Application of Thrombomodulin-Thrombin Generation to Diverse Abnormalities of the Protein C System

Background: Increased thrombin generation is an interesting candidate as a potential indicator of hypercoagulability and thrombosis risk. Increased thrombin generation using the calibrated automated thrombogram (CAT) has been reported in antithrombin deficiency but the CAT does not reflect protein C deficiency without the addition of thrombomodulin (TM). TM activates protein C (PC) in the CAT and reduces thrombin generation.

Objective: The objective of this study was to determine the capacity of the TM-augmented CAT to detect defects in various protein C system components; the lupus anticoagulant was also investigated as a cause of decreased protein C activation.

Methods: The CAT-TM was performed with reagents and methods per the manufacturer’s instructions using 5 pM tissue factor and TM (Stago). Other assays included: PC (chromogenic), free protein S (PS, LIA), activated protein C resistance (aPTT-based clotting assay), factor V Leiden (FVL PCR) and lupus anticoagulant (LA, dRVVT and Staclot LA, Stago). Platelet poor plasma samples were obtained from the biobank of a consented prospective inceptional cohort study of thrombosis and thrombophilia or a positive family history of either (ThromboPICS #05-0339); samples from healthy controls with no personal or family history of thrombosis or thrombophilia were collected on a consented protocol (#09-0816). Samples from participants on therapeutic inhibitors of factor Xa or thrombin were treated with appropriate neutralizers; no sample was tested on warfarin. Clinical data included gender, age (< 18 versus ≥ 18 years), history of thrombosis, timing of sample from most recent thrombosis (acute < 14 days; subacute 14-90 days; or chronic > 90 days) and use of anticoagulants at the time of the blood draw. Tests for violations of normality were negative. Therefore, results of cohorts versus controls were compared with independent sample t-test and subgroup analyses relative to control used One-Way ANOVA. Post-hoc comparisons of each subgroup to the controls were collected for multiple comparisons using the Bonferroni correction.

Results: Ninety-nine cases and 45 normal controls were studied. Overall half of the cases had a history of thrombosis and 66% of those with thrombosis were on no anticoagulation at the time of testing. Table 1 displays results of the CAT-TM in the 2 control and 6 study groups. Control adults and children showed a mean 50% thrombin reduction in thrombin generation with CAT-TM; overall, persons with protein C system defects showed approximately 25% reduction, with the least reduction of thrombin generation in the cohort of PC deficiency at 19%. Sensitivity of the CAT-TM was 100% for PC deficiency, 81% for LA positivity, 80% for PS deficiency and 72% for FVL positivity with no difference in degree of thrombin reduction between hetero- and homozygous FVL. In addition, we identified 14 individuals with CAT-TM results similar to protein C system defects but with confirmed normal PC, PS, FVL and LA tests. Although 9 of these unknowns had a personal (7) or family (2) history of thrombosis, five came from the healthy controls. The false positive results in 3 adult and 2 pediatric controls conferred a CAT-TM test specificity of 89%. Furthermore, analyses showed no differences in CAT-TM results related to gender, age group, history of thrombosis, age of clot at blood draw (acute, subacute or chronic) or use of anticoagulation at the time of blood draw.

Conclusions: The CAT-TM is a useful screening test for defects in the protein C system, including LA, although this test could not discriminate between heterozygous and homozygous FVL. The etiology of positive CAT-TM in normal individuals or individuals with a personal or family history of thrombosis without identified PC system defects is currently unknown and under ongoing investigation.