Spatial Structure of Plasma Coagulation Factor XIII and Changes with Activation

Introduction

Blood coagulation factor XIII (FXIII) is a precursor of the active transglutaminase (FXIIIa) that catalyzes covalent cross-linking within polymeric fibrin to increase mechanical and proteolytic stability of blood clots and thrombi. Structurally, inactive plasma FXIII (pFXIII) with a molecular mass of 326 kDa is a heterotetramer (A₂B₂) with two catalytic A subunits (FXIII-A, 83 kDa) and two non-catalytic B subunits (FXIII-B, 80 kDa).

The inactive pFXIII is converted into a catalytically active form by thrombin and Ca²⁺. Thrombin cleaves off an activation peptide (AP) from the N-terminus of each A subunit, and subsequently B subunits dissociate from thrombin-cleaved A subunits in presence of Ca²⁺. However, the oligomerization state of activated A and B subunits remain controversial.

Both activated and inactive FXIII A subunits have been characterized crystallographically, while the atomic structure of the FXIII-A₂B₂ complex remains unknown. Therefore, our goal was to visualize and characterize the molecular structure of FXIII and its activated form FXIIIa using high-resolution atomic force microscopy (AFM).

Methods

Commercially available pFXIII (Enzyme Research Laboratories, USA), non-activated or activated with thrombin and Ca²⁺, was adsorbed on the surface of graphite rendered hydrophilic with an amphiphilic hydrocarbon-glycine modifier followed by single-molecule AFM imaging, methodology developed by us to allow high-resolution imaging. Additionally, recombinant rFXIII-A₂ (Zymogenetics, USA) and recombinant rFXIII-B₂ (Zedira, Germany) were imaged to compare the structures and dimensions of FXIII and FXIIIa with individual dimeric subunits.

Results

Individual molecules of pFXIII were visualized in AFM as globules with two (67% of the molecules), one (14%), or no (19%) thread-like extensions (Fig. 1A). The size (height) of the globular portions had a symmetrical distribution with a maximum at 3.5 nm. The strands had a peak contour length of 20 nm and a height of 0.4 nm. Based on the known polypeptide composition of pFXIII-A₂B₂, the observed molecular images could be interpreted as an A₂ globule with one or two individual B subunits extending out from it. The structural diversity of FXIII forms may reflect an equilibrium between association and dissociation of B subunits from the A₂ dimer. Alternatively, the B subunits can be partially or fully wrapped around the globular A₂ dimer, making them invisible in AFM images.

Although a crystallographic structure of FXIII-A₂ is available, its AFM imaging was important to identify the A₂ dimer within inactive and activated pFXIII molecules: rFXIII-A₂ was a globule with the same height as the pFXIII globular core (Fig. 1B).

Recombinant dimeric B subunits (rFXIII-B₂) appear as thin flexible strands with an average contour length of 33 nm and a height of 0.6 nm (Fig. 1C). Thus, both the length and height of the free B₂ dimers were significantly larger compared to the strands sticking out from the globular core of pFXIII-A₂B₂ molecules, we conclude that these strands in pFXIII-A₂B₂ comprise monomeric B subunits.

In thrombin-activated pFXIIIa, the B-subunits dissociated from the A₂ dimer and self-associated to form B₂ dimers (Fig. 1D). The height distribution of globular particles remaining separated was bimodal with two peaks: one at 3.3 nm corresponded to the whole tetrameric pFXIII-A₂B₂ and, potentially, dimeric FXIII-A₂, while the second major peak at 2.1 nm likely represented FXIII-A monomers.

Conclusions

Our results provide qualitative and quantitative molecular structural characteristics of inactive and activated human pFXIII as well as dimeric A and B subunits. We have shown that inactive plasma FXIII (pFXIII-A₂B₂) consists of a globular A₂ dimer core and individual B subunits extending out as flexible strands. Recombinant inactive FXIII-A subunits in the absence of FXIII-B subunits form a globular homodimer. Recombinant FXIII-B subunits in the absence of FXIII-A subunits form flexible homodimeric strands. Upon activation of pFXIII-A₂B₂ with thrombin and Ca²⁺, the initially monomeric B subunits dissociate from the globular core of the molecule and form B₂ homodimers, while the initially dimeric activated A subunits dissociate into monomers.

Fig. 1. AFM images of A: pFXIII-A₂B₂, B: rFXIII-A₂, C: rFXIII-B₂, D: thrombin-activated pFXIIIa.

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