Interacting processes in protein coagulation.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.     

Microplate coagulation assays.

This report describes the development of microplate-based blood coagulation assays. The assays require a kinetic microplate reader to follow changes in absorbance at 405 nm caused by the coagulating plasma. Procedures for performing prothrombin time and activated partial thromboplastin time tests are described with intra- and inter-assay variability of a few percentage points. The prothrombin time of normal plasma was 64.5 +/- 3.6 s, and the activated partial thromboplastin time was 69.8 +/- 3.2 s. Clotting times were prolonged when normal plasma was mixed with plasmas deficient in particular coagulation factors, as expected. These assays take advantage of the microplate format (small sample size and multiple simultaneous assays) and can be customized for specific purposes, such as quantifying purified factor IX or assessing protein C activity in plasma.     

Review. Endotoxins and blood coagulation

The impact of blood coagulation caused by endotoxins (ET) is reported in a survey. In this connection the activation of factor XII by ET and the activation of the intrinsic system of coagulation due to it are discussed, the mechanism of blood platelet damage with subsequent thrombocytopenia is dealt with, and the induction for liberating of a thromboplastin-like procoagulant from leukocytes as well as the factors influencing this liberation are described. Furthermore, the mechanisms leading to the damage of the endothelia cell are discussed and the correlations to the complement system are described. On the basis of facts known up till now special attention is devoted to the role of the thromboplastin-like procoagulant and the activation of the extrinsic system caused by it in developing a DIC syndrome.     

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.