAMTS13 transcript is de- to unstimulated controls with unvaried creased significantly in HSCs after stimulation with TNFα (100 ng/mL), IFNγ (100 ng/mL), transcripts (hypoxanthine- guanine phos- IL-1β (100 ng/mL), IL-2 (100 ng/mL), IL-6 (100 ng/mL) and pooled serum from septic pa- phoribosyltransferase [HPRT])/disinte- tients (pooled SepSe) for 6 h or 24 h. Expression of ADAMTS13 based on Pfaffl method and grin and metalloproteinase domain-con- normalized to reference transcripts (ADAM9 and HPRT). (B) ADAMTS13 transcript is signifi- taining protein 9 [ADAM9] for cantly decreased in HMEC after stimulation with TNFα (100 ng/mL), IFNγ (100 ng/mL), IL-2 HSCs–HPRT/porphobilinogen deami- (100 ng/mL), IL-6 (100 ng/mL), cytokine mix (CM) and pooled SepSe for 6 h or 24 h. Expres- sion of ADAMTS13 is normalized to reference genes (PBGD and HPRT ). Bars represent nase [PBGD] for HMEC) and ratios ana- mean values of log -ratios ± SEM for at least four independent experiments. lyzed according to Pfaffl (42). ADAM9 2 (stability value [M] < 0.340), GUSB (M < 0.305), PBGD (M < 0.303) and HPRT (M < 0.554) were most suitable for data RESULTS Incubation with Adjuvant Septic normalization. Therapeutics Regulates ADAMTS13 Proinflammatory Cytokines and Transcription Characterization of Septic Patients Serum from Septic Patients Suppresses We assessed the role of drugs used in and Determination of Laboratory ADAMTS13 Transcription in Primary patients with septic shock that promote Parameters Cellular Sources vasoconstriction (norepinephrine, AVP) Blood was collected in sodium citrate TNFα, IL-1β and IL-6 decreased the (46), of a stabilized analogue of AVP anticoagulant. ADAMTS13 activity was normalized amount of transcript en- triggering the release of VWF from en- determined using a von Willebrand fac- coding ADAMTS13 in HSCs, which dothelial cells (DDAVP) (47), selenite tor fluorescent resonance energy transfer was found differentially regulated by supplementation with known antiox- 73 (VWF-FRETS73) substrate as previ- two fold across both time points (Fig- idative properties (48) and APC which ously described (43,44). von Willebrand ure 1A). However, IFNγ and IL-2 dis- is reported to regulate severe inflamma- factor antigen (VWF:Ag), von Willebrand played moderate effects within the cut- tory response (49). In HSCs, all tested collagen binding activity (VWF:CB), von off value, while endotoxin failed to compounds displayed nonsignificant Willebrand ristocetin cofactor assay significantly regulate ADAMTS13 tran- effects on ADAMTS13 transcription at 6 (VWF:RCo), VWF propeptides were de- scription in HSCs, but reached mini- h (Figure 2A). However, after 24 h, the termined as described previously mum values around 60% of basal value amount of mRNA encoding ADAMTS13 (9,30,45). in HMEC. Interestingly, the mixture of was significantly diminished, up to 26% prototypic mediators (cytokines and en- of basal value. Incubation with selenite Statistics dotoxin) did not impair the transcrip- reduced the expression rate to 50% (24 Statistical analyses were performed tional rate significantly. Pooled serum h). In endothelial cells, ADAMTS13 using SPSS 22 (SPSS [IBM, Armonk, NY, from patients with sepsis significantly transcription was only affected by AVP USA]). The Student t test or Mann– reduced the ADAMTS13 transcription and APC at the late observation point Whitney test was used to calculate statis- rate to 10% compared with an unstimu- while incubation with selenite in- tical significance of data sets (P < 0.05). lated control group. In endothelial cells, creased ADAMTS13 transcription rate Data are shown as means ± standard de- the effect of TNFα, IL-2, IL-6 and the after 24 h (Figure 2B). To elucidate the viation of at least four independent ex- mixture of cytokines in suppressing signaling mechanism involved in periments. Based on normalized expres- ADAMTS13 transcription was more DDAVP reduction of ADAMTS13 tran- sion data, a log2-threshold value of +1.0 pronounced. However, serum from scripts, we coincubated endothelial or –1.0 signifying a two-fold variation septic patients reduced ADAMTS13 cells with DDAVP and a V2-receptor was used to define transcripts differen- transcription rate to a minor extent antagonist (tolvaptan) for 24 h (Figure tially expressed. (Figure 1B). 5). The choice of endothelial cells was

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Selenite and APC Prevent ADAMTS13 Suppression during Proinflammatory Conditions Because APC and selenite regulate ADAMTS13 expression, we elucidated the effects of serum obtained from patients with sepsis in the presence of both therapeutic agents. In HSCs, APC completely abrogated the depressive effects of serum constituents on ADAMTS13 expression Figure 2. ADAMTS13 expression after stimulation with agents relevant for sepsis manage- while selenite stabilized ADAMTS13 ex- ment. (A) ADAMTS13 transcript is decreased significantly in HSCs after stimulation with AVP pression rate without attaining the two- and DDAVP (10 ng/mL), and activated protein C (APC, 10 ng/mL) for 6 h or 24 h. Expres- fold threshold value of differential expression of ADAMTS13 based Pfaffl method normalized to reference transcripts (ADAM9 and sion (Figure 4A). In endothelial cells, APC HPRT ). (B) ADAMTS13 transcript in HMEC is altered after stimulation with AVP and DDAVP and selenite induced an increase in (10 ng/mL), with NE (10 ng/mL), APC (10 ng/mL) and selenite (1 ng/mL) for 6 h or 24 h. Ex- ADAMTS13 expression after 24 h (Figure pression of ADAMTS13 based on Pfaffl method and normalized to reference transcripts 4B). To elucidate the mechanism of action (PBGD and HPRT ). Bars represent mean values of log2-ratios ± SEM for at least four independent experiments. of APC-mediated rescue of ADAMTS13, we used a specific blocking antibody tar- geting the endothelial protein C receptor selected because ADAMTS13 transcrip- sulted in a prominently decreased ex- (EPCR). It was evident that blocking the tion was significantly diminished in en- pression of ADAMTS13 by DDAVP and EPCR prevented APC-mediated rescue of dothelial cells and at 24 h. We observed IL-1β (see Figure 3). Stimulation with ADAMTS13 transcription in the presence that by inhibiting the V2 receptor, serum from septic patients resulted in a of septic stimuli (Figure 4C). To elucidate ADAMTS13 suppression was com- decreased expression value of a potential synergism between APC and pletely prohibited while there was no ADAMTS13 as compared with unstimu- selenite, we used a combination of both significant change of ADAMTS13 in the lated controls. APC and selenite to determine the tran- presence of the pharmacological agent alone (Figure 3).

Proinflammatory Conditions Affect ADAMTS13 Transcription in a Human Ex Vivo Liver Model To investigate the effects of proinflammatory stimulation on ADAMTS13 transcription in a human ex vivo liver model, we evaluated the expression value of disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) as a marker specific for the activation state of HSCs. We observed an increase of ADAM12 expression values at 6 h after stimulation with TNFα, endotoxin, IL-1β, IL-2 and cytokine mixture (see Figure 3). IFNγ and IL-6 displayed moderate effects that were com- Figure. 3. Decrease of ADAMTS13 expression level in human ex vivo model. HPLS were stim- pletely absent following incubation with ulated with TNFα (100 ng/mL), AVP (10 ng/mL) and DDAVP (10 ng/mL), LPS (200 ng/mL), AVP and DDAVP. Furthermore, stimula- IFNγ (100 ng/mL), IL-1β (100 ng/mL), IL-2 (100 ng/mL), IL-6 (100 ng/mL), a mix of cytokines tion with serum obtained from patients (CM), endotoxin, pooled serum from septic patients (SepSe) and pooled serum from with sepsis also exhibited an increase in healthy individuals (control) for 6 h. Expression of ADAMTS13 based on Pfaffl method and ADAM12 expression compared with un- normalized to ADAM9 and HPRT. Expression of ADAM12 as a marker for inflammatory re- stimulated controls. Additionally, in this sponse. Bars represent mean values of log2-ratios ± SEM for at least four independent model, proinflammatory stimulation re- experiments.

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DISCUSSION In this study, we demonstrate a mechanism of ADAMTS13 suppression origi- nating from a decrease in mRNA transcription encoding ADAMTS13. This suppression of transcription was induced by proinflammatory cytokines, which are abundantly secreted in the circulation of septic patients due to an overwhelming immune response during infection (3,4). HSCs and endothelial cells are primary sources of ADAMTS13, but it is unclear to what extent they regulate ADAMTS13 activity. Therefore, we explored the effect of proinflammatory stimulation in HSCs and endothelial cells as the primary cellular sources of ADAMTS13 synthesis and secretion in circulation. The transcriptional suppression of ADAMTS13 mRNA by proinflammatory cytokines Figure. 4. Decrease of ADAMTS13 expression is abrogated by selenite and APC. may explain the observed loss of prote- (A) ADAMTS13 transcripts decreased in HSCs after stimulation with serum from septic pa- olytic activity because ADAMTS13 is se- tients (SepSe). Costimulation with APC (SepSe + APC) and selenite (SepSe + selenite) for 6 h creted directly from the Golgi to the cell or 24 h nullified ADAMTS13 transcript reduction as normalized to ADAM9 and HPRT. (B) The exterior without storage (50). In addition, decrease of ADAMTS13 transcripts in HMEC is nullified after costimulation with APC (SepSe + secreted ADAMTS13 is inactivated by APC) and selenite (SepSe + selenite) for 6 h or 24 h. (C) APC-mediated rescue of ADAMTS13 is triggered via the endothelial protein C receptor (EPCR) in endothelial cells. (D) Coadmin- plasma proteases such as elastase, plas- istration of APC and selenite increased ADAMTS13 mRNA transcription rate slightly. Bars rep- min or thrombin (29,34) abundantly present in the circulation of septic patients. A resent mean values of log2-ratios ± SEM for at least four independent experiments. significant reduction in ADAMTS13 mRNA in this study is consistent with scription rate of ADAMTS13 in the pres- treatment group (Figure 6A). VWF:Ag data obtained from primary HSCs in rats ence of septic stimuli. We observed a min- ranged between 3.6 and 6.9 U/mL at d 1 and endothelial cells in humans (32). imal additive effect when both drugs with a broad spectrum in patients with- were used (Figure 4D). Use of a specific out adjunctive therapy and an intermit- inhibitor confirmed V2-receptor mediated tent decrease at d 2 and d 3 in APC- signaling as essential for DDAVP-induced treated patients (Figure 6B). To gain ADAMTS13 depression (Figure 5). insights into associations with thrombotic microangiopathy, we evaluated APC Administration Increased collagen-binding affinity (CB). VWF:CB ADAMTS13 Activity in Patients with decreased in the treatment group rang- Sepsis ing between 146% to 232% at the day of To determine if ADAMTS13 levels and enrollment. However, only a moderate related parameters are affected after ad- variation was found in patients without junctive therapy with APC, we used APC therapy (Figure 6C). As a marker of archival samples from a small character- endothelial activation, VWF propeptide ized cohort of septic patients who re- levels were found enhanced in both ceived APC over time (Table 1). groups at d 1 (APC group: 3.4 versus Figure 5. DDAVP reduction of ADAMTS13 is ADAMTS13 activity was diminished at non-APC: 5.9 U/mL, P > 0.05), which mediated via the V2-receptor signaling. the day of enrollment (range 0.19–0.53 decreased over time, however, in the Using a V2-selective receptor antagonist U/mL) without a significant difference APC-treated group to a greater extent (tolvaptan, 50 nmol/L) prevented DDAVP- as compared with the placebo group. with minor variations and with signifi- induced reduction of ADAMTS13 tran- However, ADAMTS13 levels increased cant differences from d 4 of treatment scripts in endothelial cells after 24 h of during the study period in the APC with respect to d 1 (Figure 6D). incubation.

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Table 1. Patient characteristics at the day of enrollment. and ADAMTS13 downregulation in our Adjunctive No adjunctive study supports our proposed mecha- a b APC-treatment APC-treatment nism. Given that DDAVP is mediated via Age, years 47 (40.0/66.5)c 64.5 (58.25/74.25)c the arginine vasopressin receptor 2 (V2 Number of patients 5 4 receptor) (54) and suppressed Sex distribution, male 60% 75% ADAMTS13 mRNA, it could be specu- Scores, points lated that a V2-receptor subtype might APACHE II 25 (23.5/28.5)c 25 (19.75/34.75)c be present and functionally active in c c SAPS 50 (49/59.5) 62 (47.75/67.75) HSCs. Because increased VWF:Ag levels c c ISTH 3 (3/3) 3 (3/3) following DDAVP administration are c c SOFA 9 (8/11.5) 11 (9.25/12.75) paralleled by a mild decrease of plasma Pathogen ADAMTS13 activity in human volun- Gram-positive 3 4 teers (55,56), our current observations Gram-negative 2 0 Focus provide first evidence that the decline Lung 2 2 might be caused by transcriptional re- Abdomen 2 3 pression. Urogenital 2 1 There is evidence of splice variants of Various 1 0 ADAMTS13 in a hepatoma cell line re- Laboratory findings sulting in synthesis of an intracellularly c c Procalcitonin, ng/mL 20.57 (4.79/33.09) 1.18 (0.37/29.66) accumulated protease (57). To the best of c c Leukocytes, Gpt/L 18.5 (4.4/37.25) 27.2 (4.25/40.75) our knowledge, there was no systematic Outcome report on splice variants of ADAMTS13 Length of stay at ICU, days 8 (7/24)c 13.5 (4/40.25)c in human hepatic tumor tissue. Very re- Length of stay at hospital, days 34 (22/60)c 19 (4.25/65.25)c cent reports (58) found a maintained bal- Mortality rate, % 0 50 ance of platelet activation status in pa- aPatients who received APC (n = 5). tients with cirrhosis and cancer bPatients with placebo (n = 4). compared with age-related controls. cData given as median (25th/75th percentile). We also demonstrate that the administration of human recombinant APC re- ADAMTS13 activity is reduced markedly sults in the exposure of selective epitopes sulted in preserved basal transcriptional during apoptosis of HSC following treat- of VWF, thereby enhancing proteolysis rate of ADAMTS13 in vitro as well as an ment with dimethylnitrosamine in rats, by residual ADAMTS13 as a compensa- increased plasma activity. APC treatment but not in animals treated with tetra- tory event (53). In our study, ADAMTS13 was accompanied by an improved clini- chloromethane (CCl4) or thioacetamide, transcript levels and proteolytic activity cal condition of patients, as reflected by where tissue injury is rather character- are downregulated by vasopressin in enhanced levels of VWF-related parame- ized by hepatic stellate cell activation vitro. Because it is reported that DDAVP ters and of endothelial dysfunction such and fibrogenesis (51). Transactivation of action is mediated via the V2 receptor, as VWF propeptide (59,60). Following rat HSC, triggered by cultivation on a we determined the role of the V2 recep- APC administration, the mechanism re- plastic surface after isolation or in vivo by tor using a potent V2-receptor antago- sulting in a decrease in collagen-binding administration of CCl4, only controlled nist. From our study, we identified that activity, which is the measure of de- the concordant activity of plasma- inhibition of the V2 receptor prevented creased VWF-adhesive activity, is un- secreted ADAMTS13 activity marginally ADAMTS13 mRNA suppression, sug- clear. It might be speculated that an in- (52). We also demonstrate the regulation gesting a potential role of the V2 receptor crease in activity of ADAMTS13 by the of ADAMTS13 transcription by vasoac- in mediating ADAMTS13 mRNA down- presence of APC should have induced tive agents (DDAVP and AVP). At 6 h regulation. However, the predefined this phenomenon. The interaction be- and 24 h post-stimulation, HSCs and en- two-fold threshold value for definition of tween APC and human HSCs showed an dothelial cells expressed less ADAMTS13 differentially expressed genes was not at- expression of EPCR and an abrogation of mRNA. However, ADAMTS13 mRNA tained in HPLS. This may lead to dilu- thrombin- induced TNFα expression (61). deficiency should not significantly influ- tion of HSC-specific transcripts in a As shown by our blocking experiments, ence proteolysis of circulating or en- mixed tissue sample with the reference the mechanism of action is strongly asso- dothelium-bound VWF because an in- transcripts simultaneously expressed in ciated to the activity of EPCR-receptor. It crease in shear stress during hepatocytes. Nevertheless, the variation might be speculated by which pathway administration of the vasoconstrictors re- of ADAM12 upregulation as a marker the protective effect of APC is triggered

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ing systemic response which might be limited by APC. In septic patients, the role of selenite supplementation to modulate reactive oxygen species generation, lower endoge- nous antioxidative capacity and improve clinical outcome is controversial (66–68). Here, we demonstrate that selenite abro- gates the inhibitory effect of serum constituents from septic patients on culti- vated HSCs and endothelial cells. It could be speculated that this mechanism involves genes regulating cell cycle, apoptosis and intermediary metabolism (69). In this study, we obtained a data set with respect to ADAMTS13 regulation from surgical waste material, which is not representative for healthy, naïve tissue, but clearly reflects the comorbid conditions which are decisive for a pa- Figure 6. Laboratory findings in patients with adjunctive therapy. ADAMTS13 activity and tient cohort which is susceptible for gen- related parameters were determined in patients with severe sepsis with APC treatment eralized infection compared with healthy (n = 5) and without APC (n = 4) therapy over time. (A) ADAMTS13 activity was diminished controls. For evaluation of the response at the day of enrollment (range 0.19 to 0.53 u/mL) without significance between sub- generated to inflammatory stimuli, we groups but increased during the course of APC treatment. (B) VWF:Ag ranged between determined the expression of ADAM9, 3.6 to 6.9 U/mL at d 1 with a broad spreading tendency in patients without adjunctive which is a well-established and validated therapy and intermittently decrease in APC-treated patients. (C) VWF:CB (collagen bind- approach. ADAM9 was decreased in the ing) decreased in the APC group (range: 146% to 232 %) at the day of enrollment while a moderate variation was observed in the non-APC group. (D) VWF propeptide levels were presence of inflammatory stimuli. The found enhanced in both groups at d 1 (APC group: 3.4 versus non-APC: 5.9 U/mL, not sig- use of HPLS with preserved tissue archi- nificant [n.s.]) which decreased over time. However, there was a significant difference tecture was to overcome limitations of from treatment d 4 with respect to d 1. Asterisks indicate significant differences during the cell culture experiments. This model is patients’ course in comparison to day of enrollment (*P < 0.05). an alternative to mouse models where VWF plasma concentration during the complete absence of ADAMTS13 activity along intracellular signaling events, re- transcription. The mechanisms involved is the major determinant of sepsis- sulting in a rather antiinflammatory con- warrant further investigation. Further- induced mortality, which is unrepresen- dition. Otherwise, events of oxidative more, a direct interaction between tative of the clinical situation in humans stress might be involved in the regula- ADAMTS13 activity and inflammation is (36). As observed in the mouse endotox- tion of ADAMTS13 transcription, while provided by the evidence that emia model, LPS administration affects the antioxidative properties of selenite ADAMTS13 deficiency results in in- both VWF and ADAMTS13 transcription are capable of interrupting the unfavor- creased leukocyte rolling and adhesion, in an opposite manner in hepatic and able effect with respect to ADAMTS13. as well as enhanced extravasation of renal tissue accompanied by variations in There is mounting evidence that the pre- neutrophils, where both processes are plasma concentration (35). vention of oxidative damage and the at- dependent on the presence of ulVWF tenuation of inflammatory response by and platelets (65). In accordance with CONCLUSION selenite is reflected by either a decrease these observations, our findings suggest In conclusion, we provide consistent of the transcriptional rate of endothelial that ADAMTS13 deficiency during sys- data from in vitro experiments and an ob- adhesion molecules (62) and cytokine- temic inflammation and infection accel- servational patient cohort implicating a induced cellular activation (63) or the erate inflammatory responses by slowing possible benefit of selenite and APC in restoration of antioxidative selenoen- down leukocytes and facilitating their mediating ADAMTS13 regulation during zymes (64). Strikingly, costimulation extravasation, which functions intrinsi- sepsis. We also report that the dysbalance with APC and selenite exert at least an cally as a protective mechanism, but be- ratio between VWF and ADAMTS13 additive effect on rescue of ADAMTS13 comes destructive due to an overwhelm- might be governed by treatment of en-

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