Novel Murine Models Of Fetal and Neonatal Alloimmune Thrombocytopenia Established In αIIb Deficient and Human αIIb Transgenic Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2314-2314
Author(s):  
Brian Vadasz ◽  
Pingguo Chen ◽  
Yougbare Issaka ◽  
Guangheng Zhu ◽  
Frampton Jonathan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Transgenic Mice ◽  
Conflicts Of Interest ◽  
Fetal Loss ◽  
Severe Bleeding ◽  
Severe Thrombocytopenia ◽  
Maternal Circulation ◽  
Common Cause ◽  
Knockout Mouse Model ◽  
Alloimmune Thrombocytopenia

Abstract Background Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder that often results in intracranial hemorrhage (ICH), leading to neurological impairments or death. FNAIT has also been linked with miscarriage although the incidence of fetal loss has not been adequately studied. It is the most common cause of severe thrombocytopenia in live born neonates and accounts for up to 35% of all neonates admitted into the intensive care unit. In FNAIT, maternal alloantibodies cross the placenta and target paternally-derived fetal platelet antigens, most commonly platelet GPIIbIIIa (αIIbβ3 integrin). Polymorphisms in human platelet antigen-1a (HPA-1a), located on the integrin β3 subunit are the most common cause of FNAIT and are most widely studied, however the reported incidence of anti-αIIb-mediated FNAIT has recently increased; but the pathogenesis of this disease is unclear. Methods and Results To establish mouse models of anti-αIIb antibody mediated FNAIT, αIIb deficient (αIIb-/-) and human αIIb transgenic mice were employed in this study. We first immunized female αIIb-/- mice by weekly transfusions of wild type (WT) mouse platelets 2 times or 4 times. We demonstrated that the αIIb-/- mice were immunoresponsive to the αIIb antigen and both IgG1 and IgG2 antibodies against αIIb (i.e.TH2-like and TH1-like immune response, respectively) were detected two weeks after platelet transfusion. To test whether αIIb antisera can cause platelet destruction, we injected the antisera into WT mice and found severe thrombocytopenia in the recipient mice (P<0.001). Subsequently, immunized female αIIb-/- mice were bred with WT males to generate heterozygous fetuses and to recapitulate the features of FNAIT. Fetuses and neonates generated from naïve αIIb-/- females bred with WT males were used as a control. We found the severity of symptoms was correlated with the level of antibody titers in the maternal circulation. In 2 times and 4 times immunized females, miscarriage was observed (n= 2/9; and n =3/3, respectively). Upon post mortem dissection of miscarrying mothers at embryonic day 15.5, fetuses possessed ICH in 2 times and 4 times immunized females (n=2/14; and 5/12, respectively). Live born neonates from 2 times immunized mothers showed severe thrombocytopenia (P<0.001), purpura (n=9/22) and ICH (n=4/22). In addition, neonate body weight decreased compared to naïve controls (P<0.05), suggesting intrauterine growth restriction. There were no live pups delivered from 4 times immunized mothers. We also tested the effect of antisera from 2 times immunized αIIb-/- mice on platelet function and found the antisera enhanced ADP-induced platelet aggregation. This is different from the anti-β3 sera that inhibited platelet aggregation in our previous FNAIT model. Whether the different effects on platelets may lead to different pathology in FNAIT is under investigation in the laboratory. In addition to the knockout mouse model, we also used human αIIb transgenic mice, which do not express mouse αIIb on their platelets, in order to generate an alloimmune model of FNAIT. This model may better mimic the conditions of human FNAIT caused by the polymorphisms in αIIb genes. Following the same protocol as we used in the αIIb-/- mice, we found anti-murine αIIb antibodies were induced although the antibody titer was slightly lower. After breeding with male WT mice, immunized female human αIIb transgenic mice deliver pups with thrombocytopenia and ICH. Miscarriage was also observed, however the severity and symptoms of the disease were less. Furthermore, we also established another model of mouse-anti-human FNAIT by breeding humanized αIIb male mice with immunized female WT mice. We will compare the differences in these iso- and alloimmune models to elucidate the pathogenesis of αIIb mediated FNAIT. Conclusions and Significance We established the first anti-αIIb model of FNAIT and the first alloimmune animal models of FNAIT. Our results indicate that these models recapitulate human disease symptoms such as; lower platelet counts, bleeding diasthesis, and decreased birth weight compared to controls. Both neonates and fetuses possess ICH, and some immunized mothers underwent miscarriage. These models give us the opportunity to compare iso- vs. alloimmune FNAIT, and to compare the pathology between anti- αIIb and our anti-β3 mediated FNAIT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An Authentic Murine Model of Fetal/Neonatal Alloimmune Thrombocytopenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 97-97
Author(s):  
Huiying Zhi ◽  
Maria Therese Ahlen ◽  
Trude Rasmussen ◽  
Bjørn Skogen ◽  
Peter J. Newman
Keyword(s):  
Amino Acid ◽  
Transgenic Mice ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Severe Thrombocytopenia ◽  
Wild Type ◽  
Amino Acid Polymorphism ◽  
Female Mice ◽  
Wild Type Female ◽  
Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally-inherited antigens present on the surface of fetal platelets. The human platelet alloantigen, HPA-1a (also known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the ~50 amino acid plexin/semaphorin/integrin (PSI) domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein (GP)IIIa), is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa. Neither human polyclonal nor mouse monoclonal anti-HPA-1a antibodies, however, recognize murine GPIIIa due to amino acid differences both within and surrounding the HPA-1a alloepitope. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically important platelet alloantigen. We have recently shown that humanizing residues 30, 32, 33, and 39 within the PSI domain, as well as amino acid 470 within the linearly distant but conformationally close Epidermal Growth Factor (EGF) 1 domain, of murine GPIIIa, is sufficient to re-create the epitope recognized by most maternal anti-HPA-1a alloantibodies. We therefore used CRISPR/Cas9 gene editing technology to generate transgenic mice whose platelets express human residues A30P32L33D39Q470 on a murine GPIIIa backbone - hereafter referred to as APLDQ mice. Intraperitoneal injection of an anti-HPA-1a mAb induced severe thrombocytopenia in these APLDQ, but not wild-type, mice. Furthermore, platelets from APLDQ mice, when introduced into wild-type mice, elicited a strong polyclonal immune response that was specific for, and importantly restricted to, the epitopes created by these humanized residues, demonstrating that the APLDQ humanized form of murine GPIIIa is immunogenic in mice. Wild-type female mice pre-immunized with APLDQ platelets, when bred with APLDQ male mice, gave birth to severely thrombocytopenic pups, many of whom exhibited an accompanying bleeding phenotype. Notably, administration of intravenous immunoglobulin G (IVIG) into pregnant female mice at days 10 and 17 lowered the concentration of anti-APLDQ alloantibodies in both the maternal and fetal circulation, and importantly normalized the platelet count in the pups. Taken together, these data establish a novel murine model of FNAIT that recapitulates many of the clinically important features of FNAIT. Availability of APLDQ humanized transgenic mice should pave the way for the pre-clinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans. Disclosures No relevant conflicts of interest to declare.

scholarly journals Management of alloimmune thrombocytopenia: antenatal diagnosis and in utero transfusion of maternal platelets

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 340-343
Author(s):  
C Kaplan ◽  
F Daffos ◽  
F Forestier ◽  
WL Cox ◽  
D Lyon-Caen ◽  
...  
Keyword(s):  
Antenatal Diagnosis ◽  
In Utero ◽  
Severe Bleeding ◽  
Severe Thrombocytopenia ◽  
Fetal Blood ◽  
Alternative Approach ◽  
Neurologic Sequelae ◽  
The Central Nervous System ◽  
Circulating Antibodies ◽  
Alloimmune Thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) can cause severe bleeding in the central nervous system (CNS) and death or severe neurologic sequelae. The expression of the PLA1 antigen is detectable as early as 19 weeks of gestation. Alloimmunization can therefore lead to fetal thrombocytopenia very early in pregnancy. Until recently, we have had no means of detecting and assessing the severity of fetal thrombocytopenia during pregnancy. The level of the maternal antibody is not of a predictable value since 20% of the mothers had no circulating antibodies in our series. An alternative approach is to carry out investigations on fetal blood samplings. This management leads to an exact knowledge of the fetal status and antenatal diagnosis is feasible as early as the 21st week of gestation. Early diagnosis facilitates appropriate management and makes possible such therapeutic options as in utero maternal platelet transfusions. We report our experience in the antenatal diagnosis and management of nine cases with in utero transfusion in the six cases with severe thrombocytopenia. All neonates did well, with no signs of bleeding at birth. No side effects of therapy were noted after a period ranging from 6 months to 3 years.

HLADRB3*0101 Combined with DRB4*0101 Are Potential Predictor of Neonatal Alloimmune Thrombocytopenia (NAIT) Development

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2221-2221
Author(s):  
Ophira Salomon ◽  
Ron Loewenthal ◽  
Rivka Kalt ◽  
Nurit Rosenberg ◽  
Rima Dardik ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Conflicts Of Interest ◽  
Medical Center ◽  
Control Group ◽  
Study Group ◽  
Severe Thrombocytopenia ◽  
Hla Antibodies ◽  
Hla Dr ◽  
Alloimmune Thrombocytopenia ◽  
Cross Match

Abstract Abstract 2221 Neonatal alloimmune thrombocytopenia (NAIT) is a life-threatening bleeding disorder in the fetus/ neonate caused by maternal alloantibodies directed against fetal platelet antigens inherited from the father. Of the 21 allonatigens described up to date http://www.ebi.ac.uk/ipd/hpa/table2.html, the human platelet antigen (HPA) −1 defined as a leucine /proline polymorphism at residue 33 in b3 integrin is the immune-dominant antigen leading to severe NAIT. Immunization to HPA-1a occurs only in 10% of HPA-1bb pregnant women, 90% of them carrying histocompatibility leukocyte antigen (HLA) DRB3*0101. The aims of the present study were: 1. to prospectively study whether in addition to HLA DR B3*0101 there other potential HLA DR antigens involved in the immune process. 2. To analyse whether the response to IVIgG treatment correlates with maternal HLA DR genotype 3. to determine whether, anti HLA antibodies in addition to HPA1a antibodies, resulted in a more severe thrombocytopenia. We enrolled 21 pregnant women who had previously given birth to newborns with NAIT due to HPA1 incompatibility and had antibodies directed to their spouses HPA1 phenotype (leucine or proline polymorphism ) at the Sheba Medical Center from May 2001 to January 2011. The control group consisted of 21 women who delivered at term newborns with thrombocytopenia and a diagnosis of NAIT was ruled out. Twelve women of 21 (57%) in the study group carried both HLADRB3*0101 and DRB4*0101phenotype. But in none of the women in the control group such combination was detected. Interestingly is the observation that of 20 women who gave birth once more following IVIgG treatment, four did not respond to IVIgG and 3 (75%) of them harbor both DRB3*0101 and DRB4*0101. When lymphocytotoxic cross match analysis was performed in eighteen couples of the study group seven (39%) of them had positive lymphocytotoxic cross match. However the presence of both HLA and HPA1 antibodies against their partner phenotype did not affect platelets' counts in the newborn. Taken together we postulate that the presence of both HLADRB3*0101 and HLADRB4*0101 in the mother increases the risk of NAIT development when HPA1 incompatibility between the couples is found and can serve as a diagnostic tool. The presence of HLA antibodies against the spouse did not alter response to IgG treatment. Disclosures: No relevant conflicts of interest to declare.

Fetal and Neonatal Alloimmune Thrombocytopenia: Lessons Learned from Animal Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-50-SCI-50
Author(s):  
Heyu Ni
Keyword(s):  
Mechanism Of Action ◽  
Maternal Antibodies ◽  
Fetal Mortality ◽  
Severe Bleeding ◽  
Severe Thrombocytopenia ◽  
Bleeding Tendency ◽  
Murine Models ◽  
Β3 Integrin ◽  
Alloimmune Thrombocytopenia ◽  
Deficient Mice

Abstract Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe alloimmune disorder that results from fetal/neonatal platelet opsonization by maternal antibodies, which cross the placenta and result in fetal/neonatal platelet destruction. The frequency of FNAIT has been estimated at 0.5-1.5/1,000 liveborn neonates. However, this number does not include fetuses that die from this disease, since the incidence of FNAIT miscarriage has not been adequately studied. Analogous to autoimmune thrombocytopenia (ITP), the major target antigens in FNAIT are the platelet GPIIIa (β3 integrin) and GPIbα. However, severe bleeding is much more frequent in FNAIT, particularly the occurrence of intracranial hemorrhage (ICH). It is not known why the reported incidence of FNAIT mediated by anti-GPIbα antibodies is at a much lower frequency when compared to ITP, and whether the severe bleeding tendency in FNAIT is due to β3 integrin expression on angiogenic vessels in the developing fetus, which are targeted by cross-reacting maternal anti-β3 integrin antibodies. To study the pathogenesis and to develop new strategies for prevention and treatment, we have established murine models of FNAIT using β3 integrin and GPIbα deficient (-/-) mice. We first transfused these deficient mice with wild-type (WT) platelets to induce anti-β3 or anti-GPIbα antibody responses; we then bred these immunized female mice with WT males, causing FNAIT in the offspring. We found that maternal antiplatelet antibody titer correlated with the severity of FNAIT. These two murine models have revealed fundamental differences between the pathogenesis of anti-β3 and anti-GPIbα-mediated FNAIT. In anti-β3-mediated FNAIT, we found severe thrombocytopenia, ICH, and miscarriage. We also found the impairment of angiogenesis, which may contribute to ICH and intrauterine growth retardation. In contrast, the anti-GPIbα-mediated model revealed a nonclassical form of FNAIT (e.g., miscarriage but not bleeding disorders in neonates). We found that anti-GPIbα antibodies caused thrombosis in the placentas and miscarriage in most pregnant mice, which may partially explain the rarity of anti-GPIbα-mediated FNAIT reported in humans. Despite these substantial differences, there are also similarities between anti-β3 and anti-GPIbα-mediated FNAIT, such as the role of the neonatal Fc receptor (FcRn). FcRn is important for serum IgG homeostasis and for IgG transportation across the placenta. Using FcRn-/- mice, we demonstrated that fetal (but not maternal) FcRn is required to transport maternal antibodies to the fetal circulation and is indispensable for FNAIT. Blocking FcRn with an anti-FcRn antibody markedly reduced the severity of both anti-β3 and anti-GPIbα-mediated FNAIT. This important finding may lead to the development of new therapies (e.g., anti-FcRn antibody) against FNAIT and other maternal pathogenic antibody-mediated fetal/neonatal diseases. These models of FNAIT have allowed us to investigate the efficacy and mechanism of action of several therapies, including the aforementioned anti-FcRn antibody as well as antibody-mediated immune suppression (AMIS) and intravenous IgG (IVIG). In β3-/- mice, prophylactic administration of anti-HPA-1a antibody or murine β3 antisera induced AMIS against human HPA-1a-positive or murine WT platelets, respectively. Importantly, AMIS induced by β3 antisera suppressed the antibody response, thrombocytopenia, and miscarriage in FNAIT mice. These findings support the hypothesis that anti-HPA-1a antibody administration to HPA-1a-negative women after delivery of an HPA-1a-positive child may prevent FNAIT in subsequent pregnancies. The efficacy of IVIG is inconsistent, sources are limited, and the mechanism of action is not fully understood. We demonstrated that IVIG ameliorated both anti-β3 and anti-GPIbα-mediated FNAIT. IVIG decreased antiplatelet antibodies in both maternal and neonatal circulations, fetal platelet clearance, bleeding, and fetal mortality. In summary, we have uncovered fundamental differences in the pathogenesis of anti-β3 and anti-GPIbα-mediated FNAIT and have greatly enhanced our understanding of emerging and existing therapies. We will continue to investigate the pathogenesis of FNAIT so that we may develop more tailored and accessible therapeutic strategies for patients. Disclosures: No relevant conflicts of interest to declare.

Platelet-Derived ERp57 Mediates Platelet Incorporation Into a Growing Thrombus By Regulation Of The αIIbβ3 Integrin

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3505-3505
Author(s):  
Lu Wang ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Syed S. Ahmad ◽  
Bulent Mutus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Direct Interaction ◽  
Time Frame ◽  
Human Platelets ◽  
Platelet Surface ◽  
Knockout Mouse Model ◽  
Thrombosis Model ◽  
Normal Platelet ◽  
Αiibβ3 Integrin

Abstract ERp57 is a member of protein disulfide isomerase (PDI) family. Although previous studies using antibodies have indicated ERp57 mediates platelet aggregation, the specific role of platelet-derived ERp57 in hemostasis and thrombosis is unknown. In this study we addressed this issue using a megakaryocyte/platelet specific ERp57-knockout mouse model (PF4-Cre/ERp57fl/fl). Although PF4-Cre/ERp57fl/fl mice had normal platelet counts and platelet glycoproteins expression, they had significantly prolonged tail-bleeding times, and thrombus occlusion times with FeCl3-induced carotid artery-injury. Using a mesenteric artery thrombosis model we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Upon stimulation with convulxin and thrombin, platelets lacking ERp57 had defective activation of the αIIbβ3 integrin and platelet aggregation. Addition of exogenous wild type ERp57 corrected the defect in aggregation implicating surface ERp57 in platelet aggregation. Moreover, using recombinant ERp57 mutants we found that the second active site of ERp57 targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was substantially decreased, suggesting a direct interaction of ERp57 with αIIbβ3. Surface ERp57 activity in human platelets increased in response to stimulation with thrombin, concomitant with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus. Disclosures: No relevant conflicts of interest to declare.

Non-Invasive Management of Alloimmune Thrombocytopenia: Prediction of the Fetal Status.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3690-3690
Author(s):  
Gerald Bertrand ◽  
Moustapha Drame ◽  
Corinne Martageix ◽  
Cecile Kaplan-Gouet
Keyword(s):  
Platelet Count ◽  
Predictive Value ◽  
Conflicts Of Interest ◽  
Antibody Concentration ◽  
P Value ◽  
Severe Thrombocytopenia ◽  
Non Invasive ◽  
Predictive Parameters ◽  
Therapy Effectiveness ◽  
Alloimmune Thrombocytopenia

Abstract Abstract 3690 Fetal/neonatal alloimmune thrombocytopenia (F/NAIT) is the most common cause of severe thrombocytopenia in the fetus and the newborn in maternity wards. To counter the bleeding consequences of severe fetal thrombocytopenia, antenatal therapies have been implemented for subsequent pregnancies with incompatible fetuses. Predictive parameters for fetal severe thrombocytopenia are important for the development of non-invasive strategy and tailored intervention. We report here data concerning 67 HPA-1bb women with 81 managed pregnancies with IVIG. In 51% of the cases, the diagnosis of F/NAIT was made during the first gestation, following an intracranial haemorrhage (ICH) in 8 cases (12%). Severe thrombocytopenia was recorded for 88% of the newborn. Analysis of the index cases did not show any correlation between the severity of the disease and the maternal genetic background (ABO group and HLA-DRB3 allele). Subsequent pregnancies were managed and therapy effectiveness was evaluated. The highest mean newborn plt count was observed for a combination of intravenous immunoglobulin (IVIG) and corticoids (135.109/L; 54 newborn), in comparison with IVIG alone (89.109/L; 27 newborn). No ICH was recorded in these 2 groups. The maternal anti HPA-1a antibody concentration measured before any treatment and before 28 weeks of gestation was predictive of the fetal status: a high antibody concentration (≥28 International Units/mL) was correlated with a severe thrombocytopenia of the fetus (p=0.0016). Follow-up of the antibody concentrations during 34 pregnancies with antenatal management allowed demonstrating for the first time that the areas under curves (AUC) weighted by the weeks of gestation were a predictive parameter of therapy failure. The weighted AUC was significantly higher for women who delivered severely thrombocytopenic newborn than newborn with platelet count above 50.109/L (p=0.0107). To conclude, this large retrospective survey gives new insights on maternal predictive parameters for fetal status and therapy effectiveness allowing non-invasive strategies. Table 1: Maternal anti HPA-1a alloantibody concentrations and fetal platelet counts: statistical analysis. Predictive parameter Platelet count (×109/L) p-value Se (%) Sp (%) PPV (%) NPV (%) <50 ≥50 Number of mothers with antibody concentration (<28wg) <28 IU/mL 3 Fetuses 10 0.0016* 81.2 91.7 92.3 79.9 ≥28 IU/mL 13 2 Number of pregnancies with weighted AUC <24 IU/mL/wg 5 Newborn 18 0.0153* 64.3 82.6 69.2 79.2 ≥24 IU/mL/wg 9 5 * P<0.05. AUC: area under curve; IU: international units; Se: sensitivity, Sp: specificity; PPV: positive-predictive value; NPV: negative-predictive value. Wg: weeks of gestation. Disclosures: No relevant conflicts of interest to declare.

β2-Glycoprotein 1 Antibodies Directly Activate the Platelet IgG Receptor, FcγRIIa, and Cause Thrombosis in FCGR2A Transgenic but Not in Wild Type Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 106-106
Author(s):  
Ali Amirkhosravi ◽  
Todd V Meyer ◽  
Liza Robles-Carrillo ◽  
Meghan Hatfield ◽  
Hina Desai ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Conflicts Of Interest ◽  
Degradation Products ◽  
Higher Order ◽  
Size Exclusion ◽  
Size Analysis ◽  
Severe Thrombocytopenia ◽  
Wild Type ◽  
Increased Risk ◽  
Induced Aggregation

Abstract Abstract 106 Antibodies targeting β2-glycoprotein 1 (β2-GP1; β2-Abs) are of primary importance in antiphospholipid syndrome (APS), a thrombotic autoimmune disorder. The predominance of the IgG antibody isotype in APS is conspicuously associated with increased risk of thrombosis, raising the question whether the platelet IgG receptor, FcγRIIa, may play a role in thrombosis in APS, as is the case in heparin-induced thrombocytopenia (HIT). The hypothesis that platelet FcγRIIa may contribute to thrombosis in APS has received little attention, with research emphasis instead placed on several proposed alternative mechanisms of action. We have shown that, like HIT antibodies, antibodies targeting VEGF or CD154 are also potently thrombotic in mice transgenic for human FcγRIIa but have no activity in mice lacking FcγRIIa (i.e., wild type mice). We therefore asked whether antiphospholipid antibodies can activate platelets and cause thrombosis via FcγRIIa. To this end, we tested mouse monoclonal (mAb) and goat polyclonal anti-human β2-Abs alone or complexed to human β2-glycoprotein 1 (i.e., to form immune complexes, or ICs) by the serotonin release assay (SRA) and platelet aggregation methods using washed human platelets. We found that two of three commercially available polyclonal anti-β2-Abs (pAb1 and pAb3), both alone or in IC form, induced platelet granule release and aggregation, and that this activity was abolished by anti-FcγRIIa mAb, IV.3. pAb2 and mAb were inactive. Activity analysis (SRA) of preformed ICs using constant pAb1 concentration with varied β2-GP1 stoichiometries revealed a zone-of-equivalence pattern, with maximal activity near balanced stoichiometry (1:1). Because pAb2 did not activate platelets, we sought to determine its capacity to form higher order ICs (which are known to be required for FcγRIIa activation) by size exclusion chromatography (SEC). All antibodies tested in isolation were shown by HPLC-SEC to be free of aggregates or degradation products. Antibody-antigen complex size analysis revealed that, as expected, mAb+β2-GP1 in 1:1 stoichiometry failed to form higher order ICs (i.e., complexes having ≥2 IgGs/complex). pAb2 (inactive) did form higher order ICs at 1:1 and 4:1 (IgG:Ag) stoichiometries, but less extensively than pAb1 (active), which efficiently formed higher order ICs at both 1:1 and 4:1 stoichiometries. pAb1 alone (i.e., not in IC form) caused aggregation, while pAb2 did not. Furthermore, preincubation of washed platelets with pAb2 prevented pAb1-induced aggregation, suggesting that pAb1 activity is β2-GP1-specific. A single intravenous injection of anti-β2-GP1 ICs (20 μg β2-GP1 plus 120 μg pAb1, a 1:2 molar stoichiometry) induced severe thrombocytopenia (>90%) and caused thrombotic shock in FcγRIIa-transgenic mice but not in wild type mice. Symptoms of shock occurred within 10 minutes. Pervasive occlusive thrombi were observed in the lungs of all FcγRIIa-transgenic but not in any wild type mice (H&E microscopy). Injection of pAb2 ICs produced none of these effects in transgenic or wild type mice. Finally, injection of pAb1 or pAb2 alone (neither of which bind mouse β2-GP1) had no effects in transgenic mice. In summary, these findings confirm previous in vitro studies that β2-Abs can directly activate platelets in a manner wholly dependent on the platelet IgG receptor, FcγRIIa. Additionally, we have shown for the first time in vivo that β2-Abs can also cause FcγRIIa-dependent thrombosis. This mechanism may contribute to thrombosis in APS, suggesting that further studies on the importance of FcγRIIa in APS are warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Management of alloimmune thrombocytopenia: antenatal diagnosis and in utero transfusion of maternal platelets

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 340-343 ◽  
Author(s):  
C Kaplan ◽  
F Daffos ◽  
F Forestier ◽  
WL Cox ◽  
D Lyon-Caen ◽  
...  
Keyword(s):  
Antenatal Diagnosis ◽  
In Utero ◽  
Severe Bleeding ◽  
Severe Thrombocytopenia ◽  
Fetal Blood ◽  
Alternative Approach ◽  
Neurologic Sequelae ◽  
The Central Nervous System ◽  
Circulating Antibodies ◽  
Alloimmune Thrombocytopenia

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe bleeding in the central nervous system (CNS) and death or severe neurologic sequelae. The expression of the PLA1 antigen is detectable as early as 19 weeks of gestation. Alloimmunization can therefore lead to fetal thrombocytopenia very early in pregnancy. Until recently, we have had no means of detecting and assessing the severity of fetal thrombocytopenia during pregnancy. The level of the maternal antibody is not of a predictable value since 20% of the mothers had no circulating antibodies in our series. An alternative approach is to carry out investigations on fetal blood samplings. This management leads to an exact knowledge of the fetal status and antenatal diagnosis is feasible as early as the 21st week of gestation. Early diagnosis facilitates appropriate management and makes possible such therapeutic options as in utero maternal platelet transfusions. We report our experience in the antenatal diagnosis and management of nine cases with in utero transfusion in the six cases with severe thrombocytopenia. All neonates did well, with no signs of bleeding at birth. No side effects of therapy were noted after a period ranging from 6 months to 3 years.

Evaluation of Two Structurally Distinct Novel Inhibitors of Apoptosis Signal-Regulating Kinase 1 (MAP3K5), As Potent Anti-Platelet Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3833-3833
Author(s):  
Meghna U. Naik ◽  
Xi Chen ◽  
Brendan Bachman ◽  
Ganesha Rai ◽  
David Maloney ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Receptor Activation ◽  
New Drugs ◽  
Severe Bleeding ◽  
Fibrinogen Receptor

Abstract Platelets play a critical role in both hemostasis and thrombosis. Anti-platelet drugs currently available apart from aspirin are directed against platelet agonist receptors or fibrinogen receptor integrin aIIbb3. These antagonists, although having potent anti-thrombotic activities, cause severe bleeding due to their effect on hemostasis. It is therefore of utmost important to develop new drugs that will protect from thrombosis with minimal effect on hemostasis. Apoptosis signal-regulating kinase (ASK1) is a redox sensitive serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its role in platelets is not known. We found that ASK1 is rapidly activated downstream of all platelet agonists. Ablation of Ask1 gene results in impaired platelet functions such as granule secretion, thromboxane A2 generation, as well as fibrinogen receptor activation, which translates into attenuated platelet aggregation compared to WT littermates. We also found that thrombin failed to activate p38 in Ask1 null platelets, showing that Ask1 is indispensable for p38 activation by thrombin. FeCl3-induced carotid artery injury model of thrombosis showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. These results indicated that ASK1 plays a central role in regulating platelet function, making it a potential target for combating thrombosis. We therefore synthesized two novel and highly specific ASK1 inhibitors based on the published reports, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) and GS-4997. We found that GS-4997 (500nM) and IPTB (5mM) inhibit agonist-induced ASK1 activation in human platelets. They do not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1/2. We also found that IPTB and GS-4997 dose-dependently inhibited activation of p38, a downstream effector kinase, induced by a variety of platelet agonists. Furthermore, these compounds dose-dependently inhibited ADP, collagen, convulxin and PAR4 activating peptide AYPGKF-induced platelet aggregation as well as platelet spreading on immobilized fibrinogen. In-vivo carotid artery thrombosis assay revealed that WT mice injected intraperitoneally with either IPTB (100mg/kg) of GS-4997 (100mg/kg), showed a significantly increased time of occlusion (P=0.028 and P=0.005 respectively) and thrombus formed were unstable as compared to control WT mice treated with saline alone. Furthermore, injection of either IPTB or GS-4997 protected mice against collagen/epinephrine-induced pulmonary thromboembolism. Out of 14 saline-treated mice only two survived whereas, 10 out of 11 mice treated with GS-4997 (100mg/kg) survived (P=0.0002). In case of IPTB 1 out of 12 control mice survived as compared to 9 out of 12 treated mice (P=0.0028). Interestingly, tail-bleeding studies revealed that WT mice treated with either IPTB (1mg/kg) of GS-4997 (1mg/kg), did not affect the average bleeding time (100s) seen in the WT mice treated with saline alone, suggesting that both inhibitors had no effect on in-vivo hemostasis. Moreover, pretreatment of the whole blood with these inhibitors significantly reduced thrombus formation under arterial flow (800s-1) without affecting platelet adhesion to collagen as assessed using a microfluidic device. Our results strongly suggest that both IPTB and GS-4997 protect the mice from thrombosis without affecting hemostasis. Further development of these inhibitors as a potential therapeutic agent to combat thrombotic disorders is highly warranted. Disclosures No relevant conflicts of interest to declare.

scholarly journals Neonatal Alloimmune Thrombocytopenia Detected After Traumatic Delivery: Case Report

2021 ◽  
Author(s):  
Gökhan KAya ◽  
Sema Tanrıverdi
Keyword(s):  
Human Platelet ◽  
Clinical Manifestations ◽  
Early Period ◽  
Ivig Therapy ◽  
High Dose ◽  
Severe Bleeding ◽  
Severe Thrombocytopenia ◽  
Platelet Suspension ◽  
Life Threatening ◽  
Alloimmune Thrombocytopenia

Neonatal Alloimmune Thrombocytopenia (NAIT), a complication of maternal alloimmunization against fetal platelets, is the result of immune destruction of platelets due to maternal antibodies in the early period. The cause of thrombocytopenia here is by antibodies developed against human platelet antigens frequently inherited from the mother. The clinical manifestations of NAIT extend from asymptomatic to severe bleeding. Platelet suspension can be used in the setting of severe thrombocytopenia or life-threatening bleeding. High-dose intravenous immunoglobulin (IVIG) therapy is another treatment option. A case who had no clinical finding except diffuse ecchymoses after traumatic delivery, but was considered to have NAIT due to severe thrombocytopenia and was successfully treated with platelet suspension and IVIG is presented.

Sign in / Sign up

Export Citation Format

Share Document