Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

SARS-CoV-2 RBD and Its Variants Can Induce Platelet Activation and Clearance: Implications for Antibody Therapy and Vaccinations against COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the ß3 integrin as binding was significantly reduced in ß3-/- mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbß3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbß3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19.

Dual roles of fucoidan-GPIbα interaction in thrombosis and hemostasis: implications for drug development targeting GPIbα.

BACKGROUND: Platelet GPIbα-von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb-VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. OBJECTIVE: The objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. METHODS: Natural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα-VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX-induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. RESULTS: We found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbß3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF-mediated platelet aggregation, spreading, and thrombus growth at high shear. CONCLUSION: Fucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb-VWF interaction after fucoidan is hydrolyzed.

Updated Understanding of Platelets in Thrombosis and Hemostasis: The Roles of Integrin PSI Domains and their Potential as Therapeutic Targets.

Platelets are small blood cells known primarily for their ability to adhere and aggregate at injured vessels to arrest bleeding. However, when triggered under pathological conditions, the same adaptive mechanism of platelet adhesion and aggregation may cause thrombosis, a primary cause of heart attack and stroke. Over recent decades, research has made considerable progress in uncovering the intricate and dynamic interactions that regulate these processes. Integrins are heterodimeric cell surface receptors expressed on all metazoan cells that facilitate cell adhesion, movement, and signaling, to drive biological and pathological processes such as thrombosis and hemostasis. Recently, our group discovered that the plexin-semaphorin-integrin (PSI) domains of the integrin ß subunits exert endogenous thiol isomerase activity derived from their two highly conserved CXXC active site motifs. Given the importance of redox reactions in integrin activation and its location in the knee region, this PSI domain activity may be critically involved in facilitating the interconversions between integrin conformations. Our monoclonal antibodies against the ß3 PSI domain inhibited its thiol isomerase activity and proportionally attenuated fibrinogen binding and platelet aggregation. Notably, these antibodies inhibited thrombosis without significantly impairing hemostasis or causing platelet clearance. In this review, we will update mechanisms of thrombosis and hemostasis, including platelet versatilities and immune-mediated thrombocytopenia, discuss critical contributions of the newly discovered PSI domain thiol isomerase activity, and its potential as a novel target for anti-thrombotic therapies and beyond.

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

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 IIbß3 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 αIIbß3 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.

Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP).

The activation status of platelets in Immune Thrombocytopenia (ITP) patients--which is still somewhat controversial--is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.

Fibrotic remodeling of the extracellular matrix through a novel (engineered, dual-function) antibody reactive to a cryptic epitope on the N-terminal 30 kDa fragment of fibronectin.

Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached "RGD" sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis--migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.

Investigation of the possible association between the HLA antigens and idiopathic thrombocytopenic purpura (ITP).

The etiology of idiopathic thrombocytopenic purpura (ITP), characterized by destruction of platelets, is still poorly understood. Although genetic as well as immunological factors are thought to play a role in the disease pathogenesis, genetic association studies in terms of major histocompatibility complex (MHC) polymorphisms are scarce and discrepant. Results from previous studies suggest that different populations show varying associations with MHC alleles. Since i) there are inconsistencies in HLA associations, and ii) such an association study does not exist for the Indian subcontinent, we carried out sequence specific priming (SSP)-based genotyping of HLA DRB1 alleles in the North Indian population. Data for such studies is available for two East Asian countries, Japan and China, and the association in both cases is different. Further, among the Japanese population too, there are discrepant results. It was therefore important to analyze such an association in the Indian population, belonging to Southern Asia. Our data shows that none of the alleles have any significant association with ITP. Moreover, in contrast to other studies, comparison made between patients who were responsive to steroid therapy against those who were refractory to steroids, also did not show any association of the HLA DRB1 alleles with steroid responsiveness.

Towards an indirect screening technique facilitating detection of cellular populations bearing specific cell surface markers.

We describe and demonstrate a technique for detection of cell surface antigens, with potential use in tissue antigen research. Briefly, a small volume of wetted chromatographic beads (specifically, 100 µL of Nickel-nitriloacetic acid (NTA) agarose) was bound to small quantities (specifically, ∼0.1-0.2 µg) of a single-chain Fv antibody recognizing the Class I MHC heavy chain antigen. The beads were then used to capture and detect cells bearing this antigen, through SDS polyacrylamide gel electrophoresis of boiled bead-cell complexes. A key feature of this method is its use of "signal amplification." Although the antibody-mediated binding and immobilization of intact cells involves relatively small numbers of antibodies, and cells, what is being detected is simply the presence of cells. Each bound cell contains a number of abundant proteins that are present in millions of copies per cell, and the abundance of these proteins ensures that they are detectable on SDS-PAGE, signaling cell-binding. As few as 10(10) -10(12) scFv antibodies immobilized on 100 µL of Ni-NTA beads are thus enough for the trapping of enough cells to allow visualization of their abundant proteins. Conceptually, this method could be easily developed and applied to detection of cells bearing any other cell specific antigen.