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Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

ABSTRACT: When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

SUBMITTER: Ivanov I

PROVIDER: S-EPMC5356457 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Essentials During contact system activation, factor XII is progressively cleaved by plasma kallikrein. We investigated the role of factor XII truncation in biochemical studies. Factor XII contains naturally occurring truncating cleavage sites for a variety of enzymes. Truncation of factor XII primes it for activation in solution through exposure of R353. SUMMARY: Background The contact activation system and innate immune system are interlinked in inflammatory pathology. Plasma kallikrein (PKa) is held responsible for the stepwise processing of factor XII (FXII). A first cleavage activates FXII (into FXIIa); subsequent cleavages truncate it. This truncation eliminates its surface-binding domains, which negatively regulates surface-dependent coagulation. Objectives To investigate the influence of FXII truncation on its activation and downstream kallikrein-kinin system activation. Methods We study activation of recombinant FXII variants by chromogenic assays, by FXIIa ELISA and western blotting. Results We demonstrate that FXII truncation primes it for activation by PKa in solution. We demonstrate this phenomenon in three settings. (i) Truncation at a naturally occurring PKa-sensitive cleavage site, R334, accelerates FXIIa formation in solution. A site-directed mutant FXII-R334A displays ~50% reduced activity when exposed to PKa. (ii) A pathogenic mutation in FXII that causes hereditary angioedema, introduces an additional plasmin-sensitive cleavage site. Truncation at this site synergistically accelerates FXII activation in solution. (iii) We identify new, naturally occurring cleavage sites in FXII that have so far not been functionally linked to contact system activation. As examples, we show that non-activating truncation of FXII by neutrophil elastase and cathepsin K primes it for activation by PKa in solution. Conclusions FXII truncation, mediated by either pathogenic mutations or naturally occurring cleavage sites, primes FXII for activation in solution. We propose that the surface-binding domains of FXII shield its activating cleavage site, R353. This may help to explain how the contact system contributes to inflammatory pathology.

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Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

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