AN INHIBITOR OF COLLAGEN-STIMULATED PLATELET ACTIVATION FROM THE SALIVARY-GLANDS OF THE HAEMENTERIA-OFFICINALIS LEECH .2. CLONING OF THE CDNA AND EXPRESSION

Salivary glands of the leech Haementeria officinalis contain a protein , leech antiplatelet protein (LAPP), that specifically blocks collagen -mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Co ndra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleo tides whose sequences were derived from two short peptides from V8 dig ests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated contai ning the entire deduced amino acid sequence for LAPP. Computer analysi s of the amino acid sequence predicts a peptidase cleavage site betwee n a 21-residue prepeptide and a mature protein of 126 amino acids. A D NA insert to express the predicted mature LAPP protein was generated b y PCR amplification using phage-derived cDNA clones as a substrate. Th is insert encoded a fusion protein with the leader sequence of the yea st alpha-mating factor and the mature LAPP cDNA. These PCR products we re cloned into the yeast expression vector pKH4-alpha-2. A KEX 2 Lys-A rg endopeptidase cleavage site was placed NH2-terminal to the predicte d mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its rea ctivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesi on of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/funct ion studies and to studies on the effects of an inhibitor of collagen- stimulated platelet aggregation in vivo.