Search

Background: PCSK9 (proprotein convertase subtilisin/kexin 9), mainly secreted by the liver and released into the blood, elevates plasma low-density lipoprotein cholesterol by degrading low-density lipoprotein receptor. Pleiotropic effects of PCSK9 beyond lipid metabolism have been shown. However, the direct effects of PCSK9 on platelet activation and thrombosis, and the underlying mechanisms, as well, still remain unclear.Methods: We detected the direct effects of PCSK9 on agonist-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, α-granule release, spreading, and clot retraction. These studies were complemented by in vivo analysis of FeCl3-injured mouse mesenteric arteriole thrombosis. We also investigated the underlying mechanisms. Using the myocardial infarction (MI) model, we explored the effects of PCSK9 on microvascular obstruction and infarct expansion post-MI.Results: PCSK9 directly enhances agonist-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, P-selectin release from α-granules, spreading, and clot retraction. In line, PCSK9 enhances in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model, whereas PCSK9 inhibitor evolocumab ameliorates its enhancing effects. Mechanism studies revealed that PCSK9 binds to platelet CD36 and thus activates Src kinase and MAPK (mitogen-activated protein kinase)-extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase, increases the generation of reactive oxygen species, and activates the p38MAPK/cytosolic phospholipase A2/cyclooxygenase-1/thromboxane A2 signaling pathways downstream of CD36 to enhance platelet activation, as well. Using CD36 knockout mice, we showed that the enhancing effects of PCSK9 on platelet activation are CD36 dependent. It is important to note that aspirin consistently abolishes the enhancing effects of PCSK9 on platelet activation and in vivo thrombosis. Last, we showed that PCSK9 activating platelet CD36 aggravates microvascular obstruction and promotes MI expansion post-MI.Conclusions: PCSK9 in plasma directly enhances platelet activation and in vivo thrombosis, and MI expansion post-MI, as well, by binding to platelet CD36 and thus activating the downstream signaling pathways. PCSK9 inhibitors or aspirin abolish the enhancing effects of PCSK9, supporting the use of aspirin in patients with high plasma PCSK9 levels in addition to PCSK9 inhibitors to prevent thrombotic complications. [ABSTRACT FROM AUTHOR]

Background: Severe acute respiratory syndrome corona virus 2 infection causes severe pneumonia (coronavirus disease 2019 [COVID-19]), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in patients with COVID-19.Methods: A total of 62 subjects were included in our study (n=38 patients with reverse transcriptase polymerase chain reaction-confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathologic assessment of autopsy cases, surface marker-based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions, and coagulation tests, as well.Results: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that, in COVID-19, inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. Patients with COVID-19 also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, patients severely affected with COVID-19 are characterized by excessive platelet and neutrophil activation in comparison with healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in severe acute respiratory syndrome corona virus 2 pneumonia is linked to both acute respiratory distress syndrome and systemic hypercoagulability.Conclusions: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19. [ABSTRACT FROM AUTHOR]

Background: Branched-chain amino acids (BCAAs), essential nutrients including leucine, isoleucine, and valine, serve as a resource for energy production and the regulator of important nutrient and metabolic signals. Recent studies have suggested that dysfunction of BCAA catabolism is associated with the risk of cardiovascular disease. Platelets play an important role in cardiovascular disease, but the functions of BCAA catabolism in platelets remain unknown.Methods: The activity of human platelets from healthy subjects before and after ingestion of BCAAs was measured. Protein phosphatase 2Cm specifically dephosphorylates branched-chain α-keto acid dehydrogenase and thereby activates BCAA catabolism. Protein phosphatase 2Cm-deficient mice were used to elucidate the impacts of BCAA catabolism on platelet activation and thrombus formation.Results: We found that ingestion of BCAAs significantly promoted human platelet activity (n=5; P<0.001) and arterial thrombosis formation in mice (n=9; P<0.05). We also found that the valine catabolite α-ketoisovaleric acid and the ultimate oxidation product propionyl-coenzyme A showed the strongest promotion effects on platelet activation, suggesting that the valine/α-ketoisovaleric acid catabolic pathway plays a major role in BCAA-facilitated platelet activation. Protein phosphatase 2Cm deficiency significantly suppresses the activity of platelets in response to agonists (n=5; P<0.05). Our results also suggested that BCAA metabolic pathways may be involved in the integrin αIIbβ3-mediated bidirectional signaling pathway that regulates platelet activation. Mass spectrometry identification and immunoblotting revealed that BCAAs enhanced propionylation of tropomodulin-3 at K255 in platelets or Chinese hamster ovary cells expressing integrin αIIbβ3. The tropomodulin-3 K255A mutation abolished propionylation and attenuated the promotion effects of BCAAs on integrin-mediated cell spreading, suggesting that K255 propionylation of tropomodulin-3 is an important mechanism underlying integrin αIIbβ3-mediated BCAA-facilitated platelet activation and thrombosis formation. In addition, the increased levels of BCAAs and the expression of positive regulators of BCAA catabolism in platelets from patients with type 2 diabetes mellitus are significantly correlated with platelet hyperreactivity. Lowering dietary BCAA intake significantly reduced platelet activity in ob/ob mice (n=4; P<0.05).Conclusions: BCAA catabolism is an important regulator of platelet activation and is associated with arterial thrombosis risk. Targeting the BCAA catabolism pathway or lowering dietary BCAA intake may serve as a novel therapeutic strategy for metabolic syndrome-associated thrombophilia. [ABSTRACT FROM AUTHOR]

Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role.Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro.Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbβ3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbβ3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology. [ABSTRACT FROM AUTHOR]

Background: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant.Methods: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry.Results: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation.Conclusions: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Purpose: Acute coronary syndromes and ischemic stroke are associated with arterial events involving platelets, the endothelium, and atherosclerosis. Although regular physical activity is associated with lower risk of cardiovascular events and mortality, risk is transiently increased during and immediately after participation in an acute bout of exercise. No previous study has investigated the acute impact of exercise on platelet activation and arterial function in the same participants; it is also unknown if responses are dependent on exercise modality. We hypothesized that commonly adopted, yet physiologically distinct, modalities of exercise ("aerobic" vs "resistance") have differing effects on in vivo platelet activation and conduit artery diameter. Methods: Eight apparently healthy middle-age (53.5 ± 1.6 yr) male subjects took part in four 30-min experimental interventions (aerobic exercise, resistance exercise, combined aerobic/resistance exercise, or no-exercise), in random order. Blood samples were collected, and the measurement of brachial artery diameter by ultrasound was performed before, immediately after, and 1 h after each intervention. Platelet activation was determined by the positive binding of antibodies to surface receptors exposed on activated platelets (anti-CD62P and PAC-1). Results: Brachial artery diameter increased immediately after all three exercise modalities (P < 0.001) and remained above preexercise levels 1 h after resistance exercise and after combined aerobic/resistance exercise. No changes were observed in markers of in vivo platelet activation with any experimental protocol. Conclusions: These data suggest that postexercise enhancement in arterial function may mitigate the acute impact of exercise on platelet activation. [ABSTRACT FROM AUTHOR]

Background: Integrins are heterodimeric (α/β) membrane proteins that play fundamental roles in many biological processes, for example, cell adhesion and spreading, which are important for platelet function and hemostasis. The molecular mechanism that regulates integrin activation is not completely understood.Methods and Results: Here, we show that VPS33B, a member of the Sec1/Munc18 family, binds directly to the integrin β subunit. Overexpression of VPS33B in Chinese hamster ovary cells potentiated αIIbβ3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, had normal morphology, yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose agonists were reduced in the VPS33B knockout mice. αIIbβ3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in an FeCl3-induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1-dependent pathways that lead to clot retraction and cell spreading, respectively.Conclusions: Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of arthrogryposis, renal dysfunction, and cholestasis syndrome. [ABSTRACT FROM AUTHOR]

Background--Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results--Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/ protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1β maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions--NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation. [ABSTRACT FROM AUTHOR]

Background--A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. Methods and Results--This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cy2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions--DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the role of hydrogen peroxide in promoting platelet hyperactivation and thrombosis during aging. The study uses analysis of variance (ANOVA), real-time polymerase chain reaction (PCR), and collects blood sample to determine the influence of hydrogen peroxide in thrombotic and hyperactivity of platelet in aged mice. Results show that hydrogen peroxide promotes platelet hyperactivity and thrombotic events.

The article presents study on the pathophysiology of continuous flow ventricular assist devices (CF-VADs). It offers that the CF-VAD implantation leads to the interaction of prosthetic material with blood that can be measured in terms of impact on hematology, inflammatory or immunologic parameters. It also informs that CF-VAD supported patients have increased platelet activation and leukocyte interaction.

In Panel 3H1, the platelet spreading image of wild type mice with 20 min PCSK9 treatment was a duplication of the image of CD36 -/- mice with 40 min PCSK9 treatment. DOI: 10.1161/CIR.0000000000000948 CORRECTION Correction to: PCSK9 (Proprotein Convertase Subtilisin/Kexin 9) Enhances Platelet Activation, Thrombosis, and Myocardial Infarct Expansion by Binding to Platelet CD36 In the article by Qi et al, "PCSK9 (Proprotein Convertase Subtilisin/Kexin 9) Enhances Platelet Activation, Thrombosis, and Myocardial Infarct Expansion by Binding to Platelet CD36", which published online ahead of print on September 29, 2020 and appears in the January 5, 2021 issue of the journal (Circulation. [Extracted from the article]

Introduction: Although it is known that reactive oxygen species (ROS) are generated during platelet activation and promote platelet function, the signaling mechanisms involved remain unclear. Here we investigated the role of integrin αIIbβ3 and the outside-in signaling pathway in regulating ROS during platelet activation, particularly under the shear flow present in blood. Results: Agonist-induced ROS generation under shear and activation of p47phox, the main regulator of NADPH oxidase 2 (NOX2), was significantly impaired by the integrin antagonist integrilin or knockout of β3, indicating that integrin αIIbβ3 stimulates ROS generation and p47phox activation during platelet activation. Platelet spreading on integrin αIIbβ3 ligand, fibrinogen, also generated ROS and activated p47phox, and was inhibited by knockout of β3 or the early outside-in signaling inhibitor mP6, which also blocked agonist-induced ROS production under shear. This demonstrates that the early integrin outside-in signaling pathway, mediated by Gα13-β3 interaction, is critical for ROS generation in platelets. In agreement with these results, ROS generation was defective in NOX2-/- platelets under shear and during platelet spreading, which led to defects in spreading and stable adhesion under shear. Finally, inhibition of SFKs, PI3K and Akt reduced p47phox activity during platelet activation. Conclusions: ROS generation during platelet activation is regulated by the early outside-in signaling pathway mediated by integrin αIIbβ3 and involves Gα13 and SFK/PI3K/Akt-dependent activation of p47phox and NOX2. [ABSTRACT FROM AUTHOR]

ATP-binding cassetteC4 (ABCC4) has a role in platelet activation as ABCC4 knockout mice have prolonged bleeding time and in-vivo thrombosis. ABCC4 inhibition by cilostazol reduces high on-aspirin residual platelet reactivity. ABCC4 is identified as a mediator of platelet activity in people with HIV, as they have an ABCC4-dependent hyperreactive platelet phenotype. In randomized trial no significant benefit on vascular health from low-dose aspirin in HIV patients was found. Aim of the study is verified whether in platelet with over-expression of ABCC4, the cyclic nucleotide inhibition is reduced. We evaluated the inhibitory effect of SNP, a NO donor that enhances cGMP inhibitory effect in platelets obtained from aspirinated patients (ASA-Pts; N=45) and in control population (CTR; N=47), similar for risk factors and medications, except for aspirin assumption. We also studied the effects of an ABCC4 inhibitor, cilostazol. Platelet function was evaluated by ADP-induced platelet activation (Mx%). Platelet ABCC4-expression was measured by ABCC4-mRNA levels using Q-RT-PCR. A significant reduction of SNP inhibitory effect was found in ASA-Pts compared to CTR (28±18Mx% and 14±10Mx% respectively p=0.008). The effect of ABCC4 in reducing SNP platelet inhibition is more evident in the ASA-Pts that have higher ABCC4 expression in comparison with those with low level of protein (38±22Mx% and 22±14Mx% respectively p=0.009). In vitro cilostazol treatment reduces residual platelet reactivity in ASA-Pts (41.1±15.8 Mx% vs 61.3±14.5Mx% ; p<0.0001), and enhances SNP inhibition (7.4±4.3 vs 20.6 ±10.0 p=0.0006). We demonstrate that cilostazol reduces platelet hypereactivity due to ABCC4 over expression by acting on the cyclic-nucleotide extrusion. As cilostazol is used as antiplatelet drugs the combination of aspirin plus cilostazol may be useful to reduces cardiovascular risk in HIV patients. [ABSTRACT FROM AUTHOR]

Introduction: Left ventricular assist devices (LVAD) play an increasingly important role for the medical-therapy refractory end-stage heart failure (HF) patient population, in the context of stagnant donor heart availability. Thromboembolic and hemorrhagic complications such as strokes, however, remain high, and in some cases have seen increased incidence. There are a lack of guidelines governing LVAD surgical implantation configurations, which have been shown to have a strong influence on the thrombogenicity of LVAD therapy. Methods: We use a platelet-focused approach to simulate the thrombogenicity of virtual LVAD implantations under a wide range of clinically-relevant parameters. We investigate the hemodynamic microenvironment in the LVAD inflow (left ventricle inflow cannula angulation, inflow cannula insertion depth, left ventricle size) and outflow (outflow graft anastomoses angles, outflow graft diameters and partial intermittent aortic valve opening) regions, analyzing platelet biomechanics. Rigorous statistical methods are used to analyze hundreds of thousands of platelet trajectories to characterize thrombogenic indices such as residence time (RT), shear history (SH) and shear loading (SL) for platelet activation. Results: An unfavorable hemodynamic microenvironment in the left ventricle due to malangulation of the inflow cannula predisposes platelets, to activate and agglomerate before entering the LVAD. Similarly, small left ventricles pose a markedly elevated thrombosis risk, especially in the presence of deep inflow cannula insertion depths. These intraventricular hemodynamics, coupled with non-optimal outflow graft anastomosis angles, represent an additional risk of stroke, via flow-mediated platelet activation. Thrombogenic risk can be mitigated to a significant extent by optimizing surgical configurations and patient management strategies for LVAD speed, conducive to intermittent aortic valve flow. Conclusions: The novel comprehensive simulation approach followed in this study to evaluate thrombogenicity via platelet-based metrics can be implemented in surgical implantation guidelines to reduce the rate of thromboembolic complications, improving long-term outcomes of LVAD therapy. [ABSTRACT FROM AUTHOR]

Introduction: Biological experiments revealed that glycoprotein (GP) Ibα has two binding sites for thrombin and that these binding sites are important for regulating the platelet activation and thrombus formation. Though the thrombin binding site is close to VWF binding one, the influence of thrombin on the binding of GP 1bα to von Willebrand factor (VWF) is still unknown.
Methods: We applied molecular dynamic (MD) simulation using the Chemistry at Harvard Macromolecular Mechanics (CHARMM) 36 force field to elucidate the difference in stable binding structure of GP Ibα with VWF in the presence and absence of thrombin. The calculation was done on the Ultrafast Oakforest-PACS computer using the Nanoscale Molecular Dynamics (NAMD) package. The initial structures were obtained by aligning the crystal structure of thrombin bound to exosites I and II of GP1bα to the crystal structure. The MD calculation were conducted in time step of 2 x 10-15seconds until the binding of GP Ibα and VWF in-silicobecame stable. Results: After 300 ns (150 x 106steps) of the MD calculation, the binding structure was stabilized with root mean square distance deviation within 0.5Å. The bond between GP Ibα and thrombin was apparently unchanged. However, the binding of GP Ibα to VWF was markedly different when thrombin was bound to the exosites I and II of GP Ibα compared with the binding when thrombin was absent (Fig 1). The relative position of GP Ibα to VWF in the presence of thrombin moved approximately 39Å right (in Fig 1) compared to the structure without thrombin. Conclusions: The MD simulation predicted that the stable binding structure of GP Ibα and VWF is modified by thrombin. These results suggest the additional role of thrombin for controlling thrombus formation by influencing platelet adhesion at site of vessel injury under blood flow, which is mediated exclusively by GP Ibα binding with VWF. [ABSTRACT FROM AUTHOR]

Introduction: The current anti-platelet drugs inhibit platelet function permanently and have systemic side effects including thrombocytopenia and hemorrhage. There is a significant clinical interest in developing anti-platelet therapies based on novel mechanisms. We found thymidine phosphorylase (TYMP), a platelet cytoplasmic protein, facilitates multiple agonist-induced platelet activations and enhances thrombosis. Hypothesis: Tipiracial (TPI), a potent TYMP inhibitor, is a novel and safe anti-platelet and anti-thrombotic medicine. Methods: By using platelet aggregation and adhesion assays, we examined the role of TYMP deficiency and TPI on platelet activation in vitro and thrombosis in vivo. TPI was delivered to mice via intraperitoneal injection (IP), intravenous injection (IV) or gavage (PO) and its anti-thrombotic effect were studied using a FeCl3 induced carotid artery injury thrombosis model. The potential side effects of TPI treatment on atherogenesis and hemostasis were studied on apoE-/- mice fed with high fat diet. Results: TYMP deficiency dramatically inhibited in vivo thrombosis regardless of genders. TPI dose-dependently and reversely inhibited collagen-related peptide induced wild type (WT) platelet aggregation. TYMP deficiency dramatically delayed ADP induced AKT activation. TPI significantly inhibited ADP induced WT platelet aggregation but have no effects on Tymp-/- platelets, suggesting that the inhibitory effect of TPI on platelet aggregation is TYMP dependent. Treatment of WT mice with TPI via IP (14 mg/Kg), IV (as low as 170 μg/kg) or PO (60 μg/Kg) dramatically inhibited in vivo thrombosis in WT mice. Feeding of Tymp-/-;Lyn-/- mice with 170 μg/Kg TPI did not affect in vivo thrombosis, further suggested that the inhibitory effect of TPI on thrombosis is mediated by TYMP inhibition. TYMP deficiency and inhibition of TYMP dramatically diminished platelet accumulation to the collagen-coated surface. TPI treatment did affect gain of body weight and weight of liver and visceral fat of a poE-/- mice fed with high fat diet. Area of atherosclerotic lesion was also not affected by TPI feeding. TPI treatment did not affect tail-bleeding time in all examined strains including WT, Tymp-/-;Lyn-/- and a poE-/- mice. Conclusions: This pharmacological study further demonstrated that TYMP participates in multiple platelet signaling pathways and plays a mechanistic role in regulating platelet activation and thrombosis. Tipiracil, the TYMP inhibitor, may be a novel safe anti-platelet and anti-thrombosis medicine. [ABSTRACT FROM AUTHOR]

Introduction: High circulating level of soluble protein convertase subtilisin/kexin type 9 (PCSK9) are associated to an increase risk of cardiovascular events (CVEs) in patients with atrial fibrillation (AF), especially with values > 1600pg/ml. Although a link between PCSK9 and platelet activation was recently demonstrated by in vitro experiments (Camera M, JACC 2018), no data on the in vivo relationship between PCSK9 levels and platelet activation in AF patients are available. Hypothesis: As platelet activation is mediated by NADPH oxidase activity (Nox2) (Violi F, Circulation 2012), our aim was to investigate whether PCSK9 may increase platelet function by modulating the activity of NADPH oxidase in AF. Methods: We conducted a post-hoc analysis including 31 patients with baseline PCSK9 below and 21 above the median value of 1200pg/ml from our previous prospective study (Pastori D, JACC 2018). The two groups were balanced for age and sex. In vivo platelet activation was assessed by collagen induced aggregation, platelet recruitment and plasma thromboxane A2 (TxA2) formation. As markers of oxidative stress we used sNox2-dp and plasma isoprostanes. Moreover, we performed an in vitro study where we evaluated the association of PCSK9 with Nox2 activation, evaluated by the soluble levels of sNox2-dp and plasma isoprostanes formation. For the in vitro study we used a scalar concentrations of PCSK9 as follows: 500, 1000, 2000 pg/ml. Results: In vivo study showed a significant correlation between plasma levels of PCSK9 and markers of platelet activation, such as collagen induced aggregation (R: 0.708, p<0.001), recruitment (R: 0.608, p<0.001), and plasma TxA2 (R: 0.237, p=0.002), markers of oxidative stress such as sNox2-dp (R: 0.608, p<0.001) and plasma isoprostanes (R: 0.105, p=0.02). We observed an increased level of platelet activation and oxidative stress in patients with PCSK9 levels above median compared to those below (p<0.05). In vitro study demonstrated that PCSK9 increased platelet aggregation and recruitment in a dose dependent manner. PCSK9 dose dependently induced sNox2-dp release and isoprostanes formation. PCSK9 mediated Platelet aggregation and Nox2 activity were inhibited by the specific Nox2 blocking peptide. Conclusions: We found a significant direct correlation between plasma PCSK9 levels and platelet activation in vivo and in vitro. PCSK9 dose dependently increased oxidative stress in parallel to platelet function suggesting a possible role of Nox2 in PCSK9-related platelet activation. [ABSTRACT FROM AUTHOR]