FLIM-FRET-based analysis of S100A11/annexin interactions in living cells.

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1-containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM-FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.

Measurement of maximum thrombin generation capacity in blood and plasma using the thrombin generation assay (THROGA).

Diagnosis of a hyper- or hypocoagulable state has been very difficult. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample.

Platelet adhesion assay--a new quantitative whole blood test to measure platelet function.

At this time no practical laboratory method for the measurement of the current functional state of blood platelets is available. A new innovative platelet adhesion assay (PADA) is described here. With only a short time requirement and minimal equipment, the PADA provides quantitative measurements of platelet adhesiveness. Only 0.5 mL of freshly drawn citrated whole blood is needed, to which special polymer particles are added. A defined shear grade is induced by a short period of shaking the sample. Proteins of the blood sample, especially fibrinogen, and thereafter also activated platelets, bind to the specific polymer surface. Following platelet counts both in the sample and in a control (blood without particles), the adhesion index (AI) is calculated as a quantitative measure of platelet adhesiveness. In healthy volunteers, a mean AI of 52+/-12 was measured. AI was shown to be nearly independent of the number of platelets in the sample (100 to 350 k/ microL), the hematocrit (44 to 25%), and the fibrinogen content (1.5 to 5 g/L). Age of the volunteers had only a minor influence on the AI. PADA was shown to be a simple reliable laboratory method for the detection of disturbed platelet function. The test requires low analytical efforts compared with other diagnostic platelet function tests.