SummaryA method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording of p-nitroaniline (pNA) production in a 2:3 diluted plasma. A chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ68), is used that is specifically split by thrombin but at a low rate. The thrombin that appears and disappears in the plasma does not split more than 5% of the substrate added, so the rate of substrate conversion is in good approximation proportional to the amidolytic activity in the plasma over the entire period of thrombin generation. The course of the enzyme concentration can be calculated from the amidolytic activity curve. It is shown that the thrombin generation curves obtained in this way are essentially identical to those obtained via the classical subsampling method.The presence of SQ 68 influences the amount of free thrombin that appears in plasma because it competitively inhibits the inactivation of thrombin by AT III and α2 macroglobulin. The inhibition of the thrombin peak by heparin, relative to an uninhibited control, remains unaltered by the presence of the substrate.From the course of thrombin activity and the prevailing decay constants, the course of prothrombin conversion velocity can be calculated. Prothrombin conversion was seen to be inhibited at high (>500 μM) substrate concentrations only, and experimental conditions are found under which the inhibition of the clotting process by the substrate is negligibleThe amidolytic activity is the sum of the activities of free thrombin and of the α2 macroglobulin-thrombin complex formed. Via a mathematical procedure the amount of SQ 68 that has been split by thrombin alone and not by the a2 macroglobulin-thrombin complex, can be derived from the course of the optical density.The total amount of SQ 68 eventually split by thrombin alone is proportional to the surface under the thrombin generation curve, i. e. to the time-integral of free thrombin. This value, that we call the thrombin potential (TP), directly indicates how much of any physiological substrate can potentially be split by the thrombin being generated in the plasma.

SummaryThe area under the thrombin generation curve (the endogenous thrombin potential; ETP) has been proposed as a parameter for plasma-based hypercoagulability and to monitor anticoagulant treatment. We present an ETP assay for the routine laboratory using a centrifugal analyser. Throughput is 30 samples/h, within and between run imprecision is 4-5.6%. Suitable substrates were developed for the ranges of 10-500% and 2-100% of normal.Independent of tissue factor concentration (if >4 pM), the normal value of the extrinsic ETP is 384.8 ±51.7 nM.min. The intrinsic ETP, triggered by ellagic acid, is 414 ± 41 nM.min.The ETP is decreased to 15 and 35% of normal by oral anticoagulation (INR 2.5-4.0) and by heparin administration (APTT 1.5-2.5 X control).The ETP is increased in untreated subjects with congenital antithrombin deficiency and in women using oral contraceptives. In deep vein thrombosis (phlebographically confirmed), it is increased by 29.4% (extrinsic) and 53% (intrinsic). In (angiographically assessed) coronary artery disease the increase is by 10% and 17% respectively.

Background Generation of active procoagulant cofactor FVa and its subsequent association with the enzyme FXa to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied using plasma, whole blood and purified systems. Here we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B-domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. In line, tick feeding is impaired on TIX-5 immune rabbits displaying the in vivo importance of TIX-5. Conclusions Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. Based on our data we propose a revised blood coagulation scheme wherein direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.