Inhibition of the Thrombin-Thrombomodulin by a Covalent Antithrombin-Heparin.
Bibliographic record
Abstract
Abstract Endothelial thrombin·thrombomodulin (IIa-TM) complex is capable of activating protein C anticoagulant. In vivo, TM can exist in two forms: one with a chondroitin sulfate (CS) (acidic) and one without (non-acidic). TM binding has been shown to reduce the rate of IIa inhibition by antithrombin (AT) in the presence of unfractionated heparin (UFH), especially when the TM possesses a glycosaminoglycan chain. We have recently developed a covalent antithrombin-heparin (ATH) with high potency for inhibition of IIa bound to surfaces. In the present study we examined the role of the CS chain in the inhibition of IIa-TM complex by the novel anticoagulant, ATH, in comparison to AT + UFH. For this study, discontinuous second order rate constant (k2) inhibition assays of IIa-TM were performed at 37°C. IIa was incubated with TM for 30 min and inhibited using either 20nM AT + varying concentrations (0–6000nM) of UFH or a constant concentration of 10nM ATH. Residual IIa activity was measured at 405nm after stopping the reaction using a solution of polybrene and chromogenic substrate (S-2238). Second order rate constants (mean +/− SEM; n≥5) were calculated from plots of IIa-TM activity vs. time and differences determined using two-sample t-tests. Comparison of the second order rate constants for inhibition of IIa, IIa-TM(−CS), or IIa-TM(+CS) by AT + UFH (2.015 × 108 +/− 7.079 × 106 M−1min−1, 1.633 × 108 +/− 8.922 × 106 M−1min−1, and 5.248 × 107 +/− 5.693 × 106 M−1min−1, respectively) showed that the presence of TM reduced the rate of inhibition (with respect to IIa alone; p=0.012, p<0.001, respectively). Moreover, the presence of a CS on the TM inhibited IIa neutralization to a greater degree than when the CS is absent (p<0.001). Furthermore, ATH was a much faster inhibitor of IIa, IIa-TM(−CS), and TM(+CS) (1.486 × 109 +/− 1.193 × 108 M−1min−1, 1.370 × 109 +/− 1.246 × 108 M−1min−1, and 8.869 × 108 +/− 1.246 × 108 M−1min−1, respectively) compared to AT + UFH (p<0.001). CS removal by chondroitin ABC lyase (ABCase) showed that the rate of IIa-TM(+CS) inhibition significantly increased for both AT + UFH and ATH, while no significant change due to ABCase treatment was found for the rate of inhibition of IIa-TM(−CS) by either AT + UFH or ATH. We speculate that during IIa-TM(+CS) inhibition by ATH, the covalently linked heparin moiety may be able to repel (rotate) the CS chain in order for ATH to interact with the IIa-TM complex. In the case of AT + UFH, lack of permanent UFH linkage to AT may allow the CS to displace the UFH chain from binding both AT and IIa. Steric hindrance by CS chains remains a significant factor since the k2 values were lower for the AT + UFH or ATH reactions with IIa-TM(+CS) compared to those with IIa-TM(−CS). Given the rapid reaction rate with free or bound IIa, ATH significantly limits availability of IIa for coagulant or anticoagulant functions.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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) | 0.001 | 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".