scholarly journals Bleeding Cessation in a Mouse Jugular Vein Puncture Wound Model Is Caused By Extravascular Capping, Not Hole Infill

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-14
Author(s):  
Brian Storrie ◽  
Sung Rhee ◽  
Timothy J. Stalker ◽  
Irina D Pokrovskaya ◽  
Kenny Ling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Shell Model ◽  
Mouse Models ◽  
Thrombus Formation ◽  
Vascular Damage ◽  
Puncture Wound ◽  
Platelet Aggregates ◽  
Granule Secretion ◽  
Scanning Electron

Introduction: Vascular damage comes in many forms with the puncture wound likely the longest known to humans. Experimentally, vascular damage has typically been visualized in mouse models in which there is little bleeding. Under these conditions, damage is limited mostly to the endothelial layer lining the vessel. Visualization has varied from light to scanning electron microscopy. Interpretation has been dominated by intravital microscopy outcomes in which an initial layer of p-selection-exposed, i.e., α-granule secretion-positive, platelets is deposited in association with the damaged vessel wall and extended by the accumulation of a less activated outer layer of platelets. These results have given rise to a Core and Shell model of platelet-rich thrombus formation [Tomaiuolo et al., 2017]. Recently, new mouse models have been presented in which the vessel is punctured to create a 300 to 600 micron wound hole [Tomoiuolo et al., 2019]. Bleeding is now profuse. The puncture wound results have been interpreted within a Core and Shell model. However, two important aspects of the experimental data [Tomoiuolo et al., 2019] suggest that the existing model may not explain the actual results. First, p-selectin expression as a marker for α-granule secretion and platelet activation was present in limited areas towards the periphery of the resulting thrombus, not as well-defined Core. Second, the hemostatic thrombus when viewed at early stages, ex vivo, showed a pebbly distribution of platelet aggregates suggestive of nucleated platelet accumulation rather than the smooth layers that would follow from a Core and Shell model. We hypothesize that nucleated accumulation of platelet aggregates within the puncture hole could provide pedestals upon which localized accumulation of platelets form the infrastructure of a vaulted thrombus whose extravascular capping leads to bleeding cessation. Methods: To test the proposed hypothesis, we visualized the interior and overall structure of the forming puncture wound thrombus in full 3D at sub-platelet level resolution. To achieve this end, we took our proven serial block face scanning electron microscopy (SBF-SEM) protocols for visualizing platelet organelles in 3D [Pokrovskaya et al., 2018] and adapted them to the visualization of forming thrombi over 1000s of image. To localize samples for electron microscopy, we used in vivo antibody labeling [Tomaiuolo et al., 2019]. This approach had the added advantage of enabling correlative light microscopy mapping overall p-selectin, a marker of platelet secretion, and fibrin distributions against 3D, platelet resolution, thrombus morphology. Results: We found that a 1 min thrombus, pre-bleeding cessation, was structured about the localized accumulation of pedestal-like platelet aggregates along the sides of the puncture hole, extended and spaced along the extravascular adventitia. Subsequent pedestal extension formed a "pontoon" bridge that "capped" extravascularly the puncture hole. At 5 min, full bleeding cessation, we found that forming platelet thrombus had a Swiss cheese-like interior of vaults that were continuous with the intravascular vessel lumen and framed by columns of platelets, presumed pedestal extensions. The thrombus was sealed on the extravascular side by a platelet "cap" (Figure). As expected after bleeding cessation, red blood cells accumulated on the intravascular side of the cap. Formation of a tightly sealed cap was dependent on α-granule secretion as indicated by the effect of knockout of VAMP-8, the primary SNARE protein involved in a-granule release. Based upon morphology, vaults within the forming thrombus were lined with apparent procoagulant platelets providing a potential protected surface for coagulation factor activation. Conclusions: We conclude that bleeding cessation in a true puncture wound occurs from the extravascular side of the thrombus rather than through the formation of a platelet plug that fills the hole. We propose an alternative model of bleeding cessation in which localized platelet aggregates are the starting pedestal upon which all subsequent steps in puncture thrombus formation build, i.e., "Cap and Build". The extent to which properties differ among systems remains an open question. Tomaiuolo et al. 2017. Intervent. Cardiol. Clin. 6: 1-12. Pokrovskaya et al. 2018. Blood Adv. 2: 2947-2958 Tomaiuolo et al. 2019. Proc. Natl. Acad. Sci. USA 116:2 243-2252 Figure Disclosures No relevant conflicts of interest to declare.

Neutrophil Derived Cathepsin G Induces Potentially Thrombogenic Changes in Human Endothelial Cells: a Scanning Electron Microscopy Study in Static and Dynamic Conditions

1994 ◽  
Vol 72 (01) ◽  
pp. 140-145 ◽  
Author(s):  
Valeri Kolpakov ◽  
Maria Cristina D'Adamo ◽  
Lorena Salvatore ◽  
Concetta Amore ◽  
Alexander Mironov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Thrombus Formation ◽  
Cathepsin G ◽  
Arterial Segment ◽  
Dynamic Conditions ◽  
Activated Neutrophils ◽  
Scanning Electron

SummaryActivated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer.We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions.Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment.Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an “in vivo” model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential.This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.

scholarly journals Bioengineering of Improved Biomaterials Coatings for Extracorporeal Circulation Requires Extended Observation of Blood-Biomaterial Interaction under Flow

2007 ◽  
Vol 2007 ◽  
pp. 1-10 ◽  
Author(s):  
Kris N. J. Stevens ◽  
Yvette B. J. Aldenhoff ◽  
Frederik H. van der Veen ◽  
Jos G. Maessen ◽  
Leo H. Koole
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thrombus Formation ◽  
Blood Parameters ◽  
Systematic Evaluation ◽  
Loop Model ◽  
Biomaterial Surfaces ◽  
Chandler Loop ◽  
Scanning Electron

Extended use of cardiopulmonary bypass (CPB) systems is often hampered by thrombus formation and infection. Part of these problems relates to imperfect hemocompatibility of the CPB circuitry. The engineering of biomaterial surfaces with genuine long-term hemocompatibility is essentially virgin territory in biomaterials science. For example, most experiments with the well-known Chandler loop model, for evaluation of blood-biomaterial interactions under flow, have been described for a maximum duration of 2 hours only. This study reports a systematic evaluation of two commercial CPB tubings, each with a hemocompatible coating, and one uncoated control. The experiments comprised (i) testing over 5 hours under flow, with human whole blood from 4 different donors; (ii) measurement of essential blood parameters of hemocompatibility; (iii) analysis of the luminal surfaces by scanning electron microscopy and thrombin generation time measurements. The dataset indicated differences in hemocompatibility of the tubings. Furthermore, it appeared that discrimination between biomaterial coatings can be made only after several hours of blood-biomaterial contact. Platelet counting, myeloperoxidase quantification, and scanning electron microscopy proved to be the most useful methods. These findings are believed to be relevant with respect to the bioengineering of extracorporeal devices that should function in contact with blood for extended time.

Scanning Electron Microscopy Study of Endothelial Injury and Thrombus Formation in WT and VWF Deficient Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3062-3062
Author(s):  
Justin Barr ◽  
Jennifer Barr ◽  
Marielle Meurice ◽  
David Motto
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Wild Type ◽  
Subsequent Formation ◽  
Time Points ◽  
Endothelial Surface ◽  
Deficient Mice ◽  
Scanning Electron

Abstract Abstract 3062 Poster Board II-1038 VWF is a large plasma glycoprotein required for normal hemostasis, and performs its function through binding to coagulation Factor VIII, and via interactions with both platelet surface glycoproteins and the activated and/or damaged vascular surface. We have developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation in wild-type and VWF-deficient mice. Thrombus formation is initiated by ferric chloride, and subsequently at defined time points, the circulation is rapidly flushed and aldehyde fixed. The carotid artery is removed, externally fixed, sectioned (both longitudinally and in cross-section), processed for SEM, and visualized. With this protocol we have obtained high-quality images (exceeding 100,000x) of FeCl3-induced endothelial damage and thrombus formation in C57BL/6 and VWF-deficient mice at baseline, and at 30, 60, 90, 120, 240, and 300 seconds post-injury (please access http://sites.google.com/site/mottolab/ to view images). Interestingly, we find that FeCl3 induces little, if any, endothelial denudation and collagen exposure at these time points, with the endothelium clearly appearing changed from baseline, but not damaged. Thus, initial platelet adhesion seems to be occurring in the absence of collagen exposure in this model. In wild-type mice, platelets adhere rapidly to the endothelial surface and assume a cross-linked appearance by 90 seconds, with continual inward growth of the thrombus through the 300 second time point. In VWF-deficient mice, platelets also adhere rapidly to the endothelial surface, but in contrast, remain recognizable longer without assuming a highly-activated phenotype. Compared with wild-type, at all time points examined the VWF-deficient thrombus appears smaller with considerably less cross-linking and platelet activation. Interestingly, during the course of these experiments we also have identified what appears to be red blood cells (RBCs) participating in thrombus formation. Similar to platelets, RBCs interact directly with the endothelial surface, and subsequently become elongated in the direction of blood flow. These elongated RBCs are often observed to cluster and bind platelets, with the subsequent formation of large platelet-erythrocyte complexes. Further characterization of these complexes and the role they may play in thrombus formation is currently in progress. Additionally, similar SEM studies are underway with both ADAMTS13-deficient and GPIb alpha-deficient mice, and with mice transiently expressing in vivo biotinylated VWF for visualization of this molecule at high magnification and resolution. These studies should help better define the mechanisms of endothelial activation and thrombus formation as they occur in situ. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet Aggregation and Fibrin Deposition: The Yin and Yang of Thrombin Activity Regulation By Platelet Glycoprotein Ibα

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 114-114
Author(s):  
Alessandro Zarpellon ◽  
Patrizia Marchese ◽  
Antonella Zampolli ◽  
Grazia Loredana Mendolicchio ◽  
Zaverio M. Ruggeri
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Shear Stress ◽  
Carotid Artery ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Type I ◽  
Scanning Electron

Abstract Generation of α-thrombin (FIIa) in response to vascular injury is a key mechanism influencing thrombus formation. Platelet activation by FIIa is mediated by different protease activated receptors (PARs), although the most abundant FIIa binding site on platelets, but not a substrate for proteolysis, is glycoprotein (GP) Ibα in the GPIb-IX-V complex. The functional role of GPIbα in mediating/regulation thrombin functions relative to that of different PARs remains unclear. The goal of these studies was to define how binding to GPIbα can modulate FIIa functions. In mouse platelets we replaced endogenous GPIbα with either its human wild type counterpart (huGPIbα-WT) or with huGPIbα mutated at key residues involved in thrombin binding (D277N, Y276-8-9/F). Because these two mutations resulted in an undistinguishable phenotype, they are designated collectively as huGPIbα-Mut hereon. Mice expressing huGPIbα, WT or Mut, were evaluated in intravital models of arterial thrombosis induced by a ferric chloride-induced carotid artery lesion and venous thromboembolism induced by intravenous α-thrombin injection. Moreover, the blood of huGPIbα WT or Mut mice was also tested in an ex vivo model of thrombus formation upon perfusion over a thrombogenic surface under controlled flow conditions and platelets were evaluated for their responses to FIIa-induced activation. Mice expressing huGPIbα - WT or Mut - have comparable platelet counts and GPIbα surface density. Moreover, huGPIbα-WT platelets bind FIIa similarly than their normal human control counterpart, while huGPIbα-Mut platelets have essentially no detectable FIIa binding. Upon FIIa stimulation, which on mouse platelets is mediated by PAR4, aggregation and Ca2+ transients were significantly enhanced in huGPIbα-Mut as compared to huGPIbα-WT. In contrast, blocking FIIa binding to GPIbα on human platelets essentially abolished FIIa mediated activation, which in human occurs predominantly through PAR1. These results are compatible with the conclusion that, in mice, GPIbα is a competitive inhibitor of FIIa for PAR4-mediated functions. In the presence of metabolically inactive (PGE1 treated) huGPIbα-Mut washed platelets, the clotting time of a purified fibrinogen solution was significantly shorter when triggered by relative high concentration of FIIa (4 nM), but pronouncedly prolonged at a lower FIIa concentration (0.5 nM). Clot visualization showed a much more structured fibrin mesh in the presence of huGPIbα-WT platelets, which was lost with in the presence of huGPIbα-Mut platelets. Mutant mice tested in a model of carotid artery injury exhibited a pronounced prothrombotic phenotype, with a shorter time to occlusion. However they were protected from death induced by I.V. injection of α-thrombin. In ex vivo perfusion studies, the total volume of platelet aggregates formed in huGPIbα-Mut mouse blood exposed to acid-insoluble fibrillar collagen type I was slightly bigger than in huGPIbα-WT mice, but the number of thrombi was increased and their individual size smaller. These huGPIbα-Mut platelets exhibited clear signs of increased activation, as visualized by scanning electron microscopy (SEM). Strikingly, fibrin was almost totally absent in the huGPIbα-Mut thrombi. This was in striking contrast with what observed in huGPIbα-WT mice, in which the surface of platelet thrombi with directly and tightly connected with thick fibrin fibers as visualized by scanning electron microscopy. Possibly because of the reduced platelet membrane-fibrin fibril connection in huGPIbα-Mut platelets, these mice were significantly less susceptible to death when injected with an α-thrombin dose that caused 80% mortality plus in huGPIbα-WT mice. Thus, mice whose platelets have defective α-thrombin binding to GPIbα have a prothrombotic phenotype in high shear stress flow arteries and are protected from thromboembolic death in the low shear stress venous circulation. Our findings identify GPIbα as a relevant FIIa activity modulator in hemostasis and thrombosis through distinct and opposite mechanisms affecting platelet activation (The Yin) and fibrin formation (The Yang). Disclosures No relevant conflicts of interest to declare.

Non-Thrombogenic Characteristics of Hybrid Vascular Prostheses

1979 ◽  
Author(s):  
P.N. Sawyer ◽  
B. Stanczewski
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Protein Structure ◽  
Thrombus Formation ◽  
Vascular Prosthesis ◽  
Short Term ◽  
Vascular Prostheses ◽  
Complete Occlusion ◽  
Dacron Prostheses ◽  
Scanning Electron

In our continuing efforts to develop a compound, coated vascular prosthesis, combining the best characteristics of biologic and nonbiologic materials, we have produced a hybri graft with pseudo biological coating. Five types of velour and knit dacron prostheses, have been cross linked subsequently with negatively charged proteins. Albumin and gelatin were used to coat the prosthesis or the combination of both. Following electrochemical characterization coated prostheses were implanted into the carotid and femoral arteries of dogs for 1 sec. 2 hr. and 1 mo. Preparations that showed promising short term results were implanted in the abdominal aorta of dogs for 18 months. Controls included uncoated prostheses and prostheses that had been coated but not negatively charged. Upon removal, the grafts were examined for thrombi and other luminal narrowings (photography, light and scanning electron microscopy). Fifty two uncoated and coated grafts have been examined. These exhibit a range of thrombus formation, from none to complete occlusion, in the various knit/coating combinations. We believe these hybrid prostheses will prove to be very useful in vascular reconstructive surgery due to (i) the nonthrombogenic characteristics of the negatively charged protein structure, (ii) more uniform healing and (iii) ease of implantation since preclotting is not necessary.

Recruitment of Labelled Monocytes by Experimental Venous Thrombi

2001 ◽  
Vol 85 (06) ◽  
pp. 1018-1024 ◽  
Author(s):  
C. L. McGuinness ◽  
J. Humphries ◽  
M. Waltham ◽  
K. G. Burnand ◽  
M. Collins ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Vessel Wall ◽  
Time Course ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Steady Increase ◽  
Time Interval ◽  
Venous Thrombus ◽  
Scanning Electron

Summary Objective. Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease. Design and Methods. Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction. Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval). Results. Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter.

scholarly journals Platelet Activation State Intermixing in a Venous Puncture Model Indicates Novel Patterns of Thrombus Formation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 9-9
Author(s):  
Brian Storrie ◽  
Sung W. Rhee ◽  
Irina D Pokrovskaya ◽  
Kenny Ling ◽  
Yajnesh Vedanaparti ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Activation ◽  
Vessel Wall ◽  
Jugular Vein ◽  
Thrombus Formation ◽  
Sem Images ◽  
Puncture Wound ◽  
Activated Platelets ◽  
Puncture Model ◽  
Scanning Electron

Introduction: Platelet recruitment to generate a thrombus is key to bleeding cessation. That recruitment is dependent on a series of platelet activation processes that include adhesion to the exposed vessel matrix, platelet-platelet adhesion and platelet granule release. How platelet activation is patterned to generate a thrombus has previously been studied by intravital light microscopy, two-photon microscopy and scanning electron microscopy at resolutions insufficient to infer platelet activation at the level of the individual platelet. Here, we present a collaborative effort to stratify spatially the extent of platelet activation at the cellular level in a mouse jugular vein puncture model. We used wide-area transmission electron microscopy (WA-TEM) and serial block face scanning electron microscopy (SBF-SEM) at a resolution of 3 to 100 nm across whole thrombi to determine activation state of individual platelets. Our results, indicate a pattern of platelet stratification within the puncture wound that varies in a time-dependent manner with distinct structural stages in the formation of the thrombus. Methods: Jugular vein thrombi from C57BL/6 mice were collected 1, 5, or 20 min after a 300 µm needle puncture and prepared for EM imaging. For WA-TEM, hundreds of overlapping 3 nm resolution images were acquired using a gondimeter stage. The images sets were aligned using NIH Fiji software to create a single high-res, thrombus-wide image. Individual platelets were stratified into morphologically defined activation states (1: no activation, 2: decreased granule number, 3: no visible granules left, 4: hollow inside). The spatial distribution of platelet stratification was analyzed using iVision software. For SBF-SEM, 100-nm XY-resolution SEM images were collected every 200 nm and 20 nm XY-resolution images every 20 µm. Semi-automated stratification of platelet activation state in individual slices of the thrombus were combined into a 3-D representation using Amira software. Volumetric distributions of platelets with respect to the puncture hole and the vascular wall were quantified. Results: Thrombus Formation Stage 1 (anchor and extend) -- One min post puncture, platelets were anchored in clumps along the exposed vessel wall. Near the damaged vessel wall was a peripheral layer of activated or degranulated platelets (states 3 and 4) covered by additional layers of less-activated platelets (state 1 and 2). Short cylindrical ingrowths extended into the 300 µm hole. Unexpectedly, large aggregates of platelets with a mixture of activation states (states 1 - 4) were found extending from these anchor points into the hole and vertically into the intravascular space. Aggregate surface layers were composed mostly of degranulated platelets (states 3 and 4). Less than 40% of neighboring platelets were of the same activation state as their neighbor. Surprisingly, &lt;2% of platelet-occupied volume within the puncture hole contained largely degranulated platelets (aggregates of only states 3 and 4). Stage 2 (cap and erect) -- At 5 min after injury, the puncture hole was capped. ~70% of platelets neighboring degranulated platelets (3 and 4) formed visible aggregates within the thrombus. These aggregates were found along the exposed vessel wall and encasing vertical platelet aggregate towers containing a mixture of platelets in different states (1 - 4). Towers were typically separated by large cavities. SBF-SEM images, a machine-based, unbiased sampling of the underlying platelet distribution, revealed that ~10% of the platelet volume in the puncture hole of the thrombus and the intravascular towers contained largely degranulated platelets, similar to the data from WA-TEM. Stage 3 (infill and remodeling) - At 20 min post-puncture, the thrombus was filled with a mixture of platelets of varying activation states, which surrounded central, vertical aggregates (towers) of degranulated platelets seen at 5 min. Only minor cavity space was apparent. The intravascular surface of the thrombus was covered with an ~10 platelet-thick layer of loosely packed, variably activated platelets (states 1 - 4). Conclusions: Our results demonstrate dynamic spatial patterns of platelet activation within a forming puncture-wound thrombus. Such patterns yield insights into thrombus formation and suggest the need to reference platelets defects and anti-thrombotics drugs against new models. Figure Disclosures No relevant conflicts of interest to declare.

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