A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.

Among all patients with von Willebrand disease (vWD), alloantibodies to von Willebrand factor (vWF) have been described only in severe vWD (type III). The relationship between the development of alloantibodies and the nature of the genetic lesion in vWD is not known. In hemophilia B, large deletions within the factor IX gene appear to correlate with the occurrence of alloantibodies, whereas in hemophilia A no such correlation is apparent. We have studied 19 patients with severe recessive vWD (type III) and 19 with autosomal dominant vWD (type I) by Southern blotting with probes encompassing the full 9 kilobases (kb) of the vWF cDNA. Two apparently unrelated patients were shown to have large deletions within the vWF gene. Both patients had severe vWD (type HI) and were the only patients among those studied that had inhibitory alloantibodies to vWF. The extent of deletion was similar in both patients, corresponding to at least the 3'-7.4 kb of the vWF cDNA. The deletion in each patient was estimated to exceed 110 kb. In addition, the localization of the vWF gene to chromosome 12 was confirmed, and a homologous sequence on chromosome 22 was identified.

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The recent isolation of cDNA and genomic DNA clones for human vWF by ourselves and others has finally laid to rest the historical notion that factor VIII and vWF might have a precursor-product or other complex relationship. These two hemostatic activities are clearly present on two distinct proteins, each encoded by a separate gene. Whether ther is coordinate regulation of the factor VIII and vWF genes is still unknown. The structure and structure-function relationships of the vWF protein have been elucidated by many investigators, only some of whom can be cited in this short paper. Together, these molecular biology and protein chemistry studies have shown that vWAgII is the amino-terminal propeptide of vWF, explaining the proportional deficiency of these two plasma proteins in von Willebrand disease (type I). In addition, sites of proteolytic processing, glycosylation, and disulfide bond formation have been defined, and some of the binding functions of mature vWF have been identified with specific amino acid sequences. The protein has a highly repeated structure, suggesting a complex evolutionary history. The A domains of vWF appear to be homologous to complement factor B, and perhaps to component C2, although the biological meaning of this similarity is unknown. Genomic DNA clones have been isolated corresponding to approximately one third of the vWF gene on chromosome 12, and a fragment of the cDNA also hybridizes to uncharacterized sequences on chromosome 22. Preliminary studies in von Willebrand disease have revealed two patients with very large deletions in the vWF gene.(ABSTRACT TRUNCATED AT 250 WORDS)