We analyzed the levels of circulating tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 correlated with disease severity in HCPS but not in HFRS.Peer reviewe

Avsic-Zupanc, Tatjana

Bellomo, Carla

Korva, Misa

Martinez, Valeria P.

Mustonen, Jukka

Mäkelä, Satu

Papa, Anna

Strandin, Tomas

Vaheri, Antti

Open Forum Infectious Diseases

Martinez, Valeria P

Trepo - Institutional Repository of Tampere University

BRIEF REPORT • OFID • 1Open Forum Infectious DiseasesB R I E F  R E P O R TDifferential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal SyndromeCarla Bellomo,1 Miša Korva,2 Anna Papa,3 Satu Mäkelä,4 Jukka Mustonen,4 Tatjana Avšič-Županc,2 Antti Vaheri,5 Valeria P. Martinez,1 and Tomas Strandin51Laboratorio Nacional de Referencia para Hantavirus, Servicio Biología Molecular, Departamento Virología—INEI-ANLIS “Dr C. G. Malbrán,” Buenos Aires, Argentina; 2Institute of Microbiology and Immunology, Faculty of Medicine University of Ljubljana, Ljubljana, Slovenia; 3Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece; 4Department of Internal Medicine, Tampere University Hospital and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland; 5Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland.We analyzed the levels of circulating tissue plasminogen acti-vator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 corre-lated with disease severity in HCPS but not in HFRS.Keywords.  hantavirus; HCPS; HFRS; PAI-1; tPA.Hantaviruses cause two diseases: hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The hallmark of both diseases is increased vascular permeability [1, 2]; however, the most affected organ generally varies from the lungs in HCPS to the kidneys in HFRS. The type of hantavirus disease is determined by the causative species. Andes (ANDV) and Sin Nombre (SNV) hantaviruses cause HCPS with mortality that can reach 40% in South and North America, respectively. Puumala hantavirus (PUUV) causes a mild HFRS, also called nephropathia epidemica (NE; mor-tality ~0.1%), mainly in Northern Europe and Russia, whereas Dobrava (DOBV) and Hantaan (HTNV) viruses cause severe HFRS (mortality 1%–5%) in the Balkans and East Asia, respect-ively [3].Hantavirus infection activates intravascular coagulation path-ways in the acute stage of HCPS and HFRS [4–6]. At the same time, however, PUUV has been suggested to cause enhanced fibrinolysis [5], that is, excessive solubilization of blood clots, which we recently showed to be characterized by elevated ac-tivity of circulating tissue plasminogen activator (tPA), whereas its main physiological inhibitor, plasminogen activator in-hibitor–1 (PAI-1), remains unaltered [7]. In this study, we assessed the levels of tPA and PAI-1 in HCPS and HFRS caused by ANDV and DOBV, respectively, to better understand the role hemostatic regulation in hantavirus pathogenesis in general.METHODSThe study protocol was conducted in accordance with the eth-ical standards laid down by the Declaration of Helsinki and was approved by the Ethical Committee in Research of ANLIS “Dr C.  Malbrán,” Slovenian National Medical Ethics Committee, and the Ethics Committee of the Tampere University Hospital for serologically confirmed ANDV, DOBV, and PUUV cases, respectively. The study cohort consisted of patient samples that were collected ≤10 days after the beginning of the disease, that is, after onset of fever. Severe HCPS was classified, for example, due to pulmonary failure and need of mechanical ventilation, whereas signs of lower respiratory compromise were considered mild HCPS. Non-HCPS cases with pulmonary complications were included as reference. The DOBV patients were classified as severe or mild, for example, based on the need for dialysis. Additional information on patient demographics and disease classification criteria can be found in Supplementary Table  1 and the Supplementary Methods, respectively.Protein levels of tPA and PAI-1 were quantitated from patient plasma or serum by enzyme-linked immunosorbent assays, similar to methods described previously [7].RESULTSThe levels of circulating tPA were significantly elevated above control in both severe and mild acute HCPS but not in symp-tomatic non-HCPS cases (Figure  1A). Furthermore, tPA was significantly increased in severe as compared with mild HCPS. Its main physiological inhibitor, PAI-1, was also found to be increased above control in severe HCPS. However, in contrast to tPA, PAI-1 was not elevated in the mild HCPS or non-HCPS groups (Figure  1B). Thus, simultaneously elevated circulating levels of tPA and PAI-1 distinguish severe HCPS from mild HCPS and symptomatic non-HCPS cases. Strikingly, when taking into account all HCPS cases in our cohort, a robust cor-relation between fatality and PAI-1 (r = .514; P = .001), but not tPA (r = .262; P = .1), was observed.Open Forum Infectious Diseases®© The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.comDOI: 10.1093/ofid/ofy021Received 5 December 2017; editorial decision 9 January 2018; accepted 16 January 2018.Correspondence: T. Strandin, PhD, Department of Virology, Faculty of Medicine, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland (tomas.strandin@helsinki.fi).Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by University of Tampere Library useron 10 April 20182 • OFID • BRIEF REPORTTo get further insight into the upregulation kinetics and the role of tPA and PAI-1 in HCPS disease severity, we fur-ther classified severe HCPS to fatal and nonfatal cases at early acute stage (≤5  days after onset of fever) (Figure  1C) and found either statistically significantly or close to signif-icantly elevated levels of PAI-1 in fatal HCPS as compared with mild (P = .001) or severe but nonfatal (P = .073) cases, respectively. tPA levels did not differ between groups. These BADCE F10080tPA ng/ml250******* *200150100500PAI-1 ng/ml604020010080tPA ng/ml 6040200200Severe Mild Non-HCPS**** *****ControlstPA PAI-1tPA PAI-1SevereDOBVMildMildDOBV PUUV Controls SevereDOBV-HFRSNon-fatalDevereFatal PUUV ControlsMild Non-HCPS Controls180160140120ng/ml100806040200140120PAI-1 ng/ml100806040200140120ng/ml10080604020Figure 1. Levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). Box plots of the levels of tPA (A) and PAI-1 (B) in acute (≤10 days after onset of disease) serum samples of severe (n = 18) and mild (n = 22) Andes (ANDV)-caused HCPS, non-HCPS cases (n = 5 for tPA and n = 12 for PAI-1) with similar symptoms to HCPS, and healthy volunteers (n = 10) as controls. (C) The levels of tPA and PAI-1 in early acute (≤5 days after onset of disease) serum samples of fatal (n = 7), severe but nonfatal (n = 4), and mild (n = 15) ANDV-caused HCPS. The levels of tPA (D) and PAI-1 (E) in acute (≤10 days after onset of disease) plasma samples of Dobrava (DOBV)- (n = 7) and Puumala hantavirus (PUUV)–caused (n = 16) HFRS in addition to healthy volunteers (n = 15) as controls. (F) The levels of tPA and PAI-1 in acute plasma samples of mild (n = 4) and severe (n = 3) DOBV-caused HFRS. Box plots illustrate the median value (center horizontal line), interquartile range (the lower and upper quartiles), and the highest and lowest values (whiskers) that are not outliers. Distributions across groups were compared by the Mann-Whitney U test. **Statistically highly significant difference at P < .01. *Statistically significant difference at P < .05.Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by University of Tampere Library useron 10 April 2018BRIEF REPORT • OFID • 3findings suggest a direct role for PAI-1, but not tPA, in the fatality of HCPS.The levels of tPA and PAI-1 were also compared in the plasma of acute DOBV- and PUUV-caused HFRS patients. As seen in Figure 1D, the median levels of tPA were increased significantly above control in HFRS irrespective of the causa-tive virus, but were further elevated by DOBV as compared with PUUV. While PAI-1 was not elevated in PUUV-HFRS (Figure  1E), it was significantly increased above control in DOBV-HFRS. This finding suggests a marked difference in regulation of PAI-1 based on the etiology and/or severity of HFRS. Interestingly, however, when categorizing DOBV-HFRS to mild vs severe forms of the disease based mainly on the extent of kidney failure and need for hemodialysis, PAI-1 was significantly lower in severe DOBV-HFRS, whereas no change between groups was observed for tPA.These results indicate marked differences between the severe forms of HCPS and HFRS in the regulation of PAI-1. Further corroborating this difference, tPA was found to strongly correl-ate with PAI-1 in HCPS (r = .471; P = .002) but not in DOBV-HFRS (r = .402; P = .371). No correlation was observed between tPA or PAI-1 with platelet counts in HCPS or patient age in HCPS or HFRS (data not shown).DISCUSSIONThe results of this study show for the first time that circu-lating levels of tPA are increased in acute HCPS and HFRS caused by ANDV and DOBV, respectively. In addition, higher tPA levels were found in DOBV- vs PUUV-caused HFRS and in severe vs mild HCPS, indicating a possible role for tPA in hantavirus pathogenesis. This is in accordance with our previous study, where tPA was found to positively correlate with hemorrhages in PUUV-HFRS [7]. However, no signifi-cant differences in tPA levels were observed between severe vs mild DOBV-HFRS or fatal vs nonfatal severe HCPS, indi-cating that additional factors are necessary to fully explain virulence in both diseases.In contrast to tPA, its main physiological inhibitor, PAI-1, was only increased in severe HCPS and generally in DOBV-HFRS. Furthermore, PAI-1 levels were nearly statistically sig-nificantly elevated in fatal vs nonfatal severe HCPS, indicating a possible role for this protein in the mortality of HCPS. This latter finding is in accordance with a previous report indicat-ing higher plasma PAI-1 levels in fatal SNV-caused HCPS as compared with nonfatal cases [4]. However, seemingly contra-dicting the possible role of PAI-1 in the pathogenesis of HFRS, it was reduced in severe vs mild DOBV-HFRS (Figure 1F) and not elevated at all in PUUV-HFRS (Figure 1E) [7].Due to differences in blood sampling (serum vs plasma), we were not capable of reliably comparing the quantity of PAI-1 between HCPS and HFRS cases. Platelets, which are activated during blood clotting in the preparation of serum samples, are one of the major physiological sources of PAI-1 [8]. This affects baseline PAI-1 levels, as indicated by the increased con-centration of PAI-1 in the serum of healthy volunteers (com-pare Figure 1B and E). On the other hand, this effect is probably negligible in HCPS patient serum due to the often severe thrombocytopenia associated with the disease [9], and thus the difference between PAI-1 levels in HCPS patients and controls in circulation is most likely higher than estimated in this study. In fact, thrombocytopenia might explain the slightly lower level of PAI-1 in mild HCPS vs healthy controls (Figure  1B). Another potential caveat in our study is the fact that all se-vere HCPS cases were, by definition, subjected to mechanical ventilation, which has the potential to increase the circulating levels of PAI-1 [10]. However, while we are not aware whether the analyzed samples were acquired before or after mechanical ventilation, we did not observe any changes in tPA or PAI-1 levels in non-HCPS cases receiving similar treatment as com-pared with HCPS cases (Figure  1A and B). Furthermore, by comparing fatal with nonfatal mechanically ventilated HCPS cases (Figure  1C), we observed a difference in PAI-1 levels, suggesting that ventilation per se is not a significant factor in the regulation of PAI-1 in HCPS. This is also in concordance with another study indicating that PAI-1 levels were increased in fatal vs nonfatal SNV-HCPS cases receiving mechanical ven-tilation and/or extracorporeal membrane oxygenation [4]. All severe DOBV-HFRS cases received hemodialysis, which also potentially affects hemostasis [11]. However, all DOBV samples were acquired before hemodialysis, eliminating the influence of this factor in the observed results.Taken together, it is possible that the observed increase in tPA/PAI-1 ratio accounts for elevated hemorrhagic compli-cations and kidney failure in severe as compared with mild DOBV-HFRS. This is in line with our previous findings, where tPA, in the concurrent absence of neutralizing PAI-1, correlated with hemorrhages in PUUV-HFRS [7]. Analogously, the highly elevated levels of PAI-1 could also explain the absence of hem-orrhages in severe HCPS [12], which is in striking contrast to severe HFRS cases [9]. Minor hemorrhages were reported in 20% of our HCPS patients, but no correlations between bleed-ing and disease severity, tPA, or PAI-1 were found (data not shown). Further studies on the role of tPA and PAI-1 in hanta-virus diseases are certainly warranted.Supplementary DataSupplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corre-sponding author.AcknowledgmentsFinancial support. This work is financially supported by the Academy of Finland (grant 1275597 to T.S.), Sigrid Juselius Foundation (to J.M. and A.V.), and Magnus Ehrnrooth Foundation (to A.V.).Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by University of Tampere Library useron 10 April 20184 • OFID • BRIEF REPORTPotential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.References1. Hepojoki J, Vaheri A, Strandin T. The fundamental role of endothelial cells in hantavirus pathogenesis. Front Microbiol 2014; 5:727.2. Mackow ER, Gorbunova EE, Gavrilovskaya IN. Endothelial cell dysfunction in viral hemorrhage and edema. Front Microbiol 2014; 5:733.3. Vaheri A, Strandin T, Hepojoki J, et al. Uncovering the mysteries of hantavirus infections. Nat Rev Microbiol 2013; 11:539–50.4. Bondu V, Schrader R, Gawinowicz MA, et al. Elevated cytokines, thrombin and PAI-1 in severe HCPS patients due to Sin Nombre virus. Viruses 2015; 7:559–89.5. Laine O, Mäkelä S, Mustonen J, et al. Enhanced thrombin formation and fibrin-olysis during acute Puumala hantavirus infection. Thromb Res 2010; 126:154–8.6. Lee M. Coagulopathy in patients with hemorrhagic fever with renal syndrome. J Korean Med Sci 1987; 2:201–11.7. Strandin T, Hepojoki J, Laine O, et  al. Interferons induce STAT1-dependent expression of tissue plasminogen activator, a pathogenicity factor in Puumala hantavirus disease. J Infect Dis 2016; 213:1632–41.8. Braaten JV, Handt S, Jerome WG, et al. Regulation of fibrinolysis by platelet-re-leased plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus. Blood 1993; 81:1290–9.9. Peters CJ, Simpson GL, Levy H. Spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Annu Rev Med 1999; 50:531–45.10. Schultz MJ, Millo J, Levi M, et al. Local activation of coagulation and inhibition of fibrinolysis in the lung during ventilator associated pneumonia. Thorax 2004; 59:130–5.11. Segarra A, Chacón P, Martinez-Eyarre C, et  al. Circulating levels of plas-minogen activator inhibitor type-1, tissue plasminogen activator, and throm-bomodulin in hemodialysis patients: biochemical correlations and role as independent predictors of coronary artery stenosis. J Am Soc Nephrol 2001; 12:1255–63.12. Zaki SR, Greer PW, Coffield LM, et  al. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol 1995; 146:552–79.Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by University of Tampere Library useron 10 April 2018

Differential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal Syndrome

TamPub Julkaisuarkisto - TamPub Institutional Repository

Helsingin yliopiston digitaalinen arkisto

BRIEF REPORT • OFID • 1Open Forum Infectious DiseasesB R I E F  R E P O R TDifferential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal SyndromeCarla Bellomo,1 Miša Korva,2 Anna Papa,3 Satu Mäkelä,4 Jukka Mustonen,4 Tatjana Avšič-Županc,2 Antti Vaheri,5 Valeria P. Martinez,1 and Tomas Strandin51Laboratorio Nacional de Referencia para Hantavirus, Servicio Biología Molecular, Departamento Virología—INEI-ANLIS “Dr C. G. Malbrán,” Buenos Aires, Argentina; 2Institute of Microbiology and Immunology, Faculty of Medicine University of Ljubljana, Ljubljana, Slovenia; 3Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece; 4Department of Internal Medicine, Tampere University Hospital and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland; 5Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland.We analyzed the levels of circulating tissue plasminogen acti-vator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 corre-lated with disease severity in HCPS but not in HFRS.Keywords.  hantavirus; HCPS; HFRS; PAI-1; tPA. Hantaviruses cause two diseases: hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The hallmark of both diseases is increased vascular permeability [1, 2]; however, the most affected organ generally varies from the lungs in HCPS to the kidneys in HFRS. The type of hantavirus disease is determined by the causative species. Andes (ANDV) and Sin Nombre (SNV) hantaviruses cause HCPS with mortality that can reach 40% in South and North America, respectively. Puumala hantavirus (PUUV) causes a mild HFRS, also called nephropathia epidemica (NE; mor-tality ~0.1%), mainly in Northern Europe and Russia, whereas Dobrava (DOBV) and Hantaan (HTNV) viruses cause severe HFRS (mortality 1%–5%) in the Balkans and East Asia, respect-ively [3].Hantavirus infection activates intravascular coagulation path-ways in the acute stage of HCPS and HFRS [4–6]. At the same time, however, PUUV has been suggested to cause enhanced fibrinolysis [5], that is, excessive solubilization of blood clots, which we recently showed to be characterized by elevated ac-tivity of circulating tissue plasminogen activator (tPA), whereas its main physiological inhibitor, plasminogen activator in-hibitor–1 (PAI-1), remains unaltered [7]. In this study, we assessed the levels of tPA and PAI-1 in HCPS and HFRS caused by ANDV and DOBV, respectively, to better understand the role hemostatic regulation in hantavirus pathogenesis in general.METHODSThe study protocol was conducted in accordance with the eth-ical standards laid down by the Declaration of Helsinki and was approved by the Ethical Committee in Research of ANLIS “Dr C.  Malbrán,” Slovenian National Medical Ethics Committee, and the Ethics Committee of the Tampere University Hospital for serologically confirmed ANDV, DOBV, and PUUV cases, respectively. The study cohort consisted of patient samples that were collected ≤10 days after the beginning of the disease, that is, after onset of fever. Severe HCPS was classified, for example, due to pulmonary failure and need of mechanical ventilation, whereas signs of lower respiratory compromise were considered mild HCPS. Non-HCPS cases with pulmonary complications were included as reference. The DOBV patients were classified as severe or mild, for example, based on the need for dialysis. Additional information on patient demographics and disease classification criteria can be found in Supplementary Table  1 and the Supplementary Methods, respectively.Protein levels of tPA and PAI-1 were quantitated from patient plasma or serum by enzyme-linked immunosorbent assays, similar to methods described previously [7].RESULTSThe levels of circulating tPA were significantly elevated above control in both severe and mild acute HCPS but not in symp-tomatic non-HCPS cases (Figure  1A). Furthermore, tPA was significantly increased in severe as compared with mild HCPS. Its main physiological inhibitor, PAI-1, was also found to be increased above control in severe HCPS. However, in contrast to tPA, PAI-1 was not elevated in the mild HCPS or non-HCPS groups (Figure  1B). Thus, simultaneously elevated circulating levels of tPA and PAI-1 distinguish severe HCPS from mild HCPS and symptomatic non-HCPS cases. Strikingly, when taking into account all HCPS cases in our cohort, a robust cor-relation between fatality and PAI-1 (r = .514; P = .001), but not tPA (r = .262; P = .1), was observed.Open Forum Infectious Diseases®© The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.comDOI: 10.1093/ofid/ofy021Received 5 December 2017; editorial decision 9 January 2018; accepted 16 January 2018.Correspondence: T. Strandin, PhD, Department of Virology, Faculty of Medicine, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland (tomas.strandin@helsinki.fi).Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by Department of Mathematics, University of Helsinki useron 25 April 20182 • OFID • BRIEF REPORTTo get further insight into the upregulation kinetics and the role of tPA and PAI-1 in HCPS disease severity, we fur-ther classified severe HCPS to fatal and nonfatal cases at early acute stage (≤5  days after onset of fever) (Figure  1C) and found either statistically significantly or close to signif-icantly elevated levels of PAI-1 in fatal HCPS as compared with mild (P = .001) or severe but nonfatal (P = .073) cases, respectively. tPA levels did not differ between groups. These BADCE F10080tPA ng/ml250******* *200150100500PAI-1 ng/ml604020010080tPA ng/ml 6040200200Severe Mild Non-HCPS**** *****ControlstPA PAI-1tPA PAI-1SevereDOBVMildMildDOBV PUUV Controls SevereDOBV-HFRSNon-fatalDevereFatal PUUV ControlsMild Non-HCPS Controls180160140120ng/ml100806040200140120PAI-1 ng/ml100806040200140120ng/ml10080604020Figure 1. Levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). Box plots of the levels of tPA (A) and PAI-1 (B) in acute (≤10 days after onset of disease) serum samples of severe (n = 18) and mild (n = 22) Andes (ANDV)-caused HCPS, non-HCPS cases (n = 5 for tPA and n = 12 for PAI-1) with similar symptoms to HCPS, and healthy volunteers (n = 10) as controls. (C) The levels of tPA and PAI-1 in early acute (≤5 days after onset of disease) serum samples of fatal (n = 7), severe but nonfatal (n = 4), and mild (n = 15) ANDV-caused HCPS. The levels of tPA (D) and PAI-1 (E) in acute (≤10 days after onset of disease) plasma samples of Dobrava (DOBV)- (n = 7) and Puumala hantavirus (PUUV)–caused (n = 16) HFRS in addition to healthy volunteers (n = 15) as controls. (F) The levels of tPA and PAI-1 in acute plasma samples of mild (n = 4) and severe (n = 3) DOBV-caused HFRS. Box plots illustrate the median value (center horizontal line), interquartile range (the lower and upper quartiles), and the highest and lowest values (whiskers) that are not outliers. Distributions across groups were compared by the Mann-Whitney U test. **Statistically highly significant difference at P < .01. *Statistically significant difference at P < .05.Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by Department of Mathematics, University of Helsinki useron 25 April 2018BRIEF REPORT • OFID • 3findings suggest a direct role for PAI-1, but not tPA, in the fatality of HCPS.The levels of tPA and PAI-1 were also compared in the plasma of acute DOBV- and PUUV-caused HFRS patients. As seen in Figure 1D, the median levels of tPA were increased significantly above control in HFRS irrespective of the causa-tive virus, but were further elevated by DOBV as compared with PUUV. While PAI-1 was not elevated in PUUV-HFRS (Figure  1E), it was significantly increased above control in DOBV-HFRS. This finding suggests a marked difference in regulation of PAI-1 based on the etiology and/or severity of HFRS. Interestingly, however, when categorizing DOBV-HFRS to mild vs severe forms of the disease based mainly on the extent of kidney failure and need for hemodialysis, PAI-1 was significantly lower in severe DOBV-HFRS, whereas no change between groups was observed for tPA.These results indicate marked differences between the severe forms of HCPS and HFRS in the regulation of PAI-1. Further corroborating this difference, tPA was found to strongly correl-ate with PAI-1 in HCPS (r = .471; P = .002) but not in DOBV-HFRS (r = .402; P = .371). No correlation was observed between tPA or PAI-1 with platelet counts in HCPS or patient age in HCPS or HFRS (data not shown).DISCUSSIONThe results of this study show for the first time that circu-lating levels of tPA are increased in acute HCPS and HFRS caused by ANDV and DOBV, respectively. In addition, higher tPA levels were found in DOBV- vs PUUV-caused HFRS and in severe vs mild HCPS, indicating a possible role for tPA in hantavirus pathogenesis. This is in accordance with our previous study, where tPA was found to positively correlate with hemorrhages in PUUV-HFRS [7]. However, no signifi-cant differences in tPA levels were observed between severe vs mild DOBV-HFRS or fatal vs nonfatal severe HCPS, indi-cating that additional factors are necessary to fully explain virulence in both diseases.In contrast to tPA, its main physiological inhibitor, PAI-1, was only increased in severe HCPS and generally in DOBV-HFRS. Furthermore, PAI-1 levels were nearly statistically sig-nificantly elevated in fatal vs nonfatal severe HCPS, indicating a possible role for this protein in the mortality of HCPS. This latter finding is in accordance with a previous report indicat-ing higher plasma PAI-1 levels in fatal SNV-caused HCPS as compared with nonfatal cases [4]. However, seemingly contra-dicting the possible role of PAI-1 in the pathogenesis of HFRS, it was reduced in severe vs mild DOBV-HFRS (Figure 1F) and not elevated at all in PUUV-HFRS (Figure 1E) [7].Due to differences in blood sampling (serum vs plasma), we were not capable of reliably comparing the quantity of PAI-1 between HCPS and HFRS cases. Platelets, which are activated during blood clotting in the preparation of serum samples, are one of the major physiological sources of PAI-1 [8]. This affects baseline PAI-1 levels, as indicated by the increased con-centration of PAI-1 in the serum of healthy volunteers (com-pare Figure 1B and E). On the other hand, this effect is probably negligible in HCPS patient serum due to the often severe thrombocytopenia associated with the disease [9], and thus the difference between PAI-1 levels in HCPS patients and controls in circulation is most likely higher than estimated in this study. In fact, thrombocytopenia might explain the slightly lower level of PAI-1 in mild HCPS vs healthy controls (Figure  1B). Another potential caveat in our study is the fact that all se-vere HCPS cases were, by definition, subjected to mechanical ventilation, which has the potential to increase the circulating levels of PAI-1 [10]. However, while we are not aware whether the analyzed samples were acquired before or after mechanical ventilation, we did not observe any changes in tPA or PAI-1 levels in non-HCPS cases receiving similar treatment as com-pared with HCPS cases (Figure  1A and B). Furthermore, by comparing fatal with nonfatal mechanically ventilated HCPS cases (Figure  1C), we observed a difference in PAI-1 levels, suggesting that ventilation per se is not a significant factor in the regulation of PAI-1 in HCPS. This is also in concordance with another study indicating that PAI-1 levels were increased in fatal vs nonfatal SNV-HCPS cases receiving mechanical ven-tilation and/or extracorporeal membrane oxygenation [4]. All severe DOBV-HFRS cases received hemodialysis, which also potentially affects hemostasis [11]. However, all DOBV samples were acquired before hemodialysis, eliminating the influence of this factor in the observed results.Taken together, it is possible that the observed increase in tPA/PAI-1 ratio accounts for elevated hemorrhagic compli-cations and kidney failure in severe as compared with mild DOBV-HFRS. This is in line with our previous findings, where tPA, in the concurrent absence of neutralizing PAI-1, correlated with hemorrhages in PUUV-HFRS [7]. Analogously, the highly elevated levels of PAI-1 could also explain the absence of hem-orrhages in severe HCPS [12], which is in striking contrast to severe HFRS cases [9]. Minor hemorrhages were reported in 20% of our HCPS patients, but no correlations between bleed-ing and disease severity, tPA, or PAI-1 were found (data not shown). Further studies on the role of tPA and PAI-1 in hanta-virus diseases are certainly warranted.Supplementary DataSupplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corre-sponding author.AcknowledgmentsFinancial support. This work is financially supported by the Academy of Finland (grant 1275597 to T.S.), Sigrid Juselius Foundation (to J.M. and A.V.), and Magnus Ehrnrooth Foundation (to A.V.).Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by Department of Mathematics, University of Helsinki useron 25 April 20184 • OFID • BRIEF REPORTPotential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.References1. Hepojoki J, Vaheri A, Strandin T. The fundamental role of endothelial cells in hantavirus pathogenesis. Front Microbiol 2014; 5:727.2. Mackow ER, Gorbunova EE, Gavrilovskaya IN. Endothelial cell dysfunction in viral hemorrhage and edema. Front Microbiol 2014; 5:733.3. Vaheri A, Strandin T, Hepojoki J, et al. Uncovering the mysteries of hantavirus infections. 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Spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Annu Rev Med 1999; 50:531–45.10. Schultz MJ, Millo J, Levi M, et al. Local activation of coagulation and inhibition of fibrinolysis in the lung during ventilator associated pneumonia. Thorax 2004; 59:130–5.11. Segarra A, Chacón P, Martinez-Eyarre C, et  al. Circulating levels of plas-minogen activator inhibitor type-1, tissue plasminogen activator, and throm-bomodulin in hemodialysis patients: biochemical correlations and role as independent predictors of coronary artery stenosis. J Am Soc Nephrol 2001; 12:1255–63.12. Zaki SR, Greer PW, Coffield LM, et  al. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol 1995; 146:552–79.Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy021/4812611by Department of Mathematics, University of Helsinki useron 25 April 2018

AbstractWe analyzed the levels of circulating tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)–1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 correlated with disease severity in HCPS but not in HFRS.</jats:p

Differential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal Syndrome

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