Reduced platelet hyper-reactivity and platelet-leukocyte aggregation after periodontal therapy

Abstract

Background: Platelets from untreated periodontitis patients are hyper-reactive and form more platelet-leukocyte complexes compared to cells from individuals without periodontitis. It is not known whether the improvement of the periodontal condition achievable by therapy has beneficial effects on the platelet function. We aimed to assess the effects of periodontal therapy on platelet reactivity.

Methods: Patients with periodontitis (n = 25) but unaffected by any other medical condition or medication were included and donated blood before and after periodontal therapy. Reactivity to ADP or oral bacteria was assessed by flow cytometric analysis of membrane markers (binding of PAC-1, P-selectin, CD63) and platelet-leukocyte complex formation. Reactivity values were expressed as ratio between the stimulated and unstimulated sample. Plasma levels of soluble (s) P-selectin were determined by enzyme-linked immunosorbent assay (ELISA).

Results: Binding of PAC-1, the expression of P-selectin and CD63 in response to the oral bacterium P. gingivalis were lower at recall (1.4 ± 1.1, 1.5 ± 1.2, and 1.0 ± 0.1) than at baseline (2.7 ± 4.1, P = 0.026, 6.0 ± 12.5, P = 0.045, and 2.7 ± 6.7, P = 0.042, respectively). Formation of platelet-leukocyte complexes in response to P. gingivalis was also reduced at recall compared to baseline (1.2 ± 0.7 vs. 11.4 ± 50.5, P = 0.045). sP-selectin levels were significantly increased post-therapy.

Conclusions: In periodontitis patients, the improvement of the periodontal condition is paralleled by a reduction in platelet hyper-reactivity. We suggest that periodontal therapy, as an intervention for improved oral health, can facilitate the management of thrombotic risk, and on the long term can contribute to the prevention of cardiovascular events in patients at risk.

Trial registration: Current Controlled Trials identifier ISRCTN36043780. Retrospectively registered 25 September 2013.

Keywords: Periodontal treatment; Periodontitis; Platelet reactivity; Platelet-monocyte complexes; Platelet-neutrophil Complexes.

Fig. 1

Flow cytometric features of leukocytes, neutrophils, monocytes, and lymphocytes and their complexes with platelets. Panel a is showing all events. The Neutrophils gate is characterized by high side scatter and low CD45 expression, the Monocytes gate is characterized by intermediate side scatter and intermediate CD45 expression, and the Lymphocyte gate is characterized by low side scatter and high CD45 expression. All events within the Neutrophils gate in a were represented in a CD66b and CD61 plot. Events characterized by high expression of both CD66b and CD61 designate the platelet-neutrophil complexes (PNCs, dotted box in b. Similar strategy was employed for the monocytes (using CD14, the platelet-monocyte complexes, PMCs, are in the dotted box in c and lymphocytes (using CD4, the platelet-lymphocyte complexes, PLymCs, are in the dotted box in d. An example of an unstimulated sample (incubated in HEPES buffer) is shown in b, c, and d, whereas flow cytometric analysis of a stimulated sample (incubated in ADP) is shown in e, f, and g

Fig. 2

Platelet response to stimulation with ADP or oral bacteria (Aggregatibacter actinomycetemcomitans [Aa], Porphyromonas gingivalis [Pg], Tannerella forsythia [Tf], Streptococcus sanguis [Ss] and Streptococcus mutans [Sm]). The mean fluorescence intensity (MFI) of (a) PAC-1 binding, (b) P-selectin (CD62P) and (c) CD63 in response to an agonist was recorded as a measure of reactivity. The data were plotted as fold change in MFI [MFI of individual sample after stimulation/MFI of unstimulated sample in buffer] representing the change in reactivity. Data are presented as means ± standard error of the mean (N = 25). Addition of stimuli induced an increase in platelet surface activation markers when analyzed within each timepoint (P < 0.001 - repeated measures ANOVA). The comparisons between timepoints were analyzed by paired T- test (*P < 0.05, baseline vs. recall)

Fig. 3

Formation of platelet-leukocyte complexes in response to stimulation with ADP or oral bacteria (Aggregatibacter actinomycetemcomitans [Aa], Porphyromonas gingivalis [Pg], Tannerella forsythia [Tf], Streptococcus sanguis [Ss] and Streptococcus mutans [Sm]). The number of CD61-positive (equivalent to platelet-conjugated) leukocytes (PLC) was recorded as a percentage of the total population. Data were plotted as fold change [% platelet-bound cells after stimulation/% platelet-bound cells in unstimulated sample in buffer] representing the change in number of complexes formed in response to stimulation with ADP or oral bacteria. Data are presented as means ± standard error of the mean (N = 25). The addition of stimuli induced formation of complexes when analyzed within each timepoint (P < 0.001 in repeated measures ANOVA). The comparisons between timepoints were analyzed by paired T- test (*P < 0.05 baseline vs. recall)

Fig. 4

Concentration of sP-selectin (N = 24) in citrate plasma at baseline (white bars) and recall (black bars). Data are presented as means ± standard deviation. P-value calculated by paired t- test (***P = 0.0008)