The Effects of Activated Protein C, Heparin, and Antithrombin On Thresholding Behaviour in Prothrombin Activation by Prothrombinase.

Abstract 2138

Poster Board II-115

The coagulation cascade results in considerable thrombin generation through a series of zymogen activations to enzymes initiated by factor VIIa-tissue factor complex. Thrombin generation consists of an initiation phase, where a small amount of thrombin (∼25 nM) sufficient to form a clot is made, and a propagation phase, which is characterized by a rapid increase (to ∼850 nM) and subsequent decay in the thrombin concentration. Thrombin generation is possibly described as under threshold limited control. Thresholding is observed in systems where a precursor in the presence of a stimulus gives rise to a response, which can both positively feedback and be inhibited. The threshold level of stimulus depends on the kinetics of feedback and inhibition. The coagulation cascade is intensely regulated by several mechanisms and inhibitors. In this study, prothrombin (the precursor) activation initiated by factor Xa (the stimulus) in the presence of factor V (the positive feedback loop) and antithrombin (the inhibitor) was investigated for threshold type behaviour. Reactions were started by adding factor Xa to the purified components prothrombin (140 nM), factor V (2 nM), phosphatidylcholine and phosphatidylserine vesicles (2 μM), CaCl₂ (5 mM), antithrombin (280 nM), and Z-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC, 400 μM), a fluorogenic thrombin substrate. Thrombin generation was observed as an increase in fluorescence over time due to Z-GGR-AMC hydrolysis. When thrombin generation ceased, the fluorescence plateaued. Under these conditions, the plateau in fluorescence was not a result of complete Z-GGR-AMC hydrolysis, but due to cessation of prothrombin activation. TableCurve 2D (Systat Software Inc., San Jose CA) was used to smooth the raw fluorescence data by drawing a best fit line, which then was used for further data manipulation. Three point running slopes over each time course then were calculated to produce rates of Z-GGR-AMC hydrolysis. The kinetic parameters, k_cat (2.77 s^-1), K_m (92.2 μM), and K_i (product inhibition constant, 233 μM), for thrombin mediated Z-GGR-AMC hydrolysis were used to convert each slope to a free thrombin concentration. In each experimental condition tested, there was no detectable thrombin generated at low factor Xa concentrations. At higher factor Xa concentrations, thrombin generation was transient: the thrombin concentration rapidly rose to a peak followed by its decay. Thrombin potential, defined as the area under the thrombin versus time curve, was calculated by approximating the thrombin peak as a series of trapezoids. The dose-response curve (thrombin potential versus factor Xa concentration) resembled a sigmoid: at low factor Xa concentrations, the thrombin potential was almost zero; as the factor Xa concentration increased, the thrombin potential increased rapidly to a plateau. Under the above conditions, no thrombin generation was detected from 0 pM to approximately 0.5 pM factor Xa. The thrombin potential increased from approximately 0.5 pM to 3 pM factor Xa and past 3 pM thrombin potential was relatively constant. This type of dose-response curve is expected if thresholding exists. Adding activated protein C (aPC) at concentrations ranging from 0 nM to 5 nM increased the threshold concentration of factor Xa from approximately 0.5 pM to 10 pM. In addition, increasing the antithrombin concentration from 280 nM to 560 nM increased the threshold concentration of factor Xa from approximately 0.5 pM to 3 pM. Lastly, adding heparin at concentrations ranging from 0 μg/mL to 2 μg/mL increased the threshold concentration of factor Xa from approximately 0.5 pM to 50 pM. Because the dose-response curve displays a marked increase in thrombin potential over a small range of factor Xa concentrations and this pattern shifts to higher factor Xa concentrations with added aPC, heparin or increased antithrombin, prothrombin activation by prothrombinase is likely a threshold limited occurrence with respect to the initiating factor Xa concentration and the threshold level changes with the kinetics of inhibition.