Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin.
Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.
Stamatoglou
S C
SC
National Institute for Medical Research, London, United Kingdom.
Hughes
R C
RC
Lindahl
U
U
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D005337:Fibrin / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D005353:Fibronectins / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000502:physiology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D005353:Fibronectins; D006025:Glycosaminoglycans; D006629:Hirudins; D005337:Fibrin