Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability DATA AVAILABILITY The authors declare that the data supporting the findings of this study are available within the article and from the authors upon request.

nature research | reporting summary
April 2020 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample or experiment sizes were determined empirically based on previous experience, e.g. Larsson M et al. Sci Trans Med 2014; no statistical tests were used to predetermine the size of the experiments. FXII zymogen contact activation proceeds by a PKa mediated amplification loop. This specific mechanisms with internal amplification either results in (almost) complete or no detectable activation. Therefore a gradual or partial activation is not common and the sample size can be rather limited as we essentially find activation or no activation without grading.
To check if data were normally distributed, a quantile-quantile plot was used and data were analyzed by Student's t test or, in the case of multiple comparisons, one-way analysis of variance (ANOVA) followed by post hoc analysis using Dunnett's multiple comparisons test.
Data exclusions All data for all experiments were included. In previous versions of the manuscript prior to revision we had excluded an outlier, however this experiment and the corresponding data set is not shown in the final manuscript.

Replication
Key experiments were performed and repeated from independent researchers over several years. Data was obtained in three distinct laboratories (Würzburg, Stockholm, Hamburg) with highly similar results. Key experiments were performed from two researchers independently, in a blinded fashion. Attempts for replications were all successful. We controlled quality of coagulation tests by inclusions of samples & materials that are used in an accreditated clinical diagnostic laboratory. Number of replications was based on our established experience with coagulantion assays ex vivo and in mice. The n-number of the experiment shown is given in the legend. Coagulation experiments, competition assays and thrombosis models were performed multiple times (n of 2-21) with comparable results. Our study design comprises three complementary approaches: 1. deletion mutants, 2. competition studies, 3. polyclonal and monoclonal antibodies. These independent approaches come to the same conclusion and show a critical fole of PR-III for FXII contact activation.
Randomization Animals were randomly allocated to challenges. The coagulation tests used in the study contain positive and negative controls and are highly standardized and quality controlled and do not require randomization. Factor deficient plasma from different lots and distinct sources was used as indicated in the material section. For the activity assays using chromogenic substrates allocation nor randomization is required as postive / negative controls were included and importantly findings were confirmed by independent assays (real time thrombin formation and/ or clotting).

Blinding
Observers were blinded to animal treatments, and otherwise blinding was performed for data analyses. For experiments other than animal experiments, blinding was not relevant to data collection as calibrated coagulation analyzers (Kugelkoagulometer and CAT with internal controls/standard) were used. The real time thrombin analyses were automatically recorded by the machine and means +/-SD was directly reported without calculations by the researcher. As described above the complementary study design (mutants, competition experiments and various antibodies) offers independent internal controls.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation Primary antibodies were validated using (i) plasma samples with inherited deficiency in the specific antigen, (ii) overexpression systems and (iii) comparisons with other antibodies against the same antigen. Specifically: anti-FXII antibody failed to detect a signal in plasma with inherited FXII deficiency and probed for proteolysis froducts follwing FXII contact activation (Björkqvist J et al, J Clin Invest 2015) Specificity of anti-MBP antibody has been conformed previously (Herwald H et al, J Biol Chem 1996). In our hands, anti-MBP failed to give a signal prior to IPTG-mediated stimulation of MBP-fusion protein expression. Specificity of HRP-coupled donkey anti-rabbit antibody is confirmed by Jackson Immunoresearch. Furthermore, we obtained similar results using an other HRP-coupled donkey anti-rabbit antibody obtained by DAKO company. Authentication the three typical immortalized cell lines described above were from commercial sources. Cell lines used were not authenticated since we used them for recombinant protein expression and not for cell type specific assays (e.g signalling).

Eukaryotic cell lines
Mycoplasma contamination All cells were tested negatively Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used in the study. We only used the cell lines for recombinant protein expression.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals FXII deficient (F12-/-) mice were 6-12 weeks of age and of both sexes as originally described (Pauer et al, reference 61) and used in various later studies (e.g. refs. 6, 23, 26) were used for the thrombosis studies (shown in figure 5 and supplemental figure S4). Littermate wild type controls of the same age and sex were used. All work with BALB/c mice and generation of antibodies in BALB/c mice was performed by Biogenes company Berlin (https://www.biogenes.de/custom-antibodies). F12-/-and wild type mice were kept in the animal facility of the University Medical Center Hamburg. Mice were kept with a 12 h light/12 h dark cycle and researchers and technicians did not enter the mouse room during the dark cycle. Temperatures were constantly ~19-21°C with 40-60% humidity.

Wild animals
No wild animals were used in the study.
Field-collected samples No field collected samples were used in the study

Ethics oversight
Animal experiments are approved by the University Medical Center Hamburg-Eppendorf local authorities (Tierversuchsantrag, TVA #76/16).
Note that full information on the approval of the study protocol must also be provided in the manuscript.