Abstract
Patients undergoing oral anticoagulant therapy (OAT) with coumarins have reduced plasma levels of vitamin K-dependent clotting factors. The primary laboratory test for monitoring OAT is the prothrombin time (PT), in which clotting is initiated by tissue factor (TF). Clotting factors that contribute to the PT, and whose levels respond to OAT, are factor VII (FVII), factor X (FX), and prothrombin, although they are not suppressed to the same extent. Thromboplastin reagents (the source of TF activity in PT tests) can vary dramatically in their sensitivities to the effects of OAT. A calibration system, the International Sensitivity Index (ISI), is widely used to correct the PT for variable thromboplastin sensitivity, but discrepant responses by reagents of similar ISI have been reported. We have undertaken studies aimed at understanding which factors control the sensitivity of thromboplastin reagents, with a goal of creating “designer thromboplastins” whose sensitivities to specific clotting factors can be individually tailored. Thromboplastin reagents were prepared by reconstituting recombinant human TF into phospholipid vesicles containing varying amounts of phosphatidylcholine, phosphatidylserine (PS), and phosphatidylethanolamine (PE). Thromboplastins containing low levels of PS and high ionic strength had the highest sensitivity to OAT (i.e., lowest ISI). PE shifted the dose-response such that lower levels of PS were required to obtain the same ISI value. These studies demonstrate that multiple combinations of phospholipid composition and ionic strength can be used to produce reagents of identical ISI. We hypothesized that reagents of identical ISI values but different composition could have very different responses to changes in the levels of individual coagulation factors. Accordingly, thromboplastin reagents of varying composition were evaluated for their responses to deficiencies of FVII, FX and prothrombin. PT tests were performed using pooled normal plasma mixed with individual factor-depleted plasmas to yield 10%, 3%, 1% or 0.3% of the normal level of the specific clotting factor. Responses of thromboplastin reagents to individual factors were compared by plotting the clotting times obtained with these plasmas on log-log scales versus the percent factor level and fitting lines to the data by linear regression. Interestingly, altering the composition of the thromboplastin reagents dramatically and independently altered their sensitivities to individual clotting factors. For example, increasing ionic strength had no impact on the response to FVII, but markedly enhanced the response to prothrombin deficiency. Furthermore, the effect of changes in ionic strength on specific factors levels differed depending upon the phospholipid composition. These studies demonstrate that thromboplastin reagents of dissimilar composition but nearly identical ISI values can have very different sensitivities to deficiencies in FVII, FX, or prothrombin, so reagents of identical ISI do not necessarily respond to the factor deficiencies induced by OAT in an identical fashion. These studies evaluated samples with isolated individual factor deficiency, whereas patients on OAT have combined factor deficiency and therefore have more potential for discrepancy in PT responses between reagents. Controlling the responsiveness of thromboplastin reagents to deficiencies in individual clotting factors may therefore be desirable for monitoring OAT and for the other clinical diagnostic uses to which PT tests are commonly applied.
Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact
