Thrombin Is Able To Trigger Apoptosis of Human Platelets.
Bibliographic record
Abstract
Abstract Although primarily known as a coagulation factor and as an inducer of platelet activation and aggregation, thrombin can modulate apoptosis in nucleated cells. Over the last decade, it has been recognized that apoptosis occurs not only in nucleated cells but also in anucleated cytoplasts and platelets. The current study investigated whether thrombin can impact apoptosis in anucleated human platelets. Using flow cytometry, we studied platelet apoptosis at the single cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis (Leytin et al, Biochem Biophys Res Commun320:303, 2004; Leytin et al, Br J Haematol133:78, 2006). Western blotting was also employed, in addition to flow cytometry, for determining the expression of proapoptotic Bax and Bak proteins. We found that, in comparison to untreated platelets, human alpha-thrombin (1 U/mL) significantly induced four key manifestations of platelet apoptosis: (i) mitochondrial inner transmembrane potential depolarization (P<0.01), (ii) expression of pro-apoptotic Bax (P=0.002) and Bak (P=0.04) proteins, (iii) caspase-3 activation (P=0.0009), and (iv) phosphatidylserine (PS) exposure (P<0.0001). We also compared the magnitude of thrombin effects with those of A23187 and in vitro platelet storage under standard blood banking conditions. We demonstrated that the maximal level of both caspase-3 activation and PS exposure is achieved in A23187-stimulated platelets, indicating that A23187 is a useful positive control for quantifying these apoptosis events. Thrombin triggered caspase-3 activation to a level equal to that in A23187-induced platelets and significantly higher than in platelets stimulated with control buffer (P<0.001) and stored for 0, 6 (P<0.001) and 13 days at 22°C (P<0.05). PS exposure was also markedly enhanced in thrombin-stimulated platelets resulting in increase of annexin V-positive cells from 1.2 ± 0.1% to 21.2 ± 2.5% (P=0.0002); platelet storage increased annexin V-positive cells from 1.4 ± 0.4% (Day 0) to 6.0 ± 0.6% (Day 6, P=0.006) and 47.6 ± 5.6% (Day 13 platelets, P=0.0013) and much higher PS exposure was observed with 10 μM A23187 (97.8 ± 0.4%, P<0.0001). Thus, PS exposure induced by 1 U/mL thrombin is significantly higher than in platelets stored for 6 days (P<0.001), but lower than in 13 day-old platelets (P<0.001) and A23187-stimulated platelets (P<0.0001). This study demonstrates that, aside from its ‘classical’ function as a coagulation factor and an inducer of platelet activation, thrombin can trigger platelet apoptosis. Thrombin appears to trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a pro-apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, and stimulating aberrant exposure of phosphatidylserine on the platelet surface. Thrombin-induced platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation.
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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 | 0.001 |
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".