Search

•Disruption of the GPIb-flnA interaction is sufficient to induce macrothrombocytopenia.•The macrothrombocytopenia phenotype is associated with aberrant megakaryocyte membrane budding.

BackgroundDespite restoration of epicardial blood flow in acute ST-elevation myocardial infarction (STEMI), inadequate microcirculatory perfusion is common and portends a poor prognosis. Intracoronary (IC) thrombolytic therapy can reduce microvascular thrombotic burden; however, contemporary studies have produced conflicting outcomes.ObjectivesThis meta-analysis aims to evaluate the efficacy and safety of adjunctive IC thrombolytic therapy at the time of primary percutaneous coronary intervention (PCI) among patients with STEMI.MethodsComprehensive literature search of six electronic databases identified relevant randomised controlled trials. The primary outcome was major adverse cardiac events (MACE). The pooled risk ratio (RR) and weighted mean difference (WMD) with a 95% CI were calculated.Results12 studies with 1915 patients were included. IC thrombolysis was associated with a significantly lower incidence of MACE (RR=0.65, 95% CI 0.51 to 0.82, I2=0%, p<0.0004) and improved left ventricular ejection fraction (WMD=1.87; 95% CI 1.07 to 2.67; I2=25%; p<0.0001). Subgroup analysis demonstrated a significant reduction in MACE for trials using non-fibrin (RR=0.39, 95% CI 0.20 to 0.78, I2=0%, p=0.007) and moderately fibrin-specific thrombolytic agents (RR=0.62, 95% CI 0.47 to 0.83, I2=0%, p=0.001). No significant reduction was observed in studies using highly fibrin-specific thrombolytic agents (RR=1.10, 95% CI 0.62 to 1.96, I2=0%, p=0.75). Furthermore, there were no significant differences in mortality (RR=0.91; 95% CI 0.48 to 1.71; I2=0%; p=0.77) or bleeding events (major bleeding, RR=1.24; 95% CI 0.47 to 3.28; I2=0%; p=0.67; minor bleeding, RR=1.47; 95% CI 0.90 to 2.40; I2=0%; p=0.12).ConclusionAdjunctive IC thrombolysis at the time of primary PCI in patients with STEMI improves clinical and myocardial perfusion parameters without an increased rate of bleeding. Further research is needed to optimise the selection of thrombolytic agents and treatment protocols.

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.Graphical AbstractBiological, clinical and societal interactions that impact health-related inequalities in women with peripheral artery disease (PAD).

Vaccine‐induced thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus‐based vaccines aimed to prevent and minimize COVID‐19 and related pathophysiology. 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. 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. 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. 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.

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.

Nearly all hematologic malignancies can occasionally present with or develop pleural effusions during the clinical course of disease. Among the most common disorders are Hodgkin and non-Hodgkin lymphomas, with a [...]

The αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

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.

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.

Background/Aim

•DGKKO can reverse thrombocytopenia in a HIT murine model.•DGKKO can prevent/reverse thrombosis in vitro and in a HIT murine model.

Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488–labeled anti-ERp5 antibody at 0.05 μg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 μg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to β3 integrin with an equilibrium dissociation constant (KD) of 21 µM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to β3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with αIIbβ3.

β2-Glycoprotein I (β2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in β2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of β2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of β2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within β2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within β2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol–containing β2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress–induced cell death.

Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46 and ERp5, are required for in vivothrombus 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. In the current study, we aimed to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5fl/fl. Pf4Cre+/ERp5fl/flmice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a 2-fold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kDa glucose-regulated protein (GRP78) and calreticulin. ERp5 deficient platelets showed an enhanced ER stress response to ex vivoand in vivoER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A (eIF2a) and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 (TXA2) receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as negative regulator of ER stress responses in platelets, and highlights 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 therapeutic applications of ER stress inhibitors in thrombosis.

The antiphospholipid syndrome (APS) is an acquired thrombophilia, characterized by the occurrence of venous and arterial events. This article examines the laboratory and key clinical aspects of APS. Particular focus is given to anti–beta 2-glycoprotein I (β2GPI) antibodies in view of their recent inclusion in the APS classification criteria. The clinical utility of using the β2GPI enzyme-linked immunosorbent assay, in conjunction with the established lupus anticoagulant assays and cardiolipin enzyme-linked immunosorbent assay, for diagnosing and risk stratifying patients suspected of having APS is discussed. The relative importance of the various assays in diagnosing obstetric APS (early and late gestation miscarriages) is explored. The implications of recent epidemiologic findings for possibly understanding the underlying pathophysiologic mechanisms of obstetric APS are highlighted. Insights into which patients with obstetric APS may be at most risk of thrombotic complications are presented.

The antiphospholipid syndrome (APS) is an important cause of acquired thrombophilia. It is characterized by the core clinical manifestations of thrombosis, either venous or arterial, and in women it can also be associated with recurrent fetal loss. The detection of persistently elevated levels of antiphospholipid antibodies (aPL Abs) is a requisite laboratory feature for the diagnosis to be made. The dominant antigenic targets in APS are beta 2-glycoprotein I (β2-GPI) and prothrombin. There is an accumulating body of experimental evidence that suggests that specific subgroups of aPL Abs may directly contribute to disease pathogenesis. This review critically examines the experimental evidence underlying the various propositions made to explain how these antibodies may predispose to disease in humans. Furthermore, it also examines the evidence relating to the immunologic mechanisms that may contribute to the breakage of peripheral tolerance in this disorder. Delineating the strengths and limitations of the experimental evidence accumulated thus far will hopefully stimulate further experimentation toward achieving the ultimate goal of precisely defining the dominant pathogenic mechanisms operational in APS. This may pave the way for the development of improved therapies.

There is increased scientific interest in the diagnosis of the antiphospholipid syndrome (APS), as therapeutic interventions can lead to substantial improvement in clinical outcome. As the clinical features of APS are far from specific, a sound laboratory method is needed to support or exclude the diagnosis. Two methods are currently used for the diagnosis of APS: (1) ELISA-based immunoassays for the detection of anticardiolipin (aCL) antibodies; and (2) clotting assays for determination of the lupus anticoagulant (LA). However, the first method is limited by a low specificity, and the second by low sensitivity. Furthermore, for both methods standardisation is unsatisfactory. Therefore, a number of new assays have been proposed as alternative or supplementary to aCL and LA tests. These include the anti-beta-2-glycoprotein I or antiprothrombin ELISAs, an ELISA utilising a phospholipid mixture, clotting assays with varying activators and assays utilising chromogenic substrates. This review presents a brief outline of APS, the autoantibodies associated with this syndrome, the basic principles of the standard assays used and a description of newer methods currently being validated.

Background:

Introduction

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation; however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4.

Introduction:

Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

APL represents a particular subtype of acute myeloid leukemia with characteristic clinical features, as well as specific immunophenotypic, cytogenetic and molecular findings owing to the chromosomal translocation t(15;17). Anthracycline-based chemotherapy and All-Trans Retinoid Acid (ATRA) became the cornerstone of APL treatment by improving significantly the long term outcome of patients, even though there is some controversy regarding the impact of this combination on the mortality of the induction phase. Herein, we analysed retrospectively the outcome of 16 consecutive adult APL patients who were diagnosed and treated in our Unit from 12/1998 to 10/2004. The analysis focuses more on the parameters of treatment-related mortality, cause of death and disease-free survival post AIDA chemotherapy. All patients were suffering from the classical form of APL and were homogeneously treated as follows: induction consisted of ATRA p.o. and idarubicin i.v. at conventional doses of 45mg/m2/d, from D1 to CR and 12mg/m2/d, D2,4,6,8 (total of 4 infusions), respectively. Dose modifications for elderly individuals were not allowed. Complete remitters were consolidated with 3 courses of chemotherapy without ATRA, where as non-remitters were taken off protocol and received other therapy. Patients in continuing hematological and molecular remission at the end of consolidation were administered maintenance therapy for 2 years with oral 6-MP at 90mg/m2/d, oral MTX weekly at 15mg/m2and ATRA for 15 days every 3 months. In all cases, the morphological diagnosis of APL was confirmed by chromosome and immunophenotypic analysis of blasts in addition to molecular studies. The median age of our cohort was 55 years (range 31–78) and the male/female ratio was 12/4. Three patients (3/16, 19%) were ≥ 65 years at diagnosis. Two cases (2/16, 12%) presented with a leukocyte count of ≥10 x 103/mm3while the median Wbc at presentation was 6.5 x 103/mm3. All cases had either clinical (haemorrhagic) or laboratory evidence of disseminated intravascular coagulation. Six patients (6/16, 37%) deceased during the induction phase from pulmonary bleeding (2 cases,days 8 and 13 respectively), intracerebral bleeding (1 case,day 6), myocardial infarction-cardiac arrest (1 case,day 5), respiratory distress syndrome secondary to ATRA syndrome (1 case, day 17), and sepsis-induced hemophagocytosis syndrome (1 case, day 38). All ten out of the 16 (63%) surviving patients achieved hematological and molecular CR and remain to date relapse-free in excellent clinical condition. The median overall survival and disease-free survival for the whole group is 25 months but the same parameters for the surviving patients is better at 42 months. Our results corroborate that in APL the AIDA protocol together with maintenance treatment is highly effective in producing sustained haematological and molecular remission. Despite this excellent antileukaemic activity, early mortality (37% in our cohort) caused chiefly by fatal bleeding and thrombotic events (four patients) limits considerably patient survival and deserves further research in order to improve long-term outcome.