Detection of Activation of the Contact System of Coagulation In Vitro and In Vivo: Quantitation of Activated Hageman Factor-C1-Inhibitor and Kallikrein-C1-Inhibitor Complexes by Specific Radioimmunoassays

Abstract

SummaryRadioimmunoassays (RIAs) for the detection of C1-inhihitor (C1-Inh) complexed to either kallikrein or activated Hageman factor (factor XIIa) are described. Kallikrein-C1-Inh or factor XIIa-C1-Inh complexes were bound to Scpharosc to which monospecific antibodies against (pre)kallikrein or factor XII, respectively, were coupled. Bound complexes were subsequently detected by an incubation with affinity purified 125I-labeled antibodies against Ci-Inh. These RIAs were used to detect activation of the contact system of coagulation in vitro and in vivo. Addition of dextran sulfate (DXS) (20 μg/ml) to fresh plasma resulted at 37° C in the rapid generation of amidolytic kallikrein activity, which was maximal after 1 to 2 min of incubation and subsequently decreased within a few minutes. The generation of kallikrein activity coincided with the appearance of both kallikrein-C1-Inh and factor XIIa-C1-Inh complexes. However, in contrast to kallikrein activity, both types of complexes remained detectable in the incubation mixtures during the incubation period. Experiments with purified kallikrein, C1-Inh and partly purified β-factor XIIa, and activation experiments in plasmas deficient in either factor XII or prekallikrein, demonstrated the specificity of both RIAs. The minimal amount of DXS that resulted in the generation of measurable amounts of both types of complexes in plasma was 2-3 μg per ml. Similar experiments with kaolin showed that with limiting amounts of activator (1-2 mg/ ml), only kallikrein-C1-Inh complexes were detected in plasma. When larger amounts of kaolin were added to plasma, factor XIIa-C1-Inh complexes were additionally detected in plasma. In plasma samples obtained from healthy donors under conditions that prevented activation of the contact system in vitro, very low levels of both factor XIIa-C1-Inh and kallikrein-C1-Inh complexes were measured, representing approximately 0.3% activation of both factor XII and prekallikrein. In serial plasma samples from a patient with adult respiratory distress syndrome, increased levels of both types of complexes were detected. The radioimmunoassays described in this paper provide useful tools to detect activation of the contact system in vitroas well as in vivo.