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ABSTRACT: Stimulated endothelial cells (EC) assume an activated phenotype with pro-inflammatory and prothrombotic features, requiring new gene and protein expression. New protein synthesis in activated EC is largely regulated by transcriptional events controlled by a variety of transcription factors. However, post-transcriptional control of gene expression also influences phenotype and allows the cell to alter protein expression in a faster and more direct way than is typically possible with transcriptional mechanisms. We sought to demonstrate that post-transcriptional control of gene expression occurs during EC activation. Using thrombin-activated EC and a high-throughput, microarray-based approach, we identified a number of gene products that may be regulated through post-transcriptional mechanisms, including the AP-1 transcription factor JunB. Using polysome profiling, cytoplasts and other standard cell biologic techniques, JunB is shown to be regulated at a post-transcriptional level during EC activation. In activated EC, the AP-1 transcription factor JunB, is regulated on a post-transcriptional level. Signal-dependent control of translation may regulate transcription factor expression and therefore subsequent transcriptional events in stimulated EC. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Biochemistry 01/2013; · 2.87 Impact Factor
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Bjoern F Kraemer, Robert A Campbell,
Hansjörg Schwertz,
Zechariah G Franks,
Adriana Vieira de Abreu,
Katharina Grundler,
Benjamin T Kile,
Bijaya K Dhakal,
Matthew T Rondina,
Walter H A Kahr,
Matthew A Mulvey,
Robert C Blaylock,
Guy A Zimmerman,
Andrew S Weyrich
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ABSTRACT: Bacteria can enter the bloodstream in response to infectious insults, during surgery, or when catheters enter arteries or veins. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of activated platelets. Here, we demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl x(L), an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl x(L) protein in platelets. Bcl x(L) protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli (E. coli) that produce the pore-forming toxin α-hemolysin, a response that is markedly attenuated when the gene is deleted from E. coli. We also found that non-pathogenic E. coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl x(L) protein in platelets as do clinical Staphylococcus aureus (S. aureus) isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl x(L) protein degradation in platelets co-incubated with pathogenic E. coli including α-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which gram-negative and positive pathogens might cause thrombocytopenia in patients with bloodstream infections.
Blood 10/2012; · 9.90 Impact Factor
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Clinton F Jones, Robert A Campbell,
Amanda E Brooks,
Shoeleh Assemi,
Soheyl Tadjiki,
Giridhar Thiagarajan,
Cheyanne Mulcock,
Andrew S Weyrich,
Benjamin D Brooks,
Hamidreza Ghandehari,
David W Grainger
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ABSTRACT: Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood-contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated by electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regard to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.
ACS Nano 10/2012; · 10.77 Impact Factor
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Clinton F Jones, Robert A Campbell,
Zechariah Franks,
Christopher C Gibson,
Giridhar Thiagarajan,
Adriana Vieira-de-Abreu,
Sivaprasad Sukavaneshvar,
S Fazal Mohammad,
Dean Y Li,
Hamidreza Ghandehari,
Andrew S Weyrich,
Benjamin D Brooks,
David W Grainger
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ABSTRACT: Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.
Molecular Pharmaceutics 04/2012; 9(6):1599-611. · 4.78 Impact Factor
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ABSTRACT: Platelets are anucleate cytoplasts that circulate in the bloodstream for approximately 9-11 days. Because they lack nuclei, platelets were considered incapable of protein synthesis. However, studies over the last decade have revealed that platelets use a variety of translational control pathways to synthesize proteins.A variety of protocols can be employed to assess protein synthesis by platelets. These protocols are scattered throughout the literature and, more often than not, lack critical details. In this chapter, we thoroughly outline methods used in our laboratory to assess protein synthesis by platelets.
Methods in molecular biology (Clifton, N.J.) 01/2012; 788:141-53.
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Bjoern F Kraemer, Robert A Campbell,
Hansjörg Schwertz,
Mark J Cody,
Zechariah Franks,
Neal D Tolley,
Walter H A Kahr,
Stephan Lindemann,
Peter Seizer,
Christian C Yost,
Guy A Zimmerman,
Andrew S Weyrich
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ABSTRACT: Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.
PLoS Pathogens 11/2011; 7(11):e1002355. · 9.13 Impact Factor
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ABSTRACT: Platelets are chief effector cells in hemostasis. In addition, however, their specializations include activities and intercellular interactions that make them key effectors in inflammation and in the continuum of innate and adaptive immunity. This review focuses on the immune features of human platelets and platelets from experimental animals and on interactions between inflammatory, immune, and hemostatic activities of these anucleate but complex and versatile cells. The experimental findings and evidence for physiologic immune functions include previously unrecognized biologic characteristics of platelets and are paralleled by new evidence for unique roles of platelets in inflammatory, immune, and thrombotic diseases.
Seminars in Immunopathology 08/2011; 34(1):5-30. · 6.27 Impact Factor
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ABSTRACT: The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298Q-FVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice.
We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis.
Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeak(tPA)) and increased the area under the clot formation curve (AUC(tPA)). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeak(tPA), and AUC(tPA) as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa.
These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity.
Thrombosis Research 05/2011; 128(6):570-6. · 2.44 Impact Factor
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ABSTRACT: Stroke is a common and often fatal event, and, in survivors, it is accompanied by a high risk of recurrence. Ischemic stroke is associated with abnormal platelet activity and thrombus formation. In addition to their roles in the development of acute thrombi, platelets serve as a bridge for leukocytes within the vasculature. Myeloid leukocytes are critical mediators of atherosclerosis and atherothrombosis. Interactions between platelets and leukocytes foster an inflammatory and thrombotic milieu that influences lesion progression, facilitates plaque rupture, and triggers thrombus formation and embolization. Accordingly, antiplatelet agents, including aspirin, dipyridamole, and clopidogrel, are recommended therapies for most patients with a history of stroke. In addition to mitigating thrombosis, antiplatelet drugs have direct and indirect effects on inflammation, which may translate to enhanced clinical efficacy.
Annals of the New York Academy of Sciences 10/2010; 1207:11-7. · 3.15 Impact Factor
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Thrombosis and Haemostasis 09/2010; 104(6):1281-4. · 5.04 Impact Factor
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Arteriosclerosis Thrombosis and Vascular Biology 01/2010; 30(1):7-8. · 6.37 Impact Factor
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Blood 12/2009; 114(25):5113-4. · 9.90 Impact Factor
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ABSTRACT: Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between beta3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.
Blood 10/2009; 114(23):4886-96. · 9.90 Impact Factor
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ABSTRACT: Thrombin concentration modulates fibrin structure and fibrin structure modulates clot stability; however, the impact of localized, cell surface-driven in situ thrombin generation on fibrin structure and stability has not previously been evaluated.
Human fibroblasts were incubated with factors Xa, Va, prothrombin and fibrinogen, or plasma. Fibrin formation, structure, and lysis were examined using laser scanning confocal microscopy and transmission electron microscopy. In situ thrombin generation on the cell surface produced clots with a significantly denser fiber network in a 10-microm region proximal versus distal to (40 to 50 microm) the cell surface. This morphology was not altered by addition of integrin-blocking RGDS peptide and was not apparent in clots made by exogenous thrombin addition, suggesting that spatial morphology was dictated predominantly by localized thrombin generation on the fibroblast surface. The fibrin network lysed more rapidly distal versus proximal to the cell surface, suggesting that the structural heterogeneity of the clot affected its fibrinolytic stability.
In situ thrombin generation on the cell surface modulates the three-dimensional structure and stability of the clot. Thrombus formation in vivo may reflect the ability of the local cell population to support thrombin generation and, therefore, the three-dimensional structure and stability of the fibrin network.
Arteriosclerosis Thrombosis and Vascular Biology 01/2009; 28(12):2247-54. · 6.37 Impact Factor
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ABSTRACT: Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.
Transfusion and Apheresis Science 03/2008; 38(1):15-23. · 1.25 Impact Factor
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ABSTRACT: Recombinant activated factor VII (NovoSeven, rFVIIa) is used to abrogate bleeding in haemophiliacs with inhibitors and is hypothesised to work by increasing activated factor X generation on the platelet surface. We hypothesised that rFVIIa activity could be increased by the co-addition of platelet procoagulant surface. This study characterised the ability of a rehydrated, lyophilised (RL) platelet preparation to increase rFVIIa activity in haemophilic conditions. RL platelets supported thrombin generation in the presence of factors VIII and IX but, in the absence of factors VIII and IX, thrombin generation was significantly reduced. RL platelets supported rFVIIa-mediated thrombin generation in a rFVIIa-concentration dependent manner. In a cell-based in vitro model of haemophilia, the presence of RL platelets increased the rFVIIa-dependent thrombin generation rate 2.8-fold compared with rFVIIa alone. Similarly, the addition of RL platelets plus rFVIIa to the in vitro model of haemophilia and to haemophilic platelet-rich plasma shortened the onset of clot formation and increased clot stability in a fibrinolytic environment versus rFVIIa alone. These results suggest that RL platelets can support rFVIIa-mediated thrombin generation, and that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa in some patients.
British Journal of Haematology 08/2007; 138(1):82-93. · 4.94 Impact Factor
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ABSTRACT: Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability.
In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa.
By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.
Arteriosclerosis Thrombosis and Vascular Biology 04/2007; 27(3):683-9. · 6.37 Impact Factor
