Novel mechanisms of cell-based blood coagulation: Roles of integrin β3 psi domain and its L33P polymorphism
Bibliographic record
Abstract
Abstract Integrin αIIbβ3 is the essential platelet receptor for hemostasis and thrombosis. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence located near the N-terminus of the β3 subunit. We recently reported that the PSI domain possesses two CXXC motifs and contains endogenous thiol-isomerase activity. Targeting the PSI domain via our novel monoclonal antibodies (mAbs) reduced platelet aggregation in anticoagulated platelet-rich plasma in-vitro and inhibited thrombus formation under non-anticoagulated conditions in-vivo (Blood, 2017). This suggests that these anti-PSI mAbs may attenuate the PSI domain-mediated blood coagulation. Notably, the L33P polymorphism (Human Platelet Antigen 1) within the PSI domain has been associated with a greater risk of cardiovascular disease. However, the roles of the PSI domain and its L33P polymorphism in blood coagulation were not conceived and have never been explored. Recombinant PSI (rPSI) and recombinant L33P (rL33P) proteins were generated using a BL21 E.Coli. Using a combination of reduced RNase, insulin β-chain reduction and MPB binding assays, we found that rL33P possessed greater thiol isomerase activity compared to rPSI. Using thromboelastography and clot retraction assays, we found that clot formation time was decreased in rPSI-treated blood, with rL33P further enhancing this phenomenon. Bacitracin attenuated these changes, elucidating that the role of PSI within coagulation is due to its thiol isomerase activity. Using scanning electron microscopy imaging to examine fibrin formation and structural phenotypes, we found smaller fibrin strand formation and increased branching for rPSI treatment groups. Moreover, rL33P treatment further increased branching, representing increased end-point coagulation. Interestingly, using APTT tests, an intrinsic coagulation pathway test, we found no significant difference between rPSI or rL33P-treated platelet-poor-plasma clot formation. However, PT assays, an extrinsic coagulation pathway-specific test, displayed significantly faster clotting times for rPSI and rL33P-treated platelet-poor-plasma. Recombinant tissue factor addition further enhanced extrinsic coagulation pathway activation for rPSI and further for rL33p. Excitingly, using biochemical binding assays isothermal titration calorimetry and biolayer interferometry, we discovered rPSI and rL33P directly bind tissue factor. Additionally, using circular dichroism, we found calcium to be integral in these interactions. In all models, rL33P presented tighter binding to tissue factor than rPSI, possibly through its enhanced thiol isomerase activity. Alphafold and pie-mole theoretical docking software's modeled alternative binding orientations between PSI and L33P proteins with tissue factor and cations. Our results first demonstrate the PSI domain as a novel contributor to blood coagulation (i.e. cell-based model of coagulation) with the L33P polymorphism representing a gain of function mutation likely through enhanced thiol isomerase activity. This pro-coagulant state observed by L33P may contribute to the increased risk of cardiovascular events in patients carrying this integrin polymorphism. These findings elucidate integrins as a novel regulator of the coagulation cascade and support the development of therapeutics targeting the PSI domain.
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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) | 0.001 | 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".