Poster Sessions AP02: Proteins,Amino Acids,Peptides and Metabolism. The effect of proline rich polypeptide on the interaction between activated factor X and anti‐thrombin III

The effect of the hypothalamic neurosecretory Proline Rich Polypeptide (PRP) on the interaction of human Factor Xa (Xa) with human antithrombin III (ATIII) was investigated by SDS–PAGE and Western Blot analysis. Xa runs as two bands with masses of ～51 and ～47 kDa. A fraction of the ATIII (～55 kDa) undergoes a conformational change upon interaction with Xa to run slower at ～58 kDa. Xa and ATIII form a 1° duplex complex of ～109 and ～104 kDa, a 2° duplex complex of ～99 and ～94 kDa, and a 3° duplex complex of ～66 and ～62 kDa. Additionally, a Xa degradation product (～35 kDa) is noted. Pre‐incubation of 1.5 μg of either Xa or ATIII with 8 μg PRP for 15′ at R.T., or essentially simultaneous addition of all three, increases the 2° complex upper band, 3° complex lower band, ATIII conformational change, and the Xa degradation product. It also decreases the 3° complex upper band and the native ATIII. Xa/PRP premixtures cause a decrease in unreacted Xa. ATIII/PRP premixtures produce an increase in the 1° complex lower band and a decrease in the unreacted Xa upper band. Xa/ATIII/PRP mixtures increase both bands of the 1° complex. Thus, 8 μg PRP promotes 1° complex formation as well as formation of the 2° and 3° degradation complexes. It also promotes modification of ATIII and degradation of Xa, possibly by autolysis and/or promotion of proteolysis of the 1° complex by free Xa. Data obtained indicate the role of PRP in the molecular mechanisms of blood clotting. (Supported, in part, from a grant by the Office of Sponsored Programs and Research, BGSU.)