Major bleeding during oral anticoagulant therapy associated with factor V activation by factor Xa.

OBJECTIVE: Plasma thrombin generation (TG) provides important information on coagulation status; however, current TG output parameters do not predict major bleeding of patients on anticoagulants. We recently reported that factor V (FV) activation by factor X (FX)a contributes importantly to the initiation phase of TG. Here we investigated how this pathway varies in the normal population and whether FXa-mediated activation of FV is associated with major bleeding in patients on anticoagulant therapy. APPROACH: We employed TIX-5, a specific inhibitor of FV activation by FXa, to estimate the contribution of FXa-mediated FV activation to tissue factor (TF)-initiated TG. RESULTS: We show that the contribution of this pathway to plasma TG varies considerably in the normal population, as measured by the time needed to form the first traces of thrombin (TG lag time; mean prolongation by TIX-5 40%, range 0%-116%). Comparing patients on vitamin K antagonists (VKA) of the BLEED study (263 patients with and 538 patients without major bleeding), showed a marked prolongation in the median TG lag time in the presence of TIX-5 in cases (12.83 versus 11.00 minutes, P = 0.0030), while the TG lag time without TIX-5 only showed a minor although significant difference (5.83 vs. 5.67 minutes, P = 0.0198). The TIX-5 sensitivity (lag time + TIX-5/lag time + vehicle) in the upper quartile was associated with a 1.62-fold (95% confidence interval 1.04-2.52) increased risk of major bleeding compared to the lowest quartile. CONCLUSION: A greater dependence on FXa-mediated activation of FV of TG is associated with increased risk of major bleeding during VKA therapy.

Structure-function of anticoagulant TIX-5, the inhibitor of factor Xa-mediated FV activation.

BACKGROUND: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. OBJECTIVE: We aimed to unravel the structure-function relationships of TIX-5. METHOD: We used a structure model generated based on homology with the allergen Der F7. RESULTS: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. CONCLUSION: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.

The intestinal microbiome potentially affects thrombin generation in human subjects.

BACKGROUND: The intestinal microbiome plays a versatile role in the etiology of arterial thrombosis. In venous thrombosis, driven chiefly by plasma coagulation, no such role has yet been established. We hypothesized that the intestinal microbiome composition affects coagulation in humans. METHODS: We used healthy donor fecal microbiota transplant (FMT) to experimentally change the microbiome composition in metabolic syndrome patients. Thirty-five subjects were randomized in a blinded fashion to healthy donor FMT or autologous FMT as a control in a 2:1 ratio. We measured thrombin generation at baseline and after 6 weeks using automated calibrated thrombinography, and we determined plasma abundance of 32 coagulation related proteins using a targeted mass spectrometry-based quantitative proteomics assay with heavy labeled internal standards. RESULTS: Healthy donor FMT prolonged the thrombinography lag time (median delta 0.0 versus 0.25 minutes, P = .039). The other thrombinography parameters showed no significant difference. Unsupervised cluster analysis suggested overall downregulation of coagulation related plasma proteins in subject clusters containing predominantly subjects that had a metabolic response to healthy donor FMT. FMT treatment status itself showed no clear clustering pattern with up- or downregulation, however, and proteins did not cluster according to an apparent biological grouping. DISCUSSION: A single healthy donor FMT tends to modestly suppress the onset thrombin generation in metabolic syndrome patients, representing initial proof-of-principle that the intestinal microbiota composition might affect the coagulation system in humans. The findings merit external validation as a role for intestinal microbiota in coagulation can have clinically important implications.

Use of DOAC Stop for elimination of anticoagulants in the thrombin generation assay.

Calibrated automated thrombography (CAT) is useful in monitoring the anticoagulant status of patients treated with direct oral anticoagulants (DOACs). This as well as other applications of the CAT are hampered by the wide inter-individual variation, making the diagnosis of the anticoagulant status of a patient on DOAC difficult when using normal pooled plasma as a reference. With dabigatran, the CAT is further hampered, as this direct thrombin inhibitor also inhibits the calibrator that is used in CAT. In this study we examined the added value of the universal DOAC adsorbent DOAC Stop in CAT. For this, we used normal pooled plasma spiked with apixaban, dabigatran, edoxaban or rivaroxaban, and performed CAT with 5 pM tissue factor. DOAC Stop effectively removed DOACs from plasma, leaving the DOAC Stop-treated plasma slightly more procoagulant compared to sham treated, non-anticoagulated plasma. Examining levels of natural coagulation inhibitors revealed a slight reduction in tissue factor pathway inhibitor upon DOAC Stop treatment. When DOAC Stop-treated plasma was used in the calibrator wells, normal unaffected calibration curves were observed, even when dabigatran was present. In conclusion, DOAC Stop can be used to abolish dabigatran influences of anticoagulated plasma when used in the calibrator wells. Also, the anticoagulant status of a DOAC treated patient can be diagnosed simply by comparing untreated plasma with the same plasma sample treated with DOAC Stop. Using this approach, a minor DOAC-independent increase in CAT response in the DOAC Stop-treated sample should be taken into account.