Single Amino Acid Substitution (Asp111Ala) in Coagulation Factor Va Causes Subunit Dissociation and Disrupted Calcium and Copper Binding.
Bibliographic record
Abstract
Abstract Factor V (FV) is the essential inactive precursor of blood coagulation cofactor Va (FVa). FV is synthesized as a single chain protein that is activated at sites of injury by thrombin to FVa. As a ternary complex with the serine protease factor Xa and an anionic phospholipid (aPL) membrane in the presence of calcium (prothrombinase), FVa functions to accelerate the rate of prothrombin to thrombin conversion, the critical step in hemostasis. FVa is a non-covalent heterodimer composed of a heavy (FVaH) and a light subunit (FVaL). It has been well established that FVa requires calcium to stabilize a functional subunit interaction, although copper is also known to bind. A 1:1 divalent cation:FVa stoichiometry has been reported for both metals. Despite the importance of FV few studies have investigated the specific amino acids and divalent cations that mediate the FVaH-FVaL association. In order to understand the role of divalent cations in maintaining the FVa subunit interaction and consequent function, we investigated the effect of mutating FV Asp111 to Ala (D111A), which was suggested by our previous work, others’ crystallography studies and homology to coagulation factor VIII, to potentially participate in calcium binding. Compared to wild type recombinant FV (wtFV), there was no difference in the kinetics of thrombin-mediated conversion of purified FVD111A to FVaD111A assessed by gel electrophoresis. Binding to small unilamellar aPL-containing vesicles was followed by light scattering and also showed no change. However, the cofactor activity of FVD111A was observed in clotting and purified prothrombinase chromogenic assays to be significantly inhibited (39% and 27% reduction, respectively). When the wtFV or FVD111A was pre-activated by thrombin (i.e. 2-stage assay), markedly greater inhibition was observed (94% and 73% reduction, respectively). The 1-stage/2-stage discrepancy seen here can be explained by spontaneous dissociation of subunits in FVD111A upon activation. Indeed, thrombin activation of FVD111A was shown by light scattering to result in rapid dissociation of subunits even in the presence of excess calcium. To quantitatively investigate the direct effect of the mutation on divalent cation coordination, graphite furnace atomic absorption spectrometry was utilized to measure the amount of bound calcium and copper. As predicted, calcium binding to FVD111A was severely inhibited resulting in a stoichiometry of <0.16:1. Interestingly, the stoichiometry of copper binding was also appreciably reduced to ∼0.5:1. These data demonstrate for the first time that a single point mutation can result in the spontaneous subunit dissociation of purified FVa. The finding that mutation of Asp111 disrupted not only the coordination of calcium but also that of copper, suggests that interdependent metal ion binding sites may contribute to FVa configuration and function.
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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 | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| 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".