Platelet agglutinating protein (PAP p37) from a patient with thrombotic thrombocytopenic purpura is identical with prethrombin 2

Jin, Jong Youl; Park, Soo Jeong; Lee, Jong Woo; Kim, Seung Ho

Abstract

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is characterized by widespread platelet thrombi with little fibrin in the arterioles and capillaries. Unusually large or multimeric von Willebrand factor (vWF) and one or more platelet-agglutinating factors have been implicated in the pathogenesis of TTP. However, there had not been any satisfactory explanations regarding the actual mechanisms of platelet agglutination until now. Recent studies suggested 37 kDa platelet agglutinating protein (PAP p37) to be responsible for the formation of platelet thrombi in the patients with TTP. We already purified and reported 37 kDa platelet agglutinating protein (PAP p37) in a patient with TTP. To identify PAP p37, we studied more characteristics and sequenced N-terminal 21 amino acid residues of PAP p37.
METHODS
PAP p37 was purified from the plasma which was obtained during the first plasmapheresis in a 31-year-old male Korean patient with acute TTP by ammonium sulfate fractionation, DEAE-Sephacel, concanavalin A-Sepharose and Superose 12 gel filteration chromatographies. In each step, agglutinating activity of platelet was studied by platelet aggregometer. N-terminal 21 amino acid of PAP p37 was sequenced using automatic amino acid sequence analyzer (Beckmann, USA), Sequence Homology Analysis (NCBI BLAST 2.0 from http://www.ncbi.nlm.nih.gov/BLAST1), and Multiple Sequence Alignment (GeneDoc 2.6.0 from http://www.psc.edu/biomed/genedoc/). After we found out that the amino acid sequence of PAP p37 is identical with prothrombin 285-305 amino acid sequence, prothrombin gene was sequenced by the Amersham and CircumVent thermal cycle DNA sequencing kit for detecting gene mutation.
RESULTS
The results are as follows: 1) N-terminal 21 amino acid sequence of PAP p37 was T-F-G-S-G-E-A-D-X-G-L-R-P-L-F-E-K-K-S-L-E and appeared to be identical to that of 285-305 amino acid residues of human prothrombin (prethrombin 2) Compared with thrombin by SDS-PAGE with or without beta-mercaptoethanol, PAP p37 was suggested that is unsuccessfully cleaved thrombin light chain which was not cleaved disulfied bond between A-chain and B-chain in prethrombin 2. 2) No gene DNA mutation was found in any prothrombin gene. 3) PAP p37 revealed competitive binding against anti-thrombin antibody with thrombin by ELISA method and their antigenicity was similar with thrombin.
CONCLUSION
PAP p37 has potent platelets agglutinating activity and the N-terminal 21 amino acid residues, the pattern of SDS-PAGE with beta-mercaptoethanol and the antigenicity were the same as prethrombin 2 of procoagulant. This prethrombin 2 in TTP may develop due to unsuccessful cleaving of the thrombin light chain. These results suggest that there are defects in procoagulant proteolysis of TTP.