Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal 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 (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Association between fibrinogen receptor (Glycoprotein IIb) polymorphism and the risk of venous thromboembolism: a systematic review

Background The fibrinogen receptor is an integrin on the platelet surface and is shaped from two types of glycoprotein (GP) subunits, GPIIb and GPIIIa. Membrane glycoprotein IIb/IIIa plays an important role in platelet function. The gene encoding the glycoprotein IIIa shows a common polymorphism, PLA2 that increases the binding of the receptor to fibrinogen and enhances the platelet aggregation. The clinical impact of PLA2 polymorphism has been studied in some diseases, but the definition of its exact role on venous thromboembolism complications has been challenging. The present systematic review aimed to clarify the association of PLA2 polymorphism and venous thromboembolism. Main text In this study, Electronic databases including PubMed, Embase, Scopus, Web of Science, and Cochrane Library were searched. All the assessed studies focused on the relationship between PLA2 polymorphism and venous thromboembolism. Five studies were eligible for systematic review. One study revealed a significant correlation between PLA2 polymorphism and venous thromboembolism. PLA2 polymorphism was associated with deep vein thrombosis in one study and pulmonary thromboembolism in another one. Conclusion The published data supported the hypothesis that having the PLA2 polymorphism of GPIIIa may be a risk factor for venous thromboembolism, but the association cannot be concluded; it needs more clinical investigation.

Recreation of Fetal Neonatal Alloimmune Thrombocytopenia in CRISPR/Cas9 Gene-Edited Mice Expressing the Human Platelet Alloantigen, HPA-1a

Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening alloimmune disorder caused by maternal antibodies directed against fetal platelet alloantigens. The human platelet alloantigens, (HPA)-1a/HPA-1b (also known as the PlA1 alloantigen system), residing on the integrin β3 subunit (= platelet membrane glycoprotein IIIa), is the most frequently implicated HPA for causing FNAIT in Caucasians, and a single Leu33Pro amino acid polymorphism is responsible for generating the HPA-1a and HPA-1b epitopes. Neither human nor mouse anti-HPA-1a antibodies recognize murine GPIIIa due to amino acid differences both within and surrounding the polymorphic amino acid, which lies within a long flexible loop in PSI domain of the molecule. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically-important platelet antigen. We employed CRISPR/Cas9 gene editing technology to generate mice expressing the human HPA-1a allogeneic epitope. Four critical amino acid substitutions (A30P32L33D39) were introduced into the PSI domain of murine GPIIIa. The resulting humanized form of murine GPIIIa, termed APLD, reacted in a variety of immunoassays with both murine monoclonal (mAb) and human maternal polyclonal antibodies specific for the human HPA-1a epitope. Intraperitoneal injection of an anti-HPA-1a mAb induced thrombocytopenia in APLD, but not wild-type, mice. Importantly, wild-type mice immunized with murine APLD transgenic platelets generated a high-titer HPA-1a-specific antibody response, demonstrating that the humanized form of murine GPIIIa is immunogenic in mice. Finally, pre-immunized female wild-type mice bred with APLD males gave birth to neonatal mice that exhibited profound thrombocytopenia, with the maternal titer directly related to the degree of thrombocytopenia in the neonates. Recapitulating the essential features of FNAIT in mice will facilitate the investigation of a wide-range of clinically-important issues in the diagnosis, treatment, and management of platelet alloimmune disorders. Disclosures No relevant conflicts of interest to declare.

Abstract TP210: Histopathological Evaluation of Thrombus in Acute Stroke and Correlation with Stroke Etiology

Background: While more endovascular treatments for acute stroke are being performed, few studies evaluate clot composition with variable results. We sought to evaluate the feasibility of collecting and analyzing the RBC to platelet ratios in clots, and correlated our findings with stroke etiology. Methods: This is an ongoing prospective study analyzing clots retrieved by mechanical thrombectomy in acute stroke patients at our institution. Retrieved clot material was fixed and cut at 4-m thickness. All clots were stained with hematoxylin-eosin to identify red blood cells (RBC’s), and antibodies for platelet glycoprotein IIIa with CD61 (LifeSpan Biosciences, Seattle, Washington) for platelets. Stained slides were scanned at 200x magnification by using a Scanscope XT digital scanner (Apergio, Vista, California). Image-J software (National Institutes of Health, Bethesda, Maryland) was used for semiquantitative analysis of percentage RBC’s and platelets. Correlation of RBC to Platelet ratios with stroke etiology was performed. Results: A total of 18 clots from 18 patients were analyzed. Stroke etiology was cardioembolic in 8, Large vessel atherosclerosis (LVA) in 5, undetermined in 3 and carotid dissection in 2. The mean RBC to platelet ratio was 0.51:1 in cardioembolic and 0.64:1 in LVA strokes. Patients with undetermined etiology had similar clot composition (0.53:1) to cardioembolic stroke. The highest RBC content was found in carotid dissection thrombus with a ratio of 1.73:1 as compared to other etiologies (p=0.01 cardioembolic, p=0.04 undetermined, p=0.02 LVA). Conclusion: In our study’s first phase, clot processing and analysis was found to be feasible. Although a high mean RBC content was found in carotid dissections and strokes of undetermined etiology had similar clot composition to cardioembolic stroke, ongoing collection and analysis will help support these findings.