The mechanism of fibrin-induced disorganization of cultured human endothelial cell monolayers.

Abstract

Deposition of polymerizing fibrin on the vascular endothelium is the final event in intravascular coagulation. Exposure of fibrin clots to confluent monolayers of cultured human endothelial cells for 4 to 24 hours resulted in the disappearance of their normal cobblestone morphology and in the formation of endothelial cell aggregates. The present study was designed to evaluate the conditions and structural requirements of the fibrin clot for the induction of disorganization. Even after harsh treatment with denaturing agents or loading with large amounts of fibrinogen antibodies, polymerized fibrin always induced disorganization of the monolayers. In contrast, soluble fibrin that was kept in solution by either fibrinogen, fragment D-cate, or the tetrapeptide Gly-Pro-Arg-Pro did not cause any alteration of the monolayers. The fibrinogen degradation product D-cate (M r 94,000) itself had no microscopically detectable influence on the monolayer structure. In the absence of fibrin, the effect of thrombin on endothelial cells was found to be distinct from that induced by fibrin; however, the exposure of pieces of glass coverslips caused alterations in morphology indistinguishable from the fibrin-induced disorganization of the monolayer. Experiments using protein-coated polyester films indicated that the ability of the endothelial cells to attach to the overlying material, independent of its chemical structure, is the prerequisite for the induction of disorganization, but not a defined component of the fibrin molecule. Disorganization of vascular endothelium in vivo might be important for the organization and revascularization of an occluding thrombus. (Arteriosclerosis 6:139-145, March/April 1986) T he vascular endothelium contributes anticoagulant as well as procoagulant activities to the regulation of the coagulation system. A nonthrombogenic surface and the expression of fibrinolytic activities by the endothelial cells guarantee the patency of the vascular tree. Surface-bound heparan sulphate, which accelerates the inactivation of thrombin by enhancing the formation of the antithrombin Ill/thrombin complex, 1 and the membrane cofactor thrombomodulin, which facilitates the activation of protein C in conjunction with thrombin, 2 are important surface-mediated mechanisms of the vessel wall that are anticoagulant in nature. In addition, the synthesis and release of prostacyclin 3 and of tissue plasminogen activators 4 account for the inhibition of platelet activation and the continuous lysis of small amounts of fibrin. On the other hand, the synthesis of facator VIIIR:Ag, 56 specific binding of factors IX/IXa and X/Xa, 7 ' 8 and the induction of tissue factor production in the This study was supported by the Stiftung Volkswagenwerk, Hannover, West Germany.Part of this work was presented at the 10th International Congress on Thrombosis and Haemostasis, San Diego, California, in 1985 and is published in abstract form (Thromb Haemostas 1985;54:194).Address for reprints: Dr. Ulrich Delvos, Clinical Research Unit for Blood ...

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

“…Exposure of EC to enzymatically active thrombin at a concentration of0.1 U/ml (8 X 10-10 mol/liter) or greater has been shown to cause cell contraction (12,33), and a decrease in unpolymerized actin associated with increased formation of stress fibers was found after exposure to 2 U/ml (33). In our cell spreading experiments, we prepared fibrin using a thrombin concentration of 0.5 U/ml, and < 1% bound to the surface, resulting in the incorporation of < 1 X l0-' mol (0.001 U) per well or slide.…”

“…Exposure ofthe subendothelium to fibrinogen occurs only with vessel injury which also causes activation of the coagulation system, resulting in conversion of plasma fibrinogen to fibrin. The interaction of EC with fibrin results in several effects including cell retraction and loss of cell organization (12,13), separation of the monolayer into migratory cells (13), and release ofvWffrom WeibelPalade bodies (14). However, less is known about the mechanism mediating EC adhesion or spreading on fibrin, which is essential to the processes of wound healing and revascularization.…”

Fate of fibrinogen in human arterial intima.

“…All investigators find disruption of the monolayer. 383840 Weimar and Delvos 40 consider that the damaging factor is the Intact fibrin gel and found no harmful effect of fragment D, 41 but others ascribe it to fragment B01-42 39 or fragment D. 42 Whatever the mechanism of endothelial disruption, it may be a potent factor in generating the edema that characterizes gelatinous lesions.…”

Mediation of fibrin-induced release of von Willebrand factor from cultured endothelial cells by the fibrin beta chain.

“…Exposure of a confluent endothelial monolayer to fibrin causes cell retrac-tion, disruption of cellular organization, and separation of the monolayer into migratory cells (1). Both fibrin and fibrin degradation products have been shown to stimulate EC migration (1)(2)(3) and disorganization during angiogenesis (4)(5)(6). EC exposed to fibrin increase secretion ofboth prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, and tissue plasminogen activator, an inducer of fibrinolytic activity (7).…”