Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

"Sticki-ER": Functions of the Platelet Endoplasmic Reticulum.

Significance: The primary role of platelets is to generate a thrombus by platelet activation. Platelet activation relies on calcium mobilization from the endoplasmic reticulum (ER). ER resident proteins, which are externalized upon platelet activation, are essential for the function of platelet surface receptors and intercellular interactions. Recent Advances: The platelet ER is a conduit for changes in cellular function in response to the extracellular milieu. ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Alterations in ER function and ER stress results in ER proteins externalizing to the cell surface, including members of the protein disulfide isomerase family (PDIs) and chaperones. Critical Issues: The platelet ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of PDIs in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown. Future Directions: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of PDIs in the intracellular versus extracellular space, as these differentially affect platelet function. An unresolved question is how platelet ER proteins control calcium release. Regulation of protein folding in the platelet and downstream pathways of ER stress require further evaluation. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Thrombo-inflammatory response in hospitalised patients with COVID-19: a single institution experience.

BACKGROUND: Severe COVID-19 causes acute inflammation, which is complicated by venous thromboembolism events (VTE). However, it is unclear if VTE risk has evolved over time since the COVID-19 outbreak. AIMS: To determine markers of thrombo-inflammation and rates of symptomatic VTE in patients hospitalised for COVID-19 in a metropolitan hospital in Sydney, Australia. METHODS: A retrospective, single-centre, cohort study was performed by reviewing electronic medical records of consecutive patients admitted to Royal Prince Alfred Hospital between March 2020 and September 2021. This period included three waves of COVID-19 outbreaks in Australia with the ancestral, alpha and delta variants. Standard coagulation assays and inflammatory markers were recorded over 4 weeks. RESULTS: A total of 205 patients were consecutively admitted during the study period. Activated partial thromboplastin time, neutrophil count and C-reactive protein (CRP) were significantly increased in patients hospitalised in the intensive care unit (ICU) compared with non-ICU patients. The use of anti-inflammatory medication increased in 2021 compared with 2020. The mortality rate was 7.3% in our cohort. Ninety-four per cent of patients received anticoagulation with 6.3% of patients developing VTE. CONCLUSION: We observed lower rates of VTE compared to the internationally reported rate for the same period. We conclude that in the setting of controlled hospital admission rate and standard anticoagulation guidelines, COVID-19 resulted in similar thrombo-inflammatory response and VTE rates over the first 1.5 years of the pandemic.

Sex, Endothelial Cell Functions, and Peripheral Artery Disease.

Peripheral artery disease (PAD) is caused by blocked arteries due to atherosclerosis and/or thrombosis which reduce blood flow to the lower limbs. It results in major morbidity, including ischemic limb, claudication, and amputation, with patients also suffering a heightened risk of heart attack, stroke, and death. Recent studies suggest women have a higher prevalence of PAD than men, and with worse outcomes after intervention. In addition to a potential unconscious bias faced by women with PAD in the health system, with underdiagnosis, and lower rates of guideline-based therapy, fundamental biological differences between men and women may be important. In this review, we highlight sexual dimorphisms in endothelial cell functions and how they may impact PAD pathophysiology in women. Understanding sex-specific mechanisms in PAD is essential for the development of new therapies and personalized care for patients with PAD.

Focal Anticoagulation by Somatic Gene Transfer: Towards Preventing Cardioembolic Stroke.

Cardioembolic stroke (CS) has emerged as a leading cause of ischaemic stroke (IS); distinguished by thrombi embolising to the brain from cardiac origins; most often from the left atrial appendage (LAA). Contemporary therapeutic options are largely dependent on systemic anticoagulation as a blanket preventative strategy, yet this does not represent a nuanced or personalised solution. Contraindications to systemic anticoagulation create significant unmedicated and high-risk cohorts, leaving these patients at risk of significant morbidity and mortality. Atrial appendage occlusion devices are increasingly used to mitigate stroke risk from thrombi emerging from the LAA in patients ineligible for oral anticoagulants (OACs). Their use, however, is not without risk or significant cost, and does not address the underlying aetiology of thrombosis and CS. Viral vector-based gene therapy has emerged as a novel strategy to target a spectrum of haemostatic disorders, achieving success through the adeno-associated virus (AAV) based therapy of haemophilia. Yet, thrombotic disorders, such as CS, have had limited exploration within the realm of AAV gene therapy approaches-presenting a gap in the literature and an opportunity for further research. Gene therapy has the potential to directly address the cause of CS by localised targeting of the molecular remodelling that serves to promote thrombosis.

Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

Heparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4-polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4-polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

Multiple Electrode Aggregometry (Multiplate): Functional Assay for Vaccine-Induced (Immune) Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) was first described in 2021 and represents an adverse reaction to adenoviral vector COVID-19 vaccines AstraZeneca ChAdOx1 nCoV-19 (AZD1222) and Johnson & Johnson Ad26.COV2.S vaccine. VITT is a severe immune platelet activation syndrome with an incidence of 1-2 per 100,000 vaccinations. The features of VITT include thrombocytopenia and thrombosis within 4-42 days of first dose of vaccine. Affected individuals develop platelet-activating antibodies against platelet factor 4 (PF4). The International Society on Thrombosis and Haemostasis recommends both an antigen-binding assay (enzyme-linked immunosorbent assay, ELISA) and a functional platelet activation assay for the diagnostic workup of VITT. Here, the application of multiple electrode aggregometry (Multiplate) is presented as a functional assay for VITT.

Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Vaccine-induced immune thrombotic thrombocytopenia post dose 2 ChAdOx1 nCoV19 vaccination: Less severe but remains a problem.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but established complication of 1st dose ChAdOx1 nCoV19 vaccination (AZD1222), however this complication after dose 2 remains controversial. OBJECTIVES: To describe the clinicopathological features of confirmed cases of VITT post dose 2 AZD1222 vaccination in Australia, and to compare this cohort to confirmed cases of VITT post 1st dose. METHODS: Sequential cases of clinically suspected VITT (thrombocytopenia, D-Dimer > 5x upper limit normal and thrombosis) within 4-42 days of dose 2 AZD1222 referred to Australia's centralised testing centre underwent platelet activation confirmatory testing in keeping with the national diagnostic algorithm. Final classification was assigned after adjudication by an expert advisory committee. Descriptive statistics were performed on this cohort and comparative analyses carried out on confirmed cases of VITT after 1st and 2nd dose AZD1222. RESULTS: Of 62 patients referred, 15 demonstrated presence of antibody mediated platelet activation consistent with VITT after dose 2 AZD1222. Four were immunoassay positive. Median time to presentation was 13 days (range 1-53) platelet count 116x10^9/L (range 63-139) and D-dimer elevation 14.5xULN (IQR 11, 26). Two fatalities occurred. In each, the dosing interval was less than 30 days. In comparison to 1st dose, dose 2 cases were more likely to be male (OR 4.6, 95% CI 1.3-15.8, p = 0.03), present with higher platelet counts (p = 0.05), lower D-Dimer (p = 01) and less likely to have unusual site thromboses (OR 0.14, 95% CI 0.04-0.28, p = 0.02). CONCLUSIONS: VITT is a complication of dose 2 AZD1222 vaccination. Whilst clinicopathological features are less severe, fatalities occurred in patients with concomitant factors.

A hidden problem: peripheral artery disease in women.

Peripheral artery disease (PAD) has a huge social and economic burden and is an important contributor to the global health burden. Sex differences in PAD are apparent, with recent data suggesting equal if not greater prevalence in women, and women having worse clinical outcomes. Why this occurs is not clear. To identify underlying reasons for gender inequalities in PAD, we executed a deeper exploration through a social constructive perspective. A scoping review was conducted using the World Health Organization model for analysis of gender-related needs in healthcare. Complex interacting factors, including biological, clinical, and societal variables, were reviewed to highlight gender-related inequities in the diagnosis, treatment, and management of PAD. Current gaps in knowledge were identified and insights into future directions aimed at improving these inequalities were discussed. Our findings highlight the multi-level complexities that need to be considered for strategies to improve gender-related needs in PAD healthcare.

Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model.

Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46, and ERp5, are required for in vivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only ∼10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. Here, we aim to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5floxed (fl)/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a twofold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kilodalton glucose-regulated protein (GRP78), and calreticulin. ERp5-deficient platelets showed an enhanced ER stress response to ex vivo and in vivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as a negative regulator of ER stress responses in platelets and highlight the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for the therapeutic applications of ER stress inhibitors in thrombosis.

Endothelial cell dysfunction: Implications for the pathogenesis of peripheral artery disease.

Peripheral artery disease (PAD) is caused by occluded or narrowed arteries that reduce blood flow to the lower limbs. The treatment focuses on lifestyle changes, management of modifiable risk factors and vascular surgery. In this review we focus on how Endothelial Cell (EC) dysfunction contributes to PAD pathophysiology and describe the largely untapped potential of correcting endothelial dysfunction. Moreover, we describe current treatments and clinical trials which improve EC dysfunction and offer insights into where future research efforts could be made. Endothelial dysfunction could represent a target for PAD therapy.

Assessment of immunological anti-platelet factor 4 antibodies for vaccine-induced thrombotic thrombocytopenia (VITT) in a large Australian cohort: A multicenter study comprising 1284 patients.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus-based vaccines aimed to prevent and minimize COVID-19 and related pathophysiology. OBJECTIVES: To describe patterns of testing for anti-platelet factor 4 (PF4) antibodies using various ELISA assays in a large Australian cohort and comparative functional platelet activation assays in a subset. PATIENTS/METHODS: Asserachrom HPIA IgG ELISA was performed in 1284 patients over a period of 12 months, supplemented in select cohorts by comparative ELISA using three other methods (n = 78-179), three different functional assays (flow cytometry, serotonin release assay, and/or Multiplate; n = 476), and rapid immunological chemiluminescence anti-PF4 assay (n = 460), in a multicenter study. RESULTS: For first episode presentations, 190/1284 (14.8%) ELISA tests were positive. Conversely, most (445/460; 96.7%) chemiluminescence anti-PF4 test results were negative. All functional assays showed associations of higher median ELISA optical density with functional positivity and with high rates of ELISA positivity (64.0% to 85.2%). Data also identified functional positivity in 14.8%-36.0% of ELISA negative samples, suggesting false negative VITT by HPIA IgG ELISA in upward of one third of assessable cases. CONCLUSION: To our knowledge, this is the largest multicenter evaluation of anti-PF4 testing for investigation of VITT. Discrepancies in test results (ELISA vs. ELISA or ELISA vs. functional assay) in some patients highlighted limitations in relying on single methods (ELISA and functional) for PF4 antibody detection in VITT, and also highlights the variability in phenotypic test presentation and pathomechanism of VITT.

Diffuse alveolar haemorrhage as a rare complication of antiphospholipid syndrome.

Diffuse alveolar haemorrhage (DAH) is a rare complication of antiphospholipid syndrome. With a mortality rate of 46%, early diagnosis and management remain an ongoing challenge. Case reports are limited, and management guidelines are not yet definitive. In this case report, we present a 43-year-old male with DAH who required high-dose oral steroids, intravenous methylprednisolone cyclophosphamide and rituximab over 18 months to control life-threatening episodes of pulmonary bleeding.

A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

Mechano-Redox Control of Integrins in Thromboinflammation.

Significance: How mechanical forces and biochemical cues are coupled remains a miracle for many biological processes. Integrins, well-known adhesion receptors, sense changes in mechanical forces and reduction-oxidation reactions (redox) in their environment to mediate their adhesive function. The coupling of mechanical and redox function is a new area of investigation. Disturbance of normal mechanical forces and the redox balance occurs in thromboinflammatory conditions; atherosclerotic plaques create changes to the mechanical forces in the circulation. Diabetes induces redox changes in the circulation by the production of reactive oxygen species and vascular inflammation. Recent Advances: Integrins sense changes in the blood flow shear stress at the level of focal adhesions and respond to flow and traction forces by increased signaling. Talin, the integrin-actin linker, is a traction force sensor and adaptor. Oxidation and reduction of integrin disulfide bonds regulate their adhesion. A conserved disulfide bond in integrin αlpha IIb beta 3 (αIIbß3) is directly reduced by the thiol oxidoreductase endoplasmic reticulum protein 5 (ERp5) under shear stress. Critical Issues: The coordination of mechano-redox events between the extracellular and intracellular compartments is an active area of investigation. Another fundamental issue is to determine the spatiotemporal arrangement of key regulators of integrins' mechanical and redox interactions. How thromboinflammatory conditions lead to mechanoredox uncoupling is relatively unexplored. Future Directions: Integrated approaches, involving disulfide bond biochemistry, microfluidic assays, and dynamic force spectroscopy, will aid in showing that cell adhesion constitutes a crossroad of mechano- and redox biology, within the same molecule, the integrin. Antioxid. Redox Signal. 37, 1072-1093.

Βeta-2-glycoprotein I exerts antithrombotic function through its domain V in mice.

Little is known about the physiological role of beta-2-glycoprotein I (ß2GPI) despite it being the major auto-antigen in the antiphospholipid syndrome. A systematic study of the role of ß2GPI in thrombus formation in vivo has not been performed to date. Herein, we report that ß2GPI deficient (-/-) mice have enhanced thrombus formation compared to wild type (WT) mice in a laser-induced arteriole and venule model of thrombosis. Furthermore, neutrophil accumulation and elastase activity was enhanced in thrombi of ß2GPI -/- compared with WT mice. The antithrombotic function of ß2GPI is dependent on its fifth domain (domain V); intravenous administration of the ß2GPI domain deletion mutant lacking domain V (human recombinant domain I-IV) had no effect on platelet and fibrin thrombus size in ß2GPI -/- or WT mice. On the contrary, intravenous administration of human recombinant domain V significantly inhibited platelet and fibrin thrombus size in both ß2GPI -/- mice and WT mice. These findings reveal a major role for ß2GPI as a natural anticoagulant and implicate domain V of ß2GPI as a potential antithrombotic therapy.

Development and implementation of venous thromboembolism stewardship across a hospital network.

BACKGROUND: Venous thromboembolism (VTE) is a leading cause of preventable death in hospital. Ensuring all hospitalized patients are assessed for VTE risk and given appropriate prophylaxis can reduce the burden of VTE on patients and the healthcare system. This is the first study to explore the effectiveness of a VTE stewardship program using electronic clinical decision support (eCDS) to provide oversight of hospital initiatives to prevent VTE. AIM: To determine if a VTE stewardship program can increase risk-appropriate VTE prophylaxis, VTE risk assessment using eCDS, any documented risk assessment and risk assessment within 24 h of admission, plus reduce the incidence of hospital acquired VTE (HA-VTE). METHODS: Education, daily medication chart auditing, weekly clinician performance feedback, health promotion and gamification were deployed over 6 months by two multidisciplinary VTE stewardship teams across four hospitals. Service impact was assessed through cross-sectional audits of electronic medical records every 3 months and review of HA-VTE events pre- and post-intervention. Implementation costs were calculated. RESULTS: A total of 1622 patients were audited in separate cohorts at baseline, 3, 6 and 9 months. There was significant improvement in the prescription of appropriate prophylaxis (78%, 83%, 84%, and 88%, p = 0.004), VTE risk assessment using the eCDS tool (20%, 50%, 81% and 87%, p < 0.001), any documented risk assessment (71%, 82%, 95% and 93%, p < 0.001) and any documented risk assessment within 24 h of admission (54%, 56%, 65% and 63%, p = 0.001). Use of eCDS was associated with prescription of risk-appropriate VTE prophylaxis (p < 0.001). Annual incidence of HA-VTE decreased from 7.88 to 6.99 events per 10,000 discharges pre- to post-intervention (Odds Ratio (OR) 0.89, 95 %CI 0.66-1.18, p = 0.43). The cost of implementing the program across 133,078 episodes of care during the study period was AUD$108,167 (mean cost of $0.82 per patient).