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Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins.

ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as ? IIb?3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in ?IIb?3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.

SUBMITTER: Bhoria P

PROVIDER: S-EPMC4966486 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins.

Bhoria Preeti P Varma Neelam N Malhotra Pankaj P Varma Subhash S Luthra-Guptasarma Manni M

mAbs 20150824 6

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as α IIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying a ...[more]

PMID: 26301697

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Project description:BackgroundThis is a review article on heparin-induced thrombocytopenia, an adverse effect of heparin therapy, and vaccine-induced immune thrombotic thrombocytopenia, occurring in some patients administered certain coronavirus vaccines.Main body/textImmune-mediated thrombocytopenia occurs when specific antibodies bind to platelet factor 4 /heparin complexes. Platelet factor 4 is a naturally occurring chemokine, and under certain conditions, may complex with negatively charged molecules and polyanions, including heparin. The antibody-platelet factor 4/heparin complex may lead to platelet activation, accompanied by other cascading reactions, resulting in cerebral sinus thrombosis, deep vein thrombosis, lower limb arterial thrombosis, myocardial infarction, pulmonary embolism, skin necrosis, and thrombotic stroke. If untreated, heparin-induced thrombocytopenia can be life threatening. In parallel, rare incidents of spontaneous vaccine-induced immune thrombotic thrombocytopenia can also occur in some patients administered certain coronavirus vaccines. The role of platelet factor 4 in vaccine-induced thrombosis with thrombocytopenia syndrome further reinforces the importance the platelet factor 4/polyanion immune complexes and the complications that this might pose to susceptible individuals. These findings demonstrate, how auxiliary factors can complicate heparin therapy and drug development. An increasing interest in biomanufacturing heparins from non-animal sources has driven a growing interest in understanding the biology of immune-mediated heparin-induced thrombocytopenia, and therefore, the development of safe and effective biosynthetic heparins.Short conclusionIn conclusion, these findings further reinforce the importance of the binding of platelet factor 4 with known and unknown polyanions, and the complications that these might pose to susceptible patients. In parallel, these findings also demonstrate how auxiliary factors can complicate the heparin drug development.

Project description:Introduction of vaccines against COVID-19 has provided the most promising chance to control the world-wide COVID-19 pandemic. However, the adenovirus-vector based Oxford/AstraZeneca [ChAdOx1] (AZ) and Johnson & Johnson [Ad26.CoV2.S] COVID-19 vaccines have been linked with serious thromboembolic events combined with thrombocytopenia, denominated Vaccine-induced Immune Thrombocytopenia and Thrombosis (VITT). The pathogenesis of COVID-19 VITT remain incompletely understood; especially the initial events that trigger platelet activation, platelet factor (PF)4 release, complex formation and PF4 antibody production are puzzling. This is a prospective study investigating the impact of different COVID-19 vaccines on inflammation (CRP, TNF-α, IL-1β, IL-6, IL-8, IL-10), vascular endothelial activation (syndecan-1, thrombomodulin, E-selectin, ICAM-1, ICAM-3, VCAM-1), platelet activation (P-selectin, TGF-β, sCD40L) and aggregation (Multiplate® impedance aggregometry), whole blood coagulation (ROTEM®), thrombin generation and PF4 antibodies to reveal potential differences between AZ and mRNA vaccines in individuals without VITT. The study included 80 (55 AZ and 55 mRNA) vaccinated individuals and 55 non-vaccinated age- and gender matched healthy controls. The main findings where that both vaccines enhanced inflammation and platelet activation, though AZ vaccination induced a more pronounced increase in several inflammatory and platelet activation markers compared to mRNA vaccination and that post-vaccination thrombin generation was higher following AZ vaccination compared to mRNA vaccination. No difference in neither the PF4 antibody level nor the proportion of individuals with positive PF4 antibodies were observed between the vaccine groups. This is the first study to report enhanced inflammation, platelet activation and thrombin generation following AZ vaccination compared to mRNA vaccination in a head-to-head comparison. We speculate that specific components of the AZ adenovirus vector may serve as initial trigger(s) of (hyper)inflammation, platelet activation and thrombin generation, potentially lowering the threshold for a cascade of events that both trigger complications related to excessive inflammation, platelet and coagulation activation as observed in epidemiological studies and promote development of VITT when combined with high-titer functionally active PF4 antibodies.

Dataset Information

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins.

Publications

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins.

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