Hemostasis involves a highly regulated equilibrium between pro and anticoagulant processes. It is a dynamic system comprising cellular and plasma factors that interact with damaged vascular tissue to minimize blood loss. Platelets are anucleate cellular fragments derived from mature megakaryocytes via a process called thrombopoiesis (Stegner et al., 2017). They circulate throughout the vascular system in a quiescent discoid form, interacting minimally with healthy endothelial cells. Following injury to the endothelium, platelets serve as key effector cells for the hemostatic response. They rapidly adhere to exposed extracellular matrix via tether receptor complexes such as collagen-glycoprotein (GP) VI and von Willebrand factor-GPIb-V-IX (Canobbio et al., 2004; Chen et al., 2002). Platelet attachment triggers downstream effects including the release of platelet-specific α granules and dense granules (Blair & Flaumenhaft, 2009; Li et al., 2010). Contained within these granules are important membrane bound receptors (GPIIb/IIIa and P-selectin), platelet mediators (adenosine 5ʹ-diphosphate [ADP] and calcium ions; Blair et al., 2009; Sharda & Flaumenhaft, 2018). Release of ADP, a potent platelet agonist, is crucial for the formation of an effective platelet plug. Such ADP mediated activation leads to morphological changes, transforming resting discoid platelets to an amoeboid state with numerous pseudopodia that facilitate platelet interdigitation and stabilize the developing aggregate

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 10 6 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, plateletneutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

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