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Blood coagulation can be initiated when factor VII(a) binds to its cofactor tissue factor. This factor VIIa/tissue factor complex proteolytically activates factors IX and X, which eventually leads to the formation of a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits factor Xa directly and, in a Xa-dependent manner, also inhibits the factor VIIa/tissue factor complex. Here we report the cloning of the human LACI gene and the elucidation of its intron-exon organization. The LACI gene, which spans about 70 kb, consists of nine exons separated by eight introns. As has been found for other Kunitz-type protease inhibitors, the domain structure of human LACI is reflected in the intron-exon organization of the gene. The 5' terminus of the LACI mRNA has been determined by primer extension and S1 nuclease mapping. The putative promoter was examined and found to contain two consensus sequences for AP-1 binding and one for NF-1 binding, but no TATA consensus promoter element.

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Source and Description ofClone: Cosmid 8.5 was from a human chromosome 1 lq-specific library (1). p8,5-3, a subclone of this cosmid, was sequenced and the sequences flanking a (GT)18 repeat (EMBL accession number X52290) were used to design PCR primers. PCR Primers: (# 506)-5 '-AGTTACACCGGTTCTGCAGA-3' (# 507)-5 '-GATTAATGATAGTGCTATCC-3' Polymorphism: Allelic fragments were detected on DNA sequencing gels. Lengths (nt) were: Al = 188, A2= 190, A3=192, A4=194, A5=196, A6=208. Frequencies: From 33 unrelated European Caucasians. Al =.39, A2=.36, A3=.063, A4=.063, A5=.063, A6=.063. The PIC calculated from these frequencies is 0.66. Chromosomal Localization: 1 lq23.3-q24, by fluorescent in situ hybridization (1). Mendelian Inheritance: Mendelian inheritance was observed in 2 informative CEPH families with a total of 22 children. PCR Conditions: PCR was performed in a total volume of 25 Al containing: 50 ng genomic DNA, 5 pmoles of each primer, 1.5 mM MgCl2, 200 ItM dNTPs, 50 mM KCl, 10 mM Tris-Cl-, pH 8.3, 0.6 units Taq polymerase (Perkin-Elmer/Cetus) and 0.01 % gelatin. Primer # 506 was 5' end labeled with 32p. Amplification was for 30 cycles with denaturation at 94°C for 1 min, annealing at 49°C for 1 min and extension at 72°C for 30 sec. Amplified products were resolved on DNA sequencing gels and detected by autoradiography. Acknowledgements: This work was supported by Grants GM-32500 and HD 17449 from the National Institutes of Health.