Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix.
In a blood clot, fibrin and plasma fibronectin (pFN) are covalently cross-linked by activated factor XIII (factor XIIIa) to form pFN-fibrin multimers. To determine the functional significance of covalent pFN-fibrin interactions, we have developed an in vitro model which allows the incorporation of recombinant FN (recFN) molecules into a covalently cross-linked recFN-fibrin matrix. Using the baculovirus expression system, we have expressed recFN monomers composed of the amino-terminal 70-kDa region and the first 11 type III repeats (WT) with mutations in the glutamines at positions 3 and 4 (Q2) or at 3, 4, and 16 (Q3). Examination of the covalent incorporation of these recFNs into fibrin clots confirms that glutamines 3 and 4 are major participants in FN-fibrin cross-linking as the mutation of these sites reduces cross-linking efficiency by 65%. Additional mutation of the glutamine at position 16, however, eliminates >99% of cross-linking suggesting that it also may be factor XIIIa reactive. When the Q3 recFN-fibrin clots were used as substrates for cell adhesion, there was a decrease in both cell attachment and spreading when compared with the WT recFN-fibrin clots. These data demonstrate that for maximal cell attachment to a FN-fibrin clot, FN must be cross-linked to fibrin by factor XIIIa.
Corbett
S A
SA
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.
Lee
L
L
Wilson
C L
CL
Schwarzbauer
J E
JE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D003432:Cross-Linking Reagents; D017931:DNA Primers; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D005973:Glutamine; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection
D003432:Cross-Linking Reagents; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005973:Glutamine; D005337:Fibrin