Karl Fickenscher

Anticoagulant properties of placenta protein 4 (annexin V).

A placenta protein, originally termed PP4, was found to inhibit the aPTT in a concentration-dependent manner. PP4 which turned out to be identical with a vascular anticoagulant of the annexin type, inhibits the blood clotting process by binding of the essential lipids in a reaction which is dependent on calcium ions. Also in the presence of calcium PP4 combines with platelet membranes neutralizing their procoagulant effect. By fluorescence-microscopy binding of PP4 to stimulated macrophages is shown. The antithrombotic effect of PP4 is demonstrated by means of thrombelastography of human blood. Coagulation triggered by the addition of thromboplastin/lipid-mixtures is extinguished by PP4.

The PACT—A simple screening assay for assessing the functionality of the protein C anticoagulant pathway

The protein C system is well known as an important anticoagulant pathway (1). Hereditary defects or deficiencies in protein C or protein S might account each for about 5% of otherwise unexplained thrombosis. Besides genetic disorders, disturbances of the protein C system can also be acquired under various clinical conditions, especially, if intlammatory reactions are involved (2). Although, such an acquired deficiency leads to an enhanced thrombotic risk, the routinely investigation of the protein C system of inpatients has not been common in the past.

Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma

A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 microliters sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2-4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63% and 2.78% on the Hitachi 911 and 1.51% and 7.84% on the Cobas Mira, respectively and interassay coefficients of variation of 3.57% to 9.15% (Hitachi 911) and 3.72% to 12.99% (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x-0.003, n = 323).

Determination of factor XIII activity by a new photometric assay in plasma and platelets of healthy blood donors

Crosslinking of fibrin monomers by activated factor XIII (F XIIIa) is a final event in blood coagulation. So the fibrin clot gains mechanical stability and resistance to plasmin degradation which is thought to be essential for normal blood clotting and wound healing (1-3). In addition, changes in plasma F XIII activity were found in several state of disease such as collagenoses, inflammatory bowel diseases, leukemias, subarachnoidal bleeding and delayed fracture healing (4-12). Because approximately 50% of the potential F XIII activity in plasma are present in platelets (1), the additional determination of F XIII activity in platelets is of clinical interest especially concerning platelet transfusions that may exert an additional benefit due to simultaneous substitution of platelet-bound F XIII. The latter differs from plasma F XIII as a dimer containing only a-chains (a2) compared to the plasmatic tetramer carrying additional b-chains (a2b2). We applied a recently described photometric assay suitable for the routine laboratory after adaption to an autoanalyser to determine F XIII activity in plasma and platelets of 64 healthy blood donors.

Coagulation assay with improved specificity to factor V mutants insensitive to activated protein C

The prevalence of a new hereditary defect in the protein C anticoagulant pathway, the factor V-Leiden, has been reported to range between 20% to 60% in familial thrombophilia. In addition to differences in patient groups, these very divergent numbers might also be due to the detection method applied. In most studies a modified APTT was used, where activated protein C (APC) is added simultaneously with the start of the clotting reaction. However, this method is also influenced by other factors like protein S, factor VIII or lupus anticoagulants. Furthermore, heparin or oral anticoagulant therapy might interfere. We tried to develop a coagulation assay dependent only on those mutant forms of factor V stable against proteolytic attack by APC. For this purpose, samples were first diluted with a factor V deficient plasma (f.V-dp). Then, coagulation was initiated either on the intrinsic pathway (APTT) or on the extrinsic pathway (PT) or, by directly activating factor X (RVVT). Additionally, APC was added, which prolongation of the clotting time. Deficiencies in protein S or the presence of factor V-Leiden resulted in a less pronounced clotting time prolongation. Titration of protein S-deficient plasma samples with f.V-dp diminished this effect. In contrast, in samples with factor V-Leiden the difference to the clotting time obtained with normal plasma even increased in the order APTT>>RVVT>PT. In the APTT-based method high concentrations of factor VIII shortened the clotting times, thus mimicking a factor V-Leiden defect. This could be compensated for up to 4 U/ml factor VIII by using a f.V-dp containing factor VIII at physiological concentration. Neither unfractionated nor LMW-heparin (up to 2 U/ml) interfered with the determination. In a brief investigation on 16 plasma samples from patients under oral anticoagulation 5 (30%) showed a similar behaviour as observed with normal plasma from factor V-Leiden carriers. These results let us suggest that by simply mixing the patient sample with a factor V-deficient plasma factor V-Leiden might be detected also in patients under oral anticoagulant therapy. Inherited disorders of protein C or protein S are well known as thrombotic risk factors (1). The recent investigations by Dahlbäck et al. (2) led to the discovery of a new hereditary defect in the protein C anticoagulant pathway: the factor V-Leiden (3). This mutation renders activated factor V stable against proteolytic attack by activated protein C (APC). The reports on the prevalence of this mutation in thrombophilic patients show a considerable variation between 21% (4) and 64% (5).(ABSTRACT TRUNCATED AT 400 WORDS)