The site of activation of factor X by cancer procoagulant.
Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Gordon
S G
SG
Department of Medicine and Pathology, University of Colorado Health Sciences Center, Denver 80262.
Mourad
A M
AM
D000595:Amino Acid Sequence; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D005170:Factor X / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010920:Placenta / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry
D009363:Neoplasm Proteins; D014020:Tissue Extracts; D005170:Factor X; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant