Pediatr Nephrol DOI 10.1007/s00467-015-3173-8
EDUCATIONAL REVIEW
Platelet abnormalities in nephrotic syndrome Benedicte Eneman 1,2 & Elena Levtchenko 1,2 & Bert van den Heuvel 2 & Chris Van Geet 3 & Kathleen Freson 3
Received: 6 April 2015 / Revised: 3 July 2015 / Accepted: 3 July 2015 # IPNA 2015
Abstract Nephrotic syndrome (NS) is a common kidney disease associated with a significantly increased risk of thrombotic events. Alterations in plasma levels of pro- and anti-coagulant factors are involved in the pathophysiology of venous thrombosis in NS. However, the fact that the risk of both venous and arterial thrombosis is elevated in NS points to an additional role for blood platelets. Increased platelet counts and platelet hyperactivity have been observed in nephrotic children. Platelet hyperaggregability, increased release of active substances, and elevated surface expression of activation-dependent platelet markers have been documented. The mechanisms underlying those platelet alterations are multifactorial and are probably due to changes in plasma levels of platelet-interfering proteins and lipid changes, as a consequence of nephrosis. The causal relationship between platelet alterations seen in NS and the occurrence of thromboembolic phenomena remains unclear. Moreover, the efficiency of prophylactic treatment using antiplatelet agents for the prevention of thrombotic complications in nephrotic patients is also unknown. Thus, antiplatelet medication is currently not generally recommended for routine prophylactic therapy.
* Benedicte Eneman
[email protected] 1
Pediatric Nephrology, Department of Pediatrics, University hospital of Leuven, Leuven, Belgium
2
Laboratory of Pediatric Nephrology, Department of Development & Regeneration, KU Leuven, Leuven, Belgium
3
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Keywords Nephrotic syndrome . Platelet hyperaggregability . Thrombocytosis . Thrombosis . Antiplatelet therapy
Abbreviations AA Arachidonic acid AC Adenylate cyclase ACE Angiotensin-converting enzyme ADP Adenosine diphosphate AT Angiotensin ATP Adenosine triphosphate cAMP Cyclic adenosine monophosphate COX Cyclooxygenase DAG Diacylglycerol ELISA Enzyme-linked immunosorbent assay GP Glycoprotein IP3 Inositol triphosphate IL-6 Interleukin-6 IL-7 Interleukin-7 MDA Malondialdehyde MPV Mean platelet volume NS Nephrotic syndrome NSAID Non-steroidal anti-inflammatory drugs OCS Open canalicular system PACAP Pituitary adenylate cyclase-activating polypeptide PAR Protease-activated receptor PCT Plateletcrit PDGF Platelet-derived growth factor PDW Platelet distribution width PIP2 Phosphatidylinositol bisphosphate PKC Protein kinase C
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PLCβ PLT count PRP PS TXA2 TXB2 vWF
Phospholipase C β Platelet count Platelet-rich plasma Phosphatidylserine Thromboxane A2 Thromboxane B2 von Willebrand factor
Introduction Platelets play an important role in primary hemostasis, preventing bleeding after injury. Increased platelet counts and platelet hyperactivity have been thought to result in an increased risk of thromboembolic events, as frequently observed in patients with nephrotic syndrome (NS). Although several studies have reported platelet abnormalities in patients with NS since the 70s, the pathophysiology and the clinical consequences of these platelet alterations in NS are not fully understood. In this review we provide an overview of literature reporting on diverse platelet alterations and their potential role in primary NS.
Platelet morphology and function Platelets are anucleated discoid circulating blood cells. They are 2–3 μm in diameter and have a volume of about 9–10 fl. Platelets are derived from giant precursor cells, called megakaryocytes. Megakaryocytes arise from differentiation of hematopoietic stem cells in the bone marrow and undergo fragmentation of their cytoplasm, leading to platelet production. Platelet production is controlled by thrombopoietin and other cytokines [1]. The normal range for platelet counts in human blood is 150–350×109/l [2]. Platelets have a lifespan of about 8–10 days, meaning that about 1011 platelets are produced daily [3]. The glycocalyx forms the outer layer of the platelet, consisting of glycoprotein receptors that play an important role in platelet adhesion, activation, and aggregation. The plasma membrane is located under the glycocalyx and contains phospholipids. Phospholipids help convert prothrombin to thrombin, promoting the coagulation cascade. The platelet cytoskeleton consists of contractile proteins, which are essential for maintaining the discoid shape and are responsible for the shape change after activation. Platelets contain three major types of granules: α-granules, dense granules, and lysosomes [4]. Furthermore, platelets contain a surface-connected open canalicular system (OCS) with indentations and channels throughout the inner part of the platelet. The OCS probably plays a role in the transport of external substances into the platelet and in the release of α-granule contents. Moreover, this extensive membrane reservoir may take part in the
formation of filopodia during platelet adhesion. Lastly, it serves as a storage site for plasma membrane glycoproteins, regulating which platelet receptors become available for interaction with plasma [5]. Platelets play a crucial role in thrombus formation in response to vascular endothelial injury. They form plugs (primary hemostasis), which are then covered with fibrin strands, as a result of the coagulation cascade (secondary hemostasis). An increasing number of studies indicate that platelets might also play a role in intercellular communication and as mediators of inflammation and immunomodulation [6–8]. In order to exert their biological functions, platelets need to be activated. Exposure of platelets to specific receptor agonists or physical factors leads to platelet activation with dramatic changes in platelet shape, adhesion, release of active substances from their granules, and aggregation (Fig. 1). At a site of vascular endothelial injury, circulating platelets are exposed to von Willebrand factor (vWF) and collagen in the subendothelial connective tissue matrix. Binding of vWF to the platelet GP Ib/IX/V receptor plays a crucial role in platelet adhesion [9]. Subendothelial collagen contributes to platelet adhesion via the platelet GP Ia/IIa and GP VI collagen receptors. Due to cytoskeletal changes, activated platelets change their discoid shape into an irregular morphology, with pseudopodia formation facilitating adhesion [2]. Besides serving as a surface for platelet adhesion, collagen is also a potent stimulator of platelet activation. Collagen binding leads to an increased cytosolic calcium concentration in platelets. Increased cytosolic calcium concentration plays an important role in platelet activation by stimulating activity of enzymes involved in platelet activation and improving protein–protein interactions. Other platelet stimulatory agonists besides collagen are thrombin, adenosine diphosphate (ADP), epinephrine and thromboxane A2 (TXA2). Thrombin activates the protease-activated receptor 1 (PAR1) and PAR4 receptors, ADP interacts with the P2Y1 and P2Y12 receptors and TXA2 with the TP receptor on platelets. All of these are G-protein coupled receptors, and their activation leads to intracellular protein phosphorylation, increased TXA2 formation and elevated cytosolic calcium concentrations. Epinephrine is another platelet agonist, which stimulates the Gi-coupled α2-adrenergic receptor on platelets, resulting in inhibition of cAMP formation. Intracellular cAMP antagonizes platelet activation by inhibition of calcium release. TXA2 is metabolized from arachidonic acid (AA) by cyclooxygenase (COX) in activated platelets. It stimulates activation of other platelets and induces platelet aggregation by activation of the platelet fibrinogen-binding αIIbβ3 receptors. Binding of fibrinogen to the activated αIIbβ3 receptors of different platelets leads to platelet–platelet interactions and to the formation of platelet aggregates. Activation of platelets further leads to degranulation and the release of active substances from α- and dense granules.
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Fig. 1 Mechanisms of platelet activation. Complex mechanisms underlie platelet activation. Vascular endothelial injury induces exposure of von Willebrand factor (vWF) in the subendothelial matrix, which leads to vWF binding to the platelet GP Ib/XI/V receptor. Collagen contributes to platelet adhesion via the GP Ia/IIa and GP VI receptors. Increased cytosolic calcium concentration and the release of activation mediators such as thrombin, adenosine diphosphate (ADP) and thromboxane A2 (TXA2) further stimulate platelet activation. Thrombin activates the protease-activated receptor 1 (PAR1) and PAR4, ADP interacts with P2Y1 and P2Y12 receptors and TXA2 with the TP receptor, all Gprotein coupled receptors. P2Y12 and PAR1 have a Gi α-subunit, inhibiting adenylate cyclase (AC) and as a consequence, preventing the formation of cAMP. PAR4, P2Y1, and TP have a Gq α-subunit, which
activates phospholipase C β (PLCβ). Binding of collagen to the GP VI receptor also induces activation of PLCβ. PLCβ catalyzes the production of inositol triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP2). IP3 binds endoplasmatic reticulum calcium channels, leading to an increased cytosolic calcium concentration. DAG activates protein kinase C (PKC), which promotes degranulation of dense and α-granules. The release of active substances from dense and α-granules further stimulates activation of other platelets. TXA2, ADP and increased cytosolic calcium concentration activate the platelet fibrinogen binding αIIbβ3 receptor. Binding of fibrinogen to the activated αIIbβ3 receptors of different platelets leads to platelet–platelet interactions and the formation of platelet aggregates
Table 1 provides an overview of the major substances stored in platelet granules. α-Granules are rapidly exocytosed upon
platelet activation and secrete many different proteins involved in platelet adhesion, aggregation and secretion, but
Pediatr Nephrol Table 1
Overview of contents of platelet granules
Contents of platelet granules α-granules
Lysosomes
Albumin
Aryl sulphatase
Basic fibroblast growth factor β-Thromboglobulin
β-Galactosidase β-Glucuronidase
Epidermal growth factor Factor V
Cathepsins Collagenase
Fibronectin
Elastase
Fibrinogen Hepatocyte growth factor
Heparitinase N-Acetylglucose aminidase
Insulin-like growth factor Interleukin-1γ Interleukin-7
Dense granules
Osteonectin Platelet-derived endothelial growth factor Platelet-derived growth factor
ADP ATP
Platelet-specific proteins Platelet factor 4
Histamine Norepinephrine
Thrombospondin Transforming growth factor α Transforming growth factor β Vitronectin
Pyrophosphate Serotonin
Calcium
von Willebrand factor ADP adenosine diphosphate; ATP adenosine triphosphate
also proteins that are involved in chemotaxis, proliferation and inflammatory processes. Dense granules contain ADP, adenosine triphosphate (ATP), calcium, serotonin, and other substances inducing platelet aggregation and production of inflammatory mediators. Lysosomes contain degradative enzymes that can contribute to the clearance of platelet thrombi and extracellular matrix [10]. Degranulation occurs by fusion of granule membranes with the cell membranes. This leads to expression of specific receptor proteins on the platelet plasma membrane that are normally only located in the secretory granules in resting platelets. An example of a platelet surface activation-dependent marker that is normally located in the α-granule membrane is P-selectin (CD62P). As indicated above, platelet membrane phospholipids play a role in the formation of thrombin, a central component of the coagulation system. In resting platelets, the plasma membrane aminophospholipid phosphatidylserine (PS) is almost exclusively located in the inner (cytosolic) leaflet of the cell membrane. Upon platelet activation, PS is translocated to the outer leaflet of the platelet membrane, making it accessible to circulating coagulation
factors. Moreover, platelet activation leads to the shedding of microparticles, also exposing PS in the outer membrane leaflet. PS binds and activates factors Xa and Va, which form the prothrombinase complex, catalyzing the conversion of prothrombin to thrombin [11]. While the relative amount of PS in the plasma membrane increases in response to platelet activation, the phospholipid phosphatidylinositol is reduced, as it is processed to DAG and IP3 [12]. PS exposure is not the only link between platelet activation and promotion of the coagulation system. After stimulation with collagen and thrombin, a subpopulation of so-called 'coated-platelets’ are formed, serving as a surface that promotes thrombin generation. These coated-platelets not only express PS in the outer membrane leaflet, but also retain high levels of procoagulant proteins on their surface, such as vWF, fibronectin, thrombospondin, and fibrinogen [13]. Recently, it was discovered that the degree and the mechanisms of platelet activation are not uniform throughout the hemostatic plug. The primary thrombus is a hierarchically organized structure, with a core of fully activated platelets at the site of injury, with an overlaying shell of less activated platelets. Fibrin formation and thrombin activity were found to be much more present in the core than in the shell of the primary thrombus. The core also shows a greater density of platelets than the shell does, facilitating contact-dependent signaling. ADP was found to regulate platelets in the shell, while it did much less so in the core [14].
Laboratory investigation of platelet morphology and function The platelet count (PLT count) can be measured manually or automatically. Mean platelet volume (MPV) gives information about the mean volume of individual platelets and platelet distribution width (PDW) indicates the variation in size of platelets [15]. Platelet count and morphology are investigated using a May-Grünwald stained blood smear. However, detailed ultrastructural platelet abnormalities such as granule defects can only be evaluated using electron microscopy. Platelet function tests are generally used for the diagnosis of bleeding disorders. When the role of platelets in arterial thrombosis became clear, platelet function tests were also implemented for the identification of increased risks of thrombosis. The clinical use of platelet function tests has recently been reviewed [10]. Platelet function can be tested using several methods. Light transmission aggregometry (LTA) is still the most widely used test to study platelet aggregability [10]. In this test, platelet aggregation is induced ex vivo by the addition of an agonist
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such as ADP, collagen, epinephrine, AA and thrombin [16]. Other less frequently used agonists such as thrombin-activating peptide (TRAP) and U46619 may be employed as well [17]. Spontaneous aggregation without addition of an agonist can also be measured. Ristocetininduced platelet agglutination can also be assessed by LTA. Ristocetin leads to a conformational change in vWF, inducing platelet agglutination (clumping together of platelets without platelet activation or fibrinogen binding) [18]. Much information can be obtained by LTA, as platelet function is studied directly. However, a limitation of this technique is that it is an ex vivo assay, which may not always reliably reflect in vivo conditions [4]. Moreover, LTA is quite labor-intensive, requiring a certain degree of technical expertise for performance and interpretation. Hence, its use is limited to specialized laboratories. Platelet function can also be assessed using indirect markers of in vivo platelet activity. Flow cytometry can be used to measure platelet surface expression of activation-dependent markers such as P-selectin (CD62P), the active form of the fibrinogen αIIbβ3 receptor and negatively charged phospholipids, such as PS. Platelet microparticles can also be quantified with flow cytometry, but this test is not routinely performed in clinical practice. Flow cytometry is a sensitive, but expensive technique. Whole-blood samples should be taken in a standardized way with a wide needle to prevent stasis and in vitro platelet activation. Furthermore, several soluble markers released from platelets upon activation, such as for example β-thromboglobulin, platelet-derived growth factor (PDGF), platelet factor 4, soluble P-selectin and thromboxanes can be measured in plasma, using ELISA. Those markers, however, are surrogate markers as they only indirectly reflect in vivo platelet activity. Other factors besides platelet activation, such as platelet lysis, could lead to increased levels. Table 2 summarizes laboratory investigations used to study platelet morphology and function.
Platelet morphology and function abnormalities in nephrotic syndrome NS is a disease defined by severe proteinuria (urinary protein levels above 3.5 g/1.73 m2/day), hypoalbuminemia, edema and hyperlipidemia, as a consequence of increased glomerular permeability [19]. Patients with NS have an increased risk of thromboembolic disease, including both deep venous and arterial thrombosis, which significantly increases morbidity and mortality rates. It occurs in 20–37 % of adults, and 1–27 % of children, depending on etiology and severity of the NS and on whether imaging to diagnose thromboembolism is performed in all patients or only in patients with clinical
Table 2
Laboratory investigations of platelet morphology and function
Platelet morphology -Platelet count -Platelet indices: MPV, PDW -Blood smear for light microscopic evaluation -Electron-microscopic evaluation Platelet function Direct measures of platelet function: -Light-transmission aggregometry Spontaneous aggregation Aggregation in response to ADP, collagen, epinephrine, AA, thrombin Agglutination in response to ristocetin Indirect measures of platelet function: -Flow cytometry Expression of activation-dependent markers on platelet surface Quantification of platelet microparticles -ELISA for soluble markers of platelet activation in plasma or urine MPV mean platelet volume; PDW platelet distribution width; ELISA enzyme-linked immunosorbent assay; ADP adenosine diphosphate; AA arachidonic acid
symptoms [20–22]. Underlying mechanisms of thromboembolism in NS are considered to be multifactorial. Disturbances of the coagulation system seem to play an important role for venous thrombotic events [22]. However, because the risk for arterial thrombosis is also elevated, a role has been postulated for platelets in the prothrombotic state in NS. We here provide an overview of the literature that supports a role for platelets as risk factors for thrombosis in NS. Table 3 shows a summary of platelet abnormalities that have already been reported in NS (Table 4).
Changes in platelet count and morphology during nephrotic syndrome In 1981, Walter et al. reported for the first time a statistically significant increased mean PLT count of 281×109/l in adult patients with NS in comparison to 216×109/l in the healthy control group [23]. This was later confirmed by two studies [24, 25], while other studies found a normal PLT count in adult patients with NS [26, 27]. These findings suggest that even if a significant increase in PLT count is present in adult patients with NS, the increase is limited. Its clinical significance remains questionable. For children, several studies show mean PLT counts above the reference values of 150–450×109/l [28–31], supporting evidence for thrombocytosis in children with
Pediatr Nephrol Table 3
Overview of platelet changes during nephrotic syndrome
Table 4 Drugs affecting platelet function often used in pediatric nephrotic syndrome patients
Platelet morphology Platelet count (PLT count)
↑ [23–25, 28–30], ∼ [26, 27, 32, 36]
NSAIDs
Aspirin, diclofenac, ibuprofen, indomethacin etc.
Mean platelet volume (MPV)
↑ [33], ↓ [30]
Platelet distribution width (PDW)
↑ [30]
Antibiotics Antifungals
Penicillins, cephalosporins and nitrofurantoin Miconazole
Plateletcrit (PCT)
∼ [30]
β-blockers
Propranolol
ADP
↑ [24, 25, 32, 34–37]
Calcium channel Verapamil, nifedipine, diltiazem blockers ACE-inhibitors Captopril, enalapril, lisinopril, ramipril AT receptor blockers Valsartan, losartan, olmesartan
Collagen
↑ [25, 31, 32, 35–39], ∼ [24]
Statins
Arachidonic acid
↑ [26, 34, 35], ∼ [25, 26, 32]
Epinephrine
↑ [40]
Based on Scharf [77]
Spontaneous
↑ [23–25, 28, 35, 40]
NSAID non-steroidal anti-inflammatory drugs; ACE angiotensinconverting enzyme; AT angiotensin
Platelet aggregation Aggregation after stimulation with
Agglutination after stimulation with Ristocetin
Pravastatin, rosuvastatin, simvastatin etc.
↑ [41], ∼ [32]
Plasma platelet aggregate ratio
↓ [47, 89]
Platelet–platelet aggregates
↑ [29]
Shear stress-induced platelet aggregation ↑ [90] Release of active substances from platelets
normalized during long-term remission [29]. Contradicting previous reports, Mittal et al. recently described normal PLT counts in Indian nephrotic children [32]. An elevated PDW and a decreased MPV were observed in children with NS before the start of treatment [30]. Increased MPV levels during treatment for NS in adults correlated with therapy resistance after 12 months [33].
β-thromboglobulin
↑ [23, 25, 37, 42, 43]
Platelet-derived growth factor
↑ [30]
Interleukin-7
↑ [44]
Platelet factor 4
∼ [23, 25]
Soluble P-selectin
↑ [37]
ATP, in response to collagen
↑ [38]
Thromboxane A2 metabolites in urine and plasma Thromboxane B2 release of platelets, after stimulation with Collagen
↑ [46]
Numerous studies have revealed evidence for an increased platelet function in idiopathic NS.
↑ [38], ∼ [25, 35]
Hyperaggregability of platelets
Arachidonic acid
↑ [25, 35, 47]
ADP
∼ [25]
Platelet surface expression P-selectin
↑ [29, 37]
Glycoprotein 53
↑ [37]
Phosphatidylserine
↑ [27]
Intraplatelet alterations Calcium concentration, after stimulation with STA2 cAMP concentration
↑ [46] ↓ [38]
Others Platelet microparticles
↑ [27]
Platelet adhesiveness
↑ [23, 36]
Platelet-vessel wall interactions under flow conditions
∼ [91]
↑ increased; ↓ decreased; ∼ no difference; ADP adenosine diphosphate; ATP adenosine triphosphate; STA2 9,11-Epithio-11,12-methano-thromboxane A2; cAMP cyclic adenosine monophosphate
NS. PLT counts during remission after the start of corticosteroid treatment and early after cessation of corticosteroid treatment remained high [28–31], while they
Increased platelet activation in NS
Platelet aggregability was repeatedly tested in patients during relapse of NS and was compared with platelet aggregability in healthy control subjects or in NS patients during remission. The majority of studies report increased platelet aggregability, both in adults and children in response to various agonists: ADP [24, 25, 32, 34–37], collagen [25, 31, 32, 35–39], AA [26, 34, 35] and epinephrine [40], and increased platelet agglutination after stimulation with ristocetin [41] (Table 3). However, other studies could not find increased platelet aggregability in response to collagen [24], AA [25, 26, 32] and ristocetin [32]. Spontaneous aggregation was observed during NS as well [23–25, 28, 35, 40]. Altered expression of surface markers of platelet function Platelet activation leads to cell membrane expression of different markers. As described above, these ‘activation-dependent’ markers can be used as markers for platelet activation. In NS, alterations in several platelet surface markers indicating enhanced platelet activation have been observed (Table 3). Elevated surface expression of P-selectin was found both in children and adults during relapse of NS [29, 37]. Lysosomal
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membrane glycoprotein 63 (CD63) expression in the platelet cell membrane [37] and PS exposure [27] were also increased in NS patients during relapse. Increased release of active substances Active substances released from platelets upon activation can be measured in the patient’s plasma and can then be used as surrogate markers of in vivo platelet activity. Increased levels of various active substances secreted by platelets have been reported in patients during relapse of NS, compared with healthy control subjects or with patients during remission (Table 3). Several substances released from α-granules upon platelet activation were elevated in plasma during NS. βthromboglobulin, a platelet-specific protein, was repeatedly found to be elevated in adults during NS [23, 25, 37, 42, 43]. Platelet-derived growth factor (PDGF) [30] and interleukin-7 (IL-7) [44] were increased in pediatric patients during NS, while plasma levels of platelet factor 4, another α-granule component, were normal [23, 25]. Plasma levels of the soluble form of P-selectin were elevated in both adults and children during idiopathic NS [37, 45]. Moreover, increased thromboxane formation was seen during NS. TXA2, which is a strong inducer of platelet activation, is formed from AA in activated platelets by the COX pathway. TXA2 has a short half-life and is metabolized to thromboxane B2 (TXB2) and malondialdehyde (MDA), which can be used as markers of platelet prostaglandin synthesis [40]. Increased TXA2 metabolites were found in plasma and urine of pediatric patients with NS, indicating enhanced TXA2 synthesis in platelets and the kidney in NS [46]. However, these active substances released from platelets can also be measured in the supernatant following ex vivo platelet aggregation. They then directly reflect the ex vivo platelet activity. Increased levels of active substances were also found in samples of NS patients after ex vivo platelet aggregation. Platelets from pediatric NS patients released an increased amount of ATP from dense granules in response to collagen [38]. The production of MDA and TXB2 by platelets following AA challenge was also increased in NS, indicating activation of the TXA2 signal transduction system [25, 35, 47]. TXB2 formation after stimulation with ADP and collagen was not altered in adult patients with NS [25, 35], while increased TXB2 release from platelets in response to collagen was seen in children with NS [38].
circulation. Microparticles are small (1000 × 109/l, except if there are additional prothrombotic factors [87]. As described above, several other prothrombotic risk factors can be present in NS, such as for example, increased platelet activation and hypovolemia. These may justify antiplatelet treatment in certain cases of NS with severe thrombocytosis. As guidelines are lacking, decisions should be made on an individual basis.
Conclusions and direction of future research Enhanced platelet activity has repeatedly been demonstrated in NS. However, the pathogenesis is not completely understood. Moreover, it has not been proven that platelet alterations play a role in the pathogenesis of the prothrombotic state in NS. Therefore, future research should focus on the pathogenesis of platelet alterations in NS, using an NS animal model, such as puromycin aminonucleoside-treated rats [88]. Furthermore, prospective epidemiologic studies are needed to investigate the relationship between platelet alterations and the incidence of thrombotic complications. Only if further animal and epidemiologic studies reveal an important role for platelet alterations in thrombotic complications in NS should controlled trials be performed to evaluate the effect of low-dose antiplatelet treatment on the incidence of thrombosis in NS.
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B. At least 3 days, because it causes reversible inhibition of cyclooxygenase-1 C. At least 10 days, because it causes irreversible inhibition of cyclooxygenase-1 D. At least 1 day, because it causes irreversible inhibition of cyclooxygenase-1
Conflict of interest The authors declare that they have no conflict of interest.
Summary points 1 As the risk of not only venous but also arterial thrombosis is elevated in nephrotic syndrome, a role for blood platelets is presumed in the pathogenesis. 2 Increased platelet count, platelet hyperaggregability, increased release of active substances, and elevated surface expression of activationdependent platelet markers have been documented during relapse of nephrotic syndrome. 3 The mechanisms underlying platelet abnormalities are probably due to changes in plasma levels of platelet-interfering proteins and lipids, as a consequence of nephrosis. Currently, antiplatelet medication is not generally recommended for routine prophylactic therapy in nephrotic patients.
Multiple-choice questions (Answers are provided following the reference list)
5. Which of the following methods is the best measurement of platelet function in the clinical laboratory? A. Quantification of platelet microparticles B. Measurement of platelet count and mean platelet volume C. Light transmission aggregometry after induction with an agonist such as ADP, collagen, epinephrine, arachidonic acid or thrombin D. ELISA for soluble platelet activation markers such as β-thromboglobulin and platelet-derived growth factor
References 1.
1. Which statement about platelet activation is true? A. Von Willebrand factor contributes to platelet adhesion via the platelet PAR4 receptor. B. Increased cytosolic calcium concentrations stimulate platelet activation. C. Coagulation factor VIII stimulates secretion of platelet-dense granules. D. Increased cytosolic calcium concentrations prevent degranulation of dense and α-granules. 2. Which of the following agents is not an inducer of platelet aggregation? A. Collagen B. Arachidonic acid C. cAMP D. Thrombin
2. 3.
4. 5. 6. 7. 8. 9. 10. 11.
3. Which of the following mechanisms is not involved in increased platelet activation during idiopathic nephrotic syndrome? A. Elevated plasma levels of platelet-activating substances B. Factor V Leiden thrombophilia C. Decreased plasma levels of platelet inhibitory proteins D. A reduced negative charge of the platelet surface
12.
13. 14.
15.
4. How many days should aspirin be withheld before reliable platelet aggregation tests can be performed? A. At least 5 days, because it causes reversible inhibition of cyclooxygenase-1
16.
Kaushansky K (1999) Thrombopoietin and hematopoietic stem cell development. Ann N Y Acad Sci 872:314–319 George JN (2000) Platelets. Lancet 355:1531–1539 Bautista AP, Buckler PW, Towler HM, Dawson AA, Bennett B (1984) Measurement of platelet life-span in normal subjects and patients with myeloproliferative disease with indium oxine labelled platelets. Br J Haematol 58:679–687 Kamath S, Blann AD, Lip GY (2001) Platelet activation: assessment and quantification. Eur Heart J 22:1561–1571 Thon JN, Italiano JE (2012) Platelets: production, morphology and ultrastructure. Handb Exp Pharmacol 210:3–22 Gawaz M, Langer H, May AE (2005) Platelets in inflammation and atherogenesis. J Clin Invest 115:3378–3384 Semple JW, Freedman J (2010) Platelets and innate immunity. Cell Mol Life Sci 67:499–511 Elzey BD, Sprague DL, Ratliff TL (2005) The emerging role of platelets in adaptive immunity. Cell Immunol 238:1–9 Bryckaert M, Rosa JP, Denis CV, Lenting PJ (2015) Of von Willebrand factor and platelets. Cell Mol Life Sci 72:307–326 Choi JL, Li S, Han JY (2014) Platelet function tests: a review of progresses in clinical application. Biomed Res Int 2014:456569 Lentz BR (2003) Exposure of platelet membrane phosphatidylserine regulates blood coagulation. Prog Lipid Res 42:423–438 Skeaff CM, Holub BJ (1985) Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets. Biochim Biophys Acta 834:164–171 Dale GL (2005) Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 3:2185–2192 Stalker TJ, Traxler EA, Wu J, Wannemacher KM, Cermignano SL, Voronov R, Diamond SL, Brass LF (2013) Hierarchical organization in the hemostatic response and its relationship to the plateletsignaling network. Blood 121:1875–1885 Wiwanitkit V (2004) Plateletcrit, mean platelet volume, platelet distribution width: its expected values and correlation with parallel red blood cell parameters. Clin Appl Thromb Hemost 10: 175–178 Cattaneo M, Cerletti C, Harrison P, Hayward CP, Kenny D, Nugent D, Nurden P, Rao AK, Schmaier AH, Watson SP, Lussana F,
Pediatr Nephrol Pugliano MT, Michelson AD (2013) Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost. doi:10.1111/jth. 12231 17. Gresele P, Subcommittee on Platelet Physiology (2015) Diagnosis of inherited platelet function disorders: guidance from the SSC of the ISTH. J Thromb Haemost 13:314–322 18. Kang M, Wilson L, Kermode JC (2008) Evidence from limited proteolysis of a ristocetin-induced conformational change in human von Willebrand factor that promotes its binding to platelet glycoprotein Ib-IX-V. Blood Cells Mol Dis 40:433–443 19. Orth SR, Ritz E (1998) The nephrotic syndrome. N Engl J Med 338:1202–1211 20. Singhal R, Brimble KS (2006) Thromboembolic complications in the nephrotic syndrome: pathophysiology and clinical management. Thromb Res 118:397–407 21. Mahmoodi BK, ten Kate MK, Waanders F, Veeger NJ, Brouwer JL, Vogt L, Navis G, van der Meer J (2008) High absolute risks and predictors of venous and arterial thromboembolic events in patients with nephrotic syndrome: results from a large retrospective cohort study. Circulation 117:224–230 22. Kerlin BA, Ayoob R, Smoyer WE (2012) Epidemiology and pathophysiology of nephrotic syndrome-associated thromboembolic disease. Clin J Am Soc Nephrol 7:513–520 23. Walter E, Deppermann D, Andrassy K, Koderisch J (1981) Platelet hyperaggregability as a consequence of the nephrotic syndrome. Thromb Res 23:473–479 24. Robert A, Olmer M, Sampol J, Gugliotta JE, Casanova P (1987) Clinical correlation between hypercoagulability and thromboembolic phenomena. Kidney Int 31:830–835 25. Machleidt C, Mettang T, Starz E, Weber J, Risler T, Kuhlmann U (1989) Multifactorial genesis of enhanced platelet aggregability in patients with nephrotic syndrome. Kidney Int 36:1119–1124 26. Bennett A, Cameron JS (1987) Platelet hyperaggregability in the nephrotic syndrome which is not dependent on arachidonic acid metabolism or on plasma albumin concentration. Clin Nephrol 27:182–188 27. Gao C, Xie R, Yu C, Wang Q, Shi F, Yao C, Xie R, Zhou J, Gilbert GE, Shi J (2012) Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome. Thromb Haemost 107:681–689 28. Andre E, Voisin P, Andre JL, Briquel ME, Stoltz JF, Martinet N, Alexandre P (1994) Hemorheological and hemostatic parameters in children with nephrotic syndrome undergoing steroid therapy. Nephron 68:184–191 29. Tkaczyk M, Baj Z (2002) Surface markers of platelet function in idiopathic nephrotic syndrome in children. Pediatr Nephrol 17:673– 677 30. Wasilewska AM, Zoch-Zwierz WM, Tomaszewska B, Biernacka A (2005) Platelet-derived growth factor and platelet profiles in childhood nephrotic syndrome. Pediatr Nephrol 20:36–41 31. Eneman B, Freson K, van den Heuvel L, van Hoyweghen E, Collard L, Vande Walle J, van Geet C, Levtchenko E (2015) Pituitary adenylate cyclase-activating polypeptide deficiency associated with increased platelet count and aggregability in nephrotic syndrome. J Thromb Haemost 13:755–767 32. Mittal A, Aggarwal KC, Saluja S, Aggarwal A, Sureka B (2013) Platelet functions and coagulation changes in Indian children with nephrotic syndrome. J Clin Diagn Res 7:1647–1650 33. Kocyigit I, Yilmaz MI, Simsek Y, Unal A, Sipahioglu MH, Eroglu E, Dede F, Tokgoz B, Oymak O, Utas C (2013) The role of platelet activation in determining response to therapy in patients with primary nephrotic syndrome. Platelets 24:474–479
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
Remuzzi G, Mecca G, Marchesi D, Livio M, de Gaetano G, Donati MB, Silver MJ (1979) Platelet hyperaggregability and the nephrotic syndrome. Thromb Res 16:345–354 Jackson CA, Greaves M, Patterson AD, Brown CB, Preston FE (1982) Relationship between platelet aggregation, thromboxane synthesis and albumin concentration in nephrotic syndrome. Br J Haematol 52:69–77 Panicucci F, Sagripanti A, Vispi M, Pinori E, Lecchini L, Barsotti G, Giovannetti S (1983) Comprehensive study of haemostasis in nephrotic syndrome. Nephron 33:9–13 Sirolli V, Ballone E, Garofalo D, Merciaro G, Settefrati N, Di Mascio R, Di Gregorio P, Bonomini M (2002) Platelet activation markers in patients with nephrotic syndrome. A comparative study of different platelet function tests. Nephron 91:424–430 Turi S, Bereczki C, Torday C, Havass Z (1994) Laser aggregometer studies, ATP release and thromboxane B2 release and cAMP concentration of the platelets in nephrotic syndrome. Prostaglandins Leukot Essent Fatty Acids 51:69–73 Bang NU, Trygstad W, Schroeder JE, Heidenreich RO, Csiscko BM (1973) Enhanced platelet function in glomerular renal disease. J Lab Clin Med 81:651–660 Stuart MJ, Gerrard JM, White JG (1980) The influence of albumin and calcium on human platelet arachidonic acid metabolism. Blood 55:418–423 Taira K, Okajima C, Yamashita T, Kawahara S, Matsunaga T, Nakajima M, Kamitsuji H (1990) Ristocetin induced platelet aggregation in children with nephrotic syndrome. Nihon Jinzo Gakkai Shi 32:659–666 Adler AJ, Lundin AP, Feinroth MV, Friedman EA, Berlyne GM (1980) Beta-thromboglobulin levels in the nephrotic syndrome. Am J Med 69:551–554 Goubran F, Maklady F (1988) In vivo platelet activity and serum albumin concentration in nephrotic syndrome. Blut 57:15–17 Wasilewska A, Zoch-Zwierz WM, Tomaszewska B, Zelazowska B (2005) Relationship of serum interleukin-7 concentration and the coagulation state in children with nephrotic syndrome. Pediatr Int 47:424–429 Kilis-Pstrusinska K, Medynska A, Wikiera-Magott I, Zwolinska D (2001) Levels of selected soluble adhesion molecules in blood serum of children with chronic glomerulonephritis. Pol Merkur Lekarski 10:247–249 Kobayashi T, Suzuki J, Watanabe M, Suzuki S, Yoshida K, Kume K, Suzuki H (1997) Changes in platelet calcium concentration by thromboxane A2 stimulation in patients with nephrotic syndrome of childhood. Nephron 77:309–314 Schieppati A, Dodesini P, Benigni A, Massazza M, Mecca G, Remuzzi G, Livio M, de Gaetano G, Rossi EC (1984) The metabolism of arachidonic acid by platelets in nephrotic syndrome. Kidney Int 25:671–676 Matzdorff AC, Kuhnel G, Kemkes-Matthes B, Pralle H (1998) Quantitative assessment of platelets, platelet microparticles, and platelet aggregates with flow cytometry. J Lab Clin Med 131: 507–517 Pathansali R, Smith N, Bath P (2001) Altered megakaryocyteplatelet haemostatic axis in hypercholesterolaemia. Platelets 12: 292–297 Freson K, Peeters K, De Vos R, Wittevrongel C, Thys C, Hoylaerts MF, Vermylen J, Van Geet C (2008) PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications. Blood 111:1885–1893 Yoshida N, Aoki N (1978) Release of arachidonic acid from human platelets. A key role for the potentiation of platelet aggregability in normal subjects as well as in those with nephrotic syndrome. Blood 52:969–977
Pediatr Nephrol 52.
53.
54.
55. 56.
57. 58.
59.
60. 61.
62. 63.
64.
65.
66. 67.
68.
69. 70.
71.
Kuhlmann U, Steurer J, Rhyner K, von Felten A, Briner J, Siegenthaler W (1981) Platelet aggregation and betathromboglobulin levels in nephrotic patients with and without thrombosis. Clin Nephrol 15:229–235 Ueda N (1990) Effect of corticosteroids on some hemostatic parameters in children with minimal change nephrotic syndrome. Nephron 56:374–378 Shattil SJ, Cooper RA (1978) Role of membrane lipid composition, organization, and fluidity in human platelet function. Prog Hemost Thromb 4:59–86 Gerrard JM (1988) Platelet aggregation: cellular regulation and physiologic role. Hosp Pract (Off Ed) 23:89–98, 103–104, 107–108 Akoglu H, Agbaht K, Piskinpasa S, Falay MY, Dede F, Ozet G, Odabas AR (2012) High frequency of aspirin resistance in patients with nephrotic syndrome. Nephrol Dial Transplant 27:1460–1466 Schlegel N (1997) Thromboembolic risks and complications in nephrotic children. Semin Thromb Hemost 23:271–280 Freson K, Hashimoto H, Thys C, Wittevrongel C, Danloy S, Morita Y, Shintani N, Tomiyama Y, Vermylen J, Hoylaerts MF, Baba A, Van Geet C (2004) The pituitary adenylate cyclase-activating polypeptide is a physiological inhibitor of platelet activation. J Clin Invest 113:905–912 Kerlin BA, Blatt NB, Fuh B, Zhao S, Lehman A, Blanchong C, Mahan JD, Smoyer WE (2009) Epidemiology and risk factors for thromboembolic complications of childhood nephrotic syndrome: a Midwest Pediatric Nephrology Consortium (MWPNC) study. J Pediatr 155:105–110, 110 e101 Freedman JE (2005) Molecular regulation of platelet-dependent thrombosis. Circulation 112:2725–2734 Undas A, Brummel-Ziedins K, Mann KG (2014) Why does aspirin decrease the risk of venous thromboembolism? On old and novel antithrombotic effects of acetyl salicylic acid. J Thromb Haemost 12:1776–1787 Becattini C, Agnelli G (2014) Aspirin for prevention and treatment of venous thromboembolism. Blood Rev 28:103–108 Riedl J, Hell L, Kaider A, Koder S, Marosi C, Zielinski C, Panzer S, Pabinger I, Ay C (2015) Association of platelet activation markers with cancer-associated venous thromboembolism. Platelets 13:1–6 Simanek R, Vormittag R, Ay C, Alguel G, Dunkler D, Schwarzinger I, Steger G, Jaeger U, Zielinski C, Pabinger I (2010) High platelet count associated with venous thromboembolism in cancer patients: results from the Vienna Cancer and Thrombosis Study (CATS). J Thromb Haemost 8:114–120 Zakai NA, Wright J, Cushman M (2004) Risk factors for venous thrombosis in medical inpatients: validation of a thrombosis risk score. J Thromb Haemost 2:2156–2161 Kerlin BA, Haworth K, Smoyer WE (2014) Venous thromboembolism in pediatric nephrotic syndrome. Pediatr Nephrol 29:989–997 Tuckuviene R, Christensen AL, Helgestad J, Johnsen SP, Kristensen SR (2011) Pediatric venous and arterial noncerebral thromboembolism in Denmark: a nationwide population-based study. J Pediatr 159:663–669 Lindemann S, Tolley ND, Dixon DA, McIntyre TM, Prescott SM, Zimmerman GA, Weyrich AS (2001) Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol 154:485–490 Wagner DD, Burger PC (2003) Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol 23:2131–2137 Sowa JM, Crist SA, Ratliff TL, Elzey BD (2009) Platelet influence on T- and B-cell responses. Arch Immunol Ther Exp (Warsz) 57: 235–241 Peerschke EI, Yin W, Ghebrehiwet B (2008) Platelet mediated complement activation. Adv Exp Med Biol 632:81–91
72. 73. 74.
75.
76.
77. 78.
79.
80.
81.
82. 83. 84.
85. 86.
87. 88. 89.
90.
91.
Barnes JL (1997) Platelets in glomerular disease. Nephron 77:378– 393 Zoja C, Remuzzi G (1995) Role of platelets in progressive glomerular diseases. Pediatr Nephrol 9:495–502 Barnes JL (1989) Glomerular localization of platelet secretory proteins in mesangial proliferative lesions induced by habu snake venom. J Histochem Cytochem 37:1075–1082 Barnes J, Camussi G, Tetta C, Venkatachalam MA (1990) Glomerular localization of platelet cationic proteins after immune complex-induced platelet activation. Lab Invest 63:755–761 Rachidi S, Wallace K, Day TA, Alberg AJ, Li Z (2014) Lower circulating platelet count and antiplatelet therapy independently predict better outcomes in patients with head and neck squamous cell carcinoma. J Hematol Oncol 7:65 Scharf RE (2012) Drugs that affect platelet function. Semin Thromb Hemost 38:865–883 Ueda N, Kawaguchi S, Niinomi Y, Nonoda T, Matsumoto J, Ohnishi M, Yasaki T (1987) Effect of corticosteroids on coagulation factors in children with nephrotic syndrome. Pediatr Nephrol 1: 286–289 Moraes LA, Paul-Clark MJ, Rickman A, Flower RJ, Goulding NJ, Perretti M (2005) Ligand-specific glucocorticoid receptor activation in human platelets. Blood 106:4167–4175 Liverani E, Banerjee S, Roberts W, Naseem KM, Perretti M (2012) Prednisolone exerts exquisite inhibitory properties on platelet functions. Biochem Pharmacol 83:1364–1373 Sahin G, Akay OM, Bal C, Yalcin AU, Gulbas Z (2011) The effect of calcineurin inhibitors on endothelial and platelet function in renal transplant patients. Clin Nephrol 76:218–225 George JN, Shattil SJ (1991) The clinical importance of acquired abnormalities of platelet function. N Engl J Med 324:27–39 Escolar G, Diaz-Ricart M, Cases A (2005) Uremic platelet dysfunction: past and present. Curr Hematol Rep 4:359–367 Pincus KJ, Hynicka LM (2013) Prophylaxis of thromboembolic events in patients with nephrotic syndrome. Ann Pharmacother 47:725–734 Hodson E (2003) The management of idiopathic nephrotic syndrome in children. Paediatr Drugs 5:335–349 Patrono C, Pierucci A (1986) Renal effects of nonsteroidal antiinflammatory drugs in chronic glomerular disease. Am J Med 81: 71–83 Dame C, Sutor AH (2005) Primary and secondary thrombocytosis in childhood. Br J Haematol 129:165–177 Simic I, Tabatabaeifar M, Schaefer F (2013) Animal models of nephrotic syndrome. Pediatr Nephrol 28:2079–2088 Goubran F, Maklady F, Saad M, el Ashry M (1986) In vivo platelet activity and lipoprotein patterns in coronary artery disease. Wien Klin Wochenschr 98:209–211 Takagawa K, Nakajima M, Taira K, Sakagami Y, Ueda T, Akazawa H, Maruhashi Y, Shimoyama H, Takahashi Y, Fujimura Y, Kamitsuji H, Yoshioka A (2002) Shear stress-induced platelet aggregation in children with minimal change nephrotic syndrome. Nihon Jinzo Gakkai Shi 44:380–388 Zwaginga JJ, Koomans HA, Sixma JJ, Rabelink TJ (1994) Thrombus formation and platelet-vessel wall interaction in the nephrotic syndrome under flow conditions. J Clin Invest 93:204–211
Answers to multiple-choice questions 1B - 2C - 3B - 4C - 5C
