DOWN-REGULATED BETA2-GPI IS ASSOCIATED WITH A REDUCED ABILITY TO MITIGATE COMPLEMENT ACTIVATION BY INHIBITING JNK/BID-DEPENDENT PLATELET DESTRUCTION IN THE PATHOGENESIS OF IMMUNE THROMBOCYTOPAENIA
(Abstract release date: 05/21/15)
EHA Library. Zhang X. 06/13/15; 103105; S496
Disclosure(s): Peking University People’s HospitalPeking University Institute of Hematology
Prof. Xiaohui Zhang
Contributions
Contributions
Abstract
Abstract: S496
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 15:45 to 13.06.2015 16:00
Location: Room Stolz 1
Background
It is widely accepted that immune complex-mediated classical complement pathway activation represents an important mechanism of platelet destruction in immune thrombocytopaenia (ITP) (Peerschke at al. Br J Haematol. 2010). However, not all patients diagnosed with ITP have detectable antibodies, suggesting a potential mechanism of complement activation in the plasma of ITP patients. Moreover, the mechanisms involved in complement-mediated platelets lysis are elusive. β2-glycoprotein I (β2-GPI) is a plasma glycoprotein that functions as a complement regulator (Gropp et al. Blood. 2011); however, there is no information available on the role of β2-GPI in patients with ITP to date.
Aims
The aim of the present study was to assess the contribution of β?-GPI in complement activation and the details of its underlying mechanism.
Methods
Twenty-four consecutive patients with primary ITP and 24 healthy donors were enrolled in this prospective study. The levels of plasma β2-GPI and serum C1q, C4d, C3b and C5b-9 were determined by ELISA in ITP patients and healthy donors. The deposition of the fore-mentioned complement components on immobilized heterologous platelets was quantified. The levels of C3 convertases in plasma and the phosphorylation of MAPKs (phospho-ERK, phospho-JNK, phospho-p38MAPK) and Bid in C5b-9 mediated platelet lysis were determined using Western blot. To study the effect of β2-GPI on complement activation, β2-GPI was added to ITP plasma, C3 convertase activity was assayed via the determination of C3a generation, and serum levels of C5b-9 and its deposition and signalling proteins were re-evaluated.
Results
We first explored the significantly lower concentrations of β2-GPI in plasma from patients with ITP (80±21 ng/ml) compared with healthy donors (180±22 ng/ml) (p<0.05). Increased deposition of C5b-9 was observed, indicating the complement activation in ITP plasma. Evidence for enhanced classical complement pathway activation (C1q and/or C4d deposition) was noted in 45.8% of patient plasma samples, and alternative pathway activation (C3b) was observed in 37.5% of plasma samples. Additional evidence for enhanced complement activation was noted with increased serum levels of C3 convertases (C4b2b and C3bBb). β2-GPI plasma levels and C5b-9 serum levels were negatively correlated. The intensity of phospho-MAPKs bands revealed the over-expression of phospho-JNK, and detection of Bid exhibited similar results. SP600125 (JNK) and BI-6C9 (Bid) inhibitors significantly reduced complement-mediated JNK/Bid-dependent platelet lysis. Treatment of ITP plasma with β2-GPI in vitro resulted in a dose-dependent reduction of serum C5b-9 generation and platelet deposition as well as reduced phospho-JNK and Bid. Moreover, β2-GPI inhibited C3a generation and formed complexes with C3 in plasma.
Summary
This original observation highlights the role of β2-GPI in ITP in harbouring complement activation at the level of the C3 convertase and inhibiting JNK/Bid-dependent platelet destruction. The first mention of decreased β2-GPI in ITP is suggestive of abnormal complement activation. Our data provide important insights into immune dys-regulation via decreased plasma β2-GPI, which may be exploited in the pathogenesis of ITP.
Keyword(s): Complement, Immune thrombocytopenia (ITP), JNK
Session topic: Platelet and bleeding disorders
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 15:45 to 13.06.2015 16:00
Location: Room Stolz 1
Background
It is widely accepted that immune complex-mediated classical complement pathway activation represents an important mechanism of platelet destruction in immune thrombocytopaenia (ITP) (Peerschke at al. Br J Haematol. 2010). However, not all patients diagnosed with ITP have detectable antibodies, suggesting a potential mechanism of complement activation in the plasma of ITP patients. Moreover, the mechanisms involved in complement-mediated platelets lysis are elusive. β2-glycoprotein I (β2-GPI) is a plasma glycoprotein that functions as a complement regulator (Gropp et al. Blood. 2011); however, there is no information available on the role of β2-GPI in patients with ITP to date.
Aims
The aim of the present study was to assess the contribution of β?-GPI in complement activation and the details of its underlying mechanism.
Methods
Twenty-four consecutive patients with primary ITP and 24 healthy donors were enrolled in this prospective study. The levels of plasma β2-GPI and serum C1q, C4d, C3b and C5b-9 were determined by ELISA in ITP patients and healthy donors. The deposition of the fore-mentioned complement components on immobilized heterologous platelets was quantified. The levels of C3 convertases in plasma and the phosphorylation of MAPKs (phospho-ERK, phospho-JNK, phospho-p38MAPK) and Bid in C5b-9 mediated platelet lysis were determined using Western blot. To study the effect of β2-GPI on complement activation, β2-GPI was added to ITP plasma, C3 convertase activity was assayed via the determination of C3a generation, and serum levels of C5b-9 and its deposition and signalling proteins were re-evaluated.
Results
We first explored the significantly lower concentrations of β2-GPI in plasma from patients with ITP (80±21 ng/ml) compared with healthy donors (180±22 ng/ml) (p<0.05). Increased deposition of C5b-9 was observed, indicating the complement activation in ITP plasma. Evidence for enhanced classical complement pathway activation (C1q and/or C4d deposition) was noted in 45.8% of patient plasma samples, and alternative pathway activation (C3b) was observed in 37.5% of plasma samples. Additional evidence for enhanced complement activation was noted with increased serum levels of C3 convertases (C4b2b and C3bBb). β2-GPI plasma levels and C5b-9 serum levels were negatively correlated. The intensity of phospho-MAPKs bands revealed the over-expression of phospho-JNK, and detection of Bid exhibited similar results. SP600125 (JNK) and BI-6C9 (Bid) inhibitors significantly reduced complement-mediated JNK/Bid-dependent platelet lysis. Treatment of ITP plasma with β2-GPI in vitro resulted in a dose-dependent reduction of serum C5b-9 generation and platelet deposition as well as reduced phospho-JNK and Bid. Moreover, β2-GPI inhibited C3a generation and formed complexes with C3 in plasma.
Summary
This original observation highlights the role of β2-GPI in ITP in harbouring complement activation at the level of the C3 convertase and inhibiting JNK/Bid-dependent platelet destruction. The first mention of decreased β2-GPI in ITP is suggestive of abnormal complement activation. Our data provide important insights into immune dys-regulation via decreased plasma β2-GPI, which may be exploited in the pathogenesis of ITP.
Keyword(s): Complement, Immune thrombocytopenia (ITP), JNK
Session topic: Platelet and bleeding disorders
Abstract: S496
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 15:45 to 13.06.2015 16:00
Location: Room Stolz 1
Background
It is widely accepted that immune complex-mediated classical complement pathway activation represents an important mechanism of platelet destruction in immune thrombocytopaenia (ITP) (Peerschke at al. Br J Haematol. 2010). However, not all patients diagnosed with ITP have detectable antibodies, suggesting a potential mechanism of complement activation in the plasma of ITP patients. Moreover, the mechanisms involved in complement-mediated platelets lysis are elusive. β2-glycoprotein I (β2-GPI) is a plasma glycoprotein that functions as a complement regulator (Gropp et al. Blood. 2011); however, there is no information available on the role of β2-GPI in patients with ITP to date.
Aims
The aim of the present study was to assess the contribution of β?-GPI in complement activation and the details of its underlying mechanism.
Methods
Twenty-four consecutive patients with primary ITP and 24 healthy donors were enrolled in this prospective study. The levels of plasma β2-GPI and serum C1q, C4d, C3b and C5b-9 were determined by ELISA in ITP patients and healthy donors. The deposition of the fore-mentioned complement components on immobilized heterologous platelets was quantified. The levels of C3 convertases in plasma and the phosphorylation of MAPKs (phospho-ERK, phospho-JNK, phospho-p38MAPK) and Bid in C5b-9 mediated platelet lysis were determined using Western blot. To study the effect of β2-GPI on complement activation, β2-GPI was added to ITP plasma, C3 convertase activity was assayed via the determination of C3a generation, and serum levels of C5b-9 and its deposition and signalling proteins were re-evaluated.
Results
We first explored the significantly lower concentrations of β2-GPI in plasma from patients with ITP (80±21 ng/ml) compared with healthy donors (180±22 ng/ml) (p<0.05). Increased deposition of C5b-9 was observed, indicating the complement activation in ITP plasma. Evidence for enhanced classical complement pathway activation (C1q and/or C4d deposition) was noted in 45.8% of patient plasma samples, and alternative pathway activation (C3b) was observed in 37.5% of plasma samples. Additional evidence for enhanced complement activation was noted with increased serum levels of C3 convertases (C4b2b and C3bBb). β2-GPI plasma levels and C5b-9 serum levels were negatively correlated. The intensity of phospho-MAPKs bands revealed the over-expression of phospho-JNK, and detection of Bid exhibited similar results. SP600125 (JNK) and BI-6C9 (Bid) inhibitors significantly reduced complement-mediated JNK/Bid-dependent platelet lysis. Treatment of ITP plasma with β2-GPI in vitro resulted in a dose-dependent reduction of serum C5b-9 generation and platelet deposition as well as reduced phospho-JNK and Bid. Moreover, β2-GPI inhibited C3a generation and formed complexes with C3 in plasma.
Summary
This original observation highlights the role of β2-GPI in ITP in harbouring complement activation at the level of the C3 convertase and inhibiting JNK/Bid-dependent platelet destruction. The first mention of decreased β2-GPI in ITP is suggestive of abnormal complement activation. Our data provide important insights into immune dys-regulation via decreased plasma β2-GPI, which may be exploited in the pathogenesis of ITP.
Keyword(s): Complement, Immune thrombocytopenia (ITP), JNK
Session topic: Platelet and bleeding disorders
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 15:45 to 13.06.2015 16:00
Location: Room Stolz 1
Background
It is widely accepted that immune complex-mediated classical complement pathway activation represents an important mechanism of platelet destruction in immune thrombocytopaenia (ITP) (Peerschke at al. Br J Haematol. 2010). However, not all patients diagnosed with ITP have detectable antibodies, suggesting a potential mechanism of complement activation in the plasma of ITP patients. Moreover, the mechanisms involved in complement-mediated platelets lysis are elusive. β2-glycoprotein I (β2-GPI) is a plasma glycoprotein that functions as a complement regulator (Gropp et al. Blood. 2011); however, there is no information available on the role of β2-GPI in patients with ITP to date.
Aims
The aim of the present study was to assess the contribution of β?-GPI in complement activation and the details of its underlying mechanism.
Methods
Twenty-four consecutive patients with primary ITP and 24 healthy donors were enrolled in this prospective study. The levels of plasma β2-GPI and serum C1q, C4d, C3b and C5b-9 were determined by ELISA in ITP patients and healthy donors. The deposition of the fore-mentioned complement components on immobilized heterologous platelets was quantified. The levels of C3 convertases in plasma and the phosphorylation of MAPKs (phospho-ERK, phospho-JNK, phospho-p38MAPK) and Bid in C5b-9 mediated platelet lysis were determined using Western blot. To study the effect of β2-GPI on complement activation, β2-GPI was added to ITP plasma, C3 convertase activity was assayed via the determination of C3a generation, and serum levels of C5b-9 and its deposition and signalling proteins were re-evaluated.
Results
We first explored the significantly lower concentrations of β2-GPI in plasma from patients with ITP (80±21 ng/ml) compared with healthy donors (180±22 ng/ml) (p<0.05). Increased deposition of C5b-9 was observed, indicating the complement activation in ITP plasma. Evidence for enhanced classical complement pathway activation (C1q and/or C4d deposition) was noted in 45.8% of patient plasma samples, and alternative pathway activation (C3b) was observed in 37.5% of plasma samples. Additional evidence for enhanced complement activation was noted with increased serum levels of C3 convertases (C4b2b and C3bBb). β2-GPI plasma levels and C5b-9 serum levels were negatively correlated. The intensity of phospho-MAPKs bands revealed the over-expression of phospho-JNK, and detection of Bid exhibited similar results. SP600125 (JNK) and BI-6C9 (Bid) inhibitors significantly reduced complement-mediated JNK/Bid-dependent platelet lysis. Treatment of ITP plasma with β2-GPI in vitro resulted in a dose-dependent reduction of serum C5b-9 generation and platelet deposition as well as reduced phospho-JNK and Bid. Moreover, β2-GPI inhibited C3a generation and formed complexes with C3 in plasma.
Summary
This original observation highlights the role of β2-GPI in ITP in harbouring complement activation at the level of the C3 convertase and inhibiting JNK/Bid-dependent platelet destruction. The first mention of decreased β2-GPI in ITP is suggestive of abnormal complement activation. Our data provide important insights into immune dys-regulation via decreased plasma β2-GPI, which may be exploited in the pathogenesis of ITP.
Keyword(s): Complement, Immune thrombocytopenia (ITP), JNK
Session topic: Platelet and bleeding disorders
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