Fibrinogen-γ C-Terminal Fragments Induce Endothelial Barrier Dysfunction and Microvascular Leak via Integrin-Mediated and RhoA-Dependent Mechanism

Abstract

Objectives-The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen ␥ chain (␥C) on microvascular endothelial permeability and to examine its molecular mechanism of action. Methods and Results-Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that ␥C induced time-and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated ␥C-induced barrier dysfunction. Imaging analyses demonstrated binding of ␥C to endothelial cells; the interaction was inhibited during blockage of the ␣v␤3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to ␥C was attenuated in integrin ␤3 Key Words: fibrinogen degradation products Ⅲ microvascular permeability Ⅲ signal transduction Ⅲ Rho-GTPase Ⅲ thrombosis F ibrinogen comprises double polypeptides termed ␣, ␤, and ␥ chains, which form an elongated structure with two outer globular D domains connected to central E domains through coiled-coil segments. [1][2][3] As essential steps of the coagulation cascade, thrombin cleavage of fibrinogen and conversion to cross-linked fibrin occur in response to activation of intrinsic or extrinsic factors, followed by fibrinolysis that generates fibrin degradation products (FDPs; supplemental Figure I, available online at http://atvb.ahajournals.org). Typical FDPs include D-dimer fragments and soluble monomers containing the C termini of fibrinogen-␣, ␤ and ␥ chains, clinically measured as markers of coagulation disorder. 4 -7 The fibrinolysis cascade exerts physiological functions and pathogenic impact well beyond its role in hemostasis. Increased plasma levels of fibrinogen have been correlated with atherosclerosis, stroke, myocardium infarction, and peripheral vascular disease. 5,6 Some fibrinogen proteolytic products have been shown to participate in cell proliferation, migration, and adhesion. 6 -8 An effect on endothelial barrier properties has been documented 9 -12 ; however, consensus has not been reached as to whether or not fibrinogen as intact protein increases vascular permeability, and even less is known regarding the receptor mechanism and signal transduction that mediates the endothelial response to specific subunits or degradation products, which are known to act distinctively based on their unique chemical structures and molecular conformations. 1,3,8 Investigation is often confounded by the multi-domain structure of fibrinogen and its diverse cellular targets ranging from platelets ...

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