Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.
Bibliographic record
Abstract
Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| 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) | 0.002 | 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".