ITIH3 is a secreted plasma glycoprotein processed to inter-alpha-trypsin inhibitor heavy chain H3 (HC3). HC3 is ester-linked through the chondroitin-sulfate chain of bikunin to form pre-alpha-inhibitor; bikunin, rather than HC3, supplies the complex's protease-inhibitor activity. HC2- and TSG-6-dependent transesterification can transfer HC3 from bikunin chondroitin sulfate to hyaluronan, incorporating HC3 into HA-rich extracellular matrices. HC3 also contains a cryptic N-terminal hyaluronan-binding site whose noncovalent activity was exposed by proteolytic fragmentation in vitro.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004867 serine-type endopeptidase inhibitor activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro propagates the inhibitor activity associated with the named inter-alpha-inhibitor family complex to the ITIH3 heavy-chain component. Reason: Direct human chain analysis shows that bikunin is responsible for antiprotease activity and is covalently linked to the distinct HC3 chain. This is a complex-to-component overpropagation, not an intrinsic HC3 serine-protease-inhibitor activity. Supporting Evidence: PMID:9188816 plasma. It comprises bikunin (BK) responsible for antiprotease activity, PMID:2476436 trypsin-inhibitory chain of 30,000 Da |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping correctly places the secreted HC3 precursor and mature plasma protein outside the cell. Reason: ITIH3 has a cleaved signal peptide and is a soluble secreted plasma protein, so extracellular region is correct. GO has since obsoleted GO:0005615 extracellular space, the replacement previously proposed here, and merged it into GO:0005576 (replaced_by), so this term is now the correct location term. All duplicate extracellular-region annotations are handled consistently. Supporting Evidence: file:human/ITIH3/ITIH3-uniprot.txt SUBCELLULAR LOCATION: Secreted. |
| GO:0030212 hyaluronan metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro assigns the transferable ITIH heavy-chain family to hyaluronan metabolism. Reason: Reconstitution with purified human plasma components directly demonstrates TSG-6- and HC2-dependent formation of HC3-hyaluronan complexes. Covalent transfer of HC3 onto HA constitutes direct participation in HA remodeling and is a core biological process. Supporting Evidence: PMID:18448433 the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. PMID:16873769 TSG-6 mediates the transfer of the heavy chains from IΞ±I/PΞ±I onto HA |
| GO:0005515 protein binding | IPI PMID:16873769 TSG-6 potentiates the antitissue kallikrein activity of inte... | MARK AS OVER ANNOTATED | Summary: The study analyzes the covalent HC3-bikunin pre-alpha-inhibitor assembly, TSG-6-heavy-chain intermediates, and bikunin release. Reason: The molecular associations are authentic, but generic protein binding is uninformative and obscures the covalent glycosaminoglycan linkage and HC-transfer mechanism. Hyaluronic acid binding and hyaluronan metabolism describe the demonstrated HC3 biology more specifically. Supporting Evidence: PMID:16873769 forms covalent complexes with heavy chains (HCs) from PMID:1898736 protein-glycosaminoglycan-protein (PGP) cross-link. |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-481009 | ACCEPT | Summary: Reactome places Q06033/ITIH3 in a platelet dense-granule-content set that is released extracellularly during exocytosis. Reason: The extracellular location is consistent with ITIH3 secretion and plasma occurrence. GO has since obsoleted GO:0005615 extracellular space, the replacement previously proposed here, and merged it into GO:0005576 (replaced_by), so this term is now the correct location term. This duplicate receives the same action as the IEA and NAS records. Supporting Evidence: file:human/ITIH3/ITIH3-uniprot.txt SUBCELLULAR LOCATION: Secreted. |
| GO:0031089 platelet dense granule lumen | TAS Reactome:R-HSA-481009 | KEEP AS NON CORE | Summary: The current Reactome participant set explicitly includes Q06033/ITIH3 in the defined platelet dense-granule-content set. Reason: The target-specific Reactome curation is retained, but its linked 1992 publication has no accessible abstract or ITIH3-specific text, and this compartment is peripheral to the established plasma HC3-HA matrix role. A secreted plasma protein could also be carried into or adsorb to isolated granules, so the location is not promoted into the core function. |
| GO:0070062 extracellular exosome | HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres... | MARK AS OVER ANNOTATED | Summary: Detected in an extracellular-vesicle proteome, but the detection does not establish that this protein is an EV constituent. Reason: Secreted plasma inter-alpha-trypsin inhibitor heavy chain. UniProt says only that it may carry hyaluronan, and unlike ITIH1 and ITIH2 it has no hyaluronan-binding annotation. Secreted or lumenal protein (UniProt). MISEV2018 (PMID:30637094) category 5 allows such proteins onto EVs through specific or promiscuous (proteoglycan, lipid) receptors on the EV surface, but asks that their identification be backed by evidence of that association. A bulk EV proteome cannot separate surface binding from co-isolation, and this protein's annotations record no experimental proteoglycan, heparin or lipid binding, and its own review keeps no experimental cell-surface or plasma-membrane pool. (Manual re-review for OMICS_EVIDENCE, 2026-10-10; previous action: KEEP_AS_NON_CORE.) Supporting Evidence: PMID:23533145 samples, ~900 proteins were detected. |
| GO:0005576 extracellular region | NAS PMID:14718574 The human plasma proteome: a nonredundant list developed by ... | ACCEPT | Summary: The plasma-proteome source supports extracellular occurrence of secreted ITIH3. Reason: The location is correct for a soluble secreted plasma protein. GO has since obsoleted GO:0005615 extracellular space, the replacement previously proposed here, and merged it into GO:0005576 (replaced_by), so this term is now the correct location term. This matches the actions on both duplicate annotations. Supporting Evidence: file:human/ITIH3/ITIH3-uniprot.txt SUBCELLULAR LOCATION: Secreted. |
| GO:0004866 endopeptidase inhibitor activity | TAS PMID:2465147 Human plasma inter-alpha-trypsin inhibitor is encoded by fou... | REMOVE | Summary: The cited paper identifies the distinct H3 cDNA and the multigene heavy-chain family but does not assay endopeptidase inhibition by HC3. Reason: Direct purification of human pre-alpha-inhibitor assigns antiprotease activity to bikunin, which is covalently linked to the separate H3 heavy chain. The TAS annotation conflates activity of the assembled named complex with an intrinsic activity of ITIH3. Supporting Evidence: PMID:9188816 plasma. It comprises bikunin (BK) responsible for antiprotease activity, PMID:2476436 pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 |
| GO:0005540 hyaluronic acid binding | IDA PMID:11168393 Unmasking a hyaluronan-binding site of the BX(7)B type in th... | NEW | Summary: Purified human plasma HC3 contains a cryptic N-terminal HA-binding site; a thermolysin-generated fragment bound HA strongly, whereas intact mature HC3 did not in the same assay. Reason: Direct binding by a processed human HC3 species and independent formation of covalent human HC3-HA complexes support this specific molecular function. The conditional exposure of the noncovalent site is retained explicitly and should not be read as constitutive binding by intact plasma HC3. Supporting Evidence: PMID:11168393 thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA PMID:11168393 whereas the intact H3 chain did not. PMID:18448433 the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. |
| GO:0031012 extracellular matrix | ISS PMID:18293178 Equivalent involvement of inter-alpha-trypsin inhibitor heav... | NEW | Summary: Mouse HC3 was recovered in physiological, pathological, and reconstituted SHAP-HA complexes, and purified human components independently form HC3-HA. Reason: Ortholog evidence places HC3 directly in natural HA-associated matrices, while human biochemical evidence establishes the conserved capacity for HC3-HA formation. Extracellular matrix is therefore a conservative, functionally relevant location for human ITIH3. Supporting Evidence: PMID:18293178 All three HC isoforms are found in the SHAP-hyaluronan PMID:18448433 the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. |
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Download this section (compressed HTML)Q: Which endogenous human protease or processing event exposes the cryptic N-terminal HC3 hyaluronan-binding site, and in which tissues does this occur?
Q: What molecular feature of HC2 enables TSG-6-mediated transfer of HC3 from pre-alpha-inhibitor, and does HC2 remain associated with the machinery in vivo?
Q: Does HC3 produce HA matrices with mechanical or cell-binding properties distinct from matrices containing HC1 or HC2?
Q: Is ITIH3 genuinely stored within platelet dense granules, or does its Reactome assignment reflect adsorption or co-isolation of an abundant plasma protein?
Experiment: Map native HC3 termini in plasma and inflamed-tissue extracellular matrices by N-/C-terminomics, purify the major processed species, and compare their noncovalent HA affinity with intact HC3 using SPR and orthogonal HA pull-downs. Identify candidate proteases by inhibitor profiling and cleavage-site mutagenesis.
Hypothesis: Endogenous proteolysis exposes the cryptic N-terminal HC3 HA-binding site in a regulated extracellular context.
Type: Endogenous processing and quantitative HA-binding biochemistry
Experiment: Reconstitute transfer with purified glycosylated human pre-alpha-inhibitor, TSG-6, HA, and wild-type or interface-mutant HC2. Quantify HC3-TSG-6 and HC3-HA intermediates by intact-mass spectrometry and linkage-site LC-MS/MS, with HC1 and HC3 substitutions as specificity controls.
Hypothesis: HC2 provides a distinct cofactor surface required for TSG-6-mediated HC3 transfer from pre-alpha-inhibitor to HA.
Type: Defined heavy-chain-transfer reconstitution
Experiment: Analyze washed human platelets by protease-protected immunogold electron microscopy and density-gradient granule proteomics with plasma-protein spike-in controls, then test agonist-dependent ITIH3 release and compare it with canonical dense-granule and alpha-granule markers.
Hypothesis: ITIH3 is an authentic platelet dense-granule cargo rather than a plasma contaminant of granule preparations.
Type: Platelet granule localization and regulated-release analysis
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