ResearchPad - platelet-aggregation https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A three-dimensional phase-field model for multiscale modeling of thrombus biomechanics in blood vessels]]> https://www.researchpad.co/article/elastic_article_14644 Thromboembolism is associated with detachment of small thrombus pieces from the bulk in the blood vessel. These detached pieces, also known as emboli, travel through the blood flow and may block other vessels downstream, e.g., they may plug the deep veins of the leg, groin or arm, leading to venous thromboembolism (VTE). VTE is a significant cause of morbidity and mortality and it affects more than 900,000 people in the United States and result in approximately 100,000 deaths every year. Mechanical interaction between flowing blood and a thrombus is crucial in determining the deformation of the thrombus and the possibility of releasing emboli. In this study, we develop a phase-field model that is capable of describing the structural properties of a thrombus and its biomechanical properties under different blood flow conditions. Moreover, we combine this thrombus model with a particle-based model which simulates the initiation of the thrombus. This combined framework is the first computational study to simulate the development of a thrombus from platelet aggregation to its subsequent viscoelastic responses to various shear flows. Informed by clinical data, this framework can be used to predict the risk of diverse thromboembolic events under physiological and pathological conditions.

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<![CDATA[Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome]]> https://www.researchpad.co/article/5c58d60ed5eed0c4840314e3

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation.

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<![CDATA[A simplified flow cytometric method for detection of inherited platelet disorders—A comparison to the gold standard light transmission aggregometry]]> https://www.researchpad.co/article/5c52184bd5eed0c484797a80

Background

Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score).

Methods

The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score.

Results

A two-by-two analysis using Cohen’s kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant.

Conclusions

The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.

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<![CDATA[Aspirin treatment does not increase microhemorrhage size in young or aged mice]]> https://www.researchpad.co/article/5c390bccd5eed0c48491e622

Microhemorrhages are common in the aging brain and are thought to contribute to cognitive decline and the development of neurodegenerative diseases, such as Alzheimer’s disease. Chronic aspirin therapy is widespread in older individuals and decreases the risk of coronary artery occlusions and stroke. There remains a concern that such aspirin usage may prolong bleeding after a vessel rupture in the brain, leading to larger bleeds that cause more damage to the surrounding tissue. Here, we aimed to understand the influence of aspirin usage on the size of cortical microhemorrhages and explored the impact of age. We used femtosecond laser ablation to rupture arterioles in the cortex of both young (2–5 months old) and aged (18–29 months old) mice dosed on aspirin in their drinking water and measured the extent of penetration of both red blood cells and blood plasma into the surrounding tissue. We found no difference in microhemorrhage size for both young and aged mice dosed on aspirin, as compared to controls (hematoma diameter = 104 +/- 39 (97 +/- 38) μm in controls and 109 +/- 25 (101 +/- 28) μm in aspirin-treated young (aged) mice; mean +/- SD). In contrast, young mice treated with intravenous heparin had an increased hematoma diameter of 136 +/- 44 μm. These data suggest that aspirin does not increase the size of microhemorrhages, supporting the safety of aspirin usage.

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<![CDATA[Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets]]> https://www.researchpad.co/article/5989da00ab0ee8fa60b73b74

Background

Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied.

Objectives

Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation.

Results

In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets.

Conclusion

In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.

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<![CDATA[Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?]]> https://www.researchpad.co/article/5989dadaab0ee8fa60bb97f2

Background

Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001.

Methods and results

ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y1 and P2Y12. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y12 pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used.

Conclusions

We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y12 antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.

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<![CDATA[Hemostatic function to regulate perioperative bleeding in patients undergoing spinal surgery: A prospective observational study]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be10e8

Although bleeding is a common complication of surgery, routine laboratory tests have been demonstrated to have a low ability to predict perioperative bleeding. Better understanding of hemostatic function during surgery would lead to identification of high-risk patients for bleeding. Here, we aimed to elucidate hemostatic mechanisms to determine perioperative bleeding. We prospectively enrolled 104 patients undergoing cervical spinal surgery without bleeding diathesis. Blood sampling was performed just before the operation. Volumes of perioperative blood loss were compared with the results of detailed laboratory tests assessing primary hemostasis, secondary hemostasis, and fibrinolysis. Platelet aggregations induced by several agonists correlated with each other, and only two latent factors determined inter-individual difference. Platelet aggregability independently determined perioperative bleeding. We also identified low levels of plasminogen-activator inhibitor-1 (PAI-1) and α2-plasmin inhibitor to be independent risk factors for intraoperative and postoperative bleeding, respectively. Most important independent factor to determine postoperative bleeding was body weight. Of note, obese patients with low levels of PAI-1 became high-risk patients for bleeding during surgery. Our data suggest that bleeding after surgical procedure may be influenced by inter-individual differences of hemostatic function including platelet function and fibrinolysis, even in the patients without bleeding diathesis.

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<![CDATA[RGS10 Negatively Regulates Platelet Activation and Thrombogenesis]]> https://www.researchpad.co/article/5989db10ab0ee8fa60bcc140

Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.

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<![CDATA[Serum Metabolomics Reveals Serotonin as a Predictor of Severe Dengue in the Early Phase of Dengue Fever]]> https://www.researchpad.co/article/5989da01ab0ee8fa60b74070

Effective triage of dengue patients early in the disease course for in- or out-patient management would be useful for optimal healthcare resource utilization while minimizing poor clinical outcome due to delayed intervention. Yet, early prognosis of severe dengue is hampered by the heterogeneity in clinical presentation and routine hematological and biochemical measurements in dengue patients that collectively correlates poorly with eventual clinical outcome. Herein, untargeted liquid-chromatography mass spectrometry metabolomics of serum from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) in the febrile phase (<96 h) was used to globally probe the serum metabolome to uncover early prognostic biomarkers of DHF. We identified 20 metabolites that are differentially enriched (p<0.05, fold change >1.5) in the serum, among which are two products of tryptophan metabolism–serotonin and kynurenine. Serotonin, involved in platelet aggregation and activation decreased significantly, whereas kynurenine, an immunomodulator, increased significantly in patients with DHF, consistent with thrombocytopenia and immunopathology in severe dengue. To sensitively and accurately evaluate serotonin levels as prognostic biomarkers, we implemented stable-isotope dilution mass spectrometry and used convalescence samples as their own controls. DHF serotonin was significantly 1.98 fold lower in febrile compared to convalescence phase, and significantly 1.76 fold lower compared to DF in the febrile phase of illness. Thus, serotonin alone provided good prognostic utility (Area Under Curve, AUC of serotonin = 0.8). Additionally, immune mediators associated with DHF may further increase the predictive ability than just serotonin alone. Nine cytokines, including IFN-γ, IL-1β, IL-4, IL-8, G-CSF, MIP-1β, FGF basic, TNFα and RANTES were significantly different between DF and DHF, among which IFN-γ ranked top by multivariate statistics. Combining serotonin and IFN-γ improved the prognosis performance (AUC = 0.92, sensitivity = 77.8%, specificity = 95.8%), suggesting this duplex panel as accurate metrics for the early prognosis of DHF.

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<![CDATA[The Ratio of ADP- to TRAP-Induced Platelet Aggregation Quantifies P2Y12-Dependent Platelet Inhibition Independently of the Platelet Count]]> https://www.researchpad.co/article/5989db39ab0ee8fa60bd42e1

Objective

This study aimed to assess the association of clinical factors with P2Y12-dependent platelet inhibition as monitored by the ratio of ADP- to TRAP-induced platelet aggregation and conventional ADP-induced aggregation, respectively.

Background

Controversial findings to identify and overcome high platelet reactivity (HPR) after coronary stent-implantation and to improve clinical outcome by tailored anti-platelet therapy exist. Monitoring anti-platelet therapy ex vivo underlies several confounding parameters causing that ex vivo platelet aggregation might not reflect in vivo platelet inhibition.

Methods

In a single centre observational study, multiple electrode aggregometry was performed in whole blood of patients after recent coronary stent-implantation. Relative ADP-induced aggregation (r-ADP-agg) was defined as the ratio of ADP- to TRAP- induced aggregation reflecting the individual degree of P2Y12-mediated platelet reactivity.

Results

Platelet aggregation was assessed in 359 patients. Means (± SD) of TRAP-, ADP-induced aggregation and r-ADP-agg were 794 ± 239 AU*min, 297 ± 153 AU*min and 37 ± 14%, respectively. While ADP- and TRAP-induced platelet aggregation correlated significantly with platelet count (ADP: r = 0.302; p<0.001; TRAP: r = 0.509 p<0.001), r-ADP-agg values did not (r = -0.003; p = 0.960). These findings were unaltered in multivariate analyses adjusting for a range of factors potentially influencing platelet aggregation. The presence of an acute coronary syndrome and body weight were found to correlate with both ADP-induced platelet aggregation and r-ADP-agg.

Conclusion

The ratio of ADP- to TRAP-induced platelet aggregation quantifies P2Y12-dependent platelet inhibition independently of the platelet count in contrast to conventional ADP-induced aggregation. Furthermore, r-ADP-agg was associated with the presence of an acute coronary syndrome and body weight as well as ADP-induced aggregation. Thus, the r-ADP-agg is a more valid reflecting platelet aggregation and potentially prognosis after coronary stent-implantation in P2Y12-mediated HPR than conventional ADP-induced platelet aggregation.

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<![CDATA[Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation]]> https://www.researchpad.co/article/5989da4cab0ee8fa60b8d05b

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

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<![CDATA[Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation]]> https://www.researchpad.co/article/5989db42ab0ee8fa60bd723d

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium.

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<![CDATA[Repercussion of Megakaryocyte-Specific Gata1 Loss on Megakaryopoiesis and the Hematopoietic Precursor Compartment]]> https://www.researchpad.co/article/5989da6bab0ee8fa60b92eb6

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.

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<![CDATA[Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets]]> https://www.researchpad.co/article/5989db3bab0ee8fa60bd4ae3

Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes.

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<![CDATA[The clinical efficacy and safety evaluation of ticagrelor for acute coronary syndrome in general ACS patients and diabetic patients: A systematic review and meta-analysis]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdfe57

Objective

In this study, a systematic evaluation was conducted to estimate the efficacy and safety of ticagrelor for treating acute coronary syndrome (ACS) in general ACS patients and a diabetes mellitus (DM) group.

Methods

A search of PubMed, Cochrane Central Register of Controlled Trials, Web of Science, CNKI databases was conducted to analyze relevant randomized controlled trails (RCTs) of ticagrelor treating ACS during 2007 to 2015. Article screening, quality accessing and data extracting was independently undertaken by two reviewers. A meta-analysis was performed to clarify the efficacy and safety of ticagrelor in general ACS patients, and a meta-regression analysis was taken to demonstrate the efficacy and safety of ticagrelor in DM patients compared with general ACS patients.

Result

Twenty-two studies with 35004 participants were included. The meta-analysis result implicated that ticagrelor could: 1) reduce the incidence of the composite endpoint [OR = 0.83, 95%CI (0.77, 0.90), P<0.00001] and the incidence of myocardial infarction [OR = 0.81, 95%CI (0.74, 0.89), P = 0.0001]; 2) not statistically reduce the incidence of cardiovascular death, the incidence of stroke and the incidence of bleeding events; 3) increase the incidence of dyspnea [OR = 1.90, 95%CI (1.73, 2.08), P<0.00001] compared with clopidogrel. Meanwhile, compared with prasugrel, ticagrelor could 1) reduce the platelet reactivity of patients at maintenance dose [MD = -44.59, 95%CI (-59.16, -30.02), P<0.00001]; 2) not statistically reduce the incidence of cardiovascular death, the platelet reactivity of patients 6 hours or 8 hours after administration, or the incidence of bleeding events; 3) induce the incidence of dyspnea [OR = 13.99, 95%CI (2.58, 75.92), P = 0.002]. Furthermore, the result of meta-regression analysis implicated that there was a positive correlation between DM patients and the platelet reactivity of patients 6 hours and 8 hours after administration, but there was no obvious correlation between DM patients and general ACS patients in other endpoints.

Conclusion

Ticagrelor could reduce the incidence of composite endpoint of cardiovascular death, myocardial infarction and stroke as well as platelet reactivity in DM patients with ACS, while not increasing the risk of bleeding. Because there are differences in platelet reactivity between DM patients and general ACS patients, we suggest that caution is needed when using ticagrelor in clinical applications.

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<![CDATA[Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb9c

Introduction

Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions.

Methods

We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets.

Results

In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated.

Conclusions

Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.

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<![CDATA[Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcd02

Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-65PRYH and 48ARGDWN-65PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48ARGDWN-65PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbβ3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-π interaction with the β3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.

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<![CDATA[Wdr1-Dependent Actin Reorganization in Platelet Activation]]> https://www.researchpad.co/article/5989d9e2ab0ee8fa60b69ddd

In resting platelets, the integrin αIIbβ3 is present in a low-affinity “bent” state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a “swung-out” conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation.

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<![CDATA[Toll-Like Receptor Signalling Is Not Involved in Platelet Response to Streptococcus pneumoniae In Vitro or In Vivo]]> https://www.researchpad.co/article/5989da75ab0ee8fa60b964f1

Streptococcus (S.) pneumoniae strains vary considerably in their ability to cause invasive disease in humans, which is at least in part determined by the capsular serotype. Platelets have been implicated as sentinel cells in the circulation for host defence. One of their utensils for this function is the expression of Toll-like receptors (TLRs). We here aimed to investigate platelet response to S. pneumoniae and a role for TLRs herein. Platelets were stimulated using four serotypes of S. pneumonia including an unencapsulated mutant strain. In vitro aggregation and flow cytometry assays were performed using blood of healthy volunteers, or blood of TLR knock out and WT mice. For in vivo pneumonia experiments, platelet specific Myd88 knockout (Plt-Myd88-/-) mice were used. We found that platelet aggregation was induced by unencapsulated S. pneumoniae only. Whole blood incubation with all S. pneumoniae serotypes tested resulted in platelet degranulation and platelet-leukocyte complex formation. Platelet activation was TLR independent, as responses were not inhibited by TLR blocking antibodies, not induced by TLR agonists and were equally induced in wild-type and Tlr2-/-, Tlr4-/-, Tlr2/4-/-, Tlr9-/- and Myd88-/- blood. Plt-Myd88-/- and control mice displayed no differences in bacterial clearance or immune response to pneumonia by unencapsulated S. pneumoniae. In conclusion, S. pneumoniae activates platelets through a TLR-independent mechanism that is impeded by the bacterial capsule. Additionally, platelet MyD88-dependent TLR signalling is not involved in host defence to unencapsulated S. pneumoniae in vivo.

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<![CDATA[RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets]]> https://www.researchpad.co/article/5989db46ab0ee8fa60bd8863

Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively.

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