Ihsan Gadi1, Sameen Fatima1, Berend Isermann1, Khurrum Shahzad1, Ahmed Elwakiel1, Sumra Nazir1, Moh'd Mohanad Al-Dabet1,2, Rajiv Rana1, Fabian Bock3, Jayakumar Manoharan1, Dheerendra Gupta1, Ronald Biemann1, Bernhard Nieswandt4, Ruediger Braun-Dullaeus5, Christian Besler6, Markus Scholz7, Robert Geffers8, John H Griffin9, Charles T Esmon10, Shrey Kohli1. 1. Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany (I.G., S.F., A.E., S.N., M.M.A.-D., R.R., J.M., D.G., R.B., S.K., B.I., K.S.). 2. Medical Laboratories, Faculty of Health Sciences, American University of Madaba, Amman, Jordan (M.M.A.-D.). 3. Medicine, Vanderbilt University Medical Center, Nashville, TN (F.B.). 4. Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Centre, University of Würzburg, Germany (B.N.). 5. Clinics of Cardiology and Angiology, Otto-von-Guericke-University, Magdeburg, Germany (R.B.-D.). 6. Cardiology, Leipzig Heart Center (C.B.), University of Leipzig, Germany. 7. Institute of Medical Informatics, Statistics and Epidemiology (M.S.), University of Leipzig, Germany. 8. RG Genome Analytics, Helmholtz Center for Infection Research, Braunschweig, Germany (R.G.). 9. Molecular Medicine, The Scripps Research Institute, La Jolla, CA (J.H.G.). 10. Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 (C.T.E.).
Abstract
RATIONALE: While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors. OBJECTIVES: To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury. METHODS AND RESULTS: Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl3-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect. CONCLUSIONS: We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.
RATIONALE: While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors. OBJECTIVES: To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury. METHODS AND RESULTS: Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl3-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect. CONCLUSIONS: We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.
Authors: Louis A Saddic; Martin I Sigurdsson; Tzuu-Wang Chang; Erica Mazaika; Mahyar Heydarpour; Stanton K Shernan; Christine E Seidman; Jon G Seidman; Sary F Aranki; Simon C Body; Jochen D Muehlschlegel Journal: Circ Cardiovasc Genet Date: 2017-01
Authors: Peter Ellinghaus; Elisabeth Perzborn; Peter Hauenschild; Christoph Gerdes; Stefan Heitmeier; Mayken Visser; Holger Summer; Volker Laux Journal: Thromb Res Date: 2016-04-16 Impact factor: 3.944
Authors: Moh'd Mohanad Al-Dabet; Khurrum Shahzad; Ahmed Elwakiel; Alba Sulaj; Stefan Kopf; Fabian Bock; Ihsan Gadi; Silke Zimmermann; Rajiv Rana; Shruthi Krishnan; Dheerendra Gupta; Jayakumar Manoharan; Sameen Fatima; Sumra Nazir; Constantin Schwab; Ronny Baber; Markus Scholz; Robert Geffers; Peter Rene Mertens; Peter P Nawroth; John H Griffin; Maria Keller; Chris Dockendorff; Shrey Kohli; Berend Isermann Journal: Nat Commun Date: 2022-08-27 Impact factor: 17.694