Using a range of TF and phospholipid concentrations in the presence and absence of protein C pathway inhibitors, previous studies have correlated VTE risk with elevated peak height (18
), ETP (20
) or both (19
). Other studies have focused on the LT, rate, and peak as an indicator of circulating TF and prothrombotic disease (19
). Although differences in findings between studies have been attributed to differences in assay conditions, few studies have contrasted different assay conditions within a single design, and even fewer under hyper
coagulable conditions. It has not been explicitly shown that all conditions provide equal sensitivity and
specificity to detect hypercoagulability. Given limited plasma sample sizes and limited availability of samples from large-scale epidemiologic studies of hypercoagulability, empirical evidence for the use of specific conditions and parameters is essential for designing prospective studies of plasma hypercoagulability. Our study design permitted the direct
comparison of conditions and parameters to identify those most sensitive and specific to factor-induced hypercoagulability. To our knowledge, this work is the first comprehensive, systematic comparison of these conditions in a single study.
Our analysis shows that in PFP, factors XI, IX, VIII, X, prothrombin, and fibrinogen significantly increased peak height and ETP, though increases were proportionally larger for peak height than ETP. Whereas the baseline peak height of PFP(5 pM TF) was 74% higher than that of PFP(1 pM TF) (344 nM vs. 197 nM, respectively, ), the apparent “maximum peak height” in PFP(5 pM TF) plus elevated factors XI, IX, VIII, X, or V up to 400% was only 23% higher than that in PFP(1 pM TF) plus elevated factors (396 nM vs. 322 nM, respectively). These findings suggest that the maximum observable difference between peak height in normal and “hypercoagulable” PFP was reduced in assays initiated with higher TF (15% for PFP(5 pM TF) vs. 63% for PFP(1 pM TF). Similarly, the maximum ETP in PFP(5 pM TF) with elevated factors XI, IX, VIII, X, or V (1925 nM*min) was only 3.8% higher than that in PFP(1 pM TF) (1854 nM*min). Thus, the maximum observable difference in ETP between PFP and “hypercoagulable” PFP was reduced in PFP(5 pM TF) (9.7%) versus PFP(1 pM TF) (18%). This difference is even smaller than that seen for peak height. The exception to these results is in PFP containing elevated prothrombin. Prothrombin (200%) significantly increased peak height and ETP in both in both low (1 pM) (1.3-fold and 1.6-fold, respectively) and high (5 pM) (1.4-fold and 1.7-fold, respectively) TF. These findings suggest that the mechanism limiting thrombin generation in this system results, at least in part, from the prothrombin concentration and indicates that peak height and ETP are exquisitely sensitive to hyperprothrombinemia.
It is widely accepted that PRP may provide greater physiologic relevance in assays of this nature, however studies of PFP persist, in part because of logistical limitations associated with the use of fresh PRP. Our study demonstrates that CAT assays of PFP and PRP show different trends in LT and TTP, but provide qualitatively similar data on peak height and ETP. Thus, studies comparing the presence and absence of platelets are likely to provide consistent information on peak height and ETP, but inconsistent findings on LT and TTP. Differences in response of LT or TTP in PFP and PRP may, however, provide important mechanistic information on the role of platelets and platelet abnormalities in thrombosis.
Epidemiologic studies have correlated elevated levels of factors XI, IX, VIII, X, prothrombin, or fibrinogen, but not factor V, with increased VTE risk (6
). In this regard, CAT appears specific to hypercoagulability due to these factors. It is important to note, however, that the magnitude of change in thrombin generation did not correlate with the degree of thrombotic risk associated with each factor. For example, elevated prothrombin produced a large linear increase in peak height. However, thrombotic risk associated with elevated prothrombin is relatively small (OR ~2.1) (7
). Discrepancies between the thrombin generation measurements and clinical risk in the case of elevated prothrombin may result from difficulties in measuring (pro)thrombin concentrations in excess of the antithrombin level present in the plasma. Additionally, although 200% fibrinogen increased the peak height and ETP, its role in VTE risk is controversial. It is unclear whether the effects of fibrinogen on thrombin generation parameters resulted from changes in thrombin generation, itself, or in the ability of fibrinogen (“antithrombin I”) to bind thrombin and preserve its proteolytic activity towards small molecular substrates. Thus, CAT may demonstrate disproportionately high peak height and ETP for patients with lower overall risk, reducing the specificity and predictive value of this technique in certain patients.
As a means of distinguishing effects of procoagulant factors on CAT, we note that whereas several clotting factors significantly increased peak height and ETP, these factors had different and sometimes unique effects on other parameters. For example, in PFP(1 pM TF)
, elevated levels of factors IX, X, and prothrombin significantly increased peak height and ETP. However, factor IX did not affect the LT, factor X significantly decreased the LT, and prothrombin prolonged the LT in low but not high lipid concentrations (data not shown). Thus, analysis of multiple experimental conditions (e.g.
, varied lipid concentrations) and/or parameters may be helpful in discerning factors contributing to abnormal thrombin generation in certain PFPs. Indeed, Tripodi et al.
recently suggested that use of three abnormal thrombin generation parameters (LT, peak height, and ETP) improves the identification of patients at risk of recurrent VTE versus
analysis based on a single parameter (37
Comparisons of findings between centers have been difficult, as inter-center variability is high partly due to the use of “in house” reagents and protocols. Using a particularly elegant study design, Dargaud et al. (2007)
showed that the use of different TF and phospholipid sources produces large variability in CAT, but standardising conditions significantly reduces center-to-center variability (27
). We used commercially-available reagents from the CAT manufacturer, which may offer consistency in results and enable the continued evaluation of these conditions in future studies.
This study has several limitations. First, although thrombomodulin may be helpful in characterising plasmas with proteins C or S deficiency or factor V Leiden, the physiological thrombomodulin concentration has not been established because it is primarily a cell-associated protein. Thus, the concentration of soluble thrombomodulin that provides the most clinically-useful information has not been determined. Our data suggested that thrombomodulin increased the inter-assay %CV and dampened the effects of 200% fibrinogen at low TF, but did not significantly impact the ability to detect hypercoagulability from the other factors tested. Thus, its general use for detecting protein C pathway abnormalities is compatible with assays used to detect elevated levels of clotting factors. Second, although epidemiological studies have suggested elevated levels of certain clotting factors independently increase VTE risk (6
), the factor levels we tested were generally higher than those reported in these studies. Of note, however, prothrombin levels as high as 500% of normal have been reported in patients with type 2 diabetes (38
). CAT's ability to detect elevated prothrombin suggests abnormal thrombin generation in these patients would be readily detected by this technique. Third, our study design compared frozen/thawed, pooled PFP with individual PRP, which may emphasise differences in %CV between PFP and PRP. Fourth, as opposed to tests of individual factor levels, CAT offers the advantage of testing global haemostatic potential. Effects of multiple abnormal coagulation factor levels on thrombin generation may be additive, synergistic, or reflect only the effects of the most limiting factor. Additional studies are warranted to fully appreciate the effects of multiple factor abnormalities on thrombin generation measured with this technique. Finally, it is not clear whether CAT recapitulates the in vivo
pathologic effects of procoagulant factors; other mechanisms besides thrombin generation may contribute to thrombotic risk.
In sum, we have shown that CAT's ability to detect elevated factors varies between factors and depends on the assay conditions. The largest changes in thrombin generation in response to elevated factors XI, IX, VIII, X, or prothrombin were seen in peak height and ETP in PFP(1 pM TF). Smaller changes were observed in PFP(5 pM TF) and PRP(1 pM TF). Therefore, monitoring the peak height and/or ETP following initiation of clotting in PFP with 1 pM TF is most likely to detect hypercoagulability due to increased procoagulant factor levels.
What is known about this topic?
- Thrombin generation tests can detect hypocoagulability. Significant interest lies in determining their ability to detect hypercoagulability.
- Previous studies have correlated primary or recurrent VTE risk with abnormal thrombin generation; however, the experimental conditions differ significantly in these studies.
- Standardisation of thrombin generation tests reduces the variability of results and is necessary for its continued clinical development.
- The experimental conditions providing the highest sensitivity and specificity for detecting hypercoagulability have not been identified.
What does this paper add?
- The ability to detect elevated factors varies between factors and depends on the assay conditions.
- The relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX, VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF.
- Monitoring the peak height and/or ETP following initiation of clotting in PFP with 1 pM TF is most likely to detect hypercoagulability due to increased procoagulant factor levels.
Our findings support efforts to standardise reagents (TF and lipid concentrations) (27
) to reliably achieve the assay conditions necessary for maximal sensitivity. Our data also confirm effects previously seen in normal plasma, but importantly, extend these findings to hypercoagulable situations. Identification of conditions that best identify hypercoagulability and predict VTE warrants further investigation. In vivo
, VTE risk likely depends on a combination of increased procoagulant and decreased anticoagulant activities, and/or other pathologic mechanisms. In a clinical setting, the concerted use of several different assay conditions may be necessary to identify patients with distinct clinical phenotypes, not unlike the use of both aPTT and PT to diagnose factor deficiencies.