Periodontal treatment does not result in detectable platelet activation in vivo.

OBJECTIVES: Periodontitis is associated with systemic inflammation, elevated platelet activation and enhanced risk for cardiovascular diseases, while periodontal treatment reduces tissue inflammation and shows desirable effects on the oral biofilm and dental health. However, subgingival debridement during conservative treatment can lead to local trauma and transient bacteraemia, which might affect cardiovascular risk in these patients. Therefore, we investigated the effect of periodontal treatment on systemic platelet activation. MATERIALS AND METHODS: In a prospective therapeutic trial, 26 patients underwent periodontal treatment and patient blood was analysed immediately before and immediately after intervention for platelet activation markers (flow cytometric analysis of P-selectin, CD63 and CD40L surface expression, integrin αIIbß3 activation and fibrinogen binding, intra-platelet reactive oxygen species production, platelet-leukocyte aggregate formation and intra-platelet vasodilator-stimulated phosphoprotein phosphorylation) in response to adenosine diphosphate (ADP). RESULTS: The present study shows that basal platelet activation levels remain largely unaltered in response to periodontal treatment. We also did not observe significant changes in platelet reactivity in response to different concentrations of platelet agonist ADP. CONCLUSION: Subgingival debridement does not result in relevantly elevated platelet activation. Thus, augmented platelet activation seems unlikely to be a causative triggering factor that increases the short-term risk for platelet-mediated thrombotic events in response to subgingival debridement. CLINICAL RELEVANCE: Subgingival debridement is a safe procedure and does not increase the short-term risk for platelet-mediated thrombotic events.

Periodontal treatment limits platelet activation in patients with periodontitis-a controlled-randomized intervention trial.

AIM: Periodontitis results in platelet activation and enhanced risk for cardiovascular disease. As it is currently unknown whether periodontal treatment reverses platelet hyper-reactivity, we aimed to investigate the role of periodontal treatment on platelet activation. MATERIALS AND METHODS: In a prospective controlled therapeutic trial, 52 patients were enrolled and randomly selected for periodontal treatment or monitored without treatment for 3 months. Patient blood was analysed by flow cytometry for platelet activation markers and by light transmission aggregometry for platelet aggregation in response to pro-thrombotic stimuli. RESULTS: In this study, platelet activation in the control group aggravated over the observation period of 3 months, whereas patients that underwent periodontal treatment showed unchanged levels of platelet activation, measured by surface expression of CD62P, CD40L, generation of reactive oxygen production, activation of GPIIb/IIIa and fibrinogen binding. Moreover, platelet turnover, measured by platelet RNA content and platelet aggregation in response to collagen, differed significantly between patients that were treated and those who were untreated. CONCLUSIONS: Subgingival debridement reduces the risk of aggravated platelet activation and therefore might potentially diminish subsequent diseases such as cardiovascular disease in periodontal patients.

Serum levels of 25-hydroxyvitamin D are associated with periodontal disease.

OBJECTIVES: Vitamin D plays an essential role in bone metabolism as well as in immunity. Hence, it might affect the development and extent of periodontal disease. The aim of this study was the assessment of 25-hydroxyvitamin D (25(OH)D) status in periodontal disease. MATERIALS AND METHODS: Twenty-nine patients with severe periodontal disease and 29 healthy volunteers were recruited in this case-control-study. Serum 25(OH)D levels, Periodontal Probing Depth (PPD), Clinical Attachment Level (CAL), Bleeding on Probing (BOP), Body Mass Index (BMI), and current smoking status and smoking history (packyears) were assessed in all participants. Serum 25(OH)D levels were compared between controls and cases. Multivariable logistic regression was used to determine the odds ratio (OR) and 95 % confidence interval (CI) for periodontal disease in 25(OH)D deficient probands. RESULTS: Patients with periodontal disease presented a significantly higher proportion of deficient 25(OH)D levels (i.e., <50 nmol/l) compared to healthy controls (48 vs. 14 % respectively). The adjusted OR for periodontal disease with vitamin D deficiency was 1.5 (95 % CI, 1.13-1.98). No correlation between serum 25(OH)D levels and CAL, PPD, and BOP in the group with periodontal disease was found. CONCLUSIONS: In this case-control-study 25(OH)D deficiency is significantly associated with periodontal disease. CLINICAL RELEVANCE: The assessment of vitamin D levels in patients presenting with periodontal disease seems advisable, as vitamin D deficiency might be involved in the onset and progression of periodontal disease.

Protein C inhibitor (PCI) binds to phosphatidylserine exposing cells with implications in the phagocytosis of apoptotic cells and activated platelets.

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10-30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal.

Periodontopathogens induce expression of CD40L on human platelets via TLR2 and TLR4.

INTRODUCTION: The outstanding importance of (soluble) CD40L to cardiovascular disease (CVD) is becoming increasingly apparent as CD40L is an important mediator of thrombotic and inflammatory processes. Platelets are the main source for CD40 ligand, linking platelet stimulatory events to inflammation and adverse adaptive immune responses. Periodontitis represents a chronic dental infection by distinct gram negative bacteria that is associated with an increased risk for CVD. However, the effects of periodontopathogens on CD40L expression by platelets have not been determined. MATERIAL AND METHODS: Effects of periodontopathogens A. actinomycetemcomitans Y and P. gingivalis on the expression of CD40L were determined and the underlying receptors and pathways were investigated. 26 patients with periodontitis and 19 controls were included in the clinical part of this study. RESULTS: Periodontopathogens directly induce surface expression of CD40L in human platelets. This activation depends on plasma factors like CD14 and involves TLR2 and TLR4 but not FcγRII. Inhibition of PI3K and PLC completely abolishes bacteria-induced surface expression of CD40L. TLR2 and TLR4 agonists, for example, are also able to induce expression and release of CD40L in human platelets. In patients with periodontitis, plasma levels of soluble CD40L are elevated and positivity for P. gingivalis is associated with a statistical significant increase of soluble CD40L. CONCLUSIONS: Our data indicate an involvement of periodontopathogens in increased plasma levels of soluble CD40L in periodontitis and therefore provide a novel link between periodontitis and increased risk for CVD.

Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets.

INTRODUCTION: Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in periodontitis and the effects of periodontopathogens on P-selectin release are unknown. MATERIAL AND METHODS: Soluble P-selectin was determined in 26 patients with periodontitis and 19 controls. Furthermore, human endothelial cells and platelets were investigated for their ability to elicit soluble and surface P-selectin in response to periodontopathogens A. actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression as well as NFκB translocation in response to these bacteria were determined on endothelial cells as well as the formation of platelet-leukocyte complexes. RESULTS: Plasma levels of soluble P-selectin are significantly elevated in periodontitis and correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets, bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and endothelial cells and results in increased levels of soluble P-selectin. CONCLUSIONS: Periodontopathogens are able to directly cause activation of endothelial cells and platelets within minutes. Given that transient periodontitis-associated bacteremia commonly occurs after tooth brushing or chewing, our data suggest that reduction of periodontopathogens might result in potential cardiovascular benefits.

Decreased phosphorylation of platelet vasodilator-stimulated phosphoprotein in periodontitis--a role of periodontal pathogens.

INTRODUCTION: Epidemiological studies indicate an association between periodontitis and cardiovascular disease, but the underlying mechanisms are poorly understood. Vasodilator-stimulated phosphoprotein (VASP) in its phosphorylated form represents a regulator of platelet function and an indicator for the sensitivity of platelets towards physiologically relevant antagonists of platelet function. As platelets and their activation state play a central role in the development of cardiovascular disease, this study aimed to investigate the influence of periodontal disease and periodontal pathogens on intraplatelet VASP-phosphorylation and platelet function. MATERIAL AND METHODS: Besides several markers of platelet activation, basal and PGE(1) induced intracellular VASP-phosphorylation were determined in platelets of periodontitis patients (n = 26) and healthy donors (n = 19). Furthermore, platelets from healthy donors were incubated with distinct periodontal pathogens and basal and PGE(1) induced VASP-phosphorylation was determined. RESULTS: Compared to controls, platelets of periodontitis patients showed a significant decrease in basal and PGE(1) induced VASP-phosphorylation. VASP-phosphorylation in platelets from periodontitis patients positive for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or Tannerella forsythia was significantly decreased compared to patients that were negative for these bacteria. Furthermore, VASP-phosphorylation in platelets isolated from healthy donors was affected by incubation with these periodontal pathogens. CONCLUSIONS: Our results provide evidence that periodontitis interferes with VASP-phosphorylation in human platelets, presumably as a consequence of a direct effect of periodontitis-associated bacteria. Decreased basal and PGE(1) induced VASP-phosphorylation might represent a mechanism responsible for enhanced platelet activation in periodontitis.