Hemostasis refers to the variety of mechanisms to prevent of blood loss in the body. Whenever a blood vessel is ruptured or severed, mechanisms employed to achieve homeostasis are (1) vascular constriction, (2) formation of platelet plug, (3) blood coagulation to form a blood clot, and (4) formation of fibrous tissue into the blood clot to seal off the damage in the vessel permanently (Guyton & Hall, 2006). Appropriate and adequate contributions of these mechanisms are vital for effective hemostasis to occur and defect in these mechanisms, either underactivity or overactivity, would result to increase in blood loss or increased blood coagulation, respectively. Both of which are detrimental. For the purpose of this paper, the focus of the discussion will be on the role of procoagulant microparticles on physiologic processes like platelet formation and blood coagulation and its use as marker of hypercoagulability in a variety of pathologic situations.
Cellular procoagulant microparticles (MPs) in the body fluids actually constitute a heterogenous population that differs in cellular origin, size, numbers, antigenic composition, as well as functional properties. MPs serve as a valuable hallmark of cellular damage and add to the wide array of pathogenic markers of hypercoagulability. Because of platelet's plasma membrane reactivity, platelets constitute the predominant source of circulating procoagulant microparticles not only in the blood of healthy individuals (Mackman, 2009) but also under many pathological conditions (VanWijk et al., 2003). In fact, researches showed that elevated levels of platelet-, endothelial-, and monocyte-derived microparticles are common elements of many diseases with thrombotic features (Bakouboula et al., 2008). Although researches focus on the role of procoagulant microparticles on pathologic conditions, MP is also contributing to achieve physiologic functions like hemostasis with the end effect of decreasing blood loss (Mackman, 2009).
Release of procoagulant microparticles - membrane remodeling and vesiculation
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Procoagulant microparticles (MP) are formed via cell activation and apoptosis (Gauley & Pisetsky, 2010). At present, it is not known whether cell activation and apoptosis lead to the formation of similar microparticles, in terms of lipid and protein composition, size, and their pathophysiological effects. There may be differences in the underlying mechanisms resulting in their formation but the end point of both is the disruption of the membrane skeleton (VanWijk et al., 2003). The proposed mechanism involved in microparticle formation during cell activation and apoptosis are presented schematically in Figure 1.
Figure 1. Schematic representation of general mechanisms involved in microparticle formation during cell activation (left panel) and apoptosis (right panel).
In the plasma membrane, asymmetry in the distribution of aminophospholipid is controlled by specific transporters called flippase and floppase, which govern the inward (flip) and outward (flop) translocation, respectively. In resting membrane, the flippase activity is predominant and aminophospholipids, particularly phosphatidylserine and phosphatidylethanolamine, is sequestered in the inner leaflet of the plasma membrane while phosphatidylcholine and sphingomyelin is mainly confined in the outer layer. After being stimulated, however, or during apoptosis, there is a swift egress of aminophospholipids to the outer leaflet due to the overwhelming floppase and reduced flippase activity. Membrane budding ultimately resolves by the release of MPs, which are usually referred to as 0.1 to 1Î¼m membrane fragments that exposes phosphatidylserine (Morel et al., 2006; Mackman, 2009) and other surface antigen of the parent cell.
As MPs are released into the blood stream, it acts as "messengers delivering its cargos." Among the components that MPs carry are cell surface receptors, signaling molecules, proinflammatory cytokines, and even mRNA to distal cells (Mackman, 2009). These enable MPs to exert a variety of actions on remote cells. In fact, MPs can also act as transporter of virus and prions aggravating the disease process (Mack et al., 2000; Å imák et al., 2002). Figure 2 shows the microparticles derived from different cells and the associated antigens they carry as well as their potential actions to target cells. Among its actions are the induction of expression of pro-inflammatory molecules and cytokines like ICAM-1, COX-2, and interleukins 6 and 8. It also promotes the expression of procoagulant molecules like tissue factor. In addition, Figure 2 also shows that monocyte-derived microparticles have the ability to bind activated platelets (Mackman, 2009).
Figure 2. Procoagulant microparticles derived from different cells with their associated antigens, which can induce expression of pro-inflammatory and procoagulant molecules on endothelial cells, epithelial cells, and monocytes (Mackman, 2009).
MP's as effectors of the coagulation system
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Released MP's serve as a catalyst in the formation of thrombus by providing an additional procoagulant phospholipid surface where the clotting enzyme complex can attach and assemble to complete the blood coagulation cascade. The catalytic property of MP lies on the procoagulant anionic aminophospholipid, phosphatidylserine that is translocated to the exoplasmic leaflet after membrane remodeling.
Phosphatidylserine is an anionic (negatively charged) phospholipid that binds to positively charged Gla domain of coagulation proteins (Mackman, 2009). After exposure of phosphatidylserine by MPs, it becomes accessible to the circulating blood coagulation factors and enables local concentrations required to achieve the kinetics essential for optimal thrombin generation and efficient hemostasis (Morel et al., 2006). Therefore, phosphatidylserine-positive MP facilitates coagulation more compared to its phosphatidylserine-negative counterpart (Mackman, 2009).
In addition to serving as a catalyst by binding coagulation proteins, phosphatidylserine also potentiate the effect of tissue factor (TF), which is the primary initiator of blood coagulation (Morel et al., 2006; Banfi et al., 2005). Under physiological conditions, tissue factor is expressed by adventitial cells surrounding blood vessels constitutively and initiates clotting. Blood-borne TF in the form of cell-derived microparticles and TF expression within platelets alludes that it plays a role in the amplification of the coagulation cascade. Moreover, in pathologic conditions, it was found that TF is expressed by a variety of cells including platelets, monocytes, neutrophils, and endothelial cells. Increased expression of TF leads to the elevation of the circulating TF-positive MP level and expression within the vasculature likely increases the risk of thrombosis in a variety of diseases and potentiation by phosphatidylserine may be contributory (Mackman et al., 2007).
Platelet activation by collagen and thrombin disrupts phosphatidylserine asymmetry resulting to the appearance of this phospholipid in the surface and also induces the expression of TF procoagulant activity. The concurrence of TF decryption and phosphatidylserine exposure does not necessarily mean to say that these events are coupled. There are evidences, however, that phosphatidylserine accelerates coagulation reactions on membrane surfaces, and the loss of phosphatidylserine asymmetry appears to be connected to the process of TF decryption. It was found that inhibition of expression of decrypted TF procoagulant activity occurs when an inhibitor, annexin V, is bound to phosphatidylserine on the surface of a Ca2+ ionophore-stimulated cell. In addition to that, it has also been stipulated that phosphatidylserine increases Vmax of activation of encrypted and inactive TF-FVIIa to a proteolytic active TF-FVIIa via protein substrate hydrolysis resulting to the decryption of TF procoagulant activity. Phosphatidylserine mediates this by increasing the number of functional catalytic sites strengthening the connection between this phospholipid and TF (Bach, 2006).
Hemostatic modulation of circulating procoagulant microparticles
In addition to being catalyst of the coagulation system, circulating microparticles also contain various functional membrane or cytoplasmic effectors including selectins, GPIIbIIIa, GPIb, von Willebrand factor, arachidonic acid, and thromboxane A2 that are able to amplify prothrombotic responses. Moreover, when MPs bear the appropriate counterligands, they can transfer their procoagulant capability to their target cells (Morel et al., 2006). For example, platelet derived MPs can bind to soluble and immobilized fibrinogen thus delivering procoagulant entities to the thrombus via the formation of aggregates (Holme et al., 1998). Moreover, in vitro researches also shows that interaction between monocytes and endothelial MPs promotes TF mRNA expression (Steppich et al., 2005)
The presence of tissue factor pathway inhibitor, thrombomodulin (Steppich et al., 2005) or protein C (Satta & Freyssinet, 1997) on the surface of MP suggests that although MP is implicated in increased thrombus formation, it also contributes to the anticoagulant pathway. However, because it exposes of liposaccharides, specifically phosphatidylserine, that promotes TF expression, thrombomodulin activity at monocytes and derived MP surfaces is overpowered by the activities of TF and prothrombinase (Satta & Freyssinet, 1997). During fibrinolysis in cases of myocardial infarction, the TF -driven coagulation is associated with the reduced TFPI expression at the MP surface (Steppich et al., 2005). Moreover, highly reactive oxygen species, which are greatly produced in cell-MP aggregates, may have also contributed to the reduced TFPI activity as well as increased TF expression. In essence, when TF is expressed at the MP surface, its activity prevails as a consequence of inadequate anticoagulant counterbalance (Morel et al., 2006).
In addition to the above mechanisms of MPs to modulate hemostasis, activated protein C pathways also contributes in the thrombo-resistance at the endothelial and monocyte MP surfaces. It was reported that activated protein C pathways increases shedding of endothelial and monocyte-derived MPs. Such MPs harbor functional endothelial protein C receptor, protected from metalloproteinase cleavage, and display anticoagulant ability toward factor Va inactivation.
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In summary, although MP is a procoagulant in nature mainly by exposing phosphatidylserine and increasing TF expression, it also harbors several features like the ability to express TFPI and the presence of protein C receptor as a form of counterbalance. In many disease states, however, this counterbalance mechanism is overcome by the procoagulant tendencies of MPs.
Medical implications of procoagulant microparticles
Currently, researches are undertaken to demonstrate the role of procoagulant microparticles in a variety of medical disorders (Morel et al., 2005). It is of interest to know that a defect in the ability to form procoagulant microparticles and expose phosphatidylserine can lead to a severe bleeding disorder called Scott Syndrome (Piccin et al., 2007). This finding suggests that platelet microparticles have a vital role in hemostasis in healthy individuals. This is also supported by the study of Hrachovinová et al. (2003) using a mouse model of hemophilia A infused with P-selectin-immunoglobulin (P-sel-Ig), an inducer of procoagulant micropaticles. Results showed that infusion of P-sel-Ig in hemophilia A mice produced a 20-fold increase of tissue factor-containing microparticles when compared to control. Increase in MP significantly improved the kinetics of fibrin formation in the mice with hemophilia A and normalized the mice's tail-bleeding time resonating the earlier conclusion that procoagulant microparticles are important to achieve hemostasis and that approaches that increases procoagulant microparticles should be explored to achieve control of bleeding disorders like hemophilia.
In addition, the complex role of microparticles in cardiovasular and vascular medical condition is an area of great interest that promises to yield important advances in the field of diagnosis and therapy (Piccin et al., 2007). In the study of Mallat et al. (2000) in patients with coronary heart disease that includes those with acute coronary syndrome (ACS), it was found that levels of procoagulant microparticles of endothelial origin were significantly elevated in patients with ACS compared to patients with non-coronary heart disease. This is in consonance with the case report findings of Morel et al., (2005) in a patient with anti-phospholipid antibodies (APL) presenting with myocardial infarction where levels procoagulant microparticles both of endothelial and platelet origin has been found to be significantly elevated compared to values found patients with MI but not history of anti-phospholipid syndrome (APS) with 3 and 6 fold elevation of endothelial and platelet derived MP, respectively. Moreover, when compared to control using healthy volunteers, it was found that there was a 25-fold elevation of endothelial derived MP and a 13 fold elevation of platelet derived MP in the same patient with MI. Hence, it was hypothesized that APL could have caused chronic cell activation leding to increased MP production. Above studies also suggest that high levels of cell-derived microparticles with procoagulatnt potential contributes to the initiation as well as perpetuation of thrombotic process especially in patients with recent clinical signs of plaque formation and thrombosis (Morel et al., 2005).
Another well studied area is the role of MP on sepsis, especially that which is secondary to meningococcemia. In general, patients with meningococcal sepsis suffers from dissseminated intravascular coagulation (DIC). In the study by Nieuwland et al. (2000), they analyzed the plasma samples of survivors and non-survivors for the presence of microparticles and compared it to the level of microparticle in healthy individuals. Ongoing coagulation activation in vivo was also quantified using enzyme-linked immunosorbent assay of plasma prothrombin framents F 1+2 while procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. Results of their study showed that, on admission, all meningococcal patients had elevated levels of microparticles originating from either platelets or granulocytes in comparison to the control. The patients also had increased F 1+2 and their microparticles enhances thrombin generation more intensely in vitro versus control suggesting that ongoing coagulation activation and procoagulant activity is augmented in these patients. In addition to that, it was also found that plasma samples of patient with the most fulminant disease course and exhibited severe form of DIC contained microparticles that expressed both tissue factor as well as CD14. Microparticles harboring these was said to demonstrate extreme generation of thrombin in vitro. Findings of these researches suggests that procoagulant microparticles can be potential targets of a novel therapeutic approach to oppose excessive coagulation activation in patients suffering meningococcemia (Nieuwland et al., 2000).
Procoagulant microparticles are also implicated in the development of thrombotic thrombocytopenic purpura (TTP) . The key initiating event in the pathogenesis of TTP was said to be endothelial injury, which promotes platelet activation leading to the formation of platelet-rich thrombi within the microvasculature.. However, the exact nature of endothelial injury present in TTP is still poorly defined and clinical assays that could reliably monitor the degree of endothelial injury is still not ready available. In the study by Jimenez et al. (2001) using cultured renal and brain microvascular endothelial cells (MVECs), they, instead, used the levels of endothelial microparticles (EMP) generated from these cells and their procoagulant activity as markers of endothelial injury during activation and apoptosis when these cells are exposed to TTP plasma. Results of their study demonstrated that both cell lines generated procoagulant EMPs when cultured with inducers of activation wile TNF-Î± or inducers of apoptosis like mitomycin C. Moreover, TTP plasma induced a five- to sixfold elevation of EMP and the doubling to tripling of procoagulant activityin the cultued renal and brain MVECs. Interestingly, EMP assay of blood from patients suffering acute TTP, likewise, showed marked elevation of TTP compared with normal controls and values have been shown to normalize in remission. Another study by Jimenez et al. (2003), showed that EMPs generated in TTP expresses von Willebrand factor and antigens that is consistent with activation. In conclusion, released endothelial procoagulant microparticles may have a role in the pathogenesis of TTP and assay of EMPs can also be a possible candidate as a marker of disease activity as well as endothelial injury in TTP and other disorders with thrombotic features (Jimenez et al., 2001; Jimenez et al., 2003).
Other diseases where MPs are thought to play a role includes sickle cell disease, paroxysmal nocturnal hemoglobinuria (PNH); in both cases, elevation of MPs and their pro-coagulant properties are related to the erythrocyte destruction leading to hemolysis. Meanwhile, in systemic diseases like lupus with anti-phospholipid syndrome, increased levels of MPs is attributed to the chronic endothelial damage and activation. Similar increase is found in heart failure and in pulmonary arterial hypertension (PAH) but is thought to be mediated by systemic release of inflammatory cytokines and changes hemodynamic changes that destroys the endothelium, respectively (Chironi et al., 2009).
In summary, MPs are derived from vascular endothelium and other circulating blood cells in the peripheral blood. They are released from vesiculation of cell membrane surfaces in physiological and pathological conditions and are present in low concentrations in normal plasma. Increased generation of MPs results from a number of mechanisms including platelet activation, direct vascular endothelial damage, as well as thrombin activity on the cell surface. Their exposure of phosphatidylserine and its catalytic function are mainly implicated in their procoagulant effect. Defect in the ability to form microparticles results to the severe bleeding disorder referred to as Scott syndrome providing an insight into the physiologic role of microparticles in hemostasis. The genesis and role of microparticles, derived from a variety of cells including platelets, endothelial cells and monocytes, is implicated in many medical conditions as in ACS, sepsis (especially meningococcal-induced), TTP, PNH, sickle cell disease, systemic lupus erythematous (SLE), heart failure and PAH. Procoagulant microparticles, therefore, can be a valuable marker of diseases exhibiting thrombotic features and can serve as target in the development of novel therapeutic approach (Chironi et al., 2009; Morel et al., 2006)