Monitoring of r-hirudin anticoagulation during cardiopulmonary bypass--assessment of the whole blood ecarin clotting time.

The use of recombinant (r) hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor IIa assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin-spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 micrograms/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin-spiked blood samples obtained from 50 healthy blood donors. CV-values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 micrograms/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

The extracellular matrix in human cardiac tissue. Part II: Vimentin, laminin, and fibronectin.

In continuation of our previous work describing the localization of the collagens type I, III, VI, and IV the present study describes the localization of vimentin, laminin, and fibronectin in human myocardium obtained as left ventricular needle biopsies during cardiac surgery. Myocardium from normal pigs served for comparison. Monoclonal antibodies against the various proteins were used on frozen sections, labeled with fluorescein and viewed in the fluorescence microscope. Vimentin, the intermediate filament of mesenchymal cells, is present in fibroblasts, fibrocytes, and endothelial cells. Laminin is observed in the basal membrane of myocytes, smooth muscle and endothelial cells. The staining intensity for the B1-chain is higher in and around myocytes as compared with the B2-chain antibody, but more blood vessels were stained with the latter. The antibody against the A-chain only stained the basal lamina of vascular cells but not that of myocytes. Fibronectin was localized homogeneously throughout the extracellular space as matrix material in which the cellular elements and the various other proteins such as collagens are embedded. Intracellular staining in myocytes (T-tubules) was commonly observed. Both parts of this study show the distribution of extracellular proteins in normal human cardiac tissue and are intended to be the basis for investigations of pathological changes in diseased human myocardium.

The extracellular matrix in human myocardium: Part I: Collagens I, III, IV, and VI.

The composition of the extracellular matrix in normal human myocardium obtained at open-heart surgery was investigated using monoclonal antibodies against the collagens I, III, IV, and VI, and fluorescence microscopy. The aim of the study was to provide information on normal myocardium that could be used in the evaluation of pathological changes. Porcine myocardium was used for comparison, and both tissues showed a perfect agreement of the results, apart from collagen IV. This was negative in pig myocardium, due to the species specificity of the antibody. Collagens I and III were localized in the extracellular space as either coarse or fine fibrillar structures; the cellular elements of the interstitium, except for the endothelial cells, were also stained. Labeling for collagen VI was much finer than for the other collagens, and was present throughout the interstitium. Collagen IV stained the basement membranes of myocytes and capillary endothelial cells, and also labeled the T-tubular system in the myocytes. The second part of this communication will describe the localization of fibronectin, laminin and vimentin in normal human myocardium.