Multiple ligands of von Willebrand factor-binding protein (vWbp) promote Staphylococcus aureus clot formation in human plasma

Abstract

Staphylococcus aureus secretes coagulase (Coa) and von Willebrand factor-binding protein (vWbp) to activate host prothrombin and form fibrin cables, thereby promoting the establishment of infectious lesions. The D1-D2 domains of Coa and vWbp associate with, and non-proteolytically activate prothrombin. Moreover, Coa encompasses C-terminal tandem repeats for binding to fibrinogen, whereas vWbp has been reported to associate with von Willebrand factor and fibrinogen. Here we used affinity chromatography with non-catalytic Coa and vWbp to identify the ligands for these virulence factors in human plasma. vWbp bound to prothrombin, fibrinogen, fibronectin, and factor XIII, whereas Coa co-purified with prothrombin and fibrinogen. vWbp association with fibrinogen and factor XIII, but not fibronectin, required prothrombin and triggered the non-proteolytic activation of FXIII in vitro. Staphylococcus aureus coagulation of human plasma was associated with the recruitment of prothrombin, FXIII, and fibronectin as well as the formation of cross-linked fibrin. FXIII activity in staphylococcal clots could be attributed to thrombin-dependent proteolytic activation as well as vWbp-mediated non-proteolytic activation of FXIII zymogen.

Keywords: Coagulase; Coagulation Factors; Factor XIII; Fibrin; Fibrinogen; Fibronectin; Staphylococcus aureus; vWbp.

FIGURE 1.

Identification of vWbp ligands isolated from human plasma and comparative analysis with Coa. A, human plasma (500 μl) was flowed over Strep-Tactin resin uncharged (control) or charged with _H6vWbp_strep (100 nmol). Bound proteins were eluted by boiling the resin in sample buffer, separated by SDS-PAGE, and visualized after Coomassie staining. The identity of proteins labeled 1–9 was performed by microcapillary LC/MS/MS techniques. A complete list of protein hits can be found in Table 2. PT, prothrombin; FG, fibrinogen. B and C, for the comparative analysis of Coa and vWbp ligands, human plasma (500 μl) was flowed over Strep-Tactin resin uncharged (control) or charged with either _H6vWbp_strep (100 nmol) or Coa_strep (100 nmol). Bound proteins were eluted by boiling the resin in sample buffer and separated by SDS-PAGE. Proteins in gels were visualized by Coomassie staining (B) or after transfer to PVDF membranes for immunoblotting using specific antibodies against prothrombin, FXIII subunit A (FXIII A), and fibronectin (C).

FIGURE 2.

Mapping the functional domains of vWbp. A, diagram illustrating the primary translational products of mature full-length vWbp and truncated variants. Each variant includes an N-terminal six histidyl tag (H6) and a C-terminal strep tag (WSHPQFEK). The binding sites for prothrombin, fibrinogen, FXIII, and fibronectin are delineated. Numbers indicate amino acid residues within the mature protein. B and C, human plasma (500 μl) was flowed over Strep-Tactin resin uncharged (control) or charged with 100 nmol of proteins shown in A. Samples were eluted by boiling the resin in sample buffer and examined as described in Fig. 1. Immunoblotting was performed for prothrombin (PT), FXIII subunit A (FXIII A) and B (FXIIIB), fibronectin, and vWF.

FIGURE 3.

vWbp binds purified human vWF. A, the presence of vWF in human plasma was examined by immunoblot using aliquots of plasma diluted 10, 50, and 100 times and compared with purified vWF. Aliquots were boiled in sample buffer, and proteins were separated by SDS-PAGE before transfer to the PVDF membrane. Immunoblotting with human vWF-specific antibodies reveals immunoreactive species with the expected electrophoretic mobility (225 kDa). B, _H6vWbp_strep or Coa_strep (100 nmol) were immobilized on Strep-Tactin resin, and 500 μl of human vWF (0.4 μm) was flowed over the resin. Uncharged Strep-Tactin resin was used as a control. Bound proteins were eluted by boiling the resin in sample buffer and separated by SDS-PAGE. Proteins in gels were visualized by Coomassie staining. The density of bands corresponding to vWF was determined using the Gel Analysis function of ImageJ. The percentage of bound vWF was calculated as the density of vWF retained over input. Data are representative of three independent experiments. Statistical significance was analyzed with the two-tailed t test.

FIGURE 4.

Analysis of ligand binding to vWbp. A, human fibrinogen (FG; 9 μm in 500 μl) was flowed alone or in the presence of human prothrombin (PT; 1.4 μm) over uncharged Strep-Tactin resin or resin charged with _H6vWbp_strep (100 nmol). Samples were eluted by boiling the resin in sample buffer and examined as in Fig. 1 after Coomassie staining of SDS-PAGE (A) and immunoblot against prothrombin (B). C and D, to decipher the biochemical basis for the interaction between FXIII and vWbp, human FXIII (0.03 μm in 500 μl) was flowed alone or in the presence of human fibrinogen (9 μm) and/or human prothrombin (1.4 μm) over uncharged Strep-Tactin resin (−) resin charged with _H6vWbp_strep (100 nmol), or Coa_strep (100 nmol). Samples were eluted by boiling the resin in sample buffer and examined as in Fig. 1 after Coomassie staining of SDS-PAGE (C) and immunoblot against prothrombin and FXIII subunit A (D).

FIGURE 5.

vWbp_strep-prothrombin-fibrinogen complex activates FXIII in a non-proteolytic manner. Formation of cross-linked fibrin products was monitored after incubation at 37 °C for 1 h with the following factors in various combinations: human FXIII (20 nm), human thrombin (100 nm), human prothrombin (200 nm), vWbp_strep (200 nm), fibrinogen (1.5 μm), and CaCl₂ (5 mm). Where indicated, lepirudin was added to the reaction. Reaction products were subjected to centrifugation, and sediments were washed three times with PBS and boiled in sample buffer supplemented with urea (4 m). Solubilized protein samples were examined with Coomassie-stained SDS-PAGE (A) and immunoblot against FXIII subunits A and B (B).

FIGURE 6.

FXIII and fibronectin are incorporated into staphylococcal fibrin clots. S. aureus Newman or isogenic mutants coa, vwb, or coa/vwb were grown to mid-log phase. Cells were washed, taken up in PBS, and incubated in the presence of 5 mm calcium and human plasma untreated (normal) or treated with lepirudin. A PBS control with no bacterial cell (−) was included. After incubation at 37 °C, the samples were subjected to centrifugation after 2 and 24 h. Pellets were washed three times in PBS and boiled in sample buffer supplemented with urea (4 m) to solubilize clots. Samples were examined after Coomassie staining of SDS-PAGE (A) and immunoblot against prothrombin (PT), FXIII subunit A and B, and fibronectin (B).

FIGURE 7.

FXIII depletion abolishes fibrin cross-linking in staphylococcal clots. S. aureus Newman or isogenic mutants coa, vwb, or coa/vwb were grown to mid-log phase. Cells were washed, taken up in PBS, and incubated in the presence of 5 mm calcium and human plasma that had been depleted of FXIII. A PBS control with no bacterial cell (−) was included. After incubation at 37 °C, the samples were subjected to centrifugation after 24 h. Pellets were washed 3 times in PBS and boiled in sample buffer supplemented with urea (4 m) to solubilize clots. Samples were examined after Coomassie staining of SDS-PAGE (A) and immunoblot against FXIII subunit A and B and fibronectin (B).