Page 265 - Haematologica May 2020

Different functions for FXII and PK in sepsis
Virulence factors of S. pyogenes have been studied inten- sively, and conversion of human plasminogen to plasmin by bacterial streptokinase is a mechanism which supports bacterial dissemination.4 Streptokinase-activated plasmin also activates the human contact system, an inflammatory response mechanism against artificial material and pathogens.5 The human contact system consists of two proteases, factor XII (FXII) and plasma prekallikrein (PPK), as well as the co-factor high molecular weight kininogen (HK). The proteins are produced in the liver and circulate as zymogens in the blood stream or are assembled on endothelial cells, neutrophils, and platelets. When blood is exposed to foreign artificial or biological surfaces, contact factors bind to it, and FXII becomes auto-activated and con- verts PPK to plasma kallikrein (PK). PK, which circulates in a non-covalent complex with HK,6 cleaves HK and the proinflammatory peptide bradykinin is released.7 In severe sepsis, activation of the contact system is archetypal8 and multiple animal studies with different pharmacological interventions that inhibit FXII, bradykinin receptors or the interaction of contact factors with the bacterial surface9 were carried out to evaluate potential therapeutic options.10 However, surprisingly little is known about the precise role of contact factors during microbial sepsis. Here, therefore, we studied the physiological role of FXII- and PK in a mouse model of experimental sepsis. We found that hepatic expression of F12 and Klkb1 genes after infection with S. pyogenes is quickly reduced upon streptococcal infection. Moreover, a knockdown of Klkb1 gene expression by anti- sense-oligonucleotide (ASO) technology prior to infection diminishes bacterial spreading, but knockdown of F12 did not influence bacterial dissemination. Our data indicate dif- ferent in vivo roles for FXII and PK in streptococcal sepsis.
Methods
A detailed description of materials and methods with addition- al information is provided in the Online Supplementary Appendix.
Antisense-oligonucleotides
Antisense-oligonucleotides (ASO) for Klkb1 or F12 mRNA knockdown in vivo were provided by Ionis Pharmaceuticals and have been described previously.11
Infection of HepG2 cells
Details are provided in the Online Supplementary Appendix.
mRNA analysis
Total RNA was isolated from HepG2 cells or homogenized mouse liver with RNeasy Plus Mini Kit (Qiagen). RNA quality was checked with Agilent RNA 6000 Nano Kit (Agilent Technologies) and RNA concentration determined with QubitTM RNA HS Assay Kit (Invitrogen). All analyses were performed according to the manufacturer’s instructions. 800 ng total RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the complementary DNA obtained used for real-time quantitative polymerase chain reaction (PCR). Reaction mixture (20 ML) containing gene specific nuclease assay (Taqman Universal PCR Master Mix; Applied Biosystems) and cDNA was amplified as follows: denaturation at 95°C for 10 minutes (min) and 45 cycles at 95°C for 15 seconds (sec) and 60°C for 1 min. GAPDH (human or rodent) was used as housekeeping gene. Relative expression was calculated using the 2− ct method.
Clotting assays
Details are provided in the Online Supplementary Appendix.
Clot lysis time
A clot was generated in human normal, FXII- or PK-deficient plasma by addition of PT-Reagent. In some experiments, CTI (75 mg/mL), PKSI (10 M), FXIIa (50 mg/mL) or PK (50 mg/mL) was added before clot formation was induced. The clot was incubated for 5 min at 37°C before Streptokinase (100 Units), uPA (10 mg/mL g) or tPa (10 mg) was added. Time until clot lysis was determined in a coagulometer.
Measurement of FXII and plasma kallikrein in plasma
Details are provided in the Online Supplementary Appendix.
Proteolytic potential for plasma kallikrein/factor XIIa activity in mouse plasma
Pooled plasma from four mice/group was incubated with Dapptin and FXIIa/PK activity was determined in a microplate reader by chromogenic substrate S-2302 (Chromogenix). (See Online Supplementary Appendix).
Plasma clot escape experiments
See Online Supplementary Appendix.
Light and scanning electron microscopy
See Online Supplementary Appendix and Oehmcke et al.12 and Isenring et al.13
Fibrinogen degradation
Fibrinogen was mixed with either plasmin, plasminogen and streptokinase, FXII, PPK, or PBS as negative control. (See Online Supplementary Appendix). The mix was incubated at 37°C and at indicated time points samples were analyzed by SDS-Page and western blot using fibrinogen antibody (Santa Cruz). Relative fibrinogen levels were determined by densitometry analysis (ImageStudioLite 5.2.5).
Animal experiments
Eight-week-old female BALB/c mice (weight 16-18g) (Charles River Laboratories) were treated with ASO through intraperi- toneal injections, with a dose of 800 mg/mouse, twice per week for three weeks (total 7 injections, each with 800 mg ASO/mouse).
The subcutaneous infection model with S. pyogenes AP1 strain and determination of bacterial dissemination were performed as described previously.12 (See also Online Supplementary Appendix). This study was performed in strict accordance with the recom- mendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments the Landesveterinär- und Lebensmitteluntersuchungsamt Rostock (Permit n. 7221.3-1-002/16).
Plasma proteome
The methods are provided in the Online Supplementary Appendix.
Patient samples
Patients with sepsis, severe sepsis, or septic shock were enrolled from the Intensive Care Medicine Unit at University Medical Center of Rostock, as described previously.14 The proto- col had been approved by our Institutional Ethics committee (A 201151), and informed consent was obtained from the patients or their caring relatives.
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