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Title: The influence of coagulation factor XIII-A V34L mutation on clot formation and stability
Author: Baranauskas, Adomas
ISNI:       0000 0004 8508 9752
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Background: Coagulation factor XIII (FXIII) is a key enzyme in stabilising blood clots by cross-linking fibrin molecules together, amongst other effects on fibrinolysis and matrix protein stabilisation. The human FXIII-A V34L sequence variant leads to increased activation rates, forming clots with thinner fibrin fibres and smaller pores, and has been reported to show protective effects against thrombotic diseases like venous thromboembolism. However, the complete mechanism(s) underpinning this effect has hitherto remained elusive. Aim: To establish a murine FXIII-A L34V model and study the role of this sequence variant, as well as that of FXIII itself, in thromboembolic disease. Methods: FXIII-A Knock-Out (FXIII-/-, already available in the laboratory), FXIII-A 34Val (generated by MRC Harwell for Leeds) and FXIII-A 34Leu (common variant, wild-type) mice were compared in their growth, plasma FXIII activation rates (biotin incorporation assay), turbidity for clot structure, fibrinogen concentrations (ELISA assay) and FXIII-A antigen levels (westernblotting). Whole blood clotting and lysis was measured by rotational thromboelastometry (ROTEM). Whole blood clot contraction, erythrocyte extrusion (haemoglobin) and two-hour clot weights were quantified. For pulmonary and cerebral embolism models, FeCl3 vascular injury was performed on inferior vena cava and carotid artery, respectively. Lungs or brains were optically cleared and imaged with a light sheet microscope. Pulmonary emboli volume and count were quantified using software, while cerebral emboli were quantified manually. Results: No significant differences between 34Leu and 34Val mice growth, whole blood contraction, serum haemoglobin, clot weight, ROTEM, plasma FXIII-A and fibrinogen levels were observed. FXIII-/- mice plasma contained no FXIII-A antigen, showed negligible fibrin cross-linking activity and had similar levels of fibrinogen as FXIII sufficient mice. FXIII-/- clots were less firm, easier to lyse, slower to form, retained fewer red blood cells, were lighter and formed thicker fibres than FXIII sufficient clots. 34Leu mice plasma showed increased FXIII-A activation rates over 34Val mice plasma. Increasing FXIII activation rates (34Leu > 34Val > FXIII-/-) increased both pulmonary emboli count and volume, while the trend in cerebral embolism differences was inconclusive. Conclusion: The murine FXIII-A 34Leu variant showed increased activation rates over the 34Val variant and altered thromboembolisation, particularly in the venous circulation. Potentially, increased FXIII activation may enhance venous thromboembolism by protecting emboli against fibrinolysis during their transit to the lungs. In the high shear stress arterial environment, effects of mechanical stabilisation by FXIII may counterbalance any effects on fibrinolysis. These studies show a potentially important role for FXIIImediated clot stabilisation and protection against fibrinolysis that impact on thromboembolic diseases. Pulmonary embolism dependence on FXIII could be clinically relevant, where FXIII inhibition would act as prophylaxis for patients with a high risk of pulmonary embolism.
Supervisor: Ariens, Robert ; Duval, Cedric Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available