Volume 272, Number 40, Issue of October 3, 1997 pp. 24999-25005
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Covalent Cross-linking of Fibronectin to Fibrin Is Required for Maximal Cell Adhesion to a Fibronectin-Fibrin Matrix

(Received for publication, December 27, 1996, and in revised form, May 9, 1997)

Siobhan A. Corbett ^§ , Laura Lee , Carole L. Wilson and Jean E. Schwarzbauer

From the  Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, the ^§ Department of Surgery, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08703, and the  Dermatology Division, Washington University, St. Louis, Missouri 63110

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.

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