Antiplatelet Agents Inhibit Oral Bacteria Induced Platelet Aggregation in Vitro

Objective: The purpose of this work was to compare the effect of  a single antiplatelet agent, acetylsalicylic acid (ASA), to dual antiplatelet agents, acetylsalicylic acid plus clopidogril (CLP), on in vitro platelet aggregation induced by Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). 

Method: Pg (ATCC 33277) and Tf (ATCC 43037) cells were suspended in Ca⁺⁺ free Tyrode’s buffer containing HEPES  to an optical density of 0.2 at 600 nm.  Platelet rich plasma  (PRP) was prepared from anticoagulated whole blood.  Gel filtered platelets (GFP) were obtained by applying PRP to a column of Sepharose 2B.  Platelet aggregation was measured as the absorbance of light on a dual-channel platelet aggregometer.  PRP or GFP (275,000-300,000 platelets/µl) were incubated with either ASA (final concentration 80 µM), or ASA plus CLP (final concentration 3 µM), at 37 C.  The standard assay consisted of 450 µl PRP or GFP, control or treated with antiplatelet agents, to which was added 5.0-40 µl standard bacterial cell suspension.      

Result: ASA inhibited both Pg and Tf cell-induced platelet aggregation using both PRP and GFP.  ASA inhibition became less attenuated with increasing numbers of cells (5 µl cells, 100% inhibition; 10 µl cells, 82% inhibition; 20 µl cells, 50 % inhibition; 40 µl cells,28% inhibition).  No further inhibition of platelet aggregation was noted when PRP,  incubated with ASA plus CLP, was challenged with the higher concentration of cells (20-40 µl).  In contrast, when GFP was incubated with ASA plus CLP, platelet aggregation was completely inhibited at the higher number of cells.

Conclusion: Physiologic concentration of ASA partially attenuates Pg and Tf induced platelet aggregation of PRP and GFP in vitro.  There was an inverse correlation of inhibition to the number of cells.   Aggregation of GFP was completely inhibited by a combination of ASA plus CLP, but the enhancement of inhibition  was not observed in PRP.