Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

Takagi, Junichi; Aoyama, Toshihiro; Ueki, Shoko; Ohba, Hirotsugu; Saito, Yuji; Lóránd, L.

doi:10.1111/j.1432-1033.1995.0773a.x

Cited by 4 publications

(5 citation statements)

References 29 publications

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“…Earlier studies demonstrated that specificity for factor XIIIa catalyzed cross-linking between proteins is directed by the primary structure in the vicinity of substrate glutamine residues (43,44). However attempts to find homology within the primary sequence of known FXIIIa protein substrates surrounding the Gln cross-linking site revealed no apparent sequence pattern (15,27,45). In this study we performed yet another attempt to identify common features in the sequence adjacent to the reactive Gln site.…”

Section: Discussionmentioning

confidence: 92%

“…The exo site responsible for the binding of factor XIIIa to fibrin (46) may contribute to the selection of Gln amine acceptor sites within R chains. The higher order structure (tertiary and quaternary) of the protein substrate may also play an important role in determining which Gln residue might act as an amine acceptor (27). Further studies including kinetic analysis are needed to define the role of the xQAxBxPx linear sequence in determination of the substrate specificity of factor XIIIa.…”

Section: Discussionmentioning

confidence: 99%

“…Activation was achieved by treatment of 500 µg/mL of factor XIII (Haematologic Technologies, Inc.) with 0.25 U/mL of thrombin (Sigma) in TBS, pH 7.4 buffer containing 10 mM dithiothreitol and 20 mM CaCl 2 . After incubation for 20 min at 37 °C, thrombin was inactivated by the addition of a molar excess of hirudin (Sigma) and this mixture was used as factor XIIIa (27).…”

Section: Methodsmentioning

confidence: 99%

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Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin: Identification of Gln103 as a Major Cross-Linking Site

et al. 2006

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In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin: Identification of Gln103 as a Major Cross-Linking Site

et al. 2006

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“…Multimeric vWF molecules are not cross-linked to each other by FXIIIa, but can incorporate putrescine, suggesting that only glutamine residues are available for cross-linking [94]. Studies of bovine vWF have shown that Gln 313 , Gln 509 , Gln 560 and Gln 634 are susceptible to FXIIIa cross-linking as dansylcadaverine is readily incorporated at these residues [95]; however, only Gln 313 and Gln 560 are conserved in human vWF, suggesting these may be the residues involved in cross-linking vWF.…”

Section: Cross-linking Of Vwfmentioning

confidence: 99%

Substrates of Factor XIII-A: roles in thrombosis and wound healing

et al. 2012

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FXIII (Factor XIII) is a Ca²+-dependent enzyme which forms covalent ϵ-(γ-glutamyl)lysine cross-links between the γ-carboxy-amine group of a glutamine residue and the ϵ-amino group of a lysine residue. FXIII was originally identified as a protein involved in fibrin clot stabilization; however, additional extracellular and intracellular roles for FXIII have been identified which influence thrombus resolution and tissue repair. The present review discusses the substrates of FXIIIa (activated FXIII) involved in thrombosis and wound healing with a particular focus on: (i) the influence of plasma FXIIIa on the formation of stable fibrin clots able to withstand mechanical and enzymatic breakdown through fibrin-fibrin cross-linking and cross-linking of fibrinolysis inhibitors, in particular α2-antiplasmin; (ii) the role of intracellular FXIIIa in clot retraction through cross-linking of platelet cytoskeleton proteins, including actin, myosin, filamin and vinculin; (iii) the role of intracellular FXIIIa in cross-linking the cytoplasmic tails of monocyte AT1Rs (angiotensin type 1 receptors) and potential effects on the development of atherosclerosis; and (iv) the role of FXIIIa on matrix deposition and tissue repair, including cross-linking of extracellular matrix proteins, such as fibronectin, collagen and von Willebrand factor, and the effects on matrix deposition and cell-matrix interactions. The review highlights the central role of FXIIIa in the regulation of thrombus stability, thrombus regulation, cell-matrix interactions and wound healing, which is supported by observations in FXIII-deficient humans and animals.

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“…Bovine VWFpp can bind alpha4β1-and alpha9β1-integrins, which are expressed on lymphocytes, monocytes and neutrophils, via a sequence within the VWD2 domain that is conserved in humans [45][46][47]. Another ligand for these integrins, coagulation factor FXIII, has been shown to cross link VWFpp to the extracellular matrix protein laminin [47][48][49]. Possibly, focused release of VWFpp from degranulating platelets during the initial thrombus formation and incorporation in the adhesive surface via laminin and collagen provides a mechanism to influence the adhesive properties of the exposed extracellular matrix and direct hemostatic and immune responses following vascular injury.…”

Section: Discussionmentioning

confidence: 99%

Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules

Swinkels

Hordijk

Bürgisser

et al. 2022

Preprint

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Background:Platelet alpha-granules contain Von Willebrand factor (VWF), which is stored in eccentric alpha-granule nanodomains, and VWF propeptide (VWFpp). Differential release of VWF and VWFpp has been reported from endothelial cells. It is unclear if this also occurs during platelet alpha-granule exocytosis. We have recently developed a 3D super-resolution imaging workflow for quantification of platelet alpha-granule content based on Structured Illumination Microscopy (SIM). With this we can study alpha-granule cargo release following platelet activation in hundreds of platelets simultaneously.Aims:To study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy.Methods:Platelets were activated with PAR-1 activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, SPARC and fibrinogen were imaged using 3D-SIM, followed by semi-automated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content.Results:VWF+ and VWFpp+ structures overlapped nearly completely (~90%) in resting platelets, implying they are stored in similar eccentric alpha-granule nanodomains. A subset of VWF+/VWFpp+-structures was released completely at 0.6 μM PAR-1-ap, but at higher concentration (20 μM) significantly more VWFpp (85.3±1.6%) was released than VWF (37.6±1.4%). Release of other cargo was intermediate at 20 μM (SPARC: 62.2±1.4% ; fibrinogen: 51.9±2.9%), providing further evidence for differential cargo release. Similar results were obtained using CRP-XL. Anti-VWF nanobody was taken up by VWF+/VWFpp- structures and increased with stimulus strength, demonstrating these were post-exocytotic structures.Conclusions:VWF and VWFpp are differentially released from alpha-granules. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots.

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Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

Cited by 4 publications

References 29 publications

Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin: Identification of Gln103 as a Major Cross-Linking Site

Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin: Identification of Gln103 as a Major Cross-Linking Site

Substrates of Factor XIII-A: roles in thrombosis and wound healing

Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules

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