Investigation Into Mechanisms of Prothrombinase Complex Inhibition by a Covalent Antithrombin-Heparin Complex.
Bibliographic record
Abstract
Abstract Abstract 2136 Poster Board II-113 Introduction: Formation of the prothrombinase complex results in efficient generation of thrombin (IIa) by a vast enhancement of the factor Xa (Xa) reaction with prothrombin (II). However, the integration of Xa into the complex significantly decreases its availability for inhibition by antithrombin+heparin (AT+H). Our laboratory has developed a novel anticoagulant by covalently linking antithrombin to heparin (ATH). We have shown that ATH, compared to AT+H, significantly increased the rate of inhibition of clot-bound thrombin as well as several other coagulation factors in isolation. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase system. Methods: Pseudo first-order kinetic experiments for the inhibition of Xa in the prothrombinase complex were performed by reacting AT+H or ATH with CaCl2, phospholipid vesicles (PC/PS), factor Va (Va), II, specific IIa inhibitor Pefabloc and Xa in buffer. At specific time intervals, the reactions were neutralized with Na2EDTA, polybrene and Xa substrate (S-2222) in buffer, followed by measurement of residual enzyme activity. Second-order rate constants (k2) were calculated from semi-logarithmic plots of residual Xa activity remaining versus time. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of ATH, additional experiments were performed where components of the complex (II, PC/PS or Va) were removed prior to reaction with AT+H or ATH. Results: The k2value (× 108 M−1min−1) for the inhibition of Xa alone by AT+H was 1.47 ± 0.08. Incorporation of Xa into the prothrombinase complex significantly decreased this value to 0.67 ± 0.05, p<0.001. In comparison, ATH inhibited free-Xa and prothrombinase-complexed Xa at significantly faster rates than AT+H (2.83 ± 0.15 and 2.11 ± 0.38 (p<0.01), respectively). Relative to intact prothrombinase, rates for inhibition by AT+H of Xa in complexes devoid of PC/PS, Va or II significantly increased to 1.49 ± 0.08, 1.44 ± 0.08 and 1.24 ± 0.04, p<0.001, respectively. In contrast, removal of II from prothrombinase significantly decreased the k2 for Xa inhibition by ATH to 1.57 ± 0.04, p<0.05. Although removal of PC/PS from the system resulted in a higher k2 (2.79 ± 0.14, p<0.01), removal of Va resulted in no significant change (2.09 ± 0.09, p=0.79) for ATH reactions. Conclusion: Based on these results, we conclude that the prothrombinase complex hinders the inhibitory action of AT+H on Xa, whereas ATH is less affected. In fact, ATH inhibition of the prothrombinase complex was significantly higher than the inhibition rate for either free or prothrominase-bound Xa by AT+H. We speculate that, similar to inhibition of fibrin-bound thrombin, H in the non-covalent AT+H dissociate and bind to components within the prothrombinase that repel incoming AT+H and preventing Xa inhibition. These complexes cannot occur when AT is covalently linked to H, thus allowing for enhanced inhibition of prothrominase-bound Xa by ATH. Absence of II from the prothrombinase complex removes interference of AT+H reactions by any H complexes within prothrombinase, but may antagonize inhibition by the conjugate through unproductive interactions of ATH with uncovered sites close to Xa at the phospholipid surface. These findings reveal important features involved in the enhanced anticoagulant action of ATH against prothrombinase surface-bound enzymes. 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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| 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".