The goal of this project is to characterize the genetic regulation of synthesis of the a-chain of Factor XIII, the clotting factor essential for covalent stabilization of the fibrin clot. An existing human hepatocyte cDNA library in lambdaGTll will be screened with antibodies against the Factor XIII a-chain, which is the enzymatically active subunit. If screening with antibodies is unsuccessful, cyanogen bromide fragments will be purified and sequenced. Based on amino acid sequences with limited codon degeneracy, oligonucleotide probes will be constructed to screen the library. The cloned cDNAs for Factor XIII a-chain will be characterized by restriction enzyme mapping and hybrid-selected in vitro translation. If the cloned cDNA is incomplete, full-length Factor XIII a-chain DNA will be synthesized using a neucleotide restriction fragment as a primer for extension with purified poly(A+) mRNA and reverse transcriptase. Using the Southern blot technique, the gene structure in normal individuals and in patients with cogenital deficiency of Factor XIII will be analyzed. Restriction fragments of full-length cDNA will be inserted into the sequencing vector, bacteriophage M13, and overlapping DNA inserts will be sequenced. A variety of possible physiologic mechanisms for regulating synthesis of Factor XIII a-chains will be studied. Using cDNA probes, the mRNA from cultured rat hepatocytes will be analyzed after incubation of intact cells with various components of the activated coagulation and fibrinolytic systems: fibrin degradation products, a-chains of the intermediate and active forms of plasma Factor XIII, Factor XIII b-chains, thrombin, plasmin, and coagulation Factors IXa and Xa. Similarly, the effects of defibrination or induction of fibrinolysis in animals on Factor XIII a-chain mRNA in liver tissue will be determined. The knowledge obtained from these studies will contribute to understanding the genetic regualtion of Factor XIII synthesis and eventually to understanding the coordination of synthesis of two gene products, the Factor XIII a- and b-chains.
