|Year : 2019 | Volume
| Issue : 1 | Page : 289-295
Tissue factor expression on platelets in patients with acute coronary syndrome
Amr A Fathy1, Mohamed A Soliman1, Mohamed A Helwa1, Rehab E Yasin1, Nermeen H. A. El-Sattar2
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Clinical Pathology, Shebin El-Kom Teaching Hospital, Menoufia, Egypt
|Date of Submission||22-Feb-2016|
|Date of Acceptance||13-Apr-2016|
|Date of Web Publication||17-Apr-2019|
Nermeen H. A. El-Sattar
Shebeen El-Kom, Menoufia
Source of Support: None, Conflict of Interest: None
To investigate the expression of tissue factor (TF) on platelet monocyte aggregates (PMA) and platelets as a participating factor in pathogenesis of acute coronary syndrome (ACS).
ACS is due to rupture of an arterial plaque. TF on platelets and PMA may provide potential distal sites for generation of new thrombi. In this study, the expressions of TF on PMA and platelets are investigated as participating factors in pathogenesis of ACS.
Patients and methods
Twenty-five patients with acute myocardial infarction, 25 patients with stable angina, and 20 clinically healthy patients as control are investigated for TF on PMA, as well as platelet-associated TF by flow cytometry analysis.
TF on platelets and PMA were highly significant in patients with acute myocardial infarction than those with stable angina and controls.
The greater expression of TF on platelets and PMA strengthens the link between platelet activation, blood coagulation, and thrombus formation and may further contribute to the hypercoagulability associated with the disease.
Keywords: acute coronary syndrome, stable angina, tissue factor
|How to cite this article:|
Fathy AA, Soliman MA, Helwa MA, Yasin RE, El-Sattar NH. Tissue factor expression on platelets in patients with acute coronary syndrome. Menoufia Med J 2019;32:289-95
|How to cite this URL:|
Fathy AA, Soliman MA, Helwa MA, Yasin RE, El-Sattar NH. Tissue factor expression on platelets in patients with acute coronary syndrome. Menoufia Med J [serial online] 2019 [cited 2019 May 21];32:289-95. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/289/256082
| Introduction|| |
Acute coronary syndrome (ACS) is a clinical state induced by the thrombosis consequent on the rupture of an unstable atherosclerotic plaque. In this syndrome, the procoagulant content of complex plaques triggers both platelet activation and coagulation pathways .
Circulating platelet-leukocyte aggregates (PLA) is a sensitive marker of in-vivo platelet activation . It is widely recognized that the interaction between platelets and leukocytes leads to phenotypic changes in both cell types and to the secretion of a variety of bioactive compounds .
Tissue factor (TF) is the main cellular initiator of blood coagulation, and it is also currently considered the protein that links proinflammatory and prothrombotic mechanisms in the progression of atherosclerosis. Expression of TF by leukocytes  or by PLA  may trigger the extrinsic coagulation cascade; the thrombin is generated and activates platelets, which leads to the formation of a platelet-fibrin thrombus. Previous studies have shown that plaque-associated TF, TF plasma levels, as well as TF-expressing monocytes are higher in patients with ACS than in those with stable angina (SA) .
Platelets contain TF-specific mRNA, which can be translated into protein . In addition, platelet reactivity to classical agonists results in the expression of functional TF on the platelet surface .
The aim of this study was to investigate the expression of TF on platelet monocyte aggregates (PMA) and platelets as a participating factor in pathogenesis of ACS.
| Patients and Methods|| |
This study is conducted on 70 patients, selected from Menoufiya University Hospital. Ethics rules in the form of approval of the ethics committee of Menoufia University Hospitals and patient consent are applied on the studied groups.
They are divided into three groups:
- Group I [patients with acute myocardial infarction (AMI)]: this group included 25 patients, comprising 20 males and five females, with AMI. Their ages ranged from 48 to 70 years, with a mean ± SD age of 58.8 ± 6.1 years
- Group II (patients with SA): this group included 25 patients, comprising 15 males and 10 females, with SA. Their ages ranged from 47 to 68 years, with a mean ± SD age of 59.1 ± 6.7 years
- Group III (control group): this group included 20 apparently healthy subjects, comprising 15 males and five females, who served as a control group. Their ages ranged from 48 to 68 years, with a mean ± SD age of 57.8 ± 6.3 years.
All participants in this study were subjected to the following:
- Full medical history, laying stress on family history, and past history, including history of diabetes mellitus, hypertension, smoking, recurrent chest pain, and medication use
- Clinical examination
- Laboratory investigations: (a) CBC was examined by ADVIA-2120 hematological analyzer (ADVIA-2120, Siemens Healthcare, Malvern, Pennsylvania, USA). (b) Highly sensitive C-reactive protein (hs-CRP) was assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kits (EIA-3954), supplied from DRG CRP International Inc. (USA) . (c) CK-MB mass was estimated by using the commercially available ELISA kit (EIA-4112), provided by DRG (DRG International Inc. Springfield, New Jersey, USA) . (d) Flow cytometry analysis was done for TF on platelets and on PMA; moreover, assessment of percentage of TF-positive platelets and PLA was done .
Quantitative assay of highly sensitive C-reactive protein
Hs-CRP was measured by available ELISA kit. The kit uses a double-antibody sandwich technique. CRP standards, specimens, and controls were dispensed into appropriate wells that have been precoated with a monoclonal antibody specific to hs-CRP. CRP enzyme conjugate reagent was added to each well, incubated, and washed. Then TMB solution was added to each well and incubated. The reaction was stopped by adding stop solution to each well. Wells exhibited a change in color. The color change was measured spectrphotometrically at wavelength of 450 nm. The concentration of hs-CRP in samples was then determined by comparing the OD of samples to the standard curve.
Quantitative assay of CK-MB mass
CK-MB mass was measured by available ELISA kit. The kit uses a double-antibody sandwich technique. Calibrators and sample specimens were dispensed into appropriate wells which has been precoated with a monoclonal antibody specific to CK-MB. CK-MB enzyme reagent were added to each well, incubated, and washed. Then working substrate solution were added to all wells and incubated. The reaction was stopped by adding stop solution to each well. Wells exhibited a change in color. The color change was measured spectrophotometrically at wavelength of 450 nm. The concentration of CK-MB in samples was then determined by comparing the OD of samples to the standard curve.
Assessment of tissue factor on platelet-leukocyte aggregates by immune-phenotyping
For each sample, two tubes were prepared: first one for simultaneous detection of CD142 (for TF), CD45 (for leukocytes population), and CD61 (for platelet expression) using PE (phycoerythrin)-labeled anti-CD142 Ab (provided from eBioscience Inc. San Diego, CA US), PerCP-labeled anti-CD45 Ab provided from eBioscience Inc., and FITC (fluorescein isothiocyanate)-labeled anti CD61 Ab, provided from Immunostep (Immunostep, Salamanca, Spain). In the second tube, auto control was used to exclude the cell auto-fluorescence and instrument noise.
A volume of 5 μl of monoclonal Ab CD142+, 5 μl of monoclonal Ab CD61+, and 5 μl of monoclonal AbCD45 were added to 100 μl of fresh blood, mixed well, and incubated at 2–8°C for 30 min in the dark; the cells were, then, lysed, and washed three times in 2 ml of PBS.
Finally, the cells were suspended in 200 μl of PBS for final flow cytometry analysis. All samples were analyzed using a flow cytometer (BD FACSCalibur, USA; in Clinical Pathology Department, Faculty of Medicine, Menoufia University, BD FACSCalibur, San Jose, CA. USA).
Forward scatter, and side scatter measurements, and fluorescence measurements were made with linear amplifiers. Gating was done on PMA on the basis of the forward and side scatters. Three colors and light scattering properties were applied to determine the percentage of TF-positive PLAs (CD142 +ve, CD61 +ve, and CD45 +ve events) [Figure 1].
|Figure 1: Flow cytometry gating strategy for analyzing platelet monocyte aggregates (PMA).|
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Assessment of tissue factor on platelets by immunophenotyping
Platelet-rich plasma was prepared by centrifugation of EDTA blood sample at 200g for 10 min. Separation of plasma was carried out in a separate tube, and then centrifuged at 3000 rpm for 3 min. The supernatant was decanted, and 300 μl only of the plasma was left, and mixed well with the sediment platelets.
For each sample, two tubes were prepared: first one for simultaneous detection of CD142 and CD61 expression using PE-labeled anti-CD142 Ab and FITC-labeled anti-CD61 Ab. In the second tube, auto control was used to exclude the cell auto-fluorescence and instrument noise.
A volume of 5 μl of monoclonal Ab CD142+ and 5 μl of monoclonal Ab CD61 was added to 20 μl of platelet-rich plasma, mixed well, and incubated at room temperature for 15 min in the dark. The cells were then washed three times in 2 ml of PBS. Finally, the cells were suspended in 200 μl of PBS for final flow cytometry analysis. Forward scatter and side scatter measurements were made using logarithmic amplifiers, and fluorescence measurements were made with logarithmic amplifiers. Gating was done on platelet based on forward and side scatter.
Two colors and light scattering properties were applied to determine the percentage of TF-positive platelet (CD142 +ve, CD61 +ve events). Absolute TF-positive platelet was calculated as its percentage multiplied by total number of platelet count [Figure 2].
|Figure 2: Flow cytometry gating strategy for analyzing the tissue factor expression on platelets in healthy and patient groups. (a) Flow Scatter (FSCC) vesrus Side Scatter (SCC) in analysid of Platelet Rich Plasma (PRP). (b) Flow cytometry analysis in control group. (c) Flow cytometry analysis in SA group. (d) Flow cytometry analysis in AMI group|
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The data were collected, tabulated, and analyzed by SPSS version 22.0 (SPSS version 22, IBM, Armonk, NY, USA). Data were expressed as percentage and mean and SD. χ2-Test was used to study association between two qualitative variables. Wilcoxon signed rank test (nonparametric test) is a test of significance used for comparison between two related groups not normally distributed having quantitative variables. Analysis of variance (f) test is a test of significance used for comparison between three or more groups having quantitative variables. Spearman's correlation coefficient (r) (nonparametric test) is a test used to measure the association between two quantitative variables. Level of significance was set as P value less than 0.05. The receiver operating characteristic curve is a graphic representation of relationship between sensitivity and specificity at different cutoff points for diagnostic test. It was used in this study to measure the cutoff point at which TF level predicts AMI.
| Results|| |
There was no significant difference among the studied groups (AMI, SA, and controls) regarding platelet count (P > 0.05) and highly significant difference between studied groups regarding hs-CRP (P < 0.0001). There was significant difference between the studied groups regarding lipid profile [serum cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol] [Table 1].
Comparison of lipid profile between the studied groups is shown in [Figure 3].
There was a highly significant difference between the studied groups regarding TF on platelets (P < 0.01), absolute platelet TF (P < 0.001), and TF on PMA (P < 0.0001) [Table 2].
|Table 2: Comparison of tissue factor expression among the studied groups|
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Comparison of platelet tissue factor% (PTF) and PMA TF among the studied groups is shown in [Figure 4].
|Figure 4: Comparison of platelet tissue factor (PTF%) and platelet monocyte aggregates (PMA%) among the studied groups.|
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There was a highly significant difference between smokers and nonsmokers regarding PTF and PMA TF [Table 3].
|Table 3: Comparison between tissue factor expressions in smokers versus nonsmokers|
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Comparison between PTF and PMA TF expressions in smokers versus nonsmokers is shown in [Figure 5].
|Figure 5: Comparison between platelet tissue factor (PTF) and platelet monocyte aggregates (PMA) tissue factor (TF) expressions in smokers versus nonsmokers.|
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There was a significant difference between diabetics and nondiabetics regarding PTF and PMA TF [Table 4].
|Table 4: Comparison between diabetics and nondiabetics regarding platelet tissue factor expression|
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Comparison between diabetics and nondiabetics regarding PTF and PMA TF expression is shown in [Figure 6].
|Figure 6: Comparison between diabetics and nondiabetics regarding platelet tissue factor (PTF) and platelet monocyte aggregates (PMA) tissue factor (TF) expression.|
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| Discussion|| |
ACS is a set of signs and symptoms due to the rupture of an arterial plaque which provokes platelet-rich coronary thrombus formation . Patients with ACS include unstable angina, non-ST-elevation myocardial infarction, and ST-elevation myocardial infarction . Patients with coronary artery disease have long been reported to have circulating activated platelets, platelet-derived microparticles, PMA, and increased platelet reactivity ,. Platelets are recognized as playing a major role in inflammation as well as in hemostasis and thrombosis, being the source of inflammatory mediators and able to both release and respond to chemo-attractant cytokines ,. The binding of platelets or platelet-derived micro-particles to monocytes in ACS is one of the clues to the interaction of inflammation and thrombosis: when inflammation begets local thrombosis, this in turn exacerbates inflammation, resulting in a vicious circle ,. The main mechanism of plasma coagulation cascade activation in inflammation is mediated by TF . Expression of TF by leukocytes  or by PLA  may trigger the extrinsic coagulation cascade; the thrombin thus generated can activate platelets, which leads to the formation of a platelet-fibrin thrombus. Activated platelets synthesize inflammatory proteins such as interleukin 1 , and also the synthesis of TF has been reported ,.
In light of the previous postulations, the aim of this study is to investigate the expression of TF on PMA and platelets as a participating factor in pathogenesis of ACS.
In this study, there was no significant difference in platelet count between AMI, SA, and controls. This is in agreement with Khode et al.  who reported that the comparison among AMI, stable coronary artery disease, and controls in platelet count is nonsignificant.
Moreover, this study showed highly significant difference in hs-CRP among AMI, SA, and control group. This is in agreement with Singh et al. , who reported that elevated CRP levels are an independent predictor of cardiac death and AMI; levels of hs-CRP of at least 3 mg/l also predict recurrent coronary events, suggesting that an enhanced inflammatory response at the time of hospital admission can determine subsequent plaque rupture.
There was, also, significant difference regarding LDL-c level among AMI, SA, and control group, being higher in AMI. This agreed with Gotto , who reported that reduction in the high levels of cholesterol, particularly LDL-c, decreases the risk for developing atherosclerosis, as LDL-c is associated with endothelial dysfunction, inflammation, and increased vasoconstriction.
This study, also, revealed that the percentage of TF-positive PMA was highly significant in AMI than in SA and controls. This is in agreement with Brambilla et al. , who reported that percentage of TF-positive PMA was significantly higher in patients with AMI than in patients with SA (≥3-folds higher) and in controls (≥5-folds higher). This is in agreement with Lu et al.  who reported that significantly greater number of TF-positive PMA was found by flow cytometry in blood of patients with ACS than in either patients with SA or controls. So, the expression of TF by PMA  may trigger the extrinsic coagulation cascade; the thrombin thus generated can activate platelets, and leads to the formation of a platelet-fibrin thrombus.
Moreover, in this study, the percentage of TF-positive platelets was highly significant in AMI than in SA, and in controls. This is in agreement with Brambilla et al. , who reported that the percentage of TF-expressing platelets was higher in patients with ACS than in those with SA or controls (≥3-folds). Lu et al.  reported that ACS group had a significantly higher amount of TF-positive platelets than SA or control groups. The platelet-derived TF may contribute to fibrin formation and to the propagation and stabilization of a thrombus, and it can also participate in several cellular processes that stimulate atherogenesis such as angiogenesis and cell migration, both of which are associated with plaque growth, and under certain circumstances, plaque weakening, leading to destabilization of the lesion Gawaz et al. .
There was a highly significant difference between smokers and nonsmokers regarding TF on PMA, TF on platelets, as well as absolute TF-positive platelets. This is in agreement with Steffel et al. , who revealed that patients with cardiovascular risk factors such as smoking have elevated plasma levels of TF.
Moreover, there was a significant difference between diabetics and nondiabetics regarding TF on PMA, TF on platelets, as well as absolute TF-positive platelets. This is in agreement with Steffel et al. , who revealed that patients with cardiovascular risk factors, such as diabetes mellitus, have elevated plasma levels of TF.
The correlation study between TF and hs-CRP revealed highly significant positive correlation between TF on platelets, absolute TF-positive platelets, and hs-CRP. This is in agreement with Brambilla et al. , who reported that TF mRNA levels in ACS significantly correlated with CRP levels, and this positive correlation may suggest that systemic inflammatory reaction present in patients with ACS might affect the TF content in megakaryocytes, and consequently in platelets.
There was a highly significant positive correlation between TF on PMA, TF on platelets, and absolute TF-positive platelets and LDL-c. This is in agreement with Vojacek et al. , who reported that there was a significant positive correlation between TF level and LDL-c.
Binary logistic regression analysis for independent risk factors shows that TF is an independent risk factor for cardiovascular diseases.
| Conclusion|| |
This study demonstrated that TF on PMA, as well as on platelets was highly expressed in patients with AMI than in those with SA, and healthy controls, and also, the absolute platelet TF was higher in patients with AMI than in those with SA and healthy controls.
The percentage of tissue on PMA, as well as on platelets was positively correlated with low-density lipoprotein cholesterol and hs-CRP.
TF is an independent risk factor for cardiovascular diseases.
The greater expression of TF on PMA, as well as on platelets strengthens the link between platelet activation, blood coagulation, and thrombus formation and may further contribute to the hypercoagulability state associated with the disease.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]