Jürgen Dr. Römisch

The FVII activating protease cleaves single-chain plasminogen activators.

A serine protease isolated from plasma sharing structural characteristics with a hepatocyte growth factor activator-like protease has been demonstrated recently to activate FVII. Accordingly, it was named ‘FVII activator’. Until now an impact of this protease on the fibrinolytic system has not been reported. We islolated the protease from cryo-poor plasma by subsequent ion exchange chromatography and adsorption to immobilized heparin and/or aprotinin. Incubation of single-chain plasminogen activators (sc-PAs) with the FVII activator revealed significant activation of urokinase sc-PA (scu-PA) and tissue sc-PA (sct-PA) in vitro. It was enhanced in the presence of calcium and heparin. Compared to kallikrein, a more efficient activation of scu-PA was observed, whereas sct-PA appeared to be a poorer substrate for the FVI activator. At low protease concentrations and in the presence of heparin the scu-PA activation was comparable to plasmin. Employing recalcified whole blood thrombelastography, the lysis of initially formed fibrin was observed after addition of a combination of scu-PA and the FVII activator, whereas the scu-PA alone had a negligible effect at the concentration used. The study results as presented demonstrate that the FVII activator is a potent activator of sc-PAs in vitro. Whether it plays a physiological role in fibrinolysis deserves further investigation. Its comparatively high affinity to heparin assumes a function in cell surface or matrix events.

Quantification of Antithrombin Isoform Proportions in Plasma Samples of Healthy Subjects, Sepsis Patients, and in Antithrombin Concentrates

Antithrombin (AT) circulates in plasma in two isoforms, AT-α (90–95%) and AT-β (5–10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and β-isoform content (p < 0.05). AT-β correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher β-isoform content. β-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-β content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology.

Separation of antithrombin III variants by micellar electrokinetic chromatography

The characterisation of proteins is still one of the most challenging analytical tasks in modern bioanalysis. Due to the complex structure of proteins, several analytical techniques are often required to get sufficient information. Antithrombin III (AT III), a high-molecular-mass plasma glycoprotein which is an important protease inhibitor and the main modulator of thrombin activity, circulates in plasma in two isoforms, the so-called AT III-alpha (90-95%) and -beta (5-10%). Micellar electrokinetic chromatography was used to analytically separate these AT III variants, which differ in their affinity to the polysaccharide heparin.