Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia

Experiments were conducted to investigate the effects of intravenous immunoglobulin (IVIG) in a rat model of immune thrombocytopenia (ITP). Rats were pretreated with 0 to 2 g/kg IVIG and then challenged with an antiplatelet antibody (7E3, 8 mg/kg). IVIG effects on 7E3-induced thrombocytopenia and on 7E3 pharmacokinetics were determined. IVIG pretreatment led to significant changes in the degree and time-course of 7E3-induced thrombocytopenia (P = .031). Nadir percent platelet counts were 121% to 279% greater in animals treated with IVIG (0.4-2 g/kg) than in animals receiving 7E3 alone. IVIG treatment also led to dose-dependent increases in 7E3 clearance (P < .001), with more than 2-fold increases in 7E3 clearance seen following the highest dose of IVIG. In vitro experiments showed that IVIG effects on platelet count are not likely due to anti-idiotypic inhibition of 7E3-platelet binding and that IVIG did not directly bind to 7E3. Consequently, IVIG-7E3 binding cannot explain the increase of 7E3 clearance following IVIG treatment. We propose that the observed increase in 7E3 clearance with IVIG therapy is due to saturation of the FcRn salvage receptor for IgG. The importance of the effect of IVIG on 7E3 clearance to the prevention of thrombocytopenia in these animals is unclear at present; nonetheless, these data provide experimental support for a new mechanism of IVIG action in ITP (ie, IVIG-mediated increases in antiplatelet antibody elimination). This model of ITP will be useful for further investigations of IVIG mechanism of action and for development of new therapies for ITP.

Download Full-text

Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia

Blood ◽

10.1182/blood.v100.6.2087 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2087-2093 ◽

Cited By ~ 91

Author(s):

Ryan J. Hansen ◽

Joseph P. Balthasar

Keyword(s):

Platelet Count ◽

Intravenous Immunoglobulin ◽

Rat Model ◽

Immune Thrombocytopenia ◽

Time Course ◽

Ivig Therapy ◽

Ivig Treatment ◽

Antiplatelet Antibody ◽

Dose Dependent

Abstract Experiments were conducted to investigate the effects of intravenous immunoglobulin (IVIG) in a rat model of immune thrombocytopenia (ITP). Rats were pretreated with 0 to 2 g/kg IVIG and then challenged with an antiplatelet antibody (7E3, 8 mg/kg). IVIG effects on 7E3-induced thrombocytopenia and on 7E3 pharmacokinetics were determined. IVIG pretreatment led to significant changes in the degree and time-course of 7E3-induced thrombocytopenia (P = .031). Nadir percent platelet counts were 121% to 279% greater in animals treated with IVIG (0.4-2 g/kg) than in animals receiving 7E3 alone. IVIG treatment also led to dose-dependent increases in 7E3 clearance (P < .001), with more than 2-fold increases in 7E3 clearance seen following the highest dose of IVIG. In vitro experiments showed that IVIG effects on platelet count are not likely due to anti-idiotypic inhibition of 7E3-platelet binding and that IVIG did not directly bind to 7E3. Consequently, IVIG-7E3 binding cannot explain the increase of 7E3 clearance following IVIG treatment. We propose that the observed increase in 7E3 clearance with IVIG therapy is due to saturation of the FcRn salvage receptor for IgG. The importance of the effect of IVIG on 7E3 clearance to the prevention of thrombocytopenia in these animals is unclear at present; nonetheless, these data provide experimental support for a new mechanism of IVIG action in ITP (ie, IVIG-mediated increases in antiplatelet antibody elimination). This model of ITP will be useful for further investigations of IVIG mechanism of action and for development of new therapies for ITP.

Download Full-text

Intravenous Immunoglobulin Mediates an Increase in Anti-Platelet Antibody Clearance via the FcRn Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613331 ◽

2002 ◽

Vol 88 (12) ◽

pp. 898-899 ◽

Cited By ~ 106

Author(s):

Ryan Hansen ◽

Joseph Balthasar

Keyword(s):

Animal Model ◽

Intravenous Immunoglobulin ◽

Immune Thrombocytopenia ◽

Ivig Therapy ◽

Ivig Treatment ◽

Data Support ◽

Antibody Elimination ◽

Presence And Absence ◽

Platelet Antibody

SummaryWe have recently shown that intravenous immunoglobulin (IVIG) therapy leads to an increased rate of anti-platelet antibody clearance in an animal model of immune thrombocytopenia. The present study was performed to confirm the importance of the FcRn receptor in mediating this effect of IVIG. The pharmacokinetics of an anti-platelet antibody, 7E3, were studied in mice lacking expression of FcRn and in control mice, both in the presence and absence of IVIG. IVIG increased the clearance of 7E3 in mice with functioning FcRn receptors, with an average clearance value of 14.4 ± 1.4 ml/d/kg in IVIG treated mice vs. 5.2 ± 0.3 ml/d/kg in controls (P <0.001). In mice lacking expression of FcRn, IVIG treatment did not increase 7E3 clearance (61.0 ± 3.6 ml/d/kg vs. 71.5 ± 4.0 ml/d/kg in controls). Thus, these data support the hypothesis that IVIG increases antibody elimination via saturation of FcRn.

Download Full-text

Immune Thrombocytopenia Mediated by Anti-GPIbα Antibodies May Occur Via an FcR-Independent Pathway: A Potential Explanation for Refractory Cases to IVIG Therapy.

Blood ◽

10.1182/blood.v106.11.217.217 ◽

2005 ◽

Vol 106 (11) ◽

pp. 217-217

Author(s):

Michelle L. Webster ◽

Ebrahim Sayeh ◽

Min Crow ◽

Pingguo Chen ◽

Bernhard Nieswandt ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Immune Thrombocytopenia ◽

Fc Receptors ◽

Ivig Therapy ◽

Ivig Treatment ◽

Independent Manner ◽

Platelet Surface ◽

Ivig Preparation ◽

Different Doses

Abstract Intravenous immunoglobulin G (IVIG) is used to treat immune thrombocytopenia (ITP). While benefit has been observed in many cases, for unknown reasons some patients are refractory to this therapy. ITP is characterized by platelet clearance mainly mediated by antibodies against platelet surface glycoproteins (GP) GPIIbIIIa and GPIbα. Approximately 70–80% patients have antibodies against GPIIbIIIa, 20–40% patients have antibodies against GPIbα complex, and some patients have antibodies to both. Phagocytotic macrophages activated via their Fc receptors (FcR) are thought to be responsible for the clearance of opsonized platelets. However, anti-GPIbα antibodies may be able to induce ITP in an FcR-independent manner. Since the prevailing view of the mechanism of action of IVIG is Fc receptor blockade, we hypothesized that IVIG may not be effective at preventing thrombocytopenia induced by anti-GPIbα antibodies. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against mouse GPIbα (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with different doses of IVIG (1, 2, and 4g/kg of mouse) failed to prevent ITP in all anti-GPIbα-treated mice, except for p0p4. These results were repeated with p0p3, p0p4, and p0p5 using 2g/kg IVIG in C57BL/6 mice, and using 2g/kg of a different IVIG preparation. It has been shown that F(ab)2’ fragments of the p0p antibodies can induce thrombocytopenia to the same extent as intact antibodies. To confirm that these anti-GPIbα antibodies act in an FcR-independent manner, we demonstrated that p0p3, p0p4, and p0p5 are able to induce thrombocytopenia in mice lacking activating Fc receptors (FcRγ chain −/−). Interestingly, IVIG was still able to prevent thrombocytopenia induced by p0p4 in these FcRγ chain −/− mice. We demonstrated in vitro that there are no anti-idiotype antibodies against any of the anti-GPIbα antibodies used present in IVIG. Thus, the amelioration of thrombocytopenia in p0p4-treated mice is not due to a neutralization of the antibody by IVIG. In contrast to the anti-GPIbα antibodies, anti-GPIIbIIIa monoclonal antibodies (JON1, JON2, and JON3) that induce thrombocytopenia were sensitive to IVIG treatment, which is consistent with earlier studies using other monoclonal antibodies against GPIIbIIIa. Also, JON2 was unable to induce thrombocytopenia in FcRγ chain −/− mice, confirming that this anti-GPIIbIIIa antibody is FcR-dependent. Our results suggest that ITP induced by anti-GPIbα antibodies may be mediated by FcR-independent mechanisms, many of which may be refractory to IVIG therapy. Thus, IVIG may not be as effective in treating patients with predominantly anti-GPIbα antibodies.

Download Full-text

Association between Platelet Autoantibody Specificity and Response to Intravenous Immunoglobulin G in the Treatment of Patients with Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v108.11.3987.3987 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3987-3987

Author(s):

Kaye L. Johnston ◽

Ronald S. Go

Keyword(s):

Platelet Count ◽

Intravenous Immunoglobulin ◽

Hodgkin Lymphoma ◽

Murine Model ◽

Immunoglobulin G ◽

Immune Thrombocytopenia ◽

Lymphocytic Leukemia ◽

Ivig Treatment ◽

Number Of Patients ◽

Intravenous Immunoglobulin G

Abstract Background: Recent studies in murine model of immune thrombocytopenia (ITP) suggest that autoantibodies against GPIba may cause thrombocytopenia through Fc-independent pathway and ITP mediated by anti-GPIb may be less responsive to intravenous immunoglobulin G (IVIG) treatment (Nieswandt B, et al. Blood 2000; Webster ML, et al. Blood 2006). The objective of this study was to investigate the potential association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) treatment in patients with ITP. Methods: We retrospectively reviewed the clinical history of ITP patients who had platelet autoantibody test and received IVIG for treatment of thrombocytopenia. ITP was diagnosed by usual clinical criteria. All platelet autoantibody tests were performed by the Blood Center of Wisconsin (Milwaukee, WI) using ELISA, which detects antibodies reactive with platelet GPIIb/IIIa, GPIb/IX, and GPIa/IIa. A response was defined as a platelet count of > 50 x 109/L with a minimum increment of 30 x 109/L. Results: We found 17 patients who received IVIG and had platelet autoantibody test performed. The median age was 58 years (range, 20–83) and 10 (59%) were males. ITP was considered idiopathic in 12 patients. In the remaining 5 patients, the following hematologic conditions were present: chronic lymphocytic leukemia (2), Hodgkin lymphoma (1), myelodysplastic syndrome (1), and non-Hodgkin lymphoma (1). Antibodies reactive with GPIIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. In 3 patients, none of these antibodies were detected. Among the 14 patients with antibodies, 9 (64%) had antibodies against more than 1 glycoprotein. The median pre-treatment platelet count was 9 x 109/L (range, 1–68). Overall, 10 (59%) patients responded to IVIG. A response occurred in 7 of 7 patients without anti-GPIb/IX but in only 3 of 10 patients with anti-GPIb/IX. The difference in the response rates between the 2 groups was statistically significant (P= .0098). Conclusions: Our clinical results support the murine model findings that ITP mediated by anti-GPIb may be less responsive to IVIG treatment. However, because of the small number of patients investigated and the retrospective nature of the study, our findings must be confirmed by other groups in a larger patient population.

Download Full-text

Dimethyl fumarate inhibits antibody-induced platelet destruction in immune thrombocytopenia mouse

Thrombosis Journal ◽

10.1186/s12959-021-00314-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Huan Tong ◽

Yangyang Ding ◽

Xiang Gui ◽

Zengtian Sun ◽

Guozhang Wang ◽

...

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Mononuclear Cells ◽

Dimethyl Fumarate ◽

Treg Cells ◽

Antiplatelet Antibody ◽

Platelet Destruction ◽

Immune Mediated ◽

Ifn Γ

Abstract Background Immune thrombocytopenia (ITP) is an autoimmune disease characterized as a low platelet count resulting from immune-mediated platelet destruction. Dimethyl fumarate (DMF) is widely applied for the treatment of several autoimmune diseases with immunosuppressive effect. However, whether it ameliorates ITP is unclear. This study aims to evaluate whether DMF has a preventive effect on ITP in mice. Methods DMF (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice followed by injection of rat anti-mouse integrin GPIIb/CD41antibody to induce ITP. Peripheral blood was isolated to measure platelet count and spleen mononuclear cells were extracted to measure Th1 and Treg cells along with detecting the levels of IFN-γ, and TGFβ-1 in plasma and CD68 expression in spleen by immuohistochemical staining. Additionally, macrophage cell line RAW264.7 was cultured and treated with DMF followed by analysis of cell apoptosis and cycle, and the expression of FcγRI, FcγRIIb and FcγRIV mRNA. Results DMF significantly inhibited antiplatelet antibody-induced platelet destruction, decreased Th1 cells and the expression of T-bet and IFN-γ, upregulated Treg cells and the expression of Foxp3 and TGF-β1 as well as reduced CD68 expression in the spleen of ITP mouse. DMF-treated RAW264.7 cells showed S-phase arrest, increased apoptosis and downregulated expression of FcγRI and FcγRIV. Meanwhile, in vitro treatment of DMF also decreased the expression of cyclin D1 and E2, reduced Bcl-2 level and increased Bax expression and caspase-3 activation. Conclusions In conclusion, DMF prevents antibody-mediated platelet destruction in ITP mice possibly through promoting apoptosis, indicating that it might be used as a new approach for the treatment of ITP.

Download Full-text

Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells

Biochemical Journal ◽

10.1042/bj2620449 ◽

1989 ◽

Vol 262 (2) ◽

pp. 449-456 ◽

Cited By ~ 20

Author(s):

C Hanekom ◽

A Nel ◽

C Gittinger ◽

A Rheeder ◽

G Landreth

Keyword(s):

T Cells ◽

Time Course ◽

Gel Filtration ◽

Maximal Activity ◽

Jurkat Cells ◽

Serine Kinase ◽

Transient Activation ◽

Normal Human ◽

Dose Dependent

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.

Download Full-text

VNTR2/VNTR3 genotype in the FCGRT gene is associated with reduced effectiveness of intravenous immunoglobulin in patients with myasthenia gravis

Therapeutic Advances in Neurological Disorders ◽

10.1177/1756286420986747 ◽

2021 ◽

Vol 14 ◽

pp. 175628642098674

Author(s):

Shengyao Su ◽

Qing Liu ◽

Xueping Zhang ◽

Xinmei Wen ◽

Lin Lei ◽

...

Keyword(s):

Myasthenia Gravis ◽

Intravenous Immunoglobulin ◽

Medical Records ◽

Daily Living ◽

Response Rate ◽

Predictive Marker ◽

Ivig Therapy ◽

Variable Number ◽

Ivig Treatment ◽

Therapeutic Outcomes

Background: Intravenous immunoglobulin (IVIG) has been commonly used to treat myasthenia gravis exacerbation, but is still ineffective in nearly 30% of patients. A variable number of tandem repeat (VNTR) polymorphism in the FCGRT gene has been found to reduce the efficiency of IgG biologics. However, whether the polymorphism influences the efficacy of IVIG in generalized myasthenia gravis (MG) patients with exacerbations remains unknown. Methods: The distribution of VNTR genotypes was analyzed in 334 patients with MG. Varied VNTR alleles were determined by capillary electrophoresis and confirmed by Sanger sequencing. Information of endogenous IgG levels were collected in patients without previous immunotherapy ( n = 26). Medical records of patients who received IVIG therapy were retrospectively analyzed for therapeutic outcomes of IVIG treatment ( n = 61). Patients whose Activities of Daily Living scores decreased by 2 or more points on day 14 were considered responders to the treatment. Results: The VNTR3/3 and VNTR2/3 genotypes were detected in 96.7% (323/334) and 3.4% (11/334) patients, respectively. Patients with VNTR2/3 heterozygosity had lower endogenous IgG levels than those with VNTR3/3 homozygosity (9.81 ± 2.61 g/L versus 12.41 ± 2.45g/L, p = 0.016). The response rate of IVIG therapy was 78.7% (48/61). All responders and nine non-responders were VNTR3/3 homozygotes, whereas all the patients with VNTR2/3 genotypes were non-responders ( n = 4). In patients who took IVIG treatments, endogenous IgG levels were significantly lower in non-responders compared with responders (12.93 ± 2.24 g/L versus 8.85 ± 2.69 g/L, p = 0.006), especially in VNTR2/3 heterozygotes (7.86 ± 1.78 g/L, p = 0.001). Conclusion: The VNTR2/3 genotype could influence endogenous IgG levels and serve as a predictive marker for poor responses to IVIG in MG patients.

Download Full-text

Effects of pinacidil on coronary Ca2+-myosin phosphorylation in cold potassium cardioplegia model

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.3.h882 ◽

2000 ◽

Vol 279 (3) ◽

pp. H882-H888 ◽

Cited By ~ 2

Author(s):

Naruto Matsuda ◽

Kathleen G. Morgan ◽

Frank W. Sellke

Keyword(s):

Time Course ◽

Dependent Manner ◽

Coronary Smooth Muscle ◽

Intracellular Ca ◽

No Signaling ◽

Synthase Inhibitor ◽

Mlc Phosphorylation ◽

Dose Dependent ◽

Cardioplegic Solutions

The effects of the potassium (K+) channel opener pinacidil (Pin) on the coronary smooth muscle Ca2+-myosin light chain (MLC) phosphorylation pathway under hypothermic K+cardioplegia were determined by use of an in vitro microvessel model. Rat coronary arterioles (100–260 μm in diameter) were subjected to 60 min of simulated hypothermic (20°C) K+cardioplegic solutions (K+= 25 mM). We first characterized the time course of changes in intracellular Ca2+concentration, MLC phosphorylation, and diameter and observed that the K+cardioplegia-related vasoconstriction was associated with an activation of the Ca2+-MLC phosphorylation pathway. Supplementation with Pin effectively suppressed the Ca2+accumulation and MLC phosphorylation in a dose-dependent manner and subsequently maintained a small decrease in vasomotor tone. The ATP-sensitive K+(KATP)-channel blocker glibenclamide, but not the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, significantly inhibited the effect of Pin. K+cardioplegia augments the coronary Ca2+-MLC pathway and results in vasoconstriction. Pin effectively prevents the activation of this pathway and maintains adequate vasorelaxation during K+cardioplegia through a KATP-channel mechanism not coupled with the endothelium-derived NO signaling cascade.

Download Full-text

The Effects Of Prostacyclin (PGI2) On Intravascular Platelet Aggregation

10.1055/s-0038-1653317 ◽

1981 ◽

Author(s):

G G Duncan ◽

G Mallarkey ◽

G M Smith

Keyword(s):

Platelet Count ◽

Guinea Pigs ◽

Adenosine Diphosphate ◽

Before And After ◽

Dependent Inhibition ◽

Induced Aggregation ◽

Dose Dependent ◽

Anaesthetised Rats

Intravascular aggregation can be measured by counting the number of circulating platelets before and after the injection of aggregation agents. The Technicon Autocounter was modified to count platelets continuously and connected via a double cannula in a carotid artery to an anaesthetised animal.Adenosine diphosphate (ADP) and collagen gave dose- dependent falls in the circulating platelet count when injected into rats, guinea pigs and rabbits. This enabled aggregation to be accurately quantitated in vivo.The infusion of PGI2 (0.25-1 ug/kg/min) in anaesthetised rats and rabbits produced a dose-dependent inhibition of the fall in platelet count produced by ADP and collagen. The formation of PGI2 can be inhibited in vitro by 15- hydroperoxyarachidonic acid (15HPAA). When 20 ug/kg/min of 15HPAA was infused into rats, aggregation produced by collagen was significantly increased suggesting that PGI2 is continuously formed by the rat vascular endothelium. This observation was confirmed by infusing 6-keto PGF1α antiserum. This antibody also prevented the inhibitory activity of PGI2 on collagen-induced aggregation. The study of continuous platelet counting in guinea pigs has been hampered by the occurrence of thrombocytopenia in certain animals. When 2 ug/kg/min of PGI2 was infused for 10 mins, a rise in the circulating platelet count to a steady plateau 4-5 × 105 platelets occurredThese experiments have shown that PGI2 will prevent aggregation by ADP and collagen and will reverse spontaneous thrombocytopenia and that PGI2 is continuously released from the vessels of anaesthetised rats.

Download Full-text