Extracellular matrix proteins in the regulation of thrombus formation.

PURPOSE OF REVIEW: Exposure of subendothelial extracellular matrix (ECM) proteins to the circulation is the key event that initiates platelet accumulation and clot formation after vessel injury. Soluble ECM proteins are also essential for support and regulation of these events. This review discusses the current understanding of ECM proteins in thrombosis and hemostasis. RECENT FINDINGS: Fibrinogen and von Willebrand factor (VWF) are considered essential for thrombosis and hemostasis. Interestingly, αIIbß3 integrin-dependent thrombus formation persists in the absence of fibrinogen and VWF, suggesting that other αIIbß3 ligand (s), likely ECM proteins, can still mediate thrombosis. Fibronectin increases in platelets of fibrinogen-deficient humans and mice. This ECM protein can rapidly deposit onto the injured vessel wall prior to platelet accumulation. By switching from the soluble to insoluble form, plasma fibronectin supports hemostasis and inhibits excessive thrombosis. Fibrin, fibronectin, VWF, vitronectin, neutrophil extracellular traps, and other ECM proteins in the blood clot form a de-facto ECM, which interacts with various types of blood cells to regulate the evolution and resolution of the clot. SUMMARY: ECM proteins are intricately involved in major steps of thrombus formation. Further investigations of ECM proteins will reveal new therapeutic targets for treatment of thrombosis and bleeding disorders. VIDEO ABSTRACT: http://links.lww.com/COH/A12

In vitro analysis of tantalum-containing mesoporous bioactive glass fibres for haemostasis.

Haemorrhage is the leading cause of battlefield deaths and second most common cause for civilian mortality worldwide. Biomaterials-based haemostatic agents are used to aid in bleeding stoppage; mesoporous bioactive glasses (MBGs) are candidates for haemostasis. Previously made Tantalum-containing MBG (Ta-MBG) powders' compositions were fabricated as electrospun fibres for haemostatic applications in the present study. The fibres were fabricated to address the challenges associated with the powder form: difficult to compress without gauze, getting washed away in profuse bleeding, generating dust in the surgical environment, and forming thick callus-difficult to remove for surgeons and painful for patients. Ta-MBGs were based on (80-x)SiO2-15CaO-5P2O5-xTa2O5 mol% compositions with x = 0 (0Ta), 0.5 (0.5Ta), 1 (1Ta), and 5 (5Ta) mol%. The present study details the fibres' in vitro analyses, elucidating their cytotoxic effects, and haemostatic capabilities and relating these observations to fibre chemistry and previously fabricated powders of the same glasses. As expected, when Ta addition is increased at the expense of silica, a new FTIR peak (non-bridging oxygen-silicon, Si-NBO) develops and Si-O-Si peaks become wider. Compared to 0Ta and 1Ta fibres, 0.5Ta show Si-O peaks with reduced intensity. The fibres had a weaker intensity of Si-NBO peaks and release fewer ions than powders. A reduced ion profile provides fibres with a stable matrix for clot formation. The ion release profile for 1Ta and 5Ta fibres was significantly lower than 0Ta and 0.5Ta fibres. Ta-MBGs were not found to be cytotoxic to primary rat fibroblasts using a methyl thiazolyl tetrazolium (MTT) assay. Furthermore, a modified activated partial thromboplastin time assay analysing the fibrin absorbance showed that the absorption increases from physiological clotting < 0Ta < 0.5Ta < 5Ta < commercial haemostat, Surgical SNoWTM, Ethicon, USA < 1Ta. Higher absorption signifies a stronger clot. It is concluded that Ta-MBG fibres can provide stable matrix for clot formation and 1Ta can potentially enhance clotting best among other Ta-MBGs.

Pure White Cell Aplasia and Immune Thrombocytopenia after Thymoma Resection: A Case Report and Review of the Literature.

We report a case of pure white cell aplasia (PWCA) postthymoma resection in a 74-year-old male presenting with a 2-week history of fevers, night sweats, and severe febrile neutropenia. His pure white cell aplasia was treated with intravenous immunoglobulin (IVIg), granulocyte colony-stimulating factor (G-CSF), prednisone, and cyclosporine with a mixed response. He also developed immune thrombocytopenia, which responded well to a short course of eltrombopag. With continued cyclosporine treatment, his platelet counts were stable after stopping eltrombopag. The patient's cyclosporine treatment was complicated by renal failure, resulting in cessation of cyclosporine. His PWCA and immune thrombocytopenia significantly worsened after stopping cyclosporine, and unfortunately, he died from multiorgan failure and sepsis.

Aspirin nonsensitivity in patients with vascular disease: Assessment by light transmission aggregometry (aspirin nonsensitivity in vascular patients).

BACKGROUND: Aspirin is a key antiplatelet therapy for the prevention of thrombotic events in patients with cardiovascular disease. Studies suggest that ≈20% of patients with cardiac disease suffer from aspirin nonsensitivity, a phenomenon characterized by the inability of 81 mg aspirin to inhibit platelet aggregation and/or prevent adverse cardiovascular events. OBJECTIVES: To investigate aspirin nonsensitivity in patients with vascular disease and assess the consequences of aspirin nonsensitivity. METHODS: One hundred fifty patients presenting to St. Michael's Hospital's outpatient clinics with evidence of vascular disease (peripheral arterial disease or carotid artery stenosis) and a previous prescription of 81 mg of aspirin were recruited in this study. Light transmission aggregometry with arachidonic acid induction was used to determine sensitivity to aspirin. Patients with a maximum aggregation ≥20% in response to arachidonic acid were considered aspirin nonsensitive, as per previous studies. RESULTS: Of the 150 patients recruited, 36 patients (24%) were nonsensitive to 81 mg of aspirin. Of these 36 nonsensitive patients, 30 patients provided a urine sample for urine salicyluric acid analysis (a major metabolite of aspirin). Urine analysis demonstrated that 14 patients were compliant and 16 were noncompliant with their aspirin therapy. Major adverse cardiovascular events and major adverse limb events were significantly higher in the nonsensitive patients compared to sensitive patients (hazard ratio, 3.68; P < 0.001). CONCLUSION: These data highlight the high prevalence of aspirin nonsensitivity and noncompliance in patients with vascular disease and emphasizes the urgent need for improved medical management options for this patient population.

Plateletworks® as a Point-of-Care Test for ASA Non-Sensitivity.

Aspirin (ASA) therapy is proven to be effective in preventing adverse cardiovascular events; however, up to 30% of patients are non-sensitive to their prescribed ASA dosage. In this pilot study, we demonstrated, for the first time, how ASA non-sensitivity can be diagnosed using Plateletworks®, a point-of-care platelet function test. Patients prescribed 81 mg of ASA were recruited in a series of two successive phases-a discovery phase and a validation phase. In the discovery phase, a total of 60 patients were recruited to establish a cut-off point (COP) for ASA non-sensitivity using Plateletworks®. Each sample was simultaneously cross-referenced with a light transmission aggregometer (LTA). Our findings demonstrated that >52% maximal platelet aggregation using Plateletworks® had a sensitivity, specificity, and likelihood ratio of 80%, 70%, and 2.67, respectively, in predicting ASA non-sensitivity. This COP was validated in a secondary cohort of 40 patients prescribed 81 mg of ASA using Plateletworks® and LTA. Our data demonstrated that our established COP had a 91% sensitivity and 69% specificity in identifying ASA non-sensitivity using Plateletworks®. In summary, Plateletworks® is a point-of-care platelet function test that can appropriately diagnose ASA non-sensitive patients with a sensitivity exceeding 80%.

Personalization of Aspirin Therapy Ex Vivo in Patients with Atherosclerosis Using Light Transmission Aggregometry.

Acetylsalicylic acid (ASA), also known as aspirin, appears to be ineffective in inhibiting platelet aggregation in 20-30% of patients. Light transmission aggregometry (LTA) is a gold standard platelet function assay. In this pilot study, we used LTA to personalize ASA therapy ex vivo in atherosclerotic patients. Patients were recruited who were on 81 mg ASA, presenting to ambulatory clinics at St. Michael's Hospital (n = 64), with evidence of atherosclerotic disease defined as clinical symptoms and diagnostic findings indicative of symptomatic peripheral arterial disease (PAD), with an ankle brachial index (ABI) of <0.9 (n = 52) or had diagnostic features of asymptomatic carotid arterial stenosis (CAS), with >50% stenosis of internal carotid artery on duplex ultrasound (n = 12). ASA compliance was assessed via multisegmented injection-capillary electrophoresis-mass spectrometry based on measuring the predominant urinary ASA metabolite, salicyluric acid. LTA with arachidonic acid was used to test for ASA sensitivity. Escalating ASA dosages of 162 mg and 325 mg were investigated ex vivo for ASA dose personalization. Of the 64 atherosclerotic patients recruited, 8 patients (13%) were non-compliant with ASA. Of ASA compliant patients (n = 56), 9 patients (14%) were non-sensitive to their 81 mg ASA dosage. Personalizing ASA therapy in 81 mg ASA non-sensitive patients with escalating dosages of ASA demonstrated that 6 patients became sensitive to a dosage equivalent to 162 mg ASA and 3 patients became sensitive to a dosage equivalent to 325 mg ASA. We were able to personalize ASA dosage ex vivo in all ASA non-sensitive patients with escalating dosages of ASA within 1 h of testing.

Coenzyme Q10 attenuates platelet integrin αIIbß3 signaling and platelet hyper-reactivity in ApoE-deficient mice.

Coenzyme Q10 (CoQ10) exists in a wide variety of foods and has promising cardiovascular benefits. However, its effects on platelets and integrin αIIbß3 signaling during atherosclerosis have not been previously explored. Here, apolipoprotein E-deficient (ApoE-/-) mice were fed a standard diet, high-fat diet (HFD) or CoQ10-supplemented HFD for 12 weeks. We found that CoQ10 supplementation in ApoE-/- mice significantly alleviated formation of HFD-induced atherosclerotic lesions, and attenuated platelet hyper-aggregation and granule secretion, including CD62P, CD63 and CD40 ligand (CD40L) expression and platelet factor-4, ß-thromboglobulin and activation normal T cell expressed and secreted (CCL5) release. CoQ10 supplementation decreased soluble fibrinogen and JON/A binding to αIIbß3 on activated platelets, indicating that αIIbß3-mediated inside-out signaling was attenuated. Additionally, CoQ10 down-regulated platelet αIIbß3 outside-in signaling including decreasing phosphorylation of the ß3 intracellular tail, cellular and sarcoma tyrosine-protein kinase (c-Src), and myosin light chain (MLC), and consistently attenuating platelet spreading and clot retraction. Importantly, platelet-monocyte aggregation that was primarily mediated by αIIbß3 and can be blocked using an αIIbß3-specific antagonist tirofiban was also markedly diminished by CoQ10. Thus, CoQ10 supplementation attenuates platelet hyper-reactivity via down-regulating both αIIbß3 inside-out and outside-in signaling, which may play important preventive roles in atherothrombosis.

The effect of tantalum incorporation on the physical and chemical properties of ternary silicon-calcium-phosphorous mesoporous bioactive glasses.

Synthesis and characterization of the first mesoporous bioactive glasses (MBGs) containing tantalum are reported here, along with their potential application as hemostats. Silica MBGs were synthesized using with the molar composition of (80-x)% Si, 15% Ca, 5% P, and x% Ta. It was found that incorporation of >1 mol % Ta into the MBGs changes their physical and chemical properties. Increasing Ta content from 0 to 10 mol % causes a decrease in the surface area and pore volume of ~20 and ~35%, respectively. This is due to the increase in nonbridging oxygens and mismatch of thermal expansion coefficient which created discontinuities in the ordered channel structure. However, the effect is not significant on the amount of ions (Si, Ca, P, and Ta) released, from the sample into deionized water, for short durations (<60 min). In a mouse tail-cut model, a significant decrease in bleeding time (≥50% of average bleeding time) was found for Ta-MBGs compared to having no treatment, Arista, and MBG without Ta. Further studies are proposed to determine the mechanism of Ta involvement with the hemostatic process. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2229-2237, 2019.

Coenzyme Q10 Upregulates Platelet cAMP/PKA Pathway and Attenuates Integrin αIIbß3 Signaling and Thrombus Growth.

SCOPE: Platelet integrin αIIbß3 is the key mediator of atherothrombosis. Supplementation of coenzyme Q10 (CoQ10), a fat-soluble molecule that exists in various foods, exerts protective cardiovascular effects. This study aims to investigate whether and how CoQ10 acts on αIIbß3 signaling and thrombosis, the major cause of cardiovascular diseases. METHODS AND RESULTS: Using a series of platelet functional assays in vitro, it is demonstrated that CoQ10 reduces human platelet aggregation, granule secretion, platelet spreading, and clot retraction. It is further demonstrated that CoQ10 inhibits platelet integrin αIIbß3 outside-in signaling. These inhibitory effects are mainly mediated by upregulating cAMP/PKA pathway, where CoQ10 stimulates the A2A adenosine receptor and decreases phosphodiesterase 3A phosphorylation. Moreover, CoQ10 attenuates murine thrombus growth and vessel occlusion in a ferric chloride (FeCl3 )-induced thrombosis model in vivo. Importantly, the randomized, double-blind, placebo-controlled clinical trial in dyslipidemic patients demonstrates that 24 weeks of CoQ10 supplementation increases platelet CoQ10 concentrations, enhances the cAMP/PKA pathway, and attenuates αIIbß3 outside-in signaling, leading to decreased platelet aggregation and granule release. CONCLUSION: Through upregulating the platelet cAMP/PKA pathway, and attenuating αIIbß3 signaling and thrombus growth, CoQ10 supplementation may play an important protective role in patients with risks of cardiovascular diseases.

Cyanidin-3-o-ß-Glucoside Induces Megakaryocyte Apoptosis via PI3K/Akt- and MAPKs-Mediated Inhibition of NF-κB Signalling.

Apoptotic-like phase is an essential step in thrombopoiesis from megakaryocytes. Anthocyanins are natural flavonoid pigments that possess a wide range of biological activities, including protection against cardiovascular diseases and induction of tumour cell apoptosis. We investigated the effects and underlying mechanisms of cyanidin-3-o-ß-glucoside (Cy-3-g, the major bioactive compound in anthocyanins) on the apoptosis of human primary megakaryocytes and Meg-01 cell line in vitro. We found that Cy-3-g dose-dependently increased the dissipation of the mitochondrial membrane potential, caspase-9 and caspase-3 activity in megakaryocytes from patients with newly diagnosed acute myeloid leukaemia but not in those from healthy volunteers. In Meg-01 cells, Cy-3-g regulated the distribution of Bak, Bax and Bcl-xL proteins in the mitochondria and cytosol, subsequently increasing cytochrome c release and stimulating caspase-9 and caspase-3 activation and phosphatidylserine exposure. However, Cy-3-g did not exert significant effects on factor-associated suicide (Fas), Fas ligand, caspase-8 or Bid expression. Cy-3-g inhibited nuclear factor kappa B (NF-κB) p65 activation by down-regulating inhibitor of NF-κB kinase (IKK)α and IKKß expression, followed by the inhibition of inhibitor of NF-κB (IκB)α phosphorylation and degradation and subsequent inhibition of the translocation of the p65 sub-unit into the nucleus, and finally stimulating caspase-3 activation and phosphatidylserine exposure. The inhibitory effect of Cy-3-g on NF-κB activation was mediated by the activation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinase (MAPK) and the inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling. U0126 (Erk1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and 740 Y-P (PI3K agonist) significantly reversed Cy-3-g-reduced phosphorylation of p65. Taken together, our data indicate that Cy-3-g induces megakaryocyte apoptosis via the inhibition of NF-κB signalling, which may play important roles in regulating thrombopoiesis.

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.

Platelets, immune-mediated thrombocytopenias, and fetal hemorrhage.

Platelets are small versatile blood cells generated from megakaryocytes in the bone marrow and cleared in the reticuloendothelial system. Platelet accumulation (adhesion and aggregation) at the site of injury has been considered the first wave of hemostasis. Interestingly, although fibrinogen and von Willebrand factor (VWF) are documented to be essential for hemostasis, fibrinogen/VWF-independent platelet aggregation and thrombosis still occur. Following platelet activation and phosphatidylserine expression, platelets also contribute to cell-based thrombin generation and blood coagulation - the second wave of hemostasis. Most recently, deposition of fibronectin and other plasma proteins onto the injured vessel wall was identified as a "protein wave" of hemostasis, in which platelets may release their granule proteins and thus also contribute to this very early hemostatic event. Due to the central roles of platelets in hemostasis, excessive platelet clearance may lead to bleeding disorders as observed in auto- and alloimmune-mediated thrombocytopenias. In this review, we will introduce several new pathways of thrombosis and hemostasis as well as antibody Fc-independent platelet clearance, which may play an important role in immune-mediated thrombocytopenias. We will also discuss the roles of platelets in fetal hemostasis that may deserve further investigation.

Platelets in hemostasis and thrombosis: Novel mechanisms of fibrinogen-independent platelet aggregation and fibronectin-mediated protein wave of hemostasis.

Platelets are small anucleate cells generated from megakaryocytes in the bone marrow. Although platelet generation, maturation, and clearance are still not fully understood, significant progress has been made in the last 1-2 decades. In blood circulation, platelets can quickly adhere and aggregate at sites of vascular injury, forming the platelet plug (i.e. the first wave of hemostasis). Activated platelets can also provide negatively charged phosphatidylserinerich membrane surface that enhances cell-based thrombin generation, which facilitates blood coagulation (i.e. the second wave of hemostasis). Platelets therefore play central roles in hemostasis. However, the same process of hemostasis may also cause thrombosis and vessel occlusion, which are the most common mechanisms leading to heart attack and stroke following ruptured atherosclerotic lesions. In this review, we will introduce the classical mechanisms and newly discovered pathways of platelets in hemostasis and thrombosis, including fibrinogen-independent platelet aggregation and thrombosis, and the plasma fibronectin-mediated "protein wave" of hemostasis that precedes the classical first wave of hemostasis. Furthermore, we briefly discuss the roles of platelets in inflammation and atherosclerosis and the potential strategies to control atherothrombosis.