Monitoring low molecular weight heparins at therapeutic levels: dose-responses of, and correlations and differences between aPTT, anti-factor Xa and thrombin generation assays

Abstract

Background: Low molecular weight heparins (LMWH's) are used to prevent and treat thrombosis. Tests for monitoring LMWH's include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH's varying affinities for FXa and thrombin.

Aim: To examine the effects of two different LMWH's on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests' concordance.

Method: Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents.

Results: Methods' mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62-0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80-0.92).

Conclusions: aPTT displays a linear dose-response to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa's present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH's.

Conflict of interest statement

Competing Interests: One of the authors of this manuscript has the following competing interest: N. Tynngård is a part-time consultant to MediRox. The other authors have declared that no competing interests exist. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Boxplots of the four aPTT methods’ results for enoxaparin and tinzaparin in three different concentrations.

When comparing the contiguous groups with the same aPTT-method but different concentrations of LMWH, a significant difference in aPTT was found in all cases. Statistical significances between classes are shown in S1 File.

Figure 2. A comparison of enoxaparin and tinzaparin’s relative effects on aPTT.

Bland-Altman plot showing that the aPTT’s induced by tinzaparin ranged from on average 49% more than enoxaparin (when measured using the PTT-Automat reagent) to 66% more than enoxaparin (when measured using the MRX931 reagent (FOR: free oscillation rheometry).

Figure 3. Relationship between measured anti-FXa and the concentration of low molecular weight heparin (LMWH).

There is a strong linear correlation between anti-FXa results and the concentration of LMWH (dosed in anti-FXa units per ml). For enoxaparin and tinzaparin the correlation coefficients Rs are 0.97 and 0.96 respectively.

Figure 4. Correlations between aPTT and anti-FXa at varying concentrations of enoxaparin and tinzaparin, using various reagents.

Anti-FXa activity reflects the concentration of LMWH and the aPTT correlated well to this measure. The correlation for Hemochron Jr is slightly weaker than for the other reagents (see Table 2).

Figure 5. Thrombin generation at increasing concentrations of LMWHa, measured using the TGA RC reagent.

Thrombin generation is inhibited in a negative exponential manner by increasing doses of LMWH. There is a significant difference between Endogenous Thrombin Potential (ETP) at all contiguous concentrations and between ETP for the two reagents at each individual concentration (PaLow Molecular Weight Heparin. bETP: Endogenous Thrombin Potential.

Figure 6. Relationship between aPTT measured using various reagents, and the logarithm of thrombin generation.

See Table 3 for correlation factors and regression lines. There is a negative linear relationship between aPTT and log10(ETP). Tinzaparin results in prolonged aPTT results compared to enoxaparin in the whole blood analyses (FOR and Hemochron Jr) at any given level of thrombin generation.

Figure 7. Relationship between anti-FXa activity and thrombin generation.

Anti-FXa activity is strongly negatively correlated to the logarithm of ETP (Endogenous Thrombin Potential): Rs for enoxaparin and tinzaparin are −0.93 and −0.94 respectively. Anti-FXa activity is more prolonged by enoxaparin than tinzaparin at any given level of thrombin generation.