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Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant

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Abstract

Hereditary angioedema due to pathogenic FXII variants (HAE-FXII) is a rare dominant disease caused by increased activation of the plasma contact system. The most prevalent HAE-FXII variant, c.1032C > A p.Thr309Lys (FXII309Lys), results in a smaller FXII protein with increased sensitivity to fluid-phase activation by poorly understood mechanisms. We aimed to investigate the functionality of the FXII309Lys variant in 33 HAE-FXII patients, 25 healthy controls and 46 patients with congenital disorders of glycosylation (CDG). Activation of the plasma contact system was assessed by western blot and amidolytic assay in basal conditions or after treatment with either artificial or physiological activators. Recombinant wild-type and FXII309Lys variants were expressed in S2 insect (Drosophila) cells. Amidolytic and fibrin generation assays were performed in fresh plasma samples. FXII309Lys samples exhibited an increased electrophoretic mobility comparable with N-glycan-deficient FXII from CDG patients and asialo-FXII generated by neuraminidase treatment. They presented increased sensitivity to activation by dextran sulphate and silica which resulted in the generation of an aberrant 37-kDa heavy chain. We did not observe increased susceptibility of FXII309Lys to proteolysis by exogenous or tPA-generated plasmin. However, both exogenous and endogenous thrombin cleaved the FXII309Lys variant, releasing a 37-kDa fragment and resulting in enhanced proteolytic activation on the fluid phase. This model supports a sequential proteolytic activation process involving thrombin priming of FXII309Lys, followed by kallikrein cleavage and generation of active βFXIIa. The present results and the observation that angioedema episodes in HAE-FXII patients occur predominantly during hypercoagulable situations suggest a key role for thrombin.

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Data Availability

All datasets presented in this study are included in the article/Supplementary Material.

Abbreviations

CAS :

Contact activation system

CDG :

Congenital disorders of glycosylation

DTT :

Dithiothreitol

FXII :

Coagulation factor XII

FXIIa:

Two-chain active form of coagulation factor FXII

βFXIIa:

Fluid-phase active form of coagulation factor FXII

FXII-HAE :

Hereditary angioedema with coagulation FXII variants

HAE :

Hereditary angioedema

KKS :

Kallikrein-kinin system

NHP:

Normal healthy persons

PK :

Plasma prekallikrein

PKa :

Plasma kallikrein

tPA:

Tissue plasminogen activator

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Acknowledgements

The authors thank all the patients involved for giving consent and support to this study. We would like to make a special mention to the clinicians who provided the samples and clinical data: Drs. Alfredo Reparaz (Hospital Álvaro Cunqueiro, Vigo, Spain), Tania Liñares (Complejo Hospitalario de Pontevedra, Pontevedra, Spain), Celsa Pérez (Eoxi Pontevedra-O Salnés, Pontevedra, Spain), Susana Varela and María Ángeles Álvarez-Eire (Complejo Hospitalario Universitario de Ourense, Orense, Spain), María Ángeles Lara (Servicio Andaluz de Salud, Seville, Spain), and Pilar Iriarte (Complejo Hospitalario Universitario de Ferrol, A Coruña, Spain).

Funding

AL-L is supported by the Centre for Biomedical Network Research on Rare Diseases (CIBERER). This work was funded by grant (ER19P7AC7541)—ACCI18-04 from CIBERER and Complemento II-CM network (B2017/BMD3673).

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Authors and Affiliations

  1. Servicio de Hematología Y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia, Spain

    R. López-Gálvez, M. E. de la Morena-Barrio, A. Miñano, V. Vicente & J. Corral

  2. Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, England

    M. Pathak & J. Emsley

  3. Sección Alergología, Complexo Hospitalario Universitario de Vigo. Hospital Meixoeiro, Vigo, Spain

    C. Marcos

  4. Servicio de Alergia, Hospital Universitario La Paz, Madrid, Spain

    T. Caballero

  5. Instituto de Investigación Sanitaria del Hospital La Paz (IdiPaz), Madrid, Spain

    T. Caballero & A. López-Lera

  6. Centre for Biomedical Network Research On Rare Diseases (CIBERER) U-754, Hospital Universitario La Paz, Madrid, Spain

    T. Caballero, M. López-Trascasa & A. López-Lera

  7. Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain

    M. López-Trascasa

Authors
  1. R. López-Gálvez
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  2. M. E. de la Morena-Barrio
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  3. A. Miñano
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  4. M. Pathak
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  5. C. Marcos
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  6. J. Emsley
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  8. M. López-Trascasa
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  9. V. Vicente
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  10. J. Corral
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  11. A. López-Lera
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Contributions

RL-G, MM-B and AL-L performed the experimental work. RL-G, MP and JE developed the expression system in insect cells. AL-L, MM-B and JC conceived the study and wrote the manuscript with support from JE and VV. AM, TC, CM and ML-T provided clinical data and sample management. All authors contributed to the article and approved the submitted version.

Corresponding authors

Correspondence to M. E. de la Morena-Barrio or A. López-Lera.

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López-Gálvez, R., de la Morena-Barrio, M.E., Miñano, A. et al. Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant. Clinic Rev Allerg Immunol 60, 357–368 (2021). https://doi.org/10.1007/s12016-021-08840-x

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