Mechanisms of Human Neutrophil Elastase-Catalyzed Inactivation of the Factor VIII(a).

Abstract 2125

Poster Board II-103

Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A1^1-357 and A2^375-708, respectively, by cleavages at Val³⁵⁷ and Val³⁷⁴ in A1 and Val⁷⁰⁸ in A2. The cleavages at Val³⁷⁴ and Val⁷⁰⁸ by HNE were faster than that at Val³⁵⁷. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val¹⁶⁷⁰, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val⁷⁰⁸ in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val³⁵⁷ and Val⁷⁰⁸ was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val⁷⁰⁸, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE.