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Acceleration of New Biomarkers Development and Discovery in Synergistic Diagnostics of Coronary Artery Disease

Written By

Ewa Stępien

Submitted: October 30th, 2010 Published: September 15th, 2011

DOI: 10.5772/18940

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1. Introduction

The current definition of biomarkers includes “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (Biomarkers Definitions Working Group, 2001)”. Accordingly, biomarkers are usually used for detection and establishing the magnitude of a physiological derangement as well as to monitor a treatment.

The role for imaging techniques and biomarkers in the diagnosis and treatment of myocardial infarction (MI) after percutaneous coronary intervention is well-established. Many candidate biomarkers emerging from genomics and proteomics research have the potential to serve as predictive indexes for guiding the development of interventional cardiology (Gerhardt et al. 1991; Katus et al., 1991; Lindpaintner, 1997; Kong et al., 1997). Among them the undisputed role still play cardiac proteins like troponins or creatine kinase-myocardial band (CK-MB) (Alcock et al., 2010; Lim et al., 2011). Less established, however, is the employment of biomarkers to determine long-term, progressive, or dynamic risk over time in patients with advanced coronary artery disease (CAD). Biomarkers offer a means to track differential exposure as well as impact of exposure. As such, they reflect individual vulnerability, ongoing person-environment interaction, and unmeasured environmental factors that mediate the effect of exposures (Fig. 1). Essential to a vision of synergistic diagnostics is a focus on the mechanisms of diseases. Understanding what is happening on a molecular and cellular level, how disease actually begins, how cells begin to express certain proteins, influence other cells and trigger processes (atherosclerosis, thrombosis, calcification) will allow to develop in vitrodiagnostics and imaging techniques to distinguish these processes. By characterizing a comprehensive set of measurable processes that capture diverse pathogenic aspects of CAD, a real-time systems view of disease activity can be generated to improve decision making.

This chapter summarize a current view on the development of new biomarkers as a prognostic platform among patients at risk of CAD and upcoming complications.

Figure 1.

Model showing relationship of a biomarker with internal and external factors which have an impact on measurable and unmeasurable features of biomarker.


2. Bone remodelling biomarkers

About 10 years ago, the hypothesis that bone remodelling biomarkers might be involved in the progression of coronary artery calcification seemed to be tricky and beyond any reasonable expectation. However, in 1995 Boström et al. first time proposed the possible mechanisms for bone formation in artery walls involving retention of pluripotent cells or osteoblastic immigration coupled with embryonic-like osteogenic program (Boström et al. 1995). The main reason for understanding the regulatory mechanisms of vascular calcification was firstly related to therapeutic approaches to prevent and possibly reverse vascular mineralization (Demer, 1997; Parhami et al. 1997). The data from clinical studies regularly report an association between bone remodeling biomarkers and the presence, severity and progression of a broad range of cardiovascular diseases. Whether they are biomarkers or rather play a causal role in mediating or protecting against vascular injury is not clear. The mechanisms underlying the postulated role of bone remodelling biomarkers in atherosclerosis probably involve inflammation and calcification processes.

This section will focus on the prognostic significance of plasma bone remodelling biomarkers levels in stable and unstable CAD.

2.1. Biology of bone remodelling of biomarkers

Vascular biomineralization in an atherosclerotic plaque results from an imbalance in osteoblast- and osteoclast-like cells and the induction of vascular or immune cells differentiation into osteogenic cells (Demer & Tintut, 2008). Osteobalsts, osteoclasts and inflammatory cells are firmly involved in bone remodelling (Fig. 2).

Figure 2.

Bone remodelling osteoblasts and osteoclasts differentiation. Figure was produced using Servier Medical Art.

Figure 3.

The role of osteoprotegerin (OPG) in pre-osteoclast differentiation. OPG trap and neutralize a soluble receptor activator of nuclear factor kappa-B ligand (RANKL) which activates osteoclasts by its receptor (RANK). Figure was produced using Servier Medical Art.

Mesenchymal stem cells are precursors for pre-osteoblasts. Osteoblasts activity leads to bone formation and mineralization, their differentiation and activity is mostly regulated by RANKL (receptor activator of nuclear factor kappa-B ligand) inducers, such as: vitamin D (1,25[OH]2D), glucocorticosteroids, parathormone (PTH), prostaglandins (PGE2), lipopolysaccharides (LPS), histamine and pro-inflammatory cytokines: interleukins (IL-1 and IL-11), tumor necrosisi factor alfa (TNF-α) and others (Eriksen, 2010). RANKL is a surface-bound molecule (also known as CD254). It is found on osteoblasts and serves to stimulate osteoclasts by RANK (receptor activator of nuclear factor kappa-B) activation and RANK/RANKL axis has a core regulatory role in osteoblasts and osteoclasts signalling (Fig. 3) (Caidahl et al., 2010).

2.2. Osteoprotegerin and osteopontin as risk factors of coronary artery disease

Several studies suggest the involvement of bone remodeling biomarkers in coronary artery disease and related atherosclerotic disorders (Van Campenhout & Golledge, 2009; Venuraju et al. 2010). Prime regulators of bone remodelling, such as osteoprotegerin (OPG) and osteopontin (OPN), are significantly and independently associated with inflammatory processes and arterial hypertension and may exert substantial influence on the severity of cardiovascular disease. (Stępień et al., 2011)

OPG is a soluble glycoprotein widely expressed in most human tissues including the bone (osteoblasts) and the vasculature (endothelial and vascular smooth muscle cells, VSMC) (Collin-Osdoby, 2004; Schoppet et al., 2002) that is implicated in the regulation of bone and vascular calcification. OPG is a member of the tumor necrosis factor (TNF)-related family and a part of the OPG/RANKL/RANK triad. OPG acts as a soluble secreted decay receptor for a receptor activator of nuclear factor kappa-B ligand (RANKL) and neutralize this essential cytokine required for the osteoclasts differentiation (Hsu et al., 1999) (Fig. 3).

RANKL expressed on osteoblastic, stromal and T cells binds to RANK (osteoclast differentiation factor) on the surface of osteoclasts, monocytic and dendritic cells and mediates a cell-to-cell signal responsible for osteoclastogenesis (Yasuda et al. 1998). Additional roles in immunological responses include the RANK-RANKL binding between dendritic and T cells which enhances the immunostimulatory capacity of dendritic cells and T cell proliferation (Green & Flavel 1999).

It was observed that opg-knockout mice (OPG -/-) develop early onset osteoporosis and arterial calcification (Bucay et al., 1998) and the restoration of the gene prevented osteoporosis progression and arterial calcification (Min et al., 2000). Increased OPG level has been observed in men with advanced CAD and plasma OPG level has proved to be an independent predictor of myocardial ischemia in asymptomatic diabetic patients (Avignon, 2007; Schoppet et al., 2003). Moreover, increased OPG has been related to the number and vulnerability of plaques as well as in carotid artery (Kadoglou et al., 2008; Vik et al. 2010) or coronary vessels (Palazzuoli at al., 2008), which suggests its involvement in the coronary disease progression (Mikami et al., 2008; Pedersen et al., 2010). Elevated OPG in plasma is univariable predictors of coronary artery calcification (CAC) progression (Anand et al., 2007). The sensitivity of OPG for detecting of CAC score higher than 200 Agatston units was 80% in patients with predialysis diabetic nephropathy (Schoppet et al., 2003). However, in the large Norwegian study by Pederesen et al.(Pedersen et al., 2010), adjustment for conventional risk factors attenuated the risk estimates for OPG levels. Only the subgroup of patients with stable angina pectoris (SA) with levels above the 90th percentile was at risk all-cause mortality: 1.94 (1.18, 3.18), p=0.01; CAD mortality: 2.29 (1.16, 4.49), p=0.02; and MI: 1.76 (1.02, 3.06), p=0.04.

In patients with acute coronary syndromes (ACS) the baseline OPG concentrations were strongly associated with increased long-term mortality (hazard ratio [HR] for log transformed OPG level 1.7 [range 1.5 to 1.9] p<0.0001) and heart failure hospitalizations (HR 2.0 [range 1.6 to 2.5]; p < 0.0001) but weaker with recurrent MI (HR 1.3 [range 1.0 to 1.5]; p = 0.02) and not with stroke (HR 1.2 [range 0.9 to 1.6]; p = 0.35). The association remained significant after adjustment for conventional risk markers (Omland et al., 2008). In apparently healthy individuals (the European Prospective Investigation into Cancer in Norfolk – EPIC Norfolk cohort) high serum concentrations of OPG and soluble RANKL were associated with an increased risk of future CAD (Semb et al., 2009). OPG showed a significant association with the risk of future coronary events in both sexes. This association remained statistically significant after adjustment for traditional cardiovascular risk factors (i.e. age, diabetes, systolic blood pressure, smoking, total cholesterol and HDL cholesterol).

OPN is secreted as a calcium-binding glycophosphoprotein that has been implicated in bone remodeling and inflammation as well. Similarly to OPG, osteopontin is widely distributed in different human cells including osteoblasts, lymphocytes, macrophages, endothelial cells and vascular smooth muscle cells (Brown et al., 1992).

OPN is a cytokine and has the ability to stimulate migration of macrophages and osteoclasts (Giachelli et al, 1998; Suzuki et al., 2002) and proliferation of osteoclasts and vascular smooth muscle cells (Giachelli et al, 1998; Liaw et al., 1994). A growing body of experimental evidence suggests that OPN overexpression plays an essential role in modulating compensatory cardiac fibrosis and hypertrophy (Xie et al., 2004; Singh et al. 2010). OPN acts through different integrins and thus has a great potential to regulate populations of different cells on the molecular and cellular levels (Bazzichi et al., 2009; Burke et al., 2009). OPN plays a pivotal role in inflammation and atherosclerotic plaque formation in an animal model (Scatena et al., 2007). Recent data has indicated a high predictive value of OPN for secondary manifestations of atherosclerotic disease (e.g. cardiovascular death, myocardial infarction, stroke, and endovascular interventions) in a 3-year follow-up of patients undergoing carotid surgery (de Kleijn et al., 2010).

Baseline levels of OPN are independent predictors of future adverse cardiac events in patients with chronic coronary syndrome (CCS), and may be useful for risk stratification (Minoretti et al., 2006). Recent data have indicated a high predictive value of OPN for secondary manifestations of atherosclerotic disease (e.g. cardiovascular death, MI, stroke and endovascular interventions) in a 3-year follow-up of patients undergoing carotid surgery. In a prospective study by Gogo et al. (Gogo et al., 2006), the association between angiographically quantified coronary artery calcification and OPG was not found. Detection of coronary calcification by coronary angiography may underestimate the calcification burden, thus synergistic diagnostics of coronary calcification should utilize more sensitive techniques of MSCT (Willemsen et al., 2009). However, in patients with CAD undergoing

percutaneous coronary intervention (PCI) the highest OPN levels were associated with both plaque progression and restenosis in a stent (p=0.003). In addition, OPN, IL-6, and CRP were higher in patients with ACS than in those with CCS (analysis of variance: p<0.001, p<0.05 and p<0.05, respectively) (Mazzone et al, 2011).

A question arises as to whether peripheral vascular function (calcification markers) matches the coronary arteries (calcification) and thus, whether it may serve as a surrogate marker to identify individuals with increased hazard of CAD and mortality (de Kleijn et al., 2010; Lieb et al., 2010; Scatena et al., 2007). Therefore bone-matrix proteins combined with cardiovascular imaging could be potential markers for vulnerable coronary artery plaques.


3. Microparticles

Microparticles (MP) are sub-micron sized cell membrane/cytoplasmic fragments that are released from the cell surface. There are two well-known cellular processes that can lead to the formation of MPs: chemical and physical cell activation (by agonists or shear stress, respectively), and apoptosis (Jimenez et al., 2003). However, the mechanisms that take place during MP formation are still not revealed. It seems that, the flopping of phosphatidylserine (PS) to the outer layer of the plasma membrane is pivotal. Finally, this process leads to the formation and shedding of MPs from activated or apoptotic cells. In resting condition the membrane asymmetry is maintained by an aminophospholipid translocase with flippase activity. Bilayer asymmetry is disrupted in the consequence of the inhibition of flippase activity by calcium influx. Increased calcium ions concentrations activate calcium-dependent calpains, which disturb cytoskeleton, promote the shedding of MPs (Morel et al., 2011) and stimulate scramblase and floppase activities, which lead to the collapse of the membrane asymmetry (Freyssinet & Toti, 2010).

MPs are qualitatively and quantitatively diverse and vary in diameter between 0.1 and 1.5 µm and may harbor a number of cell surface proteins (Fig. 4). MPs are released from various cell types such as circulating blood cells (platelets, lymphocytes T and B, monocytes and erythrocytes) and cells of the vessel wall (endothelial and smooth muscle cells) (Amabile et al., 2010).

Figure 4.

A platelet microparticle is carrying not only specific membrane adhesion proteins (P-selectin, integrins – e. i. GPIIbIIIa, ), but also may harbour and transfer tissue factor (TF) which has its procoagulant potential and other functional effectors (E-selectin, von Willebrand factor, arachidonic acid, thromboxane A2), that can regulate aggregation, adhesion molecule expression, cell proliferation, apoptosis and endothelial migration. MPs may capsule messenger molecules (miRNA, DNA ?), cytokines, growth factors and calpains. Figure was produced using Servier Medical Art.

MPs from numerous cellular sources have been described in human plasma. They have received increasing attention as potential biomarkers of cell damage and activation or biovectors in blood coagulation, inflammation and cancer (Benameur et al., 2009; Hoyer et al., 2010). In several pathological states like dilated cardiomyopathy, chronic renal failure or cerebrovascular disease, MPs were used as biomarkers to identify a disease or to detect complications linked to a given disease (Bulut et al. 2011; Faure et al., 2006; Jung et al., 2009). Numerous clinical studies have evaluated their usefulness in the stratification of patients at risk for vascular disorders and to monitor response to treatment. Circulating MPs may serve as a marker for cardiovascular events in CAD patients or as a predictor of acute allograft rejection after heart transplantation (Morel et al., 2008; Sinning et al., 2010).

3.1. Microparticles discrimination and enumeration

The high level of microparticles’ diversity may create a problem with compatible masurement of MPs using different analytical methods. The number of microparticles depend on the detection technique and a wide range of pre-analytical variables, i.e. blood collecting and handling, plasma preparation and storage conditions. Therefore, optimization and standardization of detection methods are important to define microparticles correctly and to avoid falsely high or low quantification. Even minor protocol changes significantly affected MP levels (Ayers et al., 2011).

3.1.1. Flow cytometry in MP analysis

Several research have evaluated the impact of these different parameters to propose a pre-analytical protocol for MP analysis. Three ISTH Scientific and Standardization Subcommittees (SSC Vascular Biology, DIC, and Haemostasis &Malignancy) have initiated a project aimed at standardizing the enumeration of cellular MPs by means of flow cytometry method (FCM). The first collaborative workshop was set to establish the resolution and a threshold levels of the flow cytometers currently used in laboratories. Additionally, the interinstrument reproducibility of platelet MP enumeration in human plasma was analyzed (Lacroix et al., 2010). The study included 40 laboratories and 59 flow instruments were validated according to the protocol based on Megamix beads calibration to discriminate microparticles between 0.5 μm and 0.9 μm using the forward scatter (FS) channeling (FSC) parameter (FS/FSC). After that, selected laboratories received PFP samples prepared as frozen aliquots by the core laboratory, to avoid any preanalytic-linked variability. The authors found high discrepancy among Becton Dickinson instruments, as well within low, medium and high values of MP: coefficients of variation were 78%, 60% and 91%, respectively. Whereas interlaboratory reproducibilities were 30%,15% and 17% for low, medium and high values among Beckmann Coulter instruments. These data indicate that standardization of platelet MPs enumeration by FCM dependents on intrinsic characteristics of instruments. Moreover, standardization by calibrated beads such is useful tool for MP enumeration, however, calibrated beads do not reflect real condition of MPs in human plasma.

3.1.2. Indirect methods for MP enumeration

Alternative methods for MP enumeration based on TF-activity/antigen or platelet glycoprotein GPIb-integrin have been already described (Huisse et al, 2009; Kuriyama et al., 2010). The activity of tissue factor is evaluated using a chromogenic substrate for factor Xa, thus the ability of MPs to promote factor X activation in the presence of factor VII using a chromogenic activity assay is utilized (Huise et al, 2009). Alternatively, TF antigen or activity can be measured in plasma or whole blood (Key NS & Mackman N, 2010). However, determination of microparticle size is not possible by such approaches.

3.1.3. Pre-analytical variability in MP determination

The analysis of different protocols used in MP preparation showed that washing, centrifugation, filtration of buffer and long-term freezing influenced significantly the MP quantification (Ayers et al., 2011; Dey-Hazra et al., 2010). Freezing samples at -80°C decreased MP levels (Ayers et al., 2011; Shah et al., 2009). The second collaborative workshop was dedicated to propose a common pre-analytical protocol useful for standardization of pre-analytical variables in determination of MPs (Scientific and Standardization Committee 2010).

3.1.4. Specific antigens in MP discrimination

There are two main features of native MPs: the small size and the anionic phospholipid - PS on the outer leaflet of their membrane. In addition, MPs carry surface membrane antigens reflecting their cell of origin, including those induced by cellular activation, cell injury or apoptosis. These properties permit detection of specific subpopulations, such as endothelial, leucocyte or platelet-derived MPs (Diamant et al., 2004).

PS is specifically bound to annexin V and is recommended as a distinguish marker for MP enumeration (Bulut et al., 2011; Shah et al. 2009). However, a number of evidence suggests that some vesicles derived from endothelial cells are PS-negative by annexin-V labelling (Jimenez et al., 2003; Sekuła et al., 2011). In platelet-poor plasma obtained from healthy donors, 80% of platelet-derived MPs failed to bind annexin V (Connor et al., 2010). In this case, a phalloidin-staining of actin filaments could be helpful in discrimination of MPs and other cell fragments (Mobarrez et al., 2010). Washing samples as well as double centrifugation result in decreased annexin-V (Ayers et al. 2011). Platelet MPs

Platelets constitute the main source of circulating procoagulant MPs under many physiological and pathophysiological situations (Geiser, et al., 1998; Huise et al, 2009; Kuriyama et al., 2010). Procoagulant platelet derived MPs are enriched in P-selectin (CD62P), cell surface protein (CD63), integrins: GPIIbIIIa (α2bβ3), GPIIb (α2b, CD41), GPIIIa (β3, CD61) and GPIb (CD42b), tissue factor (CD142, TF) or calpains (Figure 4).

Patients with unstable angina (UA) and AMI had a significantly increased number of procoagulant MPs: GPIIbIIIa-positive, CD62P-positive and CD41-positive (Huisse et al., 2009; Morel et al., 2004; Stankiewicz et al., 2007; van der Zee et al. 2006). The total number of platelet-derived MPs were numerically higher in patients with no recanalisation compared to patients with recanalisation (Huisse et al., 2009). However, we observed paradoxically lower number of CD62P-positive platelets in whole blood obtained from patients with ACS, than from SA patients, but the level of soluble P-selectin in plasma was significantly higher than in those with ACS (Figure 5). We may suspect that soluble P-selectin levels are derivatives of platelet origin MPs (Chung, 2009). Tissue factor-bearing MPs

It was shown by cell sorting with the specific marker CD42b that under resting conditions, blood-borne TF was mainly harbored by platelet-derived MPs (Müller et al, 2008). In acute coronary syndromes, TF triggers the formation of intracoronary thrombi following endothelial injury, activation of macrophages and apoptotosis of smooth muscle cells (SMCs) and macrophages (Morel et al, 2008). Apoptotic (annexin V-positive) MPs support a number of TF-positive MPs from different origin. Apoptotic macrophages and SMCs are the main source of membrane-bound TF and they contribute to TF accumulation. Formation of TF triggering MPs rich in PS provides a suitable anionic phospholipid surface for assembly of the tenase and prothrombinase complexes and thrombin activation (Del Conde et al., 2005).

Figure 5.

Platelet activation measured as a percentage of surface P-selectin-positive (CD62+) platelet (PLT), and by monocyte/platelet aggregates (MO/CD61+) and neutrophil/platelet aggregates (N/CD61+) in peripheral blood from patients with stable angina (SA) and acute myocardial infarction (AMI), and by levels of soluble P-selectin in patients with stable angina (SA), and acute myocardial infarction (AMI). Data are expressed as medians. *p<0.05,***p<0.00001 for the comparison.

Additionally, an increased number of TF-positive (CD142-positive) MPs in patients with ACS was observed (Figure 6) (Huisse et al., 2009; Steppich et al., 2005). Moreover, elevated levels of different origin TF-bearing MPs were significantly higher within the occluded coronary artery than in peripheral blood samples (Morel et al. 2009). It suggests their contrubution in coronary atherothrombosis and in situformation of procoagulat MPs. Endothelial microparticles

Endothelial microparticles (EMPs) are an emerging marker of endothelial cell (EC) activation and dysfunction and their circulating numbers are elevated in a number of pathologic states including cardiovascular disease. Many studies suggest that endothelial cell-derived MPs have a paracrine role and contribute to the development of endothelial dysfunction in most cardiovascular diseases: CAD, ACS, MI, hypertension and congestive heart failure. Moreover, diabetes, end-stage renal failure and pulmonary or venous embolism are strong factors bringing about EMP shedding [Bal et al., 2010; Chirinos et. al., 2005; Faure et al., 2006; Morel et al., 2004]. In this case patients have marked activation of endothelial, platelet, and leukocyte MPs.

Endothelial-derived microparticles (EMPs) may carry different endothelial originating coagulation factors, for example TF, which contribute to the clot formation and lysis (Chou et al., 2004; Stępień et al., 2007b). Patients with AMI displayed higher levels of all MPs than patients with SA and CD31-positive EMPs appeared the main source of procoagulant MPs (Morel et al., 2004). In patients with ACS significant correlations between both the total

Figure 6.

Representative dot plot of circulating microparticles (MPs) in a patient with acute coronary syndrome (ACS) and in a control voluntary. A, C - flow cytometry gating logic, MPs were initially gated by forward (FCS-H) and side scatter (SSC-H) in logarithmic scale; B, D - fluorescence plots show MPs binding of annexin V-FITC (FL1-H) and anti-CD142-PE (FL2-H) monoclonal antibody.

number as well as the level of CD34, CD51 and CD142 were observed (Stankiewicz et al., 2007). Moreover, increased number of EMPs (E-cadherin/CD144-positive MPs) was an independent predictor of future cardiovascular events (HR 2.42 [range 1.03 to 5.68), p=0.04), but not for all-cause mortality (HR 2.10 [range 0.83 to 5.32] p=0.12) in patients with heart failure (Nozaki et al., 2010) and the assessment of EMPs improved prediction of future cardiovascular events in patients with CAD (Nozaki et al., 2009).


4. Clotting

Clotting is a rapid and highly dynamic process, which involves both platelets and coagulation factors. To monitor the clotting process a lot of instrumentations and methods are engaged: i) clotting times: the activated partial thromboplastin time (aPTT) and the prothrombin time (PT); ii) thromboelastography; iii) assessment of thrombin generation markers and thrombin inhibitors; iv) the real-time monitoring of thrombin generation. This section will focus on the prognostic significance of thrombin generation markers in stable and unstable CAD.

4.1. Markers of thrombin generation in CAD patients

Antithrombin (AT) appears to be the most important stoichiometric inhibitor which forms equimolar complexes with thrombin molecules – TAT (thrombin-antithrombin) complexes. A concentration of TAT complexes measured in peripheral venous blood and in blood collected at the site of microvascular injury reflect thrombin generation. TAT complexes are expressed during clot formation and there are (alike fibrinopeptide A and F 1+2 fragments) markers of thrombin activation (Pelzer et al., 1988; Pelzer et al., 1991). These markers are elevated in pro-thrombotic conditions In patients with cardiovascular disease, the detection of a prothrombotic state may have two major implications: i) to extend the duration and ii) to monitor the dose of anticoagulation after cardiac intervention. The thrombin plasma activity is very firmly associated with CAD.

The potential coagulation activity in plasma can be evaluated by the rate of thrombin formation and the total amount of formed thrombin is measured by means of chromogenic or fluorescence methods (Devreese et al., 2007; Hemker et al., 2002). This thrombin potential in plasma can be assessed by different methods and the Calibrated Automated Thrombogram (CAT) applies a fluorogenic substrate. A chromogenic substrate is used in Behring Coagulation System (BCS). In both methods thrombin generation is activated by diluted recombinant tissue factor (TF), but in the BCS method a non-defined fibrin aggregation inhibitor is present. Both methods are applied in diagnostics. In CAT a calibration factor is measured in a plasma sample identical to that in which thrombin generation is being determined and the course of the calibration factor is assessed during the entire measurement (Figure 7). Thrombin generation assays seem to be useful in endogenous TF assessment (Ollivier et al. 2010; Stępień et al., 2007a).

4.2. Blood sampling for coagulation markers assessment

The most important think in coagulation diagnostics is to apply a reliably sampling method. To ensure accurate measurement samples must be collected in the circumstances under which false elevations of molecular markers of hemostatic and fibrinolytic activation will not occur. Thus, atraumatic antecubital venipuncture into vacutainer containing buffered sodium citrate is essential and the contamination with calcium or magnesium should be avoided (van den Besselaar et al., 2007; Stegnar et al., 2007). To avoid activation of coagulation by tissue thromboplastin, each collection of citrated plasma should be preceded by a serum tube. Duration of needle puncture, rather than duration of tourniquet use, produced the greatest elevation in plasma levels of TAT and F1+2 (Omote et al., 2008).

Figure 7.

The rate of thrombin formation is presented as the thrombin concentration against time curve. Three parameters are presented: lag time (Tlag), peak height (Cmax) and endogenous thrombin potential (ETP).

4.3. Prognostic value of thrombin generation in cardiac events

Increased circulating levels of thrombin and its markers characterize ACS (Ardissino et al., 2003; Takano et al., 1991). Plasma F1+2, normally about 1 nM, is roughly 1.5-2-fold higher than observed in SA patients, reaching maximum values in AMI (Ardissino et al., 2001). U-shaped relationship between plasma prothrombin fragment 1+2 levels and the risk of developing cardiac death or renewed myocardial infarction was observed. Intermediate levels (1.5-1.9 nM) were associated with the lowest risk, whereas both higher (>1.9 nM) and lower (< 1.5 nM) values were associated with an increased risk (RR 1.56 [range 1.25 to 2.28] and RR 1.35 [range 1.11 to 1.86], respectively) (Ardissino et al., 2003). Hypercoagulable state measured as thrombin-antithrombin complexes (TAT) levels and as calibrated automated thrombogram reflects vascular impairment in CAD patients (Stępień et al., 2007a). It was observed that high TAT levels may predict mortality in chronic heart disease group after adjustment for classic risk factors (Marcucci et al., 2006). In empirical reconstruction, simulated maximum thrombin levels (p<0.01) and rates (p<0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter than in patients with stable CAD (Brummel-Ziedins et al., 2008). Elevated levels of thrombin derivatives are associated with clinical risk factors for stroke (Lane et al., 1983; Takano et al., 1991). Elevated thrombin concentration reflects hypercoagulable state in patients with hypertension (Hoeper et al, 1998; Kłoczko et al., 1996), hyperglycaemia (Undas et al., 2008) and hypercholesterolemia (Wada et al., 1992; Sanguigni et al., 2005; Undas et al., 2005).


5. Conclusion

Endothelial and platelets activation leading to cardiovascular complications can be evaluated quantitatively by measurement of plasma levels of circulating MPs. Moreover, a multiple biomarkers strategy that includes bone remodeling biomarkers (OPG, OPN) and clotting properties can provide better risk stratification of cardiovascular events. Development and discovery of new biomarkers may improve clinical assessment of patients

who might benefit more from treatment. Synergistic strategies in diagnostics seem to be more advantageous than routine method in prognosis and patients’ management.



The author is a Secretary of the Board of the Polish College of Laboratory Medicine (KMLP). KMLP is a multispecialty society dedicated to the advancement of education, development and management in clinical biochemistry, hematology, immunology, toxicology, pathology and cytology, clinical genetics, microbiology and molecular biology.


  1. 1. AlcockR. F.RoyP.AdoriniK.LauG. T.KritharidesL.LoweH. C.BriegerD. B.FreedmanS. B.2010Incidence and determinants of myocardial infarction following percutaneous coronary interventions according to the revised Joint Task Force definition of troponin T elevation.Int J Cardiol.1401April 2010),6672
  2. 2. AmabileN.RautouP. E.TedguiA.BoulangerC. M.2010Microparticles: key protagonists in cardiovascular disorders.Semin Thromb Hemost.368November 2010),907916
  3. 3. AnandD. V.LimE.DarkoD.BassettP.HopkinsD.LipkinD.CorderR.LahiriA.2007Determinants of progression of coronary artery calcification in type 2 diabetes role of glycemic control and inflammatory/vascular calcification markers.J Am Coll Cardiol.5023December 2007),22182225ISSN
  4. 4. ArdissinoD.MerliniP. A.BauerK. A.BramucciE.FerrarioM.CoppolaR.FetiveauR.LucreziottiS.RosenbergR. D.MannucciP. M.2001Thrombogenic potential of human coronary atherosclerotic plaques.Blood.989November 2001),27262729
  5. 5. ArdissinoD.MerliniP. A.BauerK. A.GalvaniM.OttaniF.FranchiF.BertocchiF.RosenbergR. D.MannucciP. M.2003Coagulation activation and long-term outcome in acute coronary syndromes.Blood.1028October 2003),27312735
  6. 6. AvignonA.SultanA.PiotC.Mariano-GoulartD.ThuanDit.DieudonnéJ. F.CristolJ. P.DupuyA. M.2007Osteoprotegerin: a novel independent marker for silent myocardial ischemia in asymptomatic diabetic patients.Diabetes Care.3011November 2007),29342939ISSN
  7. 7. AyersL.KohlerM.HarrisonP.SargentI.DragovicR.SchaapM.NieuwlandR.BrooksS. A.FerryB.2011Measurement of circulating cell-derived microparticles by flow cytometry: Sources of variability within the assay.Thromb Res.1274April 2011),370377
  8. 8. BalL.EderhyS.Di AngelantonioE.TotiF.ZobairiF.DufaitreG.MeulemanC.MallatZ.BoccaraF.TedguiA.FreyssinetJ. M.CohenA.2010Circulating procoagulant microparticles in acute pulmonary embolism: a case-control study.Int J Cardiol.1452November 2010),321322
  9. 9. BazzichiL.GhiadoniL.RossiA.BernardiniM.LanzaM.De FeoF.GiacomelliC.MencaroniI.RaimoK.RossiM.MazzoneA. M.TaddeiS.BombardieriS.2009Osteopontin is associated with increased arterial stiffness in rheumatoid arthritis.Mol Med.1511-12November-December 2009),402406
  10. 10. BenameurT.AndriantsitohainaR.MartínezM. C.Therapeutic potential of plasma membrane-derived microparticles.Pharmacol Rep.611January-February20094957
  11. 11. van den BesselaarA. M.HoekstraM. M.WitteveenE.DiddenJ. H.van der MeerF. J.2007Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human and rabbit thromboplastin reagents.Am J Clin Pathol.1275May 2007),724729
  12. 12. Biomarkers Definitions Working Group.2001Biomarkers and surrogate endpoints: preferred definitions and conceptual framework.Clin Pharmacol Ther.693March 2001),8995
  13. 13. BoströmK.WatsonK. E.StanfordW. P.DemerL. L.1995Atherosclerotic calcification: relation to developmental osteogenesis.Am J Cardiol.756February 1995),88B91B
  14. 14. BrownL. F.BerseB.Van de WaterL.Papadopoulos-SergiouA.PerruzziC. A.ManseauE. J.DvorakH. F.SengerD. R.1992Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.Mol Biol Cell310October 1992),11691180
  15. 15. Brummel-ZiedinsK.UndasA.OrfeoT.GisselM.ButenasS.ZmudkaK.MannK. G.2008Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition.J Thromb Haemost.61January 2008),104110
  16. 16. BucayN.SarosiI.DunstanC.MoronyS.TarpleyJ.CapparelliC.ScullyS.TanH. L.XuW.LaceyD. L.BoyleW. J.SimonetW. S.(1998Osteoprotegerin deficient mice develop early onset osteoporosis and arterial calcification.Genes Develop.129May 1998),12601268
  17. 17. Bulut,D.Tüns,H.Mügge,A.(2009). CD31+/Annexin V+ microparticles in healthy offsprings of patients with coronary artery disease. Eur. J. Clin. Invest. Vol.39, No.1, (January 2009), pp. 17-22,
  18. 18. BulutD.ScheelerM.NiedballaL. M.MiebachT.MüggeA.2011Effects of immunoadsorption on endothelial function, circulating endothelial progenitor cells and circulating microparticles in patients with inflammatory dilated cardiomyopathy.Clin Res Cardiol.(February 2011), [Epub ahead of print],
  19. 19. BurkeD. L.FridM. G.KunrathC. L.KaroorV.AnwarA.WagnerB. D.StrassheimD.StenmarkK. R.2009Sustained hypoxia promotes the development of a pulmonary artery-specific chronic inflammatory microenvironment.Am J Physiol Lung Cell Mol Physiol.2972May 2009),L238L250
  20. 20. CaidahlK.UelandT.AukrustP.2010Osteoprotegerin: a biomarker with many faces.Arterioscler Thromb Vasc Biol.309September 2010),16841686
  21. 21. ChirinosJ. A.HeresiG. A.VelasquezH.JyW.JimenezJ. J.AhnE.HorstmanL. L.SorianoA. O.ZambranoJ. P.AhnY. S.2005Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism.J Am Coll Cardiol.459May 2005),14671471
  22. 22. ChouJ.MackamnN.Merrill-SkoloffG.PedersenB.FurieC.FurieB.2004Hematopoietic cell-derived microparticles tissue factor contributes to fibrin formation during thrombus propagation.Blood.10410November 2004),31903197
  23. 23. ChungI.ChoudhuryA.PatelJ.LipG. Y.2009Soluble, platelet-bound, and total P-selectin as indices of platelet activation in congestive heart failure.Ann Med.411January 2009),4551
  24. 24. Collin-OsdobyP.2004Regulation of vascular calcification by osteoclast regulatory factors RANKL and osteoprotegerin.Circ Res.9511November 2004),10461057
  25. 25. ConnorD. E.ExnerT.MaD. D.JosephJ. E.2010The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib.Thromb Haemost.1035May 2010),10441052
  26. 26. DemerL. L.1997Lipid hypothesis of cardiovascular calcification.Circulation.952January 1997),297298
  27. 27. DemerL. L.TintutY.2008Vascular calcification: pathobiology of a multifaceted disease.Circulation.11722June 2008),29382948
  28. 28. DevreeseK.WijnsW.CombesI.Van kerckhovenS.HoylaertsM. F.2007Thrombin generation in plasma of healthy adults and children: chromogenic versus fluorogenic thrombogram analysis.Thromb Haemost.983September 2007),600613
  29. 29. Dey-HazraE.HertelB.KirschT.WoywodtA.LovricS.HallerH.HaubitzM.ErdbrueggerU.2010Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing.Vasc Health Risk Manag.66December 2010),11251133
  30. 30. DiamantM.TushuizenM. E.SturkA.NieuwlandR.2004Cellular microparticles: new players in the field of vascular disease?Eur J Clin Invest.346June 2004),392401
  31. 31. EriksenE. F.2010Cellular mechanisms of bone remodeling.Rev Endocr Metab Disord.114December 2010),219227
  32. 32. FaureV.DouL.SabatierF.CeriniC.SampolJ.BerlandY.BrunetP.Dignat-GeorgeF.2006Elevation of circulating endothelial microparticles in patients with chronic renal failure.J Thromb Haemost.43March 2006),566573
  33. 33. FreyssinetJ. M.TotiF.2010Formation of procoagulant microparticles and properties.Thromb Res.125Suppl.1, (April 2010),S46S48
  34. 34. GeiserT.SturzeneggerM.GeneweinU.HaeberliA.BeerJ. H.1998Mechanisms of cerebrovascular events as assessed by procoagulant activity, cerebral microemboli, and platelet microparticles in patients with prosthetic heart valves.Stroke.299September 1998),17701777
  35. 35. GerhardtW.KatusH.RavkildeJ.HammC.JørgensenP. J.PeheimE.LjungdahlL.LöfdahlP.1991S-troponin T in suspected ischemic myocardial injury compared with mass and catalytic concentrations of S-creatine kinase isoenzyme MB.Clin Chem.378August 1991),14051411
  36. 36. GiachelliC. M.LombardiD.JohnsonR. J.MurryC. E.AlmeidaM.1998Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo.Am J Pathol.1522February 1998),353358
  37. 37. GogoP. B.Jr SchneiderD. J.TerrienE. F.SobelB. E.DauermanH. L.2006Osteoprotegerin is not associated with angiographic coronary calcification.J Thromb Thrombolysis.223December 2006),177183
  38. 38. GreenE. A.FlavellR. A.1999TRANCE-RANK, a new signal pathway involved in lymphocyte development and T cell activation.J Exp Med.1897April 1999),10171020
  39. 39. HemkerH. C.GiesenP.AlDieri. SmedE.WagenvoordR.LecompteT.BéguinS.2002The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability.Pathophysiol Haemost Thromb.325-6September-December 2002),249253
  40. 40. HoeperM. M.SosadaM.FabelH.1998Plasma coagulation profiles in patients with severe primary pulmonary hypertension.Eur Respir J.126December 1998),14461449
  41. 41. HoyerF. F.NickenigG.WernerN.2010Microparticles- messengers of biological information.J Cell Mol Med.149September 2010),22502256
  42. 42. HsuH.LaceyD. L.DunstanC. R.SolovyevI.ColomberoA.TimmsE.TanH. L.ElliottG.KelleyM. J.SarosiI.WangL.XiaX. Z.ElliottR.ChiuL.BlackT.ScullyS.CapparelliC.MoronyS.ShimamotoG.BassM. B.BoyleW. J.1999Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci U S A.967March 1999),35403545
  43. 43. HuisseM. G.AjzenbergN.FeldmanL.GuillinM. C.StegP. G.2009Microparticle-linked tissue factor activity and increased thrombin activity play a potential role in fibrinolysis failure in ST-segment elevation myocardial infarction. (2009).Thromb Haemost.1014April 2009),734740
  44. 44. JimenezJ. J.JyMauro. W.SoderlandL. M.HorstmanC. L. L.AhnY. S.2003Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.Thromb Res.1094February 2003),175180
  45. 45. JungK. H.ChuK.LeeS. T.ParkH. K.BahnJ. J.KimD. H.KimJ. H.KimM.KunLee. S.RohJ. K.2009Circulating endothelial microparticles as a marker of cerebrovascular disease.Ann Neurol.662August 2009),191199
  46. 46. KadoglouN. P.GerasimidisT.GolematiS.KapelouzouA.KarayannacosP. E.LiapisC. D.2008The relationship between serum levels of vascular calcification inhibitors and carotid plaque vulnerability.J Vasc Surg.471January 2008),5562
  47. 47. KatusH. A.RemppisA.ScheffoldT.DiederichK. W.KueblerW.1991Intracellular compartmentation of cardiac troponin T and its release kinetics in patients with reperfused and nonreperfused myocardial infarction.Am J Cardiol.6716June 1991),13601367
  48. 48. KeyN. S.MackmanN.Tissue factor and its measurement in whole blood, plasma, and microparticles.Semin Thromb Hemost.368November2010865875
  49. 49. de KleijnD. P.MollF. L.HellingsW. BruinP.DoevendansP. A.VinkA.CatanzaritiL. M.SchoneveldA. H.AlgraA.DaemenM. J.BiessenE. VriesJ. P.FalkE.LimS. K.van der SpekP. J.SzeS. K.PasterkampG.2010Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular events.Arterioscler Thromb Vasc Biol303March 2010),612619
  50. 50. KłoczkoJ.WojtukiewiczM. Z.GalarM.TarasówE.JarominJ.BielawiecM.1996Prothrombin activation fragment 1 + 2 and thrombin-antithrombin-III complexes in plasma of patients with essential arterial hypertension.Pol J Pharmacol.482March-April 1996),233235
  51. 51. KongT. Q.DavidsonC. J.MeyersS. N.TaukeJ. T.ParkerM. A.BonowR. O.1997Prognostic implication of creatine kinase elevation following elective coronary artery interventions.JAMA.2776February 1997),461466
  52. 52. KuriyamaN.NagakaneY.HosomiA.OharaT.KasaiT.HaradaS.TakedaK.YamadaK.OzasaK.TokudaT.WatanabeY.MizunoT.NakagawaM.2010Evaluation of factors associated with elevated levels of platelet-derived microparticles in the acute phase of cerebral infarction.Clin Appl Thromb Hemost.161February 2010),2632
  53. 53. LacroixR.RobertS.PonceletP.KasthuriR. S.KeyN. S.Dignat-GeorgeF.WorkshopI. S. T. H. S. S. C.2010Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop.J Thromb Haemost.811November 2010),25712574
  54. 54. LaneD. A.WolffS.IrelandH.GawelM.FoadiM.1983Activation of coagulation and fibrinolytic systems following stroke.Br J Haematol.534April 1983),655658
  55. 55. LiebW.GonaP.LarsonM. G.MassaroJ. M.LipinskaI.KeaneyJ. F.Jr RongJ.CoreyD.HoffmannU.FoxC. S.VasanR. S.BenjaminE. J.O’DonnellC. J.KathiresanS.2010Biomarkers of the osteoprotegerin pathway. Clinical correlates, subclinical disease, incident cardiovascular disease, and mortality.Arterioscler Thromb Vasc Biol.309September 2010),18491854
  56. 56. LiawL.AlmeidaM.HartC. E.SchwartzS. M.GiachelliC. M.1994Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro.Circ Res.742February 1994),214224
  57. 57. LimC. C.van GaalW. J.TestaL.CuculiF.ArnoldJ. R.KaramitsosT.FrancisJ. M.PetersenS. E.DigbyJ. E.WestabyS.AntoniadesC.KharbandaR. K.BurrellL. M.NeubauerS.BanningA. P.2011With the "universal definition," measurement of creatine kinase-myocardial band rather than troponin allows more accurate diagnosis of periprocedural necrosis and infarction after coronary intervention.J Am Coll Cardiol.57February 2011)653661
  58. 58. LindpaintnerK.1997Genetics of interventional cardiology. Old principles, new frontiers.Circulation.961July 1997),1214
  59. 59. MarcucciR.GoriA. M.GiannottiF.BaldiM.VerdianiV.Del PaceS.NozzoliC.AbbateR.2006Markers of hypercoagulability and inflammation predict mortality in patients with heart failure.J Thromb Haemost.45May 2006),10171022
  60. 60. MazzoneA.ParriM. S.GiannessiD.RavaniM.VaghettiM.AltieriP.CasalinoL.MaltintiM.BalbiM.BarsottiA.BertiS.2011Osteopontin plasma levels and accelerated atherosclerosis in patients with CAD undergoing PCI: a prospective clinical study.Coron Artery Dis.(March 2011), [Epub ahead of print],
  61. 61. MikamiS.HamanoT.FujiiN.NagasawaY.IsakaY.MoriyamaT.MatsuhisaM.ItoT.ImaiE.HoriM.2008Serum osteoprotegerin as a screening tool for coronary artery calcification score in diabetic pre-dialysis patients.Hypertens Res.316June 2008),11631170
  62. 62. MinH.MoroS.SarosiI.DunstanC. R.CapparelliC.ScullyS.VanG.KaufmanS.KostenuikP. J.LaceyD. L.BoyleW. J.SimonetW. S.2000Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med.1924August 2000),463474
  63. 63. MinorettiP.FalconeC.CalcagninoM.EmanueleE.BuzziM. P.CoenE.GeroldiD.2006Prognostic significance of plasma osteopontin levels in patients with chronic stable angina.Eur Heart J.277April 2006),802807
  64. 64. MobarrezF.AntovicJ.EgbergN.HanssonM.JörneskogG.HultenbyK.WallénH.2010A multicolor flow cytometric assay for measurement of platelet-derived microparticles.Thromb Res.1253March 2010),e110e116
  65. 65. MorelO.HugelB.JeselL.LanzaF.DouchetM. P.ZupanM.ChauvinM.CazenaveJ. P.FreyssinetJ. M.TotiF.2004Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus.Thromb Haemost.912February 2004),345353
  66. 66. MorelO.HugelB.JeselL.MallatZ.LanzaF.DouchetM. P.ZupanM.ChauvinM.CazenaveJ. P.TedguiA.FreyssinetJ. M.TotiF.2004Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb-IIIa antagonists.J Thromb Haemost.27July 2004),11181126
  67. 67. MorelO.OhlmannP.EpaillyE.BakouboulaB.ZobairiF.JeselL.MeyerN.ChenardM. P.FreyssinetJ. M.BareissP.MazzucotelliJ. P.TotiF.2008Endothelial cell activation contributes to the release of procoagulant microparticles during acute cardiac allograft rejection.J Heart Lung Transplant.271January 2008),3845
  68. 68. MorelO.PereiraB.AverousG.FaureA.JeselL.GermainP.GrunebaumL.OhlmannP.FreyssinetJ. M.BareissP.TotiF.2009Increased levels of procoagulant tissue factor-bearing microparticles within the occluded coronary artery of patients with ST-segment elevation myocardial infarction: role of endothelial damage and leukocyte activation.Atherosclerosis.2042June 2009),636641
  69. 69. MorelO.JeselL.FreyssinetJ. M.TotiF.2011Cellular mechanisms underlying the formation of circulating microparticles.Arterioscler Thromb Vasc Biol.311January 2011),1526
  70. 70. MüllerI.KlockeA.AlexM.KotzschM.LutherT.MorgensternE.ZiesenissS.ZahlerS.PreissnerK.EngelmannB.2003Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets.FASEB J.17(3 March 2003),476847
  71. 71. NozakiT.SugiyamaS.KogaH.SugamuraK.OhbaK.MatsuzawaY.SumidaH.MatsuiK.JinnouchiH.OgawaH.2009Significance of a multiple biomarkers strategy including endothelial dysfunction to improve risk stratification for cardiovascular events in patients at high risk for coronary heart disease. J Am Coll Cardiol.547August 2009),601608
  72. 72. NozakiT.SugiyamaS.SugamuraK.OhbaK.MatsuzawaY.KonishiM.MatsubaraJ.AkiyamaE.SumidaH.MatsuiK.JinnouchiH.OgawaH.2010Prognostic value of endothelial microparticles in patients with heart failure.Eur J Heart Fail.1211November 2010),12231228
  73. 73. OllivierV.WangJ.ManlyD.MachlusK. R.WolbergA. S.Jandrot-PerrusM.MackmanN.2010Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay.Thromb Res.1051January 2010),9096
  74. 74. OmlandT.UelandT.JanssonA. M.PerssonA.KarlssonT.SmithC.HerlitzJ.AukrustP.HartfordM.CaidahlK.2008Circulating osteoprotegerin levels and long-term prognosis in patients with acute coronary syndromes.J Am Coll Cardiol.516February 2008),627633
  75. 75. OmoteM.AsakuraH.TakamichiS.ShibayamaM.YoshidaT.KadohiraY.MaekawaM.YamazakiM.MorishitaE.NakaoS.WadaT.2008Changes in molecular markers of hemostatic and fibrinolytic activation under various sampling conditions using vacuum tube samples from healthy volunteers.Thromb Res.1232February 2008),390395
  76. 76. PalazzuoliA.RizzelloV.CalabròA.GallottaM.MartiniG.QuatriniI.CampagnaM. S.FranciB.NutiR.2008Osteoprotegerin and B-type natriuretic peptide in non-ST elevation acute coronary syndromes: relation to coronary artery narrowing and plaques number.Clin Chim Acta3911-2May 2008),7479
  77. 77. ParhamiF.MorrowA. D.BalucanJ.LeitingerN.WatsonA. D.TintutY.BerlinerJ. A.DemerL. L.1997Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients.Arterioscler Thromb Vasc Biol.174April 1997),680687
  78. 78. PedersenE. R.UelandT.SeifertR.AukrustP.Schartum-HansenH.EbbingM.BleieØ.IglandJ.SvingenG.NordrehaugJ. E.NygårdO.2010Serum osteoprotegerin levels and long-term prognosis in patients with stable angina pectoris.Atherosclerosis.2122October 2010),644649
  79. 79. PelzerH.SchwarzA.HeimburgerN.1988Determination of human thrombin-antithrombin III complex in plasma with an enzyme-linked immunosorbent assay.Thromb Haemost.591February 1988),101106
  80. 80. PelzerH.SchwarzA.StüberW.1991Determination of human prothrombin activation fragment 1 + 2 in plasma with an antibody against a synthetic peptide. Thromb Haemost.652February 1991),153159
  81. 81. SanguigniV.PignatelliP.LentiL.FerroD.BelliaA.CarnevaleR.TesauroM.SorgeR.LauroR.VioliF.2005Short-term treatment with atorvastatin reduces platelet CD40 ligand and thrombin generation in hypercholesterolemic patients.Circulation.1114February 2005),4129
  82. 82. ScatenaM.LiawL.GiachelliC. M.2007Osteopontin: a multifunctional molecule regulating chronic inflammation and vascular disease.Arterioscler Thromb Vasc Biol.2711November 2007),23022309
  83. 83. SchoppetM.PreissnerK. T.HofbauerL. C.2002RANK ligand and osteoprotegerin: paracrine regulators of bone metabolism and vascular function.Arterioscler Thromb Vasc Biol224April 2002),549553
  84. 84. SchoppetM.SattlerA. M.SchaeferJ. R.HerzumM.MaischB.HofbauerL. C.2003Increased osteoprotegerin serum levels in men with coronary artery disease.J Clin Endocrinol Metab.883March 2003),10241028
  85. 85. Scientific and Standardization Committee of International Society on Thrombosis and Haemostasis2010Standardization of Pre-analytical Variables in Plasma Microparticle Determination,
  86. 86. SekułaM.JanawaG.StankiewiczE.StępieńE.2011Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro.Cell Mol Biol Lett.161March 2011;),6978
  87. 87. SembA. G.UelandT.AukrustP.WarehamN. J.LubenR.GullestadL.KasteleinJ. J.KhawK. T.BoekholdtS. M.2009Osteoprotegerin and soluble receptor activator of nuclear factor-kappaB ligand and risk for coronary events: a nested case-control approach in the prospective EPIC-Norfolk population study 1993-2003.Arterioscler Thromb Vasc Biol.296June 2009),975980
  88. 88. ShahM. D.BergeronA. L.DongJ. F.LópezJ. A.2009Flow cytometric measurement of microparticles: pitfalls and protocol modifications.Platelets.195August 2008),365372
  89. 89. SinghM.FosterC. R.DalalS.SinghK.2010Role of osteopontin in heart failure associated with aging.Heart Fail Rev.155September 2010),487494
  90. 90. SinningJ. M.LoschJ.WalentaK.BöhmM.NickenigG.WernerN.CirculatingC.D31AnnexinV.microparticlescorrelate.withcardiovascular.outcomesEur Heart J.(December2010Epub ahead of print]
  91. 91. StankiewiczE.StępieńE.UndasA.ZalewskiJ.GodlewskiJ.ZmudkaK.2007Platelet activation is associated with generation of microparticles of different origin in patients with acute myocardial infarction.Eur J Clin Invest.37Suppl. 1, (April 2007),137
  92. 92. StegnarM.CudermanT. V.BozicM.2007Evaluation of pre-analytical, demographic, behavioural and metabolic variables on fibrinolysis and haemostasis activation markers utilised to assess hypercoagulability.Clin Chem Lab Med.451January 2007),4046
  93. 93. SteppichB.MattisekC.SobczykD.KastratiA.SchömigA.OttI.2005Tissue factor pathway inhibitor on circulating microparticles in acute myocardial infarction.Thromb Haemost.931January 2005),3539
  94. 94. StępieńE.PlicnerD.BranickaA.StankiewiczE.PazdanA.Sniezek-MaciejewskaM.GórkiewiczI.KapelakB.SadowskiJ.2007Factors influencing thrombin generation measured as thrombin-antithrombin complexes levels and using calibrated automated thrombogram in patients with advanced coronary artery disease.Pol Arch Med Wewn.1177July 2007),297305
  95. 95. StępieńE.StankiewiczE.SzuldrzynskiK.ZmudkaK.UndasA.2007Platelet- and endothelial-derived microparticles associate with the fibrin clot resistance to lysis.E Heart J.28Abstract suppl. (September 2007),667
  96. 96. StępieńE.WypasekE.StopyraK.KonieczyńskaM.PrzybyłoM.PasowiczM.2011Increased levels of bone remodeling biomarkers (osteoprotegerin and osteopontin) in hypertensive individuals.Clin Biochem.PubMed PMID: 21539822. (April 2011).
  97. 97. SuzukiK.ZhuB.RittlingS. R.DenhardtD. T.GoldbergH. A.Mc CullochC. A.SodekJ.2002Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts.J Bone Miner Res.178August 2002),14861497
  98. 98. TakanoK.YamaguchiT.KatoH.OmaeT.1991Activation of coagulation in acute cardioembolic stroke.Stroke.221January 1991),1216
  99. 99. UndasA.Celinska-LöwenhoffM.DomagalaT. B.IwaniecT.DropinskiJ.LöwenhoffT.SzczeklikA.2005Early antithrombotic and anti-inflammatory effects of simvastatin versus fenofibrate in patients with hypercholesterolemia.Thromb Haemost.941July 2005),193199
  100. 100. UndasA.WiękI.StępieńE.ZmudkaK.TraczW.2008Hyperglycemia is associated with enhanced thrombin formation, platelet activation, and fibrin clot resistance to lysis in patients with acute coronary syndrome. Diabetes Care.318August 2008),15901595
  101. 101. Van CampenhoutA.GolledgeJ.2009Osteoprotegerin, vascular calcification and atherosclerosis.Atherosclerosis.2042June),321329
  102. 102. VenurajuS. M.YerramasuA.CorderR.LahiriA.2010Osteoprotegerin as a predictor of coronary artery disease and cardiovascular mortality and morbidity.J Am Coll Cardiol.5519May 2010),20492061
  103. 103. VikA.MathiesenE. B.JohnsenS. H.BroxJ.WilsgaardT.NjølstadI.HansenJ. B.2010Serum osteoprotegerin, sRANKL and carotid plaque formation and growth in a general population--the Tromsø study.J Thromb Haemost.85May 2010),898905
  104. 104. WadaH.MoriY.KanekoT.WakitaY.MinamikawaK.OhiwaM.TamakiS.YokoyamaN.KobayashiT.DeguchiK.1992Hypercoagulable state in patients with hypercholesterolemia: effects of pravastatin.Clin Ther.146November-December 1992),829834
  105. 105. WillemsenH. JongG.TioR. A.NieuwlandW.KemaI. P.van der HorstI. C.OudkerkM.ZijlstraF.2009Quick identification of acute chest pain patients study (QICS).BMC Cardiovasc Disord.924June 2009),
  106. 106. XieZ.SinghM.SinghK.2004Osteopontin modulates myocardial hypertrophy in response to chronic pressure overload in mice.Hypertension.446December 2004),826831
  107. 107. YasudaH.ShimaN.NakagawaN.YamaguchiK.KinosakiM.MochizukiS.TomoyasuA.YanoK.GotoM.MurakamiA.TsudaE.MorinagaT.HigashioK.UdagawaN.TakahashiN.SudaT.1998Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.Proc Natl Acad Sci U S A.957March 1998),35973602
  108. 108. van der ZeeP. M.Biró WinterR. J.HackC. E.SturkA.NieuwlandR.2006P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction.Clin Chem.524April 2006),657664

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Ewa Stępien

Submitted: October 30th, 2010 Published: September 15th, 2011