Palmitoylation Augments the Association of Tetraspanin CD63 with the αIIbβ3-CD9 Complex and the Actin Cytoskeleton in Thrombin-Activated Platelets
Bibliographic record
Abstract
Abstract CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. In resting platelets, CD63 is located in the membranes of lysosomes and dense granules, and CD9 complexes with the αIIbβ3 integrin on the platelet surface. Following platelet activation and granule exocytosis, CD63 translocates to the plasma membrane, where it co-localizes with the αIIbβ3-CD9 complex and is incorporated in the Triton-insoluble actin cytoskeleton. Tetraspanin complexes cluster dynamically in unique cholesterol-rich membrane microdomains (tetraspanin-enriched microdomains, TEMs) that differ from prototypic lipid rafts. The assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some of their partner proteins. Protein palmitoylation most commonly involves the thioester linkage of palmitate to a cysteine residue and, because the process is reversible, can regulate cellular functions. In cell lines, palmitoylation of tetraspanin juxta-membrane cysteine residues affects subcellular distribution, complex stability, cellular signaling and motility. Recently we demonstrated that tetraspanins and their partner proteins form TEMs in platelets, however the role of palmitoylation in platelet TEMs assembly and maintenance has not been studied. 3[H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1U/ml). CD63 and CD9 were isolated by immunoprecipitation, separated by density gradient centrifugation, and 3[H]-palmitate quantitated. Palmitate levels increased in all fractions however the relative inter-fraction distribution did not change, consistent with previous results showing that the distribution of CD63 and CD9 does not change following platelet activation. 2-bromopalmitate (2- BP), which blocked palmitoylation as demonstrated by decreased 3[H]-palmitate-labeling of platelets, inhibited both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen, in a dose and time-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9 and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton in thrombin-activated platelets. In contrast 2-BP had minimal effect on either the integrity of the α IIb β3-CD9 complex or its agonist-induced association with the cytoskeleton. In summary, inhibition of palmitoylation blocked the activation-dependent association of CD63 with the αIIbβ3-CD9 complex and with the actin cytoskeleton but did not alter the tetraspanin-integrin association present in resting platelets. The effect of 2-BP on platelet spreading on fibrinogen mirrored that previously seen with anti-CD63 MoAbs, and supports the hypothesis that the association of CD63 with αIIbβ3-CD9 modulates outside-in signaling in adherent platelets.
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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".