Role for ADAP in shear flow–induced platelet mechanotransduction

AbstractBinding of platelets to fibrinogen via integrin αIIbβ3 stimulates cytoskeletal reorganization and spreading. These responses depend on tyrosine phosphorylation of multiple proteins by Src family members and Syk. Among Src substrates in platelets is adhesion- and degranulation-promoting adapter protein (ADAP), an adapter with potential binding partners: SLP-76, VASP, and SKAP-HOM. During studies of platelet function under shear flow, we discovered that ADAP−/− mouse platelets, unlike ADAP+/+ platelets, formed unstable thrombi in response to carotid artery injury. Moreover, fibrinogen-adherent ADAP−/− platelets in shear flow ex vivo showed reduced spreading and smaller zones of contact with the matrix. These abnormalities were not observed under static conditions, and they could not be rescued by stimulating platelets with a PAR4 receptor agonist or by direct αIIbβ3 activation with MnCl2, consistent with a defect in outside-in αIIbβ3 signaling. ADAP+/+ platelets subjected to shear flow assembled F-actin–rich structures that colocalized with SLP-76 and the Rac1 exchange factor, phospho-Vav1. In contrast, platelets deficient in ADAP, but not those deficient in VASP or SKAP-HOM, failed to form these structures. These results establish that ADAP is an essential component of αIIbβ3-mediated platelet mechanotransduction that promotes F-actin assembly and enables platelet spreading and thrombus stabilization under fluid shear stress.

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Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

Biology ◽

10.3390/biology10080748 ◽

2021 ◽

Vol 10 (8) ◽

pp. 748

Author(s):

Elisa Wirthgen ◽

Melanie Hornschuh ◽

Ida Maria Wrobel ◽

Christian Manteuffel ◽

Jan Däbritz

Keyword(s):

Blood Flow ◽

Shear Flow ◽

Ex Vivo ◽

Culture Conditions ◽

Inflammatory Signaling ◽

Gm Csf ◽

Beneficial Effects ◽

Migratory Capacity ◽

Ex Vivo Culture ◽

Static Conditions

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

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Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.56 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jenya Zilberman-Rudenko ◽

Chantal Wiesenekker ◽

Asako Itakura ◽

Owen J McCarty

Keyword(s):

Shear Flow ◽

Platelet Activation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Coagulation Factor ◽

Thrombin Inhibitor ◽

Dependent Manner ◽

Factor Xi ◽

Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

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Evaluation of bone formation on orthopedic implant surfaces using an ex-vivo bone bioreactor system

Scientific Reports ◽

10.1038/s41598-021-02070-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rupak Dua ◽

Hugh Jones ◽

Philip C. Noble

Keyword(s):

Culture System ◽

Ex Vivo ◽

Calcium Phosphates ◽

Wire Mesh ◽

Bioreactor Culture ◽

Static Culture ◽

Group 4 ◽

The Matrix ◽

Bioreactor System ◽

Fiber Mesh

AbstractRecent advances in materials and manufacturing processes have allowed the fabrication of intricate implant surfaces to facilitate bony attachment. However, refinement and evaluation of these new design strategies are hindered by the cost and complications of animal studies, particularly during early iterations in the development process. To address this problem, we have previously constructed and validated an ex-vivo bone bioreactor culture system that can maintain the viability of bone samples for an extended period ex-vivo. In this study, we investigated the mineralization of a titanium wire mesh scaffold under both static and dynamic culturing using our ex vivo bioreactor system. Thirty-six cancellous bone cores were harvested from bovine metatarsals at the time of slaughter and divided into five groups under the following conditions: Group 1) Isolated bone cores placed in static culture, Group 2) Unloaded bone cores placed in static culture in contact with a fiber-mesh metallic scaffold, Group 3) Bone cores placed in contact with a fiber-mesh metallic scaffold under the constant pressure of 150 kPa, Group 4) Bone core placed in contact with a fiber-mesh metallic scaffold and exposed to cyclic loading with continuous perfusion flow of media within the ex-vivo culture system and Group 5) Bone core evaluated on Day 0 to serve as a positive control for comparison with all other groups at weeks 4 and 7. Bone samples within Groups 1–4 were incubated for 4 and 7 weeks and then evaluated using histological examination (H&E) and the Live-Dead assay (Life Technologies). Matrix deposits on the metallic scaffolds were examined with scanning electron microscopy (SEM), while the chemical composition of the matrix was measured using energy-dispersive x-ray spectroscopy (EDX). We found that the viability of bone cores was maintained after seven weeks of loading in our ex vivo system. In addition, SEM images revealed crystallite-like structures on the dynamically loaded metal coupons (Group 4), corresponding to the initial stages of mineralization. EDX results further confirmed the presence of carbon at the interface and calcium phosphates in the matrix. We conclude that a bone bioreactor can be used as an alternate tool for in-vivo bone ingrowth studies of new implant surfaces or coatings.

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An in vitro Perfused Macroencapsulation Device to Study Hemocompatibility and Survival of Islet-Like Cell Clusters

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.674125 ◽

2021 ◽

Vol 9 ◽

Author(s):

Stephanie A. Fernandez ◽

Lisa Danielczak ◽

Gabrielle Gauvin-Rossignol ◽

Craig Hasilo ◽

André Bégin-Drolet ◽

...

Keyword(s):

Ex Vivo ◽

Blood Perfusion ◽

International Normalized Ratio ◽

Convective Transport ◽

Cell Clusters ◽

Human Scale ◽

Doppler Ultrasound Imaging ◽

Long Term Treatment ◽

Static Conditions

Transplantation of hydrogel-encapsulated pancreatic islets is a promising long-term treatment for type 1 diabetes that restores blood glucose regulation while providing graft immunoprotection. Most human-scale islet encapsulation devices that rely solely on diffusion fail to provide sufficient surface area to meet islet oxygen demands. Perfused macroencapsulation devices use blood flow to mitigate oxygen limitations but increase the complexity of blood-device interactions. Here we describe a human-scale in vitro perfusion system to study hemocompatibility and performance of islet-like cell clusters (ILCs) in alginate hydrogel. A cylindrical perfusion device was designed for multi-day culture without leakage, contamination, or flow occlusion. Rat blood perfusion was assessed for prothrombin time and international normalized ratio and demonstrated no significant change in clotting time. Ex vivo perfusion performed with rats showed patency of the device for over 100 min using Doppler ultrasound imaging. PET-CT imaging of the device successfully visualized metabolically active mouse insulinoma 6 ILCs. ILCs cultured for 7 days under static conditions exhibited abnormal morphology and increased activated caspase-3 staining when compared with the perfused device. These findings reinforce the need for convective transport in macroencapsulation strategies and offer a robust and versatile in vitro system to better inform preclinical design.

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Collagen-dependent platelet dysfunction and its relevance to either mitochondrial ROS or cytosolic superoxide generation: a question about the quality and functional competence of long-stored platelets

10.21203/rs.3.rs-29731/v2 ◽

2020 ◽

Author(s):

Ehteramolsadat Hosseini ◽

Saba Hojjati ◽

Safoora Afzalniaye gashti ◽

Mehran Ghasemzadeh

Keyword(s):

Platelet Adhesion ◽

Oxidant Stress ◽

Ros Generation ◽

Reverse Correlation ◽

Platelet Dysfunction ◽

Mitochondrial Ros ◽

Ros Accumulation ◽

The Matrix ◽

Subendothelial Matrix ◽

Platelet Spreading

Abstract Background: Upon vascular damage, the exposed subendothelial matrix recruits circulating platelets to site of injury while inducing their firm adhesion mainly via GPVI-collagen interaction. GPVI also supports aggregatory and pro-coagulant functions in arterial shear rate even on the matrix other than collagen. Reactive oxygen species (ROS) modulate these stages of thrombosis; however augmented oxidant stress also disturbs platelet functions. Stored-dependent platelet lesion is associated with the increasing levels of ROS. Whether ROS accumulation is also relevant to collagen-dependent platelet dysfunction is the main interest of this study. Methods: Fresh PRP-PCs (platelet concentrates) were either stimulated with potent ROS-inducers PMA and CCCP or stored for 5 days. Intra-platelet superoxide (O2--) or mitochondrial-ROS and GPVI expression were detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy respectively. Results: Mitochondrial-ROS levels in 5 days-stored PCs were comparable to those induced by mitochondrial uncoupler, CCCP while O2-- generations were higher than those achieved by PMA. Shedding levels in 5 days-stored PCs were higher than those induced by these potent stimuli. GPVI expressions were reduced comparably in CCCP treated and 5 days-stored PCs. Platelet adhesion was also diminished during storage while demonstrating significant reverse correlation with GPVI shedding. However, only firm adhesion (indicated by spreading or platelet adhesion surface area) was relevant to GPVI expression. Platelet adhesion and aggregation also showed reverse correlations with both O2-- and mitochondrial-ROS formations; nonetheless mitochondrial-ROS was only relevant to firm adhesion. Conclusion: As a sensitive indicator of platelet activation, GPVI shedding correlated with either simple adhesion or spreading to collagen, while GPVI expression was only relevant to platelet spreading. Thereby, if the aim of GPVI evaluation is to examine platelet firm adhesion, expression seems to be a more specific choice. Furthermore, the comparable levels of ROS generation in 5 days-stored PCs and CCCP treated platelets, indicated that these products are significantly affected by oxidative stress. Reverse correlation of accumulating ROS with collagen-dependent platelet dysfunction is also a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer stored products.

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Abstract 2876: Early Retention and Subsequent Engraftment of Transplanted Progenitor Cells is Improved by Delivery within Collagen Matrix

Circulation ◽

10.1161/circ.118.suppl_18.s_790-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Yan Zhang ◽

Marc Lamoureux ◽

Stephanie Thorn ◽

Vincent Chan ◽

Joel Price ◽

...

Keyword(s):

Cell Therapy ◽

Progenitor Cells ◽

Pet Imaging ◽

Ex Vivo ◽

Collagen Matrix ◽

Cell Labeling ◽

Type I ◽

The Matrix ◽

Early Retention

Background: To investigate the mechanisms involved in the potentiation of cell therapy by delivery matrices, we evaluated the retention and engraftment of transplanted human circulating progenitor cells (CPCs) injected in a collagen matrix by using in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and immunohistochemistry. Methods: CPCs were labeled with 18 F-FDG and injected with or without a collagen type I-based matrix in the ischemic hindlimb muscle (IM) of rats (2x10 6 cells; n=15/group). Localization of cells was acquired by PET imaging (15 min) at 150 min post-injection. In addition, radionuclide biodistribution, immunofluorescence, and immunohistochemical examination of transplanted CPCs were performed at up to 14 days. Results: Cell labeling efficiency was CPC-concentration dependent (r=0.61, p <0.001), but not 18 F-FDG-dose dependent. Labeled CPCs exhibited excellent short-term stability and viability. Persistence of 18 F-FDG radioactivity in cells was markedly greater than non-specific retention in the matrix. Wholebody (WB) PET images revealed better CPC retention in the IM and less non-specific leakage to other tissues when CPCs were delivered within the matrix (IM/WB retention ratio of 43.9±8.2%), compared to cells injected alone (22.3±10.4%; p =0.040) and to 18 F-FDG injected with or without the matrix (9.7±5.5% and 11.0±5.5%, respectively; p <0.005). Radioactivity biodistribution confirmed that accumulation was increased (by 92.5%; p =0.024) in the IM and reduced (by 1.1 to 23.8%; p <0.05) in non-specific tissues when cells were injected within the matrix, compared to cells injected alone. Anti-human mitochondria staining showed increased cell retention in the IM with use of matrices (3.0±2.1%) versus cells only (1.9±0.8%; p =0.048). At 14 days the number of CD31 + transplanted human cells was greater (1.6±0.1%) when injected within the matrix than injected alone (0.7±0.1%; p =0.004). Conclusions: Collagen-based delivery matrices improve the early retention of transplanted CPCs, which in turn favors subsequent cell engraftment in the ischemic tissue. This mechanism conferred by the matrix has potential implications for the optimization of cell therapy at the early stages after cell delivery.

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Effect of nickel content in the corrosion process of TiC/Cu-Ni composites immersed in synthetic seawater

MRS Advances ◽

10.1557/adv.2019.492 ◽

2019 ◽

Vol 4 (63) ◽

pp. 3475-3484

Author(s):

Miguel A. Téllez-Villaseñor ◽

Carlos A. León Patino ◽

Ricardo Galván Martínez ◽

Ena A. Aguilar Reyes

Keyword(s):

Corrosion Rate ◽

Electrochemical Impedance ◽

Corrosion Behaviour ◽

Nickel Content ◽

Corrosion Mechanism ◽

Composite Surface ◽

Synthetic Seawater ◽

Ni Content ◽

The Matrix ◽

Static Conditions

ABSTRACTThe work presents an electrochemical study of the corrosion behaviour of two TiC/Cu-Ni metal matrix composites with a content of 10 and 20 wt.% Ni immersed in synthetic seawater. The composites were synthesized by a capillary infiltration technique, obtaining dense materials TiC/Cu-10Ni and TiC/Cu-20 Ni with a residual porosity of 1.8 and 1.7%, respectively. The corrosion rate (CR) was evaluated from the techniques of polarization curves (PC), linear polarization resistance (LPR) and electrochemical impedance spectroscopy (EIS). Electrochemical measurements were carried out under static conditions, ambient temperature and atmospheric pressure at 24 hours exposure in the electrolytic medium. The corrosion rate is affected by the Ni content in the matrix, with less corrosion in the composite with a higher Ni content. The higher content of Ni in the Cu-Ni alloy provides higher passivation and stability to the corrosion products film that are absorbed on the composite surface. Microscopic examination (SEM) showed a characteristic morphology of a corrosion mechanism of the localized type (pits and crevices) generated by a differential aeration, where the TiC/Cu-10Ni composite showed greater degradation.

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Characterization of the Cellular Reaction to a Collagen-Based Matrix: An In Vivo Histological and Histomorphometrical Analysis

Materials ◽

10.3390/ma13122730 ◽

2020 ◽

Vol 13 (12) ◽

pp. 2730 ◽

Cited By ~ 1

Author(s):

Samuel Ebele Udeabor ◽

Carlos Herrera-Vizcaíno ◽

Robert Sader ◽

C. James Kirkpatrick ◽

Sarah Al-Maawi ◽

...

Keyword(s):

Mononuclear Cells ◽

Ex Vivo ◽

Test Group ◽

Tissue Reaction ◽

Implantation Site ◽

Control Group ◽

The Matrix ◽

Tissue Reactions ◽

Post Implantation

The permeability and inflammatory tissue reaction to Mucomaix® matrix (MM), a non- cross-linked collagen-based matrix was evaluated in both ex vivo and in vivo settings. Liquid platelet rich fibrin (PRF), a blood concentrate system, was used to assess its capacity to absorb human proteins and interact with blood cells ex vivo. In the in vivo aspect, 12 Wister rats had MM implanted subcutaneously, whereas another 12 rats (control) were sham-operated without biomaterial implantation. On days 3, 15 and 30, explantation was completed (four rats per time-point) to evaluate the tissue reactions to the matrix. Data collected were statistically analyzed using analysis of variance (ANOVA) and Tukey multiple comparisons tests (GraphPad Prism 8). The matrix absorbed the liquid PRF in the ex vivo study. Day 3 post-implantation revealed mild tissue inflammatory reaction with presence of mononuclear cells in the implantation site and on the biomaterial surface (mostly CD68-positive macrophages). The control group at this stage had more mononuclear cells than the test group. From day 15, multinucleated giant cells (MNGCs) were seen in the implantation site and the outer third of the matrix with marked increase on day 30 and spread to the matrix core. The presence of these CD68-positive MNGCs was associated with significant matrix vascularization. The matrix degraded significantly over the study period, but its core was still visible as of day 30 post-implantation. The high permeability and fast degradation properties of MM were highlighted.

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α7β1-Integrin regulates mechanotransduction and prevents skeletal muscle injury

AJP Cell Physiology ◽

10.1152/ajpcell.00317.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. C1660-C1665 ◽

Cited By ~ 78

Author(s):

Marni D. Boppart ◽

Dean J. Burkin ◽

Stephen J. Kaufman

Keyword(s):

Skeletal Muscle ◽

Muscle Damage ◽

Intracellular Signaling ◽

Ex Vivo ◽

Negative Regulator ◽

Blue Dye ◽

The Matrix ◽

Mitogen Activated Protein ◽

Exercise Induced

α7β1-Integrin links laminin in the extracellular matrix with the cell cytoskeleton and therein mediates transduction of mechanical forces into chemical signals. Muscle contraction and stretching ex vivo result in activation of intracellular signaling molecules that are integral to postexercise injury responses. Because α7β1-integrin stabilizes muscle and provides communication between the matrix and cytoskeleton, the role of this integrin in exercise-induced cell signaling and skeletal muscle damage was assessed in wild-type and transgenic mice overexpressing the α7BX2 chain. We report here that increasing α7β1-integrin inhibits phosphorylation of molecules associated with muscle damage, including the mitogen-activated protein kinases (JNK, p38, and ERK), following downhill running. Likewise, activation of molecules associated with hypertrophy (AKT, mTOR, and p70S6k) was diminished in mice overexpressing integrin. While exercise resulted in Evans blue dye-positive fibers, an index of muscle damage, increased integrin protected mice from injury. Moreover, exercise leads to an increase in α7β1 protein. These experiments provide the first evidence that α7β1-integrin is a negative regulator of mechanotransduction in vivo and provides resistance to exercise-induced muscle damage.

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Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation

Blood ◽

10.1182/blood.v98.4.1038 ◽

2001 ◽

Vol 98 (4) ◽

pp. 1038-1046 ◽

Cited By ~ 85

Author(s):

Sylvie Moog ◽

Pierre Mangin ◽

Nadège Lenain ◽

Catherine Strassel ◽

Catherine Ravanat ◽

...

Keyword(s):

Ex Vivo ◽

Adenosine Diphosphate ◽

Collagen Type I ◽

Platelet Glycoprotein ◽

Lag Phase ◽

Type I ◽

Platelet Surface ◽

Static Conditions ◽

Induced Aggregation

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I–coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist.

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