Impact of Triton X-100 on alpha 2-antiplasmin (SERPINF2) activity in solvent/detergent-treated plasma.

Large-pool solvent/detergent (SD) plasma for transfusion exhibits reduced alpha 2-antiplasmin (alpha2-AP; SERPINF2) functional activity. The reason for the loss of alpha2-AP has not been described and could be due to the SD incubation itself and/or to the processing steps implemented to remove the solvent and the detergent. We have studied alpha2-AP activity during six down-scale preparations of plasma virally-inactivated by 1% (v/v) TnBP combined with two different non-ionic detergents, either 1% Triton X-100 or 1% Triton X-45, at 31 degrees C for 4h. The SD-treated plasmas were then extracted with 7.5% (v/v) soybean oil, centrifuged at 3800 x g for 30 min, and subjected to hydrophobic interaction chromatography (HIC) to remove the SD agents. Control runs without TnBP and Triton were performed to evidence possible impacts of each process step on alpha2-AP activity. TnBP, Triton X-100, and Triton X-45 were measured at all stages of the processes to evaluate potential interferences with the alpha2-AP assay. Alpha 2-AP activity was about 10% that of starting plasma after 1% TnBP-1% Triton X-100 incubation and about 50% after oil extractions, centrifugation, and HIC. By contrast about 73% of the antiplasmin activity was found after the incubation with 1% TnBP and 1% Triton X-45, 88% after removal of the SD agents by oil extractions, 90% after centrifugation and 92% after HIC. The control runs performed without SD agents showed that the process steps did not affect the alpha2-AP activity. In conclusion, the agent altering alpha2-AP activity in SD-plasma is Triton X-100. The choice of detergents for the SD viral inactivation of therapeutic plasma fractions used in patients at risk of fibrinolysis should consider the impact on alpha2-AP activity.

Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met(374)-Ser(375) (P3-P2), Glu(416)-Leu(417) and Ser(432)-Leu(433) peptide bonds in human alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37 degrees C, alpha(2)-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. alpha(2)-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro(19)-Leu(20) peptide bond in alpha(2)-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional alpha(2)-AP variant (Ala-alpha(2)-AP or alpha(2)-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37 degrees C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of alpha(2)-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.