thromboplastin and tertiary-butyloxycarbonyl-valyl-prolyl-arginyl-7-amino-4-methylcoumarin

Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.

Topics: Amino Acid Sequence; Animals; Biocompatible Materials; Blood; Calibration; Coumarins; Enzyme Activation; Glass; Heparin; Humans; Kinetics; Materials Testing; Models, Theoretical; Molecular Sequence Data; Oligopeptides; Phosphatidylethanolamines; Platelet Activation; Polypropylenes; Polystyrenes; Polyvinyl Alcohol; Prothrombin; Rabbits; Rheology; Spectrometry, Fluorescence; Thrombin; Thromboembolism; Thromboplastin

A sensitive and quantitative amidolytic assay for thromboplastin (tissue factor) coupled to thrombin formation was established. A fluogenic peptide substrate, Boc-Val-Pro-Arg-MCA, was found to be suitable for the coupled amidolytic assay. The amidolytic assay was applied to measure TF activity of endotoxin-stimulated mononuclear leukocytes and monocytes. The amidolytic assay showed good correlation of 0.97 with the currently used clotting assay upon measuring TF activity of the cellular samples.

Topics: Animals; Blood Coagulation Tests; Coumarins; Fluorescent Dyes; Leukocytes; Macaca; Oligopeptides; Spectrometry, Fluorescence; Thromboplastin