Factor Xa-Antithrombin Complex Enhances Tissue Plasminogen Activator-Mediated Clot Lysis
Bibliographic record
Abstract
Abstract Abstract 1180 Introduction: The current model of fibrinolysis accepts fibrin as the only cofactor involved in accelerating tissue plasminogen activator (tPA)-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, intact FXa-alpha is converted to FXa-beta and then to non-covalently associated fragments of 33kDa and 13kDa (Xa33/13). Contrary to fibrinolysis dogma, Xa33/13 is a potent accelerator of tPA-dependent dissolution of purified fibrin because localized C-terminal lysine binding sites for plasminogen and tPA are exposed. Here we investigated whether Xa33/13 is produced in plasma during clot formation and dissolution. The normal fate of FXa produced in plasma is rapid irreversible association with its inhibitor, antithrombin (AT). Therefore, the effect of complex formation with AT on FXa fibrinolysis accelerator function was also evaluated. Methods: Purified FXa and Xa-AT plasmin cleavage profiles were monitored by Coomassie blue protein staining or by western blot using FX and AT-specific monoclonal antibodies. N-terminal sequencing was performed by automated Edman degradation to map FXa and AT cleavage sites.125I-plasminogen was used to probe plasminogen binding sites by ligand blotting. To follow purified plasminogen activation by tPA, plasmin generation was evaluated chromogenically (S2251). Fibrinolysis of plasma clots were measured by turbidity. Results: FX western blot analysis of plasma induced to clot showed the rapid formation of a band corresponding to a Xa-AT complex that correlated to the appearance of Xa33 antigen. An AT western blot detected the simultaneous appearance of a band of approximately 13 kDa higher apparent molecular weight than AT. Xa13 is known to contain the AT covalent linkage site, suggesting a Xa13-AT adduct. Purified Xa-AT was cleaved at least10-fold faster by plasmin compared to FXa and generated a Xa33/13 species. By electophoresis the resulting Xa33 derived from Xa-AT or FXa was not distinguishable, but sequencing revealed different cleavage sites at Lys-Met339 and Lys-Gly331, respectively. Cleavage of Xa-AT by FXa also liberated a Xa33 fragment with cleavage at Arg-Thr337. Sequencing confirmed the identity of the covalent, Xa13-AT, and non-covalent, Xa33/13-AT, complexes. Ligand blots revealed 125I-plasminogen binding to the Xa33 subunit of Xa33/13-AT generated by plasmin cleavage, which is consistent with the predicted exposure of a new C-terminal Lys338. Chromogenic assays demonstrated Xa-AT enhanced tPA-dependent plasmin generation by 7-fold. An excess of Xa-AT to overcome the endogenous FX and AT in plasma significantly enhanced the rate of fibrinolysis. Conclusions: For the first time we demonstrate the generation of Xa33/13 in plasma, supporting its physiological relevance as a tPA cofactor. Within this context, a novel function for AT is furthermore suggested by our results. In complex with FXa, AT accelerates the rate of conversion of FXa into the fibrinolytic form, Xa33/13. The subsequent exposure of new C-terminal lysine binding sites for plasminogen enhances fibrinolysis. These results are consistent with an auxiliary cofactor model of fibrinolysis in addition to the accelerating role of fibrin. Disclosures: No relevant conflicts of interest to declare.
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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 | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 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".