Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Liquid flow and control without solid walls.

When miniaturizing fluidic circuitry, the solid walls of the fluid channels become increasingly important1 because they limit the flow rates achievable for a given pressure drop, and they are prone to fouling2. Approaches for reducing the wall interactions include hydrophobic coatings3, liquid-infused porous surfaces4-6, nanoparticle surfactant jamming7, changes to surface electronic structure8, electrowetting9,10, surface tension pinning11,12 and use of atomically flat channels13. A better solution may be to avoid the solid walls altogether. Droplet microfluidics and sheath flow achieve this but require continuous flow of the central liquid and the surrounding liquid1,14. Here we demonstrate an approach in which aqueous liquid channels are surrounded by an immiscible magnetic liquid, both of which are stabilized by a quadrupolar magnetic field. This creates self-healing, non-clogging, anti-fouling and near-frictionless liquid-in-liquid fluidic channels. Manipulation of the field provides flow control, such as valving, splitting, merging and pumping. The latter is achieved by moving permanent magnets that have no physical contact with the liquid channel. We show that this magnetostaltic pumping method can be used to transport whole human blood with very little damage due to shear forces. Haemolysis (rupture of blood cells) is reduced by an order of magnitude compared with traditional peristaltic pumping, in which blood is mechanically squeezed through a plastic tube. Our liquid-in-liquid approach provides new ways to transport delicate liquids, particularly when scaling channels down to the micrometre scale, with no need for high pressures, and could also be used for microfluidic circuitry.

Loss of α4A- and ß1-tubulins leads to severe platelet spherocytosis and strongly impairs hemostasis in mice.

Native circulating blood platelets present with a discoid flat morphology maintained by a submembranous peripheral ring of microtubules, named marginal band. The functional importance of this particular shape is still debated, but it was initially hypothesized to facilitate platelet interaction with the injured vessel wall and to contribute to hemostasis. The importance of the platelet discoid morphology has since been questioned on the absence of clear bleeding tendency in mice lacking the platelet-specific ß1-tubulin isotype, which exhibits platelets with a thinner marginal band and an ovoid shape. Here, we generated a mouse model inactivated for ß1-tubulin and α4A-tubulin, an α-tubulin isotype strongly enriched in platelets. These mice present with fully spherical platelets completely devoid of a marginal band. In contrast to the single knockouts, the double deletion resulted in a severe bleeding defect in a tail-clipping assay, which was not corrected by increasing the platelet count to normal values by the thrombopoietin-analog romiplostim. In vivo, thrombus formation was almost abolished in a ferric chloride-injury model, with only a thin layer of loosely packed platelets, and mice were protected against death in a model of thromboembolism. In vitro, platelets adhered less efficiently and formed smaller-sized and loosely assembled aggregates when perfused over von Willebrand factor and collagen matrices. In conclusion, this study shows that blood platelets require 2 unique α- and ß-tubulin isotypes to acquire their characteristic discoid morphology. Lack of these 2 isotypes has a deleterious effect on flow-dependent aggregate formation and stability, leading to a severe bleeding disorder.

The relative importance of platelet integrins in hemostasis, thrombosis and beyond.

Integrins are heterodimeric transmembrane receptors composed of α and ß chains, with an N-terminal extracellular domain forming a globular head corresponding to the ligand binding site. Integrins regulate various cellular functions including adhesion, migration, proliferation, spreading and apoptosis. On platelets, integrins play a central role in adhesion and aggregation on subendothelial matrix proteins of the vascular wall, thereby ensuring hemostasis. Platelet integrins belong either to the ß1 family (α2ß1, α5ß1 and α6ß1) or to the ß3 family (αIIbß3 and αvß3). On resting platelets, integrins can engage their ligands when the latter are immobilized but not in their soluble form. The effects of various agonists promote an inside-out signal in platelets, increasing the affinity of integrins for their ligands and conveying a modest signal reinforcing platelet activation, called outside-in signaling. This outside-in signal ensures platelet adhesion, shape change, granule secretion and aggregation. In this review, we examine the role of each platelet integrin in hemostatic plug formation, hemostasis and arterial thrombosis and also beyond these classical functions, notably in tumor metastasis and sepsis.

Biochemical and functional characteristics of stored (double-dose) buffy-coat platelet concentrates treated with amotosalen and a prototype UVA light-emitting diode illuminator.

BACKGROUND: Pathogen reduction of platelet concentrates (PCs) using amotosalen and broad-spectrum UVA illumination contributes to the safety of platelet transfusion by reducing the risk of transfusion-transmitted infections. We evaluated the in vitro quality of stored buffy-coat (BC) PCs treated with amotosalen and a prototype light-emitting diode (LED) illuminator. METHODS: Double-dose BC-PCs collected into PAS-III/plasma or SSP+ /plasma (55/45%) were treated with amotosalen in combination with either conventional UVA lamps (INT100 Illuminator 320-400 nm) or LED illuminators at 350 nm. Platelet quality and function were evaluated over 7 days. RESULTS: Platelet counts were conserved during storage in all groups, as was platelet swirling without appearance of macroscopic aggregates. Integrin αIIbß3 and glycoprotein (GP) VI expression remained stable, whereas GPIbα and GPV declined similarly in all groups. UV lamp- and LED-treated PCs displayed similar glucose consumption, lactate generation, and pH variation. Comparable spontaneous and residual P-selectin and phosphatidylserine exposure, activated αIIbß3 exposure, mitochondrial membrane potential, lactate dehydrogenase release, and adhesive properties under flow conditions were observed during storage. The use of SSP+ /plasma compared with PAS-III/plasma better preserved most of these parameters, especially during late storage, irrespective of the type of illuminator. CONCLUSION: Replacing the UVA lamp for photochemical treatment by LED illuminators had no impact on platelet metabolism, spontaneous activation, apoptosis or viability, or on the in vitro function of BC-PCs stored for 7 days in SSP+ or PAS-III/plasma. These findings support improved procedures for the pathogen reduction and storage of PCs, to ensure transfusion safety and retention of platelet functional properties.

The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling.

The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing both VWF-GPIbGGand collagen-GPVI signaling events in platelets.

Glenzocimab does not impact glycoprotein VI-dependent inflammatory haemostasis.

Not available.

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin.

OBJECTIVE: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kß, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). CONCLUSIONS: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

The basement membrane protein nidogen-1 supports platelet adhesion and activation.

The core structure of the extracellular basement membrane is made up of self-assembling networks of collagen and laminin which associate with each other through the bridging adapter proteins including the sulfated monomeric glycoprotein nidogen. While collagen and laminin are known to support platelet adhesion and activation via ß1 integrins and glycoprotein (GP) VI, respectively, whether nidogen contributes to platelet activation and hemostasis is unknown. In this study, we demonstrate that recombinant human nidogen-1 supports platelet adhesion and stimulates platelet activation in a phospholipase-C γ-2 (PLCγ2), Src and Syk kinase-dependent manner downstream. Platetet adhesion to nidogen-1 was inhibited by blocking the platelet receptors GPVI and ß1 integrins. Platelet adhesion to nidogen-1 activated the IκB kinase (IKK) complex, while pharmacological inhibition of IKK blocked platelet spreading on nidogen. Taken together our results suggest that nidogen may play a redundant role in hemostasis by activating platelets downstream of GPVI.

Shear rate gradients promote a bi-phasic thrombus formation on weak adhesive proteins, such as fibrinogen in a VWF-dependent manner.

Blood flow profoundly varies throughout the vascular tree due to its pulsatile nature and to the complex vessel geometry. While thrombus formation has been extensively studied in vitro under steady flow, and in vivo under normal blood flow conditions, the impact of complex hemodynamics such as flow acceleration found in stenosed arteries has gained increased appreciation. We investigated the effect of flow acceleration, characterized by shear rate gradients, on the function of platelets adhering to fibrinogen, a plasma protein which plays a key role in hemostais and thrombosis. While we confirmed that under steady flow, fibrinogen only supports single platelet adhesion, we observed that under shear rate gradients, this surface becomes highly thrombogenic, supporting efficient platelet aggregation leading to occlusive thrombus formation. This shear rate gradient-driven thrombosis is biphasic with an initial step of slow platelet recruitment supported by direct plasma VWF adsorption to immobilized fibrinogen and followed by a second phase of explosive thrombosis initiated by VWF fiber formation on platelet monolayers. In vivo experiments confirmed that shear rate gradients accelerate thrombosis in a VWF-dependent manner. Together, this study characterizes a process of plasma VWF-dependent accelerated thrombosis on immobilized fibrinogen in the presence of shear rate gradients.

Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface.

Objective- After activation at the site of vascular injury, platelets differentiate into 2 subpopulations, exhibiting either proaggregatory or procoagulant phenotype. Although the functional role of proaggregatory platelets is well established, the physiological significance of procoagulant platelets, the dynamics of their formation, and spatial distribution in thrombus remain elusive. Approach and Results- Using transmission electron microscopy and fluorescence microscopy of arterial thrombi formed in vivo after ferric chloride-induced injury of carotid artery or mechanical injury of abdominal aorta in mice, we demonstrate that procoagulant platelets are located at the periphery of the formed thrombi. Real-time cell tracking during thrombus formation ex vivo revealed that procoagulant platelets originate from different locations within the thrombus and subsequently translocate towards its periphery. Such redistribution of procoagulant platelets was followed by generation of fibrin at thrombus surface. Using in silico model, we show that the outward translocation of procoagulant platelets can be driven by the contraction of the forming thrombi, which mechanically expels these nonaggregating cells to thrombus periphery. In line with the suggested mechanism, procoagulant platelets failed to translocate and remained inside the thrombi formed ex vivo in blood derived from nonmuscle myosin ( MYH9)-deficient mice. Ring-like distribution of procoagulant platelets and fibrin around the thrombus observed with blood of humans and wild-type mice was not present in thrombi of MYH9-knockout mice, confirming a major role of thrombus contraction in this phenomenon. Conclusions- Contraction of arterial thrombus is responsible for the mechanical extrusion of procoagulant platelets to its periphery, leading to heterogeneous structure of thrombus exterior.

Impact of PI3Kα (Phosphoinositide 3-Kinase Alpha) Inhibition on Hemostasis and Thrombosis.

Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kß and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged αIIbß3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kß, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.

Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets.

The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production.

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting.

Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (∼1 µm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.

Immobilized fibrinogen activates human platelets through glycoprotein VI.

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

The role of extracellular matrix stiffness in megakaryocyte and platelet development and function.

The extracellular matrix (ECM) is a key acellular structure in constant remodeling to provide tissue cohesion and rigidity. Deregulation of the balance between matrix deposition, degradation, and crosslinking results in fibrosis. Bone marrow fibrosis (BMF) is associated with several malignant and nonmalignant pathologies severely affecting blood cell production. BMF results from abnormal deposition of collagen fibers and enhanced lysyl oxidase-mediated ECM crosslinking within the marrow, thereby increasing marrow stiffness. Bone marrow stiffness has been recently recognized as an important regulator of blood cell development, notably by modifying the fate and differentiation process of hematopoietic or mesenchymal stem cells. This review surveys the different components of the ECM and their influence on stem cell development, with a focus on the impact of the ECM composition and stiffness on the megakaryocytic lineage in health and disease. Megakaryocyte maturation and the biogenesis of their progeny, the platelets, are thought to respond to environmental mechanical forces through a number of mechanosensors, including integrins and mechanosensitive ion channels, reviewed here.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbß3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization.

Bioreactivity of stent material: Activation of platelets, coagulation, leukocytes and endothelial cell dysfunction in vitro.

Outcome of patients with coronary artery disease has been significantly improved by percutaneous coronary interventions with stent implantation. However, despite progress made on devices and antithrombotic treatments, stent thrombosis remains an important issue because of serious adverse consequences. Several mechanisms are assumed to favor stent thrombosis as platelet aggregation, fibrin formation, defective healing and local inflammation. The objective of this study was to evaluate in vitro the thrombogenicity, proinflammatory properties and healing capacities of cobalt-chromium (CoCr), an alloy commonly used for cardiovascular implants. Platelet adhesion was quantified in static and flow conditions. Thrombin generation was performed using the calibrated automated thrombogram. Neutrophil adhesion and formation of extracellular traps were visualized by scanning electron microscopy and by immunofluorescence. The phenotype of endothelial cells grown on CoCr was analyzed using specific antibodies, whereas the procoagulant potential was analyzed by measuring thrombin generation and protein C activation. Our results show that human blood platelets adhere to and are activated on CoCr in static and flow conditions. Overall, CoCr significantly induced thrombin generation in the presence or absence of platelets by 1.5- and 4.8-fold, respectively, involving activation of the contact pathway and activation of platelets. CoCr triggered leukocyte adhesion and behaved as a scaffold for the formation of neutrophil extracellular traps in the presence of platelets. Endothelial cells adhered and formed a monolayer covering CoCr. However, they switched from an anticoagulant phenotype to a procoagulant one with a significant 2.2-fold increase in thrombin generation due to a combined 30% reduced capacity to trigger protein C activation and 30% increased expression of tissue factor. Moreover, endothelial cells grown on CoCr acquired an inflammatory phenotype as indicated by the increased expression of ICAM-1 and VCAM-1. These data show that bare CoCr is prothrombotic and proinflammatory due to its capacity to activate platelets and coagulation and to induce leukocyte adhesion and activation. More importantly, even if endothelialization is achievable, the switch in endothelial phenotype prevents effective healing. Furthermore, we propose our methodology for future preclinical in vitro evaluation of the thrombogenicity of stent materials.

Genome-wide association mapping of partial resistance to Aphanomyces euteiches in pea.

BACKGROUND: Genome-wide association (GWA) mapping has recently emerged as a valuable approach for refining the genetic basis of polygenic resistance to plant diseases, which are increasingly used in integrated strategies for durable crop protection. Aphanomyces euteiches is a soil-borne pathogen of pea and other legumes worldwide, which causes yield-damaging root rot. Linkage mapping studies reported quantitative trait loci (QTL) controlling resistance to A. euteiches in pea. However the confidence intervals (CIs) of these QTL remained large and were often linked to undesirable alleles, which limited their application in breeding. The aim of this study was to use a GWA approach to validate and refine CIs of the previously reported Aphanomyces resistance QTL, as well as identify new resistance loci. METHODS: A pea-Aphanomyces collection of 175 pea lines, enriched in germplasm derived from previously studied resistant sources, was evaluated for resistance to A. euteiches in field infested nurseries in nine environments and with two strains in climatic chambers. The collection was genotyped using 13,204 SNPs from the recently developed GenoPea Infinium® BeadChip. RESULTS: GWA analysis detected a total of 52 QTL of small size-intervals associated with resistance to A. euteiches, using the recently developed Multi-Locus Mixed Model. The analysis validated six of the seven previously reported main Aphanomyces resistance QTL and detected novel resistance loci. It also provided marker haplotypes at 14 consistent QTL regions associated with increased resistance and highlighted accumulation of favourable haplotypes in the most resistant lines. Previous linkages between resistance alleles and undesired late-flowering alleles for dry pea breeding were mostly confirmed, but the linkage between loci controlling resistance and coloured flowers was broken due to the high resolution of the analysis. A high proportion of the putative candidate genes underlying resistance loci encoded stress-related proteins and others suggested that the QTL are involved in diverse functions. CONCLUSION: This study provides valuable markers, marker haplotypes and germplasm lines to increase levels of partial resistance to A. euteiches in pea breeding.

Dehydration of blood platelets by zeodration: in vitro characterization and hemostatic properties in vivo.

BACKGROUND: Platelets (PLTs) are currently stored at room temperature (RT) for 5 to 7 days. So far, there exists no validated method for the preparation and long-term storage of dehydrated PLTs suitable for transfusion after rehydration. In this study, a desiccation process, zeodration, was applied to PLTs. STUDY DESIGN AND METHODS: A complete procedure of dehydration at RT by zeodration was employed. Zeodrated human and mouse PLTs were characterized in vitro. Zeodrated mouse PLTs were transfused into clopidogrel-treated mice to evaluate their hemostatic properties. RESULTS: The optimal conditions for dehydration of PLTs at RT in a laboratory scale zeodrator were defined as 145 mbar and 20.2 ± 1.5 °C. The recovery rate was 85 ± 2% and the dryness of zeodrated PLTs (Z_PLTs) indicated that they were sufficiently stable for long-term storage. Rehydrated Z_PLTs were round, were not aggregated, and expressed the glycoproteins required for PLT function. Z_PLTs agglutinated in the presence of von Willebrand factor (VWF) and aggregated in response to thrombin or collagen independently of an active metabolism. In a flow system, Z_PLTs could adhere to VWF and form aggregates on a collagen matrix. Thrombin was generated at the surface of Z_PLTs as efficiently as on fresh PLTs. In clopidogrel-treated mice, which exhibited a severely prolonged bleeding time, continuous perfusion of Z_PLTs restored normal hemostasis. CONCLUSION: Zeodration represents a new strategy to prepare PLTs with partly preserved aggregative properties after storage and rehydration. Z_PLTs have potential hemostatic properties provided it is possible to improve their transfusion efficacy.