A Novel GPIbα Binding Protein on Streptococcus Gordonii Induces Platelet Rolling at Low Shear.

Infective endocarditis is associated with considerable morbidity and mortality and oral Streptococci are the priniciple causative organism. Platelet adhesion and subsequent aggregation play a critical role in the pathogenesis and dissemination of the infective process. The mechanism by which S. gordonii interacts with and subsequently leads to platelet activation is currently unknown. Previously we described the ability of Streptococcus sanguis to induce platelet aggregation via the platelet vonWillebrand factor receptor, GPIbα, however identification of the bacterial protein involved was not established. Here we describe the novel interaction between a Streptococcal protein that triggers aggregation via platelet GPIbα, in a unique fashion.

Wildtype S. sanguis (133–79) and S. gordonii (DL1) support platelet adhesion and induce αIIbβ3 dependent platelet aggregation. Pre-incubation of platelets with an anti-GPIbα antibody inhibited the adhesion (61±6%, p<0.001) and abolished platelet aggregation (0% of control, p<0.001) induced by S. gordonii. These results suggest that GPIbα is a key receptor for recognition of platelets by streptococci. Passing a mutanolysin cell wall extract of S. sanguis through a GPIb affinity column identified a highly glycosylated protein, Hsa, also present on S. gordonii. S. gordonii DL-1 deficient in the Hsa gene, was generated by allelic exchange with an antibiotic resistance cassette. Platelet adhesion to S. gordonii ΔHsa was reduced by 41±5%, (p<0.001), however aggregation was unaffected (49±7% vs 55±5% wt). To confirm that Hsa was binding to platelet GPIbα we immobilised soluble GPIbα (glycocalicin, 0.2μg/ml) on a 96 well plate. Wildtype S. gordonii bound to immobilised glycocalicin, however, the Hsa mutant adhered significantly less (9±3% of wildtype, p<0.001). Furthermore, wildtype S. gordonii induced αIIbβ3 dependent platelet aggregation in washed platelets in the absence of vWf, suggesting a direct interaction between Hsa and GPIbα. We further investigated the interaction between GPIbα and Hsa under shear. Experiments were recorded in real time for a period of 8 minutes. Upon commencement of shear (50s-1), platelets interacted with S. gordonii DL-1 with a rolling fashion followed by firm adhesion. This adhesion was completely inhibited by addition of an anti-GPIbα antibody. When platelets were sheared over immobilized S. gordonii ΔHsa no interaction was observed. In addition, S. gordonii DL-1 failed to interact with platelets at the higher shear rate of 500s-1.

Collectively, these results identify a unique interaction between S. gordonii Hsa and platelet GPIbα. This interaction occurs at static and low shear but not at high shear, which is in direct contrast to the interaction between vwf and GPIbα. Furthermore, we propose that the platelet interaction with S. gordonii appears to be a 2 step process. Firstly, platelets roll across the S. gordonii via a GPIbα - Hsa interaction. Following this a second more stable interaction firmly immobilizes the platelet, thereby facilitating the intravascular colonization of a septic plaque.