13 FACTOR IXa-ATIII COMPLEX FORMATION IS INHIBITED BY PROTAMINE SULFATE

This laboratory has studied the effect of 1 and 5 μg protamine sulfate (PS) (0.2 and 1.0 nmol) upon the formation of covalent complexes between (excess) factor IXa (5 μg) and ATIII (1.92 μg) under three sets of conditions: preincubation of IXa with PS for 15 minutes prior to the addition of ATIII; preincubation of ATIII with PS for 15 minutes prior to the addition of IXa; and mixing each of the reagents within 1-minute intervals. Complex formation was detected by SDS-PAGE and quantitated. A major band for the enzyme was observed at about 45 kDa, and a minor degradation product band of the enzyme was seen at about 31 kDa. A single ATIII band of about 55 kDa was also noted. Upon mixing enzyme with inhibitor, two bands of complexes were observed, at approximately 115 kDa and 100 kDa. It was suggested that the 100 kDa band was a degradation product of the 115 kDa band. Upon preincubation of IXa, ATIII, or both with PS prior to addition of ATIII, Ixa, or buffer to the mixtures, it was noted in all three cases, and in the presence of both levels of PS, that there was a decrease in the amounts of 115 kDa and 100 kDa complexes observable. The decrease was greater in the presence of 5 μg, suggesting inhibition of complex formation. Concomitantly, with the decrease in complex formation, an increase in unreacted ATIII was noted. At the same time a decrease in free IXa was observed commensurate with an increase in the IXa fragment. The latter corresponds most likely to a promotion of autolysis of factor IXa by the presence of PS, in agreement with other proteolytic stimulations reported from this laboratory upon addition of PS to other coagulation factors.