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 cr oss linked by activated factor XIII (factor XIIIa) to form pFN-fibrin multimers, To determine the functional significance of covalent pFN-fi brin interactions, we have developed an in vitro model which allows th e incorporation of recombinant FN (recFN) molecules into a covalently cross-linked recFN-fibrin matrix, Using the baculovirus expression sys tem, we have expressed recFN monomers composed of the amino-terminal 7 0-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), Exam ination of the covalent incorporation of these recFNs into fibrin clot s confirms that glutamines 3 and 4 are major participants in FN-fibrin cross-linking as the mutation of these sites reduces cross-linking ef ficiency 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 attach ment 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.