Mass spectrometry–based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots

Schmitt, Lauren R.; Henderson, Rachel; Barrett, Alexander S.; Darula, Zsuzsanna; Issaian, Aaron; D’Alessandro, Angelo; Clendenen, Nathan; Hansen, Kirk C.

doi:10.1074/jbc.ac119.007981

Cited by 25 publications

(39 citation statements)

References 28 publications

(26 reference statements)

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“…These studies provide invaluable insights into the structural organization of fibrin oligomers; however, to circumvent technological limitations, they were not performed on full clots. A recent mass spectrometry-based study mapped endogenously cross-linked peptide species, suggesting a more compact organization of protein blocks than so far assumed (48) and, importantly, highlights the potential of mass-spectrometry based investigations of fibrin clots. Here, we employ in situ cross-linking mass spectrometry on purified clots extracted from plasma specifically to delve into the mechanisms underlying lateral aggregation (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 91%

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Klykov

Zwaan

Heck

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots which assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and the insoluble character of fibrin clots, have restricted our ability to develop novel treatments for clotting diseases. The, so far resolved, disparate structural details have provided insights into linear elongation; however, molecular details like the C-terminal domain of the α-chain, the heparin-binding domain on the β-chain, and other functional domains remain elusive. To illuminate these dark areas, we applied cross-linking mass spectrometry (XL-MS) to obtain biochemical evidence in the form of over 300 distance constraints and combined this with structural modeling. These restraints additionally define the interaction network of the clots and provide molecular details for the interaction with human serum albumin (HSA). We were able to construct the structural models of the fibrinogen α-chain (excluding two highly flexible regions) and the N termini of the β-chain, confirm these models with known structural arrangements, and map how the structure laterally aggregates to form intricate lattices together with the γ-chain. We validate the final model by mapping mutations leading to impaired clot formation. From a list of 22 mutations, we uncovered structural features for all, including a crucial role for βArg’169 (UniProt: 196) in lateral aggregation. The resulting model can potentially serve for research on dysfibrinogenemia and amyloidosis as it provides insights into the molecular mechanisms of thrombosis and bleeding disorders related to fibrinogen variants. The structure is provided in the PDB-DEV repository (PDBDEV_00000030).

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Section: Resultsmentioning

confidence: 91%

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Klykov

Zwaan

Heck

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…This strategy, coupled to high-resolution MS 3 mass spectrometry, allows to chemically stabilize protein-protein interactions by bridging epsilon amine residues of K residues and carboxylic groups in the side chains of acidic amino acids D and E, provided they face each other within a given range (e.g., 11.4 to 24 Å for the zero-length cross-linking agents we used in this study). 18,20,38 Through this approach we mapped over 5597 peptide-peptide unique inter-or intra-molecular cross-links, involving a total of 134 unique RBC proteins, generating a preliminary map of the RBC experimental interactome.…”

Section: Supplementary Results and Discussion On The Rbc Interactomementioning

confidence: 99%

“…33 Units from these donors were stored for up to 42 days prior to determination of the RBC susceptibility to hemolysis following osmotic insults. 38 Genome wide association studies (GWAS) revealed that the highly polymorphic region on chromosome 17 coding for AE1 (gene name SLC4A1) ranked amongst the top correlates to osmotic fragility (Fig. 3.E).…”

Section: Single Nucleotide Polymorphisms (Snps) In the Region Coding For Ae1 1-56 Correlate With Increased Osmotic Fragility Of Stored Humentioning

confidence: 99%

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

et al. 2021

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Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion – a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of its role in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide a map of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As a proof-of-principle and further mechanistic evidence of the role of AE1 in the regulation of redox homeostasis of stored RBCs, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice.

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“…These studies provide invaluable insights into the structural organization of fibrin oligomers, but to circumvent technological limitations were not performed on full clots. A recent study mapped endogenously crosslinked peptide species detected by mass spectrometry, which suggested a more compact organization of protein blocks then so far assumed (45) and, importantly, highlights the potential of mass-spectrometry based investigations of fibrin clots. Here we employ in situ crosslinking mass spectrometry on purified blood clots to specifically delve into the mechanisms underlying lateral aggregation (Supplementary Figure 2).…”

Section: Resultsmentioning

confidence: 89%

Flexible regions in the molecular architecture of Human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Klykov

Zwaan

Heck

et al. 2019

Preprint

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Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots that assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and insoluble character of fibrin clots, have however restricted our ability to develop novel treatments for clotting diseases. The so far resolved disparate structural details did provide insights into linear elongation; however, molecular details like the C-terminal domain of the α-chain, the heparin-binding domain on the β -chain, and others involved in lateral aggregation are lacking. To illuminate these dark areas, we applied crosslinking mass spectrometry (XL-MS) to obtain biochemical evidence in the form of over 300 distance constraints and combined this with structural modeling. These restraints additionally define the interaction network of the clots and e.g. provide molecular details for the interaction with Human Serum Albumin (HSA). We were able to construct the models of fibrinogen α(excluding two highly flexible regions) and β , confirm these models with known structural arrangements and map how the structure laterally aggregates to form intricate lattices together with fibrinogen γ. We validate the final model by mapping mutations leading to impaired clot formation. From a list of 22 mutations, we uncovered structural features for all, including a crucial role for β Arg'196 in lateral aggregation. The resulting model will be invaluable for research on dysfibrinogenemia and amyloidosis, as it provides insights into the molecular mechanisms of thrombosis and bleeding disorders related to fibrinogen variants. The structure is provided in the PDB-DEV repository.

show abstract

Mass spectrometry–based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots

Cited by 25 publications

References 28 publications

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Flexible regions in the molecular architecture of Human fibrin clots structurally resolved by XL-MS and integrative structural modeling

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