Enhanced Fibrinolysis by Proteolysed Coagulation Factor Xa Is Inhibited by Rivaroxaban
Bibliographic record
Abstract
Abstract Abstract 3352 Introduction: Our lab has previously demonstrated that protease-modulated clotting factor Xa (FXa) acts as an accelerator of tissue plasminogen activator (tPA) to enhance clot lysis in vitro. This activity is facilitated by two specific plasmin cleavages of FXa in the protease domain. The first excises a 3 kDa C-terminal peptide to yield FXa-beta. The second cleavage is at Lys330, yielding non-covalently associated fragments of 33kDa and 13kDa, termed Xa33/13. When we incorporated a chloromethyl ketone (cmk) into the active site of FXa, Xa33/13 was not produced and tPA cofactor function was attenuated. Rivaroxaban (rxbn) is a new generation anticoagulant now approved for treatment of deep vein thrombosis (DVT) in Canada. Similar to the cmk but with higher specificity, rxbn binds directly to the active site of FXa. Here we addressed the possibility that rxbn may comparably affect the proteolytic modulation of FXa and its subsequent participation in fibrinolysis. Methods: Purified FXa with or without rxbn was incubated with plasmin and visualized by Coomassie blue protein staining. The FXa cleavage profile was also evaluated by western blot analysis in normal plasma, plasma from a patient taking rxbn for treatment of DVT, and normal plasma spiked with rxbn (5μM). Innovin was used to activate FX to FXa and initiate clot formation in plasma supplemented with therapeutic tPA (50 nM). The effect of rxbn on FXa-enhanced purified fibrin clot lysis in the presence of tPA (0.1 ρM) and plasminogen (0.6 μM) was monitored by Rayleigh scattering. In the absence of fibrin, chromogenic assays (S-2251) were used to evaluate the effect of rxbn on FXa during the tPA-dependent conversion of plasminogen to plasmin. The enhancement of plasmin generation was furthermore characterized according to the FXa proteolytic profile by western blot. Results: The FXa fragments produced by plasmin-mediated proteolysis were clearly altered in the presence of rxbn, such that generation of the fibrinolytic species, Xa33/13, was inhibited. As expected, western blot analysis of normal plasma samples showed that there was a rapid conversion of FXa-beta to Xa33/13. This differed in rxbn patient plasma in which the conversion to Xa33/13 was attenuated and FXa remained, as supported by the purified FXa cleavage experiment. Due to occupancy of the active site, formation of the FXa-antithrombin (XaAT) complex was reduced. This physiological inhibition complex is generated immediately after FX activation and has been recently shown by our lab to be involved in the generation of Xa33/13 in plasma. Confirming the results observed with patient plasma, normal plasma spiked with excess rxbn (∼5 μM), also showed reduced XaAT generation and subsequent inhibition of FXa conversion to Xa33/13. Both plasmas containing rxbn had a 28 kDa Xa-derived fragment. This fragment was previously reported by us to appear after prolonged treatment with plasmin and correlated to loss of tPA cofactor function. As predicted from the altered cleavage pattern caused by rxbn, turbidity assays showed the enhancement of fibrinolysis by FXa was profoundly inhibited in the presence of saturating rxbn. Chromogenic assay results suggest that the enhancement of tPA-dependent plasminogen activation by FXa was significantly delayed in the presence of rxbn causing this reduction of fibrinolytic FXa cofactor function. Western blot analysis of the chromogenic assay samples linked this delay to the inhibition of Xa33/13 generation. These observations suggested that rxbn interferes with the initial phase of plasmin generation and subsequent fibrinolysis, by altering the proteolytic modulation of FXa by plasmin. Conclusions: In our “auxiliary cofactor model” of fibrinolysis, FXa and its plasmin-derived fragment, Xa33/13, act as tPA accelerators in addition to fibrin to enhance plasmin generation. Here we have shown that the incorporation of rxbn into the active site of FXa prevents the conversion of FXa to Xa33/13 and consequently inhibits enhancement of tPA-dependent plasmin generation and clot lysis. Molecular modeling suggests that the rxbn binding site is very near Lys330, which may explain why plasmin-mediated proteolysis to Xa33/13 is altered. While these data do not have bearing on the favourable anticoagulant properties of rxbn, they may highlight an unforeseen inhibitory effect on fibrinolysis. Disclosures: Lee: Bayer Inc.: Honoraria.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".