A novel sensitive assay for functional factor XII based on the generation of kallikrein-C1-inhibitor complexes in factor XII-deficient plasma by glass-bound factor XII.

We developed a very sensitive assay for functional factor XII. This assay is based on the property of glass-bound factor XII to activate prekallikrein (PK) into kallikrein in factor XII-deficient plasma, which is assessed by measuring the formation of kallikrein-C1-inhibitor complexes in this plasma by radioimmunoassay. Incubation of varying amounts of factor XII in glass tubes led to a dose-dependent increase in kallikrein-C1-inhibitor complexes in factor XII-deficient plasma with a lower limit of detection of +/- 20 pg of factor XII. The specificity of the assay for factor XII was demonstrated by experiments with plasmas deficient for factor XII, PK or high molecular weight kininogen (HK) and by incubation of factor XII with Polybrene or with a monoclonal antibody that inhibits the amidolytic and procoagulant activity of factor XII. The high sensitivity of the assay appeared to be due to the inability of C1-inhibitor to inhibit factor XII bound to glass, which resulted in a molar ratio of generated kallikrein to glass-bound factor XII of at least 100:1. This assay for factor XII may be a feasible tool in studies on structural and functional aspects of (recombinant) factor XII species synthesized by cultured cells.

Plasma elastase alpha 1-antitrypsin and lactoferrin in sepsis: evidence for neutrophils as mediators in fatal sepsis.

Increased vasopermeability and vasodilation, presumably the result of endothelial perturbation, are considered among the basic pathogenetic mechanisms in septic shock. Neutrophils have been implicated as a source for mediators in endothelial injury. We measured elastase-alpha 1-antitrypsin (alpha 1AT) complexes and lactoferrin as markers for release of neutrophil granule contents in plasma from patients with sepsis on admission to the Intensive Care Unit, and we delineated the relationship of neutrophil activation to other inflammatory parameters and to hemodynamic and biochemical parameters. Levels of elastase-alpha 1AT and lactoferrin significantly correlated with each other (r = 0.58; p less than 0.008), and were increased (greater than 3.33 and 5 nmol/L, respectively) in 96% and 71% of the patients, respectively. Lactoferrin, but not elastase-alpha 1AT, correlated with the number of white blood cells (r = 0.38; p = 0.008). Elastase-alpha 1 AT levels were significantly higher (p = 0.008), whereas white blood cell counts were lower (p = 0.015) in patients with shock when compared with patients without abnormal blood pressure. Both elastase-alpha 1AT and lactoferrin levels correlated with lactate levels (r = 0.33; p = 0.024 and r = 0.30; p = 0.04), suggesting a role for neutrophil activation in the pathogenesis of hypoxygenation. In addition, elastase-alpha 1AT correlated with the concentrations of interleukin 6 (IL-6) (r = 0.46; p = 0.001) and C3a (r = 0.38; p = 0.009), suggesting that cytokines and complement may contribute to the degranulation of neutrophils in sepsis. Elastase-alpha 1AT complexes were inversely related to C1-inhibitor (r = -0.33; p = 0.028) and to platelet numbers (r = -0.42; p = 0.003). Levels of elastase-alpha 1AT complexes in plasma appeared to be of prognostic significance; levels were higher in 27 patients who died than in 21 patients who survived (p = 0.01). The mortality in 27 patients with concentrations below 10 nM was 37%, whereas it was 81% in 21 patients with higher levels. The overall mortality in this study was 56%. These results provide further evidence that activation and degranulation of neutrophils, induced by multiple agonists, are involved in the development of fatal complications in patients with sepsis.