OC 25.2 - Amyloid fibrils trigger plasma clotting by activating the intrinsic pathway of blood coagulation and can explain intracardiac thrombosis in Transthyretin (ATTR) and Ig-Light Chain Amyloidosis (AL)

Background: ATTR and AL are amyloid diseases characterized by amyloid deposition in several organs, including heart chambers.They are associated with cardiomyopathy and intracardiac thrombosis.Amyloidosis-associated cardiomyopathy has a prevalence of 1–3%in elderly people.Histopathological investigations reveal exposure of amyloid fibrils in heart chambers,with the presence of intracardiac thrombi(Fig.1).However,the link between amyloidosis and coagulation is still unclear.

Aims: Demonstrate the ability of amyloid fibrils to induce coagulation, and establish the molecular mechanisms.The effect of net charge of the fibril components was investigated by comparing the effect of fibrils composed of genetically-similar precursors with opposite charges.

Methods: Fibrils were isolated from autopsies of ATTR or AL patients.Control amyloids were made with recombinant hTTR,hTTR(49-127),λ1,λ6. Fibrils(characterized by thioflavin-T binding, DLS,TEM)were added to plasma or whole blood(WB),monitoring clotting by turbidimetry and thrombin generation(plasma), or by thromboelastometry(WB).Role of coagulation factors was established by incubating fibrils either with factor-depleted plasma samples,or with isolated zymogens and chromogenic substrates.Binding of coagulation factors tofibrils was quantified by ELISA.

Results: Assays on plasma and WB indicate that both natural and artificial amyloid fibrils similarly induce clotting.Addition of either protein precursors in non-fibrillar state or amorphous precipitate of ß-casein does not trigger clotting, while λ1 amyloid fibrils, carrying a positive net charge at pH 7.4, partially lose the ability to trigger blood clotting . Furthermore, all amyloid fibrils induce autoactivation of the coagulation factor XII, both in the presence of factor XI and prekallikrein.Fibrils are also able to increase the efficiency of prothrombin activation by FXa of 40 times in the presence of FVa (Fig.2),leading to cleavage at R155.

Conclusion(s): Amyloid fibrils represent an ordered surface where coagulation factors are recruited.The ability to trigger blood coagulation seems correlated to the net charge of protein components,with negatively-charged fibrils being the most efficient.These mechanisms explain the hypercoagulable state that lead to the formation of thrombi,observed in most patients with cardiac amyloidosis.