Journal of Indian Society of Periodontology

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 18  |  Issue : 3  |  Page : 293--300

Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study


Ramesh Perumal1, Maheashwari Rajendran1, Malathi Krishnamurthy1, Kiran Kumar Ganji2, Sunil Dattuji Pendor2,  
1 Department of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Periodontics, Sharad Pawar Dental College, Sawangi Meghe, Wardha, Maharashtra, India

Correspondence Address:
Kiran Kumar Ganji
M5 3, Meghdoot Staff Quarters, Sawangi Meghe, Wardha, Maharashtra
India

Abstract

Background: The primary etiologic factor of periodontitis is the subgingival infection with a group of Gram negative pathogens. Transient bacteremia in periodontitis patients underlie chronic production and systemic increases of various proinflammatory mediators, including Interleukin (IL)-1α, IL-6, C-reactive protein and Tumor necrosis factor (TNF)-α. P- selectin is a member of selectin family of cell surface receptor which is located in the membrane of the secretory granules (alpha granules) of platelets and in the membrane of the Weibel-Palade bodies of the vascular endothelial cells. P selectin redistributes from the membrane of the granules to the plasma membrane when platelets and endothelial cells are activated and thus degranulated. Aim: To compare the level of platelet activation, soluble P Selectin level and morphological changes and aggregation of platelets in patients in periodontitis patients compared to healthy controls. Materials and Methods: 80 patients were included in the study with the age group of 35-60. The patients were divided into 2 groups, 40 subjects with generalized chronic periodontitis and 40 healthy subjects taken as control. Periodontal Examination using clinical parameters namely, Bleeding Index, Plaque Index, Probing Pocket Depth and Clinical Attachment Level were recorded. Collection of blood samples for estimation of serum soluble P- selectin level by ELISA method. Evaluation of Platelet morphology and grading the platelet aggregation. Results: P-selectin expression shows that the mean value for control group is 4.97 ± 16.56 ng/mL and study group 13.05 ± 29.94 ng/mL which was significantly higher than control group with P value 0.001. Platelet morphological changes shows small form - mean value for control group is 75.83% ± 14.24% while for study group is 39.08%. ± 21.59; Big form - mean value for control group 0.80% ± 0.35% while for study group 0.48% ± 1.3%and Spider form- mean value for control group 23.88% ± 14.13 while study group 59.32% ±. 23.42.The observation showed high statistical significance with P- value < 0.001 for small and spider form and no statistical significance for big form P = 0.075. Conclusion: Increased expression of P-selectin, spider form of platelets and pathological aggregation pattern which indicates that platelet activation may be associated with chronic periodontitis. The results of the study showed, higher number of spider forms and significant pathological aggregation pattern in periodontitis patients which indicates activation of platelets thus emphasized that periodontitis can be an contributing factor in the development of cardiovascular disease.



How to cite this article:
Perumal R, Rajendran M, Krishnamurthy M, Ganji KK, Pendor SD. Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study.J Indian Soc Periodontol 2014;18:293-300


How to cite this URL:
Perumal R, Rajendran M, Krishnamurthy M, Ganji KK, Pendor SD. Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study. J Indian Soc Periodontol [serial online] 2014 [cited 2019 Aug 24 ];18:293-300
Available from: http://www.jisponline.com/text.asp?2014/18/3/293/134563


Full Text

 INTRODUCTION



Periodontitis is an inflammatory disease of the supportive tissues of the teeth, characterized by gradual loss of tooth supporting alveolar bone, and affects up to 10% of the population in its most severe form. [1] The primary etiologic factor of periodontitis is the subgingival infection with a group of Gram-negative pathogens. The major bacterial species associated with periodontitis are Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Tannerella forsythia (Tf). [2] Transient bacteremia in periodontitis patients underlie chronic production and systemic increases of various proinflammatory mediators, including Interleukin (IL)-1α, IL-6, C-reactive protein, and Tumor necrosis factor (TNF)-α. [3],[4] Systemic inflammation can cause increase in the number of platelets and platelet activation. [5] Activation of platelets leads to the release of pro-inflammatory mediators and exposure of pro-inflammatory receptors, resulting in platelets binding to leukocytes and endothelial cells. These functions make platelets essential participants in both thrombotic and inflammatory reactions across the vasculature. [6] Platelet activation comprises a change in platelet shape, platelet aggregation, and the release of platelet constituents. Platelet shape change is an early event in the activation of platelets. When platelets adhere to the subendothelial matrix, they change their shape from discoid to spherical with the extrusion of the pseudopods. [7] Also, studies have demonstrated that periodontal pathogen like P. gingivalis can activate blood platelets and induce platelet aggregation through hemagglutinin domain protein HgP44. [8] P-selectin is a member of selectin family of cell surface receptor, which is located in the membrane of the secretory granules (alpha granules) of platelets and in the membrane of the Weibel-Palade bodies of the vascular endothelial cells.[9] P-selectin redistributes from the membrane of the granules to the plasma membrane when platelets and endothelial cells are activated and thus degranulated. [10] Platelet activation has been implicated in the development of atherosclerosis, atherothrombosis, and subsequent coronary vascular and cerebro-vascular diseases.[11] Abnormal platelet activation has also been associated with deep vein thrombosis, [12] inflammatory bowel disorders, [13] cancer, peripheral vascular disease, Alzheimer disease, [14] and atrial fibrillation. [15] Epidemiological and intervention studies have associated periodontitis with atherosclerosis and cardiovascular diseases (CVD). The underlying mechanisms of this relationship are still obscure. [16] Since only very few studies have been documented regarding association of periodontitis and platelet activation, the present study was undertaken to evaluate whether periodontitis patients have higher state of platelet activation by estimating the serum P-selectin expression and platelet morphological changes and aggregation pattern compared to healthy controls. The aim of the present study was to investigate the level of platelet activation in periodontitis patients compared to healthy controls, to estimate soluble P-selectin level in control and patients with chronic periodontitis by ELISA method, and to compare morphological changes and aggregation of platelets by spreading analysis method using light microscope in control and patients with chronic periodontitis.

 MATERIALS AND METHODS



Study design and subject selection

The study was approved by the Institutional Ethical committee. About 80 patients in the age group of 35 to 60 years from the Department of Periodontics, Tamil Nadu Government Dental College, Chennai between June and October 2010 participated in the study. The patients were divided into 2 groups, 40 subjects with generalized chronic periodontitis and 40 healthy subjects taken as control.

Patients had generalized chronic periodontitis with minimum 20 teeth were willing for voluntary participation with the age group of 35 to 60 years; no gender prediction were included in the study. A written informed consent was obtained from all patients. A complete history along with complete blood analysis (liver function test, lipid profile, ECG) was done to rule out the presence of cardiac and liver diseases. Prior to blood sample collection for the study purpose, plaque index, bleeding index, probing depth, and clinical attachment loss were recorded. About 10 ml of venous blood samples were collected from the patients with or without periodontitis for ELISA and spreading analysis test.

Exclusion criteria

Trauma or tooth extraction within 2 weeksPatients drug history (antibiotics, anticoagulants) for past 2 monthsAny chronic infectious diseasesPregnancyAnemia, bleeding disorders, or any other hematological disorderCardiac diseasesStrokeHabits like smoking, pan chewing, and alcohol.

The selected patients were divided into two groups (Control and Study groups) based on the following criteria:

Control group

This included 40 healthy subjects who exhibited the following features:

No Bleeding on ProbingAbsence of Clinical Attachment loss as determined by CAL (Clinical Attachment Level) measurements, i.e. CAL = 0Good oral hygienic status.

Study group

The diagnosis of chronic periodontitis was established on the basis of Classification of Periodontal Diseases, AAP 1999.

Bleeding on ProbingProbing pocket depth in mmPresence of signs of Clinical Attachment Loss as determined by CAL measurements ≥5 mmAt least a minimum 20 teeth should be presentPoor oral hygiene.

Study protocol

Institutional ethical committee approvalMedical History and Informed ConsentPeriodontal Examination using clinical parameters namely, Bleeding Index, Plaque Index, Probing Pocket Depth, and Clinical Attachment LevelCollection of blood samplesEstimation of serum soluble P-selectin level by ELISA methodEvaluation of Platelet morphology and grading the platelet aggregation.

Blood sample collection and storage

Fasting venous blood samples of about 10 ml was collected without stasis using 20 G needle by venipuncture in the antecubital fossa after skin preparation. About 5 ml of blood was allowed to stand in plain vacutainer tube for 30 minutes to separate the serum and centrifuged at 3000 rpm for 10 minutes. Then, 500 μl of serum sample was divided in aliquots and stored at −20˚C unit analysis. Remaining 5 ml of blood was divided into 2 parts. About 4 ml of blood was immediately transferred into 3.8% sodium citrate solution containing vacutainer tubes for PRP preparation, and about 1 ml of blood was transferred into EDTA containing vacutainer tubes and used for whole blood analysis.

Procedure

I. Elisa method

A soluble form of P-selectin, which might represent a proteolytic fragment or a soluble splice variant lacking the trans membrane domain, is found in serum which is estimated by Diaclone ELISA Kit, France.

Assay procedure summary

About 100 μl of sample added.



Incubated for 1 hour at room temperature⇓

Washed three times



About 50 μl of biotinylated detection antibody added



Incubated for 1 hour at room temperature



Washed three times



About 100 μl of Streptavidin-HRP added



Incubated for 30 minutes at room temperature



Washed three times



About 100 μl of ready-to-use TMB added

(Protect from light. Let the color develop for 5-15 minutes)



Added 100 H2SO4 (stop reagent)



Read Absorbance at 450 nm

Calculation of results

The optical density of each sample was plotted against its concentration and a curve was drawn through the points.

II. Spreading analysis test

This test was adapted from the method introduced by Marx 1960 and modified by Breddin 1965. [17]

Preparation of citrated platelet rich plasma (cPRP): About 4 ml was blood drawn and transferred into vacutainer tube mixed with sodium citrate in 1:9 ratio. Then, sample was centrifuged at 2000 rpm for 15 minutes without break at room temperature. The resulted cPRP was transferred carefully by micro-pipette (using new disposable plastic tips) into a plastic collection tube to get ready for further analysis. The aspiration of the cPRP was performed with a specific care not to disturb the lower layers of leukocytes and erythrocytes. The cPRP was pipetted gently on the inner wall of the collection tube.

Procedure

Platelet counts were assessed with a Sysmex Automated Haematology Analyzer KX-21 and each PRP sample was adjusted to 0.3 × 108/ml with 0.3% sodium citrate.

Platelet size and morphology

The preparations of Platelet spreading were examined by light microscopy.

According to Scharrer, [18] five types of PLT can be distinguished in healthy individuals:

Giant forms with a surface of more than 200 μmBig forms with minimum one diameter more than 10 μmSmall forms with an average diameter less than 10 μmIntermediate forms (round, etc.,) which are not completely spread and have little hyalomere between pseudopodiaSpider forms which are not spread Platelet with one or more short and relative thick pseudopodia.

The forms 1 to 3 are defined as spread Platelet forms, while the forms 4 and 5 are not spread. In this study, we examined only big, small, and spider forms for percentage calculation. During microscopic examination, hundred Platelets were counted in each Platelet spreading preparation. The findings were recorded on an application that was designed to include the complete descriptions of the examined preparations as follows:

The technical quality of the preparation (thickness, coloring, dirts if any, etc.) was differentiated. The thickness of the preparations reflects firstly the Platelet count of the tested sample and secondly the adhesion of PlateletThe relative presence in percentages (%) of the spread (big and small) and non spread (spiders)The grades of Platelet aggregates (Aggr) were assessed semi quantitatively, and differentiated according to grading system given by Breddin HK, Bauke J. [17]

Platelets aggregation grading

In this test, reversible platelet aggregation means the sticking of platelets to each other without fusion, with their membranes intact. In irreversible aggregation, platelets are fused and membranes are partly destroyed. The platelet aggregation is graded according to the following characteristics:

Grade 1: Platelets are seen single with only a few reversible aggregates and no irreversible aggregatesGrade 2: More reversible aggregates are found, but there are no or only very few irreversible aggregates; there is no obvious reduction in single plateletsGrade 3: An increase in reversible aggregates is evident, often with irreversible aggregation at the centre; the number of platelet is clearly diminishedGrade 4: There are more irreversible than reversible aggregates; single platelets are rareGrade 5: There is complete irreversible aggregation; single platelets are practically missing.

The grades 1 and 2 after rotation were taken as normal, and the grades 3, 4, and 5 as pathological aggregability.

WHOLE BLOOD ANALYSIS: Citrated blood was used for whole blood analysis by using Sysmex Automated Haematology Analyzer. Total WBC, platelets, neutrophils, lymphocytes, and RBCs cell were estimated in both control and Periodontitis subjects.

Statistical analysis

The statistical analysis was done using the computer software program SPSS version 12. Mean and Standard Deviation were estimated for different variables in each study group. Mean values were compared between two study groups by using either Mann-Whitney U-Test. Pearson's Chi-square test was done to compare the proportions in two study groups. In the present study, P value < 0.05 was considered as the level of significance.

 RESULTS



WBC counts for study group (8.04 ± 1.84 × 103) in contrast to the control group (5.46 ± 1.84 × 1.23 × 103) with statistical significance (P < 0.001). Mean value of RBC count (control 3.99 ± 0.58 × 106 study group 4.41 ± 0.51 × 106) with P value 0.001and Hb% (control 11.43 ± 1.32 g % study group 12.20 ± 1.87 g % with P value 0.035). Statistically, the mean value of RBC count, Hb% among control and study group is significant [Figure 1].{Figure 1}

In the study group, mean platelet count was found to be 281.25 ± 84.5 and the control group is about 200.98 ± 56.9, which is found to be statistically highly significant (P=<0.001). Mean value of neutrophil count in control group is 2.94 ± 0.68 × 103 and in study group is 4.09 ± 1.50 × 103; and lymphocyte count in control is 1.95 ± 0.55 × 103 and study group is 2.83 ± 0.72 × 103. It was found to be statically highly significant (P < 0.001) among the study group [Figure 2] and [Figure 3].{Figure 2}{Figure 3}

Statistical analysis shows that the mean value for control group is 4.97 ± 16.56 ng/mL and study group is 13.05 ± 29.94 ng/mL, which was significantly higher than control group with P value being 0.001 [Figure 4]. Result shows that increased P-selectin expression is positively associated with CAL level (P < 0.001) [Figure 5]. Small form - mean value for control group is 75.83 ± 14.24% while for study group is 39.08 ± 21.59; Big form - mean value for control group is 0.80 ± 0.35% while for study group is 0.48 ± 1.3%; and Spider form - mean value for control group is 23.88 ± 14.13% while study group is 59.32 ± 23.42%. The observation showed high statistical significance with P < 0.001 for small and spider form and no statistical significance for big form (P = 0.075) [Figure 6] and [Figure 7] Grade I in control group shows 77.8% and study group shows 22.2%. Grade II - control group: 75.5%; study group: 24.3%; Grade III - 40 control group: 15.6%; study group: 84.4%; Grade IV - control group: 0% and 100% for study group. Within the group correlation showed, grade II (70%) found maximum among the control group followed by grade I (17.5%), Grade III (12.5%), and Grade IV (0%). For study group, grade III (67.5%) was found to be maximum followed by grade II (22.3%), and grade I and grade IV (5.0% each) with P < 0.001 [Figure 8]. Pearson's correlation shows a positive correlation between these two variables with the P = 0.005 among the study and control group [Figure 9].{Figure 4}{Figure 5}{Figure 6}{Figure 7}{Figure 8}{Figure 9}

 DISCUSSION



Periodontal disease may be related to a number of systemic diseases including increased incidence of atherosclerosis, coronary heart diseases, stroke, diabetes mellitus, pre-term low birth weight delivery, and respiratory diseases.[19] Recently, the relationship between periodontitis and CVD is getting much attention particularly toward the role of putative periodontal pathogens like P. gingivalis and A. actinomycetemcomitans. Either local inflammatory stimuli (leaking from infected periodontal tissues into circulation) or systemic inflammatory stimuli (resulting from immune responses to periodontitis-associated bacteremia) can induce activation of endothelial cells and/or platelets. Furthermore, direct interaction between pathogens and endothelial cells and/or platelets might contribute to the development of CVD. [20] Platelet activation has been implicated in the pathogenesis of a number of diseases, which include atherosclerosis, coronary vascular disease, and cerebrovascular disease. [11] Abnormal platelet activation has also been associated with atrial fibrillation,[15] cancer, peripheral vascular disease, Alzheimer disease, [14] inflammatory bowel disorders, [21] and deep vein thrombosis. [22] Platelet activation seems to be influenced by diabetes mellitus, smoking, hypertension, and also the use of oral contraceptives. [23] Platelet activation comprises a change in platelet shape, platelet aggregation, and the release of platelet constituents. According to Kamath, no single test for the quantification of platelet activation is perfect and each has its own merits and demerits.

In the present study, platelet activation was evaluated by two methods. A quantitative analysis was done in order to estimate the serum P-selectin level by ELISA method and a qualitative analysis was done to evaluate the morphological changes and aggregation pattern of platelets by spreading analysis test using light microscopy in both study and control group. All the subjects were thoroughly examined and medical history was recorded. Subjects with systemic diseases like Diabetes mellitus, hypertension, CVD, stroke, systemic lupus erythematous, and habits like smoking, tobacco, cocaine abuse were excluded. [24] Subjects under medications like Aspirin, NSAIDS, Ticlopidine, Clopidogrel, Moxalactam, Losartan, Procaine, Dibucaine, Mithramycin, Diphenhydramine, and Chlorpheniramine were excluded as they cause impairment of platelet aggregation. Also, subjects with history of trauma or tooth extraction 2 weeks prior to the study and pregnant women were excluded from the study. For blood sample collection, 20 Guage needle was used. The use of needle with narrow diameter may activate the platelets due to shear stress against its wall. Fasting venous blood sample was collected from both study and control groups because some of the food contents may activate platelets. Foods like Omega-3 fatty acids, fish oil, onion, cumin, turmeric, and clove decrease platelet thromboxane production thereby may inhibit the platelet aggregation. Vitamin E and onion may induce inhibition of arachidonic acid metabolism and garlic inhibits the fibrinogen binding to platelets and impairs the platelet aggregation. In the present study, the mean age group for control group is 42.43 years and for study group is 43.20 years. The mean age of the study and control groups was similar. The gender distribution of the study group was Males - 19, Females - 21, and control Males - 17; Females - 23. In the present study, the gender distribution between both the study and control group was similar. The Mean PPD value of control group is 2.11 mm and of the study group is about 4.9 mm; and the Mean CAL value of study group is 5.8 mm. According to AAP classification 1999, the severity of Periodontitis is divided into 3 groups.

Mild periodontitis - CAL 1-2 mmModerate periodontitis - CAL 3-4 mmSevere periodontitis - CAL > 5 mm.

An elevated WBC count (reactive leukocytosis) in whole blood analysis was observed among the study group (8.04 ± 1.84) in contrast to the control group (5.46 ± 1.84) with statistical significance of P < 0.001 [Figure 1]. Fredriksson et al. [25] and Loos et al. [26] found significantly higher leukocyte count in patients with periodontitis. The leukocyte count has been demonstrated in several epidemiological studies to be an independent predictor of future coronary heart diseases. Considering the differential blood count, the neutrophil count is higher in study group (control 2.94 ± 0.68 and study group 4.09 ± 1.50). Lymphocyte count is elevated (control 1.95 ± 0.55 and study group 2.83 ± 0.72) among the study group and is found to be statistically significant (P < 0.001) [Figure 2]. Also, data reveal an elevated RBC count (control: 3.99 ± 0.58; study group: 4.41 ± 0.51) with P = 0.001 and Hb% (control: 11.43 ± 1.32 gm %; study group: 12.20 ± 1.87 gm% with P = 0.035) among the study group which is statistically significant.

Reactive thrombocytosis was observed in the study group compared to the control group. The mean Platelet count in the study group was 281.25 ± 84.5 and in the control group was 200.98 ± 56.9, which is found to be statistically highly significant (P=<0.001). This finding concurs with the study of Wakai et al., [27] who stated significant association between platelet counts and periodontitis, and that systemic inflammation causes an increase in the number of platelets and platelet activation.[28] Most stimuli cause changes in the shape of platelets. This change involves first the formation of very fine (0.1 μm diameter) pseudopodia (i.e. filopodia) from the 64 rim of the disc, followed by a general "rounding up" of the platelet so that it becomes a spiny sphere, often with much broader pseudopodia. [29] So in the present study, small, big, and spider forms were considered for percentage calculation. Small forms were higher in control group (about 75.83%) compared to the study group (39.08%). There was no significant difference in the big forms between the study and control group. Study group showed a higher percentage of spider forms (59.32%) while in the control group it is 23.88%. The observation showed high statistical significance with P < 0.001 for small and spider forms and no statistical significance for the big forms (P = 0.075). Spider form is considered as an activated form of platelet. This indicates that platelet activation is higher among the study group compared to the control group [Figure 6] and [Figure 7]. Higher percentage value of spider forms in the study group may infer that stimulation of platelets due to the persistent periodontal bacterial toxins causes activation of platelets and their shape changes. Compared to electron microscope, light microscope does not faithfully register local differences in their thickness, but do accurately show changes in their length and orientation. Several studies have demonstrated that putative periodontal pathogen P. gingivalis can activate blood platelets and induce platelets aggregation using platelet aggregometry method. Gingipains (Rgp and Kgp) from P. gingivalis was able to induce platelets aggregation [30] and this result is in agreement with [31] that P. gingivalis vesicles posses potent platelet aggregation activity due to active concentration of many virulence factors. P. gingivalis-induced platelet aggregation in platelet-rich plasma (PRP) depends on hemagglutinin A (HagA)-encoding genes that intragenically code for adhesions, such as hemagglutinin domain proteins Hgp44, but not Arg-gingipain (Rgp) proteinase. Naito et al. [32] and Keber irena et al. [33] evaluated the anti-aggregating property of propananol based on Breddin [34] classification on platelet aggregation. The present study is also according to the classification given by Breddin [34] under light microscopy. Observation revealed that Grade I and II aggregation patterns were more in control group, whereas Grade III pattern was higher in the study group. Grade IV aggregation is seen only in the study group. None of the group showed grade V aggregation pattern [Figure 8]. According to Breddin's classification, Grade I and Grade II are considered as physiological aggregations (reversible aggregations) and grade III and IV are considered as pathological aggregations (Irreversible aggregations). Result of the present study showed higher percentage prevalence for pathological aggregation in the study group (grade III: 67.5%; grade IV: 5%) compared to the control group (grade III: 12.5%; grade IV: 0%). The limitation of spreading analysis test is that it cannot define the aggregation size like platelet aggregometry. In platelet aggregometry, the aggregation can be classified as small (diameter: 9-25 μm), medium (diameter: 26-50 μm), and large-sized aggregates (diameter: >50 μm) based on their size. [34] However, spreading analysis test shows the grade of platelet aggregation and helps us to decide the pathological aggregation. Several studies have proved that activated platelets expressed increased P-selectin level which has a vital role in atherosclerotic lesion development. [35],[36] Raised levels of soluble P-selectin are found in a variety of thrombotic disorders, including ischemic heart disease, [36] cerebrovascular disease, congestive cardiac failure, [37] and hypertension. [38] Measurement of plasma levels of beta thromboglobulin and Platelet Factor 4 is specific to platelet release [39] and has been suggested as a means for detecting increased platelet activation in vivo. [40] However, some problems are evident with respect to measurement of these proteins. Thromboglobulin and Platelet Factor 4 are theoretically present in platelets in similar amounts and are released in similar quantities, but plasma levels of beta thromboglobulin greatly exceed plasma levels of Platelet Factor 4. This is possibly due to the more rapid binding of Platelet Factor 4 to the endothelial cells. Therefore, a higher ratio of beta thromboglobulin to Platelet Factor 4 is always maintained in vivo. [40]

Moreover, activation of α-granules needs higher threshold value compared to GPIIb/IIIa which need lower threshold value [41] and hence the influence of extraneous factors like speed of centrifugation and temperature can be eliminated. Michelson et al.'s [42] study showed that activated platelets shed their membrane bound P-selectin in the plasma. Papapannagiotou et al. [43] were the first to document increased plasma levels of P-selectin in chronic periodontitis. The result of the study showed that P-selectin was statistically highly significant when compared with control group and positively associated with severity of periodontitis. According to this study, increased level of P-selectin expression is from platelets and not from endothelium, as vWf (marker of endothelial activation) was normal in their study. However, since platelet-derived P-selectin is identical to P-selectin derived from endothelial cells, the cell type specific contribution was not known. In our study, P-selectin expression is significantly higher in the study group when compared with control. The mean value for control group is 4.97 ± 16.56 ng/ml and that of study group is 13.05 ± 29.94 ng/ml, which is found to be highly significant (P < 0.001) [Figure 4]. The level of P-selectin expression is higher in severe periodontitis (CAL > 5 mm) compared to moderate periodontitis (CAL < 5 mm) [Figure 5]. Results of this study concur with the reports of Papapannagiotou et al. [43] However, we have not evaluated the source of P-selectin. In the present study, 3 of the healthy controls had elevated soluble P-selectin levels even when their pocket depth was ≤2 mm.

The influence of P-selectin on platelet aggregation was correlated with Pearson's correlation coefficient test which showed a positive correlation (P = 0.005) [Figure 9]. According to Michelson et al., [42] P-selectin determines the size and stability of platelet aggregation. P-selectin, which is progressively expressed on the platelet surface, binds by means of the lectin domain to its binding site on adjacent platelets, stabilizing interactions between already bridged platelets, thereby allowing the formation of large stable platelet aggregates.[44] Moreover, the role of P-selectin in atherosclerotic lesion development is very important. According to Rinder et al., [45] P-selectin expressed by platelet activation causes the formation of platelet-neutrophil and platelet-monocyte aggregates in blood. Berger [36] states that P-selectin on microparticles could coat monocytes and contribute to their recruitment into atherosclerotic lesions. Study done in P-selectin-deficient mice has proved that both atheroselerotic lesion formation [46],[47] and neointimal growth upon arterial injury were attenuated in apoE (apoE-/-) and LDL -receptor (LDLR-/-) deficient mice. In the present study, both quantitative (estimation of serum P-selectin level) and qualitative analysis (morphological changes and pathological aggregation pattern) findings are positively correlated with chronic periodontitis and their disease severity. Therefore, we infer that periodontitis associated with platelet activation and disease severity may aggravate the further platelet activation.

 Conclusion



The results of the study showed higher number of spider forms and significant pathological aggregation pattern in study group, which indicates activation of platelets. An elevated serum P-selectin level was seen in periodontitis patients compared to the control. Also, P-selectin expression increased in severe periodontitis than moderate periodontitis. These products have the potential to activate platelets which in turn can release an arsenal of potent inflammatory and mitogenic substances leading to an altered endothelial function (chemotaxis and adhesion). Results of the present study show an increased expression of P-selectin, spider form of platelets, and pathological aggregation pattern which indicate that platelet activation may be associated with chronic periodontitis though the exact mechanism could not be established. Chronic periodontitis leads to platelet activation; it may be emphasized that periodontitis can be a contributing factor in the development of CVD.

References

1Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005;366:1809-20.
2Van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas Gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002; 29:1023-8.
3Loos BG. Systemic markers of inflammation in periodontitis. J Periodontol 2005;76:2106-15.
4Paraskevas S, Huizinga JD, Loos BG. A systematic review and meta-analyses on C-reactive protein in relation to periodontitis. J Clin Periodontol 2008; 35:277-90.
5Klinger MH, Jelkmann W. Role of blood platelets in infection and infammation. J Interferon Cytokine Res 2002;22:913-22.
6Weyrich AS, Lindemann S, Tolley ND, Kraiss LW, Dixon DA, Mahoney TM, et al. Change in protein phenotype without a nucleus: Translational control in platelets. Semin Thromb Hemost 2004;30:491-8.
7White JG. Electronmicroscopic studies of platelet secretion. Prog Hemost Thromb 1974;2:49-98.
8Naito M, Sakai E, Shi Y, Ideguchi H, Shoji M, Ohara N, et al. Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase. Mole Microbiol 2006;59:152-67.
9McEver RP. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood Cells 1990;16:73-80.
10Johnston GI, Kurosky A, McEver RP. Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells. J Biol Chem 1989;264:1816-23.
11Cahill MR, Newland AC. Platelet activation in coronary artery disease. Br J Biomed Sci 1993;50:221-34.
12De Boer AC, Han P, Turpie AG, Butt R, Zielinsky A, Genton E. Plasma and urine beta thromboglobulin concentrations in patients with deep vein thrombosis. Blood 1981;58:693-8.
13Collins CE, Cahill MR, Newland AC, Rampton DS. Platelets circulate in an activated state in inflammatory bowel disease. Gastroenterology 1994;106:840-5.
14Sevush S, Jy W, Horstman LL, Mao WW, Kolodny L, Ahn YS. Platelet activation in Alzheimer Disease. Arch Neurol 1998;55:530-6.
15Minamino T, Kitakaze M, Asanuma H, Ueda Y, Koretsune Y, Kuzuya T, et al. Plasma adenosine levels and platelet activation in patients with atrial fibrillation. Am J Cardiol 1999;83:194-8.
16Tonetti MS, D'Aiuto F, Nibali L, Donald A, Storry C, Parkar M, et al. Treatment of periodontitis and endothelial function. N Engl J Med 2007;356:911-20.
17Breddin HK, Bauke J. Thrombozytenagglutination and Gefa ß krankheiten. J Clin Exp Hematop 1965;11:144- 64.
18Scharrere I Methods of the Thrombozyten functions and diagnostics Haemotology. 1985
19Mealey BL. Influence of periodontal infections on systemic health. Periodontol 2000 1999;21:197.
20Li L, Messas E, Batista EL Jr, Levine RA, Amar S. Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 2002:105:861-7.
21Collins CE, Cahill MR, Newland AC, Rampton DS. Platelets circulate in an activated state in inflammatory bowel disease. Gastroenterology 1994;106:840-5.
22De Boer AC, Han P, Turpie AG, Butt R, Zielinsky A, Genton E. Plasma and urine beta thromboglobulin concentrations in patients with deep vein thrombosis. Blood 1981;58:693-8.
23Kamath S, Blann AD, Lip G Y. Platelet activation: Assessment and quantification. Eur Heart J 2001;22:1561-71.
24Blann AD, Lip GY. Hypothesis: Is soluble P-selectin a new marker of platelet activation? Atherosclerosis 1997;128:135-8.
25Fredriksson M, Gustafsson A, Asman B, Bergström K. Hyperreactive peripheral neutrophils in adult periodontitis: Generation of chemiluminescence and intracellular hydrogen peroxide after in vitro priming and FcgammaR- stimulation. J Clin Periodontol 1998;25:394-8.
26Loos BG, Craandijk J, Hoek FJ, Wertheim-van Dillen PM, van der Velden U. Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J Periodontol 2000;71:1528-34.
27Wakai K, Kawamura T, Umemura O, Hara Y, Machida J, Anno T, et al. Associations of medical status and physical fitness with periodontal disease. J Clin Periodontol 1999;26:664-72.
28Klinger MH, Jelkmann W. Role of blood platelets in infection and infammation. J Interferon Cytokine Res 2002;22:913-22.
29Nachmlas VT. Platelet and megakaryocyte shape change: Triggered alterations in the cytoplasm. Sem Hematol 1983;20:261-81.
30Curtis MA, Macey M, Slaney JM, Howell GL. Platelet activation by Protease I of Porphyromonas gingivalis W83. FEMS Microbio Lett 1993;110:167-73.
31Sharma A, Novak EK, Sojar HT, Swank RT, Kuramitsu HK, Genco RJ. Porphyromonas gingivalis platelet aggregation activity: Outer membrane vesicles are potent activators. Oral Microbiol Immunol 2000;15:393-6.
32Naito M, Sakai E, Shi Y, Ideguchi H, Shoji M, Ohara N, et al. Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase. Mole Microbiol 2006;59:152-67.
33Keber I, Jerse M, Keber D, Stegnar M. The influence of combined treatment with propranololand Acetylsalicylic acid on platelet Aggregation in coronary heart disease. Br J Clin Pharmacol 1979;7:287-91.
34Ozaki Y, Satoh K, Yatomi T, Yamamoto T, Shirasawa Y, Kume S. Detection of platelet aggregates with a particle counting method using light scattering. Anal Biochem 1994;218:284-94.
35Burger PC, Wagner DD. Platelet P-selectin facilitates atherosclerotic lesion development. Blood 2003;101:2661-6.
36Ikeda H, Nakayama H, Oda T, Kuwano K, Muraishi A, Sugi K, et al. Soluble form of P-selectin in patients with acute myocardial infarction. Coron Artery Dis 1994;5:515-8.
37Connor CM, Gurbel PA, Serebruany VL. Usefulness of soluble and surface bound P-selectin in detecting heightened platelet activity in patients with congestive heart failure. Am J Cardiol 1999;83:1345-9.
38Lip GY, Blann AD, Zarifis J, Beevers M, Lip PL, Beevers DG. Soluble adhesion molecule P-selectin and endothelial dysfunction in essential hypertension: Implications for atherogenesis? A preliminary report. J Hypertension 1995;13;1674-8.
39Schernthaner G, Silberbauer K, Muhlhauser I, Willvonseder R. Clinical significance of the radioimmunological determination of beta-thromboglobulin and platelet factor 4. Acta Med Austriaca Suppl 1979;6:375-9.
40Kaplan KL, Owen J. Plasma levels of beta-thromboglobulin and platelet factor as indices of platelet activation in vivo. Blood 1981;57:199-202.
41Holmes MB, Sobel BE, Howard DB, Schneider DJ. Differences between Activation Thresholds for Platelet P-Selectin and Glycoprotein IIb-IIIa Expression and Their Clinical Implications. Thromb Res 1999;95:75-82.
42Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol 1999;6:342-8.
43Papapanagiotou D, Nicu EA, Bizzarro S, Gerdes VE, Meijers JC, Nieuwland R, et al. Periodontitis is associated with platelet activation. Atherosclerosis 2009;202:605-11.
44Boukerche H, Ruchaud-Sparagano MH, Rouen C, Brochier J, Kaplan C, McGregor JL. A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation. Br J Haematol 1996;92:442-51.
45Rinder HM, Bonan JL, Rinder CS, Ault KA, Smith BR. Dynamics of leukocyte platelet adhesion in whole blood. Blood 1991;78:1730-7.
46Johnson RC, Chapman SM, Dong ZM, Ordovas JM, Mayadas TN, Herz J, et al. Absence of P-selectin delays fatty streak formation in mice. J Clin Invest 1997;99:1037-43.
47Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaud AL. P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 2000;191:189-94.