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.

Anti-inflammatory activity of CD44 antibodies in murine immune thrombocytopenia is mediated by Fcγ receptor inhibition.

Monoclonal immunoglobulin G (IgG) antibodies to CD44 (anti-CD44) are anti-inflammatory in numerous murine autoimmune models, but the mechanisms are poorly understood. Anti-CD44 anti-inflammatory activity shows complete therapeutic concordance with IV immunoglobulin (IVIg) in treating autoimmune disease models, making anti-CD44 a potential IVIg alternative. In murine immune thrombocytopenia (ITP), there is no mechanistic explanation for anti-CD44 activity, although anti-CD44 ameliorates disease similarly to IVIg. Here, we demonstrate a novel anti-inflammatory mechanism of anti-CD44 that explains disease amelioration by anti-CD44 in murine ITP. Macrophages treated with anti-CD44 in vitro had dramatically suppressed phagocytosis through FcγRs in 2 separate systems of IgG-opsonized platelets and erythrocytes. Phagocytosis inhibition by anti-CD44 was mediated by blockade of the FcγR IgG binding site without changing surface FcγR expression. Anti-CD44 of different subclasses revealed that FcγR blockade was specific to receptors that could be engaged by the respective anti-CD44 subclass, and Fc-deactivated anti-CD44 variants lost all FcγR-inhibiting activity. In vivo, anti-CD44 functioned analogously in the murine passive ITP model and protected mice from ITP when thrombocytopenia was induced through an FcγR that could be engaged by the CD44 antibody's subclass. Consistent with FcγR blockade, Fc-deactivated variants of anti-CD44 were completely unable to ameliorate ITP. Together, anti-CD44 inhibits macrophage FcγR function and ameliorates ITP consistent with an FcγR blockade mechanism. Anti-CD44 is a potential IVIg alternative and may be of particular benefit in ITP because of the significant role that FcγRs play in human ITP pathophysiology.

The Antiviral Effect of Isatis Root Polysaccharide against NADC30-like PRRSV by Transcriptome and Proteome Analysis.

(1) Background: In recent years, the porcine reproductive and respiratory syndrome virus (PRRSV) has become a virulent pathogen that has caused devastating diseases and economic losses worldwide in the swine industry. IRPS has attracted extensive attention in the field of virology. However, it is not clear that IRPS has an antiviral effect on PRRSV at gene and protein levels. (2) Methods: We used transcriptomic and proteomic analysis to investigate the antiviral effect of IRPS against PRRSV. Additionally, a microbiome was used to explore the effects of IRPS on gut microbes. (3) Results: IRPS significantly extenuated the pulmonary pathological lesions and inflammatory response. We used transcriptomic and proteomic analysis to investigate the antiviral effect of IRPS against PRRSV. In the porcine model, 1669 differentially expressed genes (DEGs) and 370 differentially expressed proteins (DEPs) were identified. Analysis of the DEG/DEP-related pathways indicated immune-system and infectious-disease (viral) pathways, such as the NOD-like receptor (NLR) signaling pathway, toll-like receptor (TLR) signaling pathway, and Influenza A-associated signaling pathways. It is noteworthy that IRPS can inhibit NLR-dependent gene expression, then reduce the inflammatory damage. IRPS could exert beneficial effects on the host by regulating the structure of intestinal flora. (4) Conclusions: The antiviral effect of IRPS on PRRSV can be directly achieved by omics techniques. Specifically, the antiviral mechanism of IPRS can be better elucidated by screening target genes and proteins using transcriptome and proteome sequencing, and then performing enrichment and classification according to DEGs and DEPs.

Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets.

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

GPIbα is required for platelet-mediated hepatic thrombopoietin generation.

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα-/- mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients. We showed that lower TPO levels in GPIbα-deficient conditions were not due to increased TPO clearance by GPIbα-/- platelets but rather to decreased hepatic TPO mRNA transcription and production. We found that WT, but not GPIbα-/-, platelet transfusions rescued hepatic TPO mRNA and circulating TPO levels in GPIbα-/- mice. In vitro hepatocyte cocultures with platelets or GPIbα-coupled beads further confirm the disruption of platelet-mediated hepatic TPO generation in the absence of GPIbα. Treatment of GPIbα-/- platelets with neuraminidase caused significant desialylation; however, strikingly, desialylated GPIbα-/- platelets could not rescue impaired hepatic TPO production in vivo or in vitro, suggesting that GPIbα, independent of platelet desialylation, is a prerequisite for hepatic TPO generation. Additionally, impaired hepatic TPO production was recapitulated in interleukin-4/GPIbα-transgenic mice, as well as with antibodies targeting the extracellular portion of GPIbα, demonstrating that the N terminus of GPIbα is required for platelet-mediated hepatic TPO generation. These findings reveal a novel nonredundant regulatory role for platelets in hepatic TPO homeostasis, which improves our understanding of constitutive TPO regulation and has important implications in diseases related to GPIbα, such as BSS and auto- and alloimmune-mediated thrombocytopenias.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

CD8+ T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by autoantibodies targeting platelet surface proteins, most commonly GPIIbIIIa (αIIbß3 integrin), leading to platelet destruction. Recently, CD8(+) cytotoxic T-lymphocytes (CTLs) targeting platelets and megakaryocytes have also been implicated in thrombocytopenia. Because steroids are the most commonly administered therapy for ITP worldwide, we established both active (immunized splenocyte engraftment) and passive (antibody injection) murine models of steroid treatment. Surprisingly, we found that, in both models, CD8(+) T cells limited the severity of the thrombocytopenia and were required for an efficacious response to steroid therapy. Conversely, CD8(+) T-cell depletion led to more severe thrombocytopenia, whereas CD8(+) T-cell transfusion ameliorated thrombocytopenia. CD8(+) T-regulatory cell (Treg) subsets were detected, and interestingly, dexamethasone (DEX) treatment selectively expanded CD8(+) Tregs while decreasing CTLs. In vitro coculture studies revealed CD8(+) Tregs suppressed CD4(+) and CD19(+) proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8(+) Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8(+) Tregs in immune thrombocytopenia.

Plant-based Food Cyanidin-3-Glucoside Modulates Human Platelet Glycoprotein VI Signaling and Inhibits Platelet Activation and Thrombus Formation.

Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 µM) of Cy-3-g. The expression of activated integrin αIIbß3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-µM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-µM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-µM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-µM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk-linker for activation of T cells (LAT)-SLP76 (Syk: -39.08%, LAT: -32.25%, SLP76: -40.00%) and the expression of Lyn (-31.89%), Fyn (-36.27%), and phospholipase C-γ2 (-39.08%).Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.

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.

Desialylated Platelet Clearance in the Liver is a Novel Mechanism of Systemic Immunosuppression.

Platelets are small, versatile blood cells that are critical for hemostasis/thrombosis. Local platelet accumulation is a known contributor to proinflammation in various disease states. However, the anti-inflammatory/immunosuppressive potential of platelets has been poorly explored. Here, we uncovered, unexpectedly, desialylated platelets (dPLTs) down-regulated immune responses against both platelet-associated and -independent antigen challenges. Utilizing multispectral photoacoustic tomography, we tracked dPLT trafficking to gut vasculature and an exclusive Kupffer cell-mediated dPLT clearance in the liver, a process that we identified to be synergistically dependent on platelet glycoprotein Ibα and hepatic Ashwell-Morell receptor. Mechanistically, Kupffer cell clearance of dPLT potentiated a systemic immunosuppressive state with increased anti-inflammatory cytokines and circulating CD4+ regulatory T cells, abolishable by Kupffer cell depletion. Last, in a clinically relevant model of hemophilia A, presensitization with dPLT attenuated anti-factor VIII antibody production after factor VIII ( infusion. As platelet desialylation commonly occurs in daily-aged and activated platelets, these findings open new avenues toward understanding immune homeostasis and potentiate the therapeutic potential of dPLT and engineered dPLT transfusions in controlling autoimmune and alloimmune diseases.

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.

Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner.

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.

Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined ß3 integrin-deficient and FcRn-deficient (ß3(-/-)FcRn(-/-)) mice. We found that ß3(-/-)FcRn(-/-) mice are immunoresponsive to ß3(+/+)FcRn(-/-) platelets. The generated antibodies were ß3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult ß3(+/+)FcRn(-/-) mice. FNIT was observed when immunized ß3(-/-)FcRn(+/+) females were bred with ß3(+/+)FcRn(+/+) males, while no FNIT occurred in ß3(-/-)FcRn(-/-) females bred with ß3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.

Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor.

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre(+), Fn(flox/flox), Fg/VWF(-/-)) mice and littermate control (Cre(-), Fn(flox/flox), Fg/VWF(-/-)) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti-mouse beta(3) integrin antibodies but was not affected by anti-mouse GPIbalpha antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl(3)-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Biofilm characteristics and transcriptomic analysis of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a conditional pathogen with the ability to form biofilms which can lead to ineffective drug treatment and severe chronic infections resulting in significant economic losses to the pig industry. Currently, knowledge of biofilm formation by H. parasuis is not well developed. The objective of this study was to investigate the three-dimensional morphology of biofilms and perform transcriptomic analysis on H. parasuis cells in biofilm versus planktonic forms. The results showed that proteins and DNA accounted for a large proportion of the H. parasuis biofilm extracellular matrix. Here, we have traced the entire biofilm formation process of H. parasuis from beginning to end for the first time. These biofilms grew rapidly in the first 48 h and became stable at 60 h. According to GO and KEGG analysis, the differentially expressed genes (DEG) artM, artQ, ssrS, pflA and HutX were implicated as being involved in bacterial colonisation and adhesion; these are the most likely genes to affect biofilm formation. Most functional gene enrichments were of those involved in metabolic pathways, biosynthesis of secondary metabolites, ATP-binding cassette (ABC) transporters, and starch and sucrose metabolism. Thus, in the present pilot study, the composition and characteristics of these biofilms were explored, and the genes related to biofilm formation were screened for. This research lays the foundation for further studies on mechanisms regulating biofilm formation, in order to find new drug targets and develop new therapeutic drugs against H. parasuis.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

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.

Apolipoprotein A-IV binds αIIbß3 integrin and inhibits thrombosis.

Platelet αIIbß3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet ß3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbß3 integrin, and the direct apoA-IV-αIIbß3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbß3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbß3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.

Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia.

Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal-fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a gestational disease caused by maternal immune responses against fetal platelets. Using a FNAIT mouse model and human trophoblast cell lines, here the authors show that uterine natural killer cell-mediated trophoblast apoptosis contributes to FNAIT pathogenesis.