Abstract 38: Identification of Thrombomodulin Binding Sites on Thrombin Activatable Fibrinolysis Inhibitor that Mediate Accelerated Activation by Thrombin
Bibliographic record
Abstract
Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM), or plasmin to generate the enzyme activated TAFI (TAFIa). TAFIa possesses basic carboxypeptidase activity which down-regulates fibrin clot lysis by removing carboxyl-terminal lysine residues from partially degraded fibrin thereby attenuating the positive feedback in the fibrinolytic cascade. Accumulating evidence from several studies suggests that TM and TAFI make direct contacts such that the enhancement of TAFI activation by TM is through binding at sites remote from the activation site (Arg92-Ala93). The elements of TAFI structure that allow accelerated activation of thrombin by TM are largely unknown. Therefore, alanine scanning mutagenesis of surface-exposed charged residues on TAFI was used to identify sites that mediate acceleration of activation by TM and which thus may indicate sites where TM binds to TAFI directly. The rates of activation by thrombin of the variants was measured in the presence or absence of TM. The variants R12A, E28A and R15A exhibited the lowest enhancement of catalytic efficiency in the presence of TM with decreases of 3.0-fold, 3.2-fold and 2.6-fold, respectively, compared to wild-type. The variants D75A/E77A/D78A, E106A, E112A/E116A, R12A/R15A and D54A/D56A showed about half the extent of rate enhancement of wild-type. On the other hand, the variants E99A and E116G exhibited approximately 2.0 to 2.5- fold increases in rate enhancement which may indicate increased binding between TAFI and TM. We determined the antifibrinolytic potential of each variant using an in vitro plasma clot lysis assay. The variants that showed reduced activation by thrombin/TM correspondingly showed impaired stimulation of antifibrinolytic potential by TM. Overall, the data indicate that the variants that showed the strongest TM dependence were focused around the activation peptide which would be near where the C-loop of EGF-3 of TM would contact TAFI. These residues contribute to the increased efficiency of TAFI activation by thrombin.
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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".