Korean J Med.  2001 Apr;60(4):373-382.

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

Affiliations
  • 1Department of Internal Medicine, Holy Family Hospital, College of Medicine, The Catholic University of Korea.
  • 2Department of Internal Medicine, Our Lady of Mercy Hospital, College of Medicine, The Catholic University of Korea.
  • 3School of Life Science, The Jeonju University
  • 4Protein Engineering Research Group, Genetic Engineering Research Institute, KIST. jyjin@hfh.cuk.ac.kr

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.

Keyword

Purpura; Thrombotic Thrombocytopenic; PAP p37

MeSH Terms

Adult
Agglutination
Amino Acid Sequence
Ammonium Sulfate
Arterioles
Binding, Competitive
Blood Platelets*
Capillaries
Chromatography
DNA
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Fibrin
Humans
Male
Plasma
Plasmapheresis
Proteolysis
Prothrombin
Purpura
Purpura, Thrombotic Thrombocytopenic*
Sequence Alignment
Sequence Analysis, DNA
Sequence Homology
Thrombin
von Willebrand Factor
Ammonium Sulfate
DNA
Fibrin
Prothrombin
Thrombin
von Willebrand Factor
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