Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin–Thrombomodulin Complex
Bibliographic record
Abstract
Thrombin activity, inhibition, and localization are regulated by two exosites that flank the active site. Substrates, cofactors, and inhibitors bind to exosite 1 to promote active site access, whereas exosite 2 interactions hold thrombin on cells, platelets, and proteins. The exosites also serve allosteric roles, whereby ligand binding alters thrombin activity. Previously, we showed that ligands that bind exosite 2 attenuate the exosite 1-mediated interaction of thrombin with fibrin, demonstrating allosteric connection between the exosites. To determine the functional consequences of these inter-exosite interactions, we examined the effect of exosite 2 ligands on thrombin's interaction with thrombomodulin, a key cofactor that binds exosite 1 and redirects thrombin activity to the anticoagulant protein C pathway. Exosite 2-directed ligands, which included the HD22 aptamer, glycoprotein 1bα-derived peptide, and fibrinogen γ'-chain peptide, reduced the level of exosite 1-mediated thrombin binding to the thrombomodulin peptide consisting of the fourth, fifth, and sixth epidermal-like growth factor-like domains, decreasing affinity by >10-fold, and attenuated thrombomodulin-dependent activation of protein C by 60-80%. The ligands had similar effects on thrombin-mediated protein C activation with intact soluble thrombomodulin and with thrombomodulin on the surface of cultured endothelial cells. Their activity was exosite 2-specific because it was attenuated when RA-thrombin, a variant lacking exosite 2, was used in place of thrombin. These results indicate that additional reactions mediated by exosite 1 are amenable to regulation by exosite 2 ligation, providing further evidence of inter-exosite allosteric regulation of thrombin activity.
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
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".