scholarly journals Counting the platelets: a robust and sensitive quantification method for thrombus formation

2016 ◽  
Vol 115 (06) ◽  
pp. 1178-1190 ◽  
Author(s):  
Tomas Lindahl ◽  
Lars Faxälv ◽  
Kjersti Claesson
Keyword(s):  
Platelet Count ◽  
Fluorescence Intensity ◽  
Thrombus Formation ◽  
Time Lapse ◽  
Flow Chamber ◽  
Volume Estimation ◽  
Research Groups ◽  
Study Objective ◽  
The Stability

SummaryFlow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, yand z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

scholarly journals Platelet-derived microparticles associate with fibrin during thrombosis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4651-4663 ◽  
Author(s):  
P Siljander ◽  
O Carpen ◽  
R Lassila
Keyword(s):  
Type I Collagen ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Type I ◽  
Binding Studies ◽  
Vitro Binding ◽  
Large Platelet ◽  
Platelet Aggregates ◽  
Thrombin Antithrombin Iii Complex

Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P- selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P- selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Flow chamber-based assays to measure thrombus formation in vitro: requirements for standardization

2011 ◽  
Vol 9 (11) ◽  
pp. 2322-2324 ◽  
Author(s):  
M. ROEST ◽  
A. REININGER ◽  
J. J. ZWAGINGA ◽  
M. R. KING ◽  
J. W. M. HEEMSKERK ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Flow Chamber

scholarly journals Quantification of Platelet Contractile Movements during Thrombus Formation

2018 ◽  
Vol 118 (09) ◽  
pp. 1600-1611 ◽  
Author(s):  
Kjersti Tunströmer ◽  
Lars Faxälv ◽  
Niklas Boknäs ◽  
Tomas Lindahl
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Time Lapse ◽  
Flow Chamber ◽  
Systematic Analysis ◽  
Time Points ◽  
Time Lapse Microscopy ◽  
Dynamic Events ◽  
Staining Protocol ◽  
Detailed Mathematical Analysis

AbstractImaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways.

scholarly journals Platelet interaction with activated endothelium: mechanistic insights from microfluidics

Blood ◽  
2017 ◽  
Vol 130 (26) ◽  
pp. 2819-2828 ◽  
Author(s):  
Daniëlle M. Coenen ◽  
Tom G. Mastenbroek ◽  
Judith M. E. M. Cosemans
Keyword(s):  
Molecular Mechanisms ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Coagulation System ◽  
Intercellular Adhesion Molecule ◽  
Platelet Glycoprotein ◽  
Intercellular Adhesion Molecule 1 ◽  
Chamber Studies

Abstract Traditionally, in vitro flow chamber experiments and in vivo arterial thrombosis studies have been proved to be of vital importance to elucidate the mechanisms of platelet thrombus formation after vessel wall injury. In recent years, it has become clear that platelets also act as modulators of inflammatory processes, such as atherosclerosis. A key element herein is the complex cross talk between platelets, the coagulation system, leukocytes, and the activated endothelium. This review provides insight into the platelet-endothelial interface, based on in vitro flow chamber studies and cross referenced with in vivo thrombosis studies. The main mechanisms of platelet interaction with the activated endothelium encompass (1) platelet rolling via interaction of platelet glycoprotein Ib-IX-V with endothelial-released von Willebrand factor with a supporting role for the P-selectin/P-selectin glycoprotein ligand 1 axis, followed by (2) firm platelet adhesion to the endothelium via interaction of platelet αIIbβ3 with endothelial αvβ3 and intercellular adhesion molecule 1, and (3) a stimulatory role for thrombin, the thrombospondin-1/CD36 axis and cyclooxygenase 1 in subsequent platelet activation and stable thrombus formation. In addition, the molecular mechanisms underlying the stimulatory effect of platelets on leukocyte transendothelial migration, a key mediator of atheroprogression, are discussed. Throughout the review, emphasis is placed on recommendations for setting up, reporting, interpreting, and comparing endothelial-lined flow chamber studies and suggestions for future studies.

scholarly journals How we discovered fluorescent speckle microscopy

2011 ◽  
Vol 22 (21) ◽  
pp. 3940-3942 ◽  
Author(s):  
E. D. Salmon ◽  
Clare M. Waterman
Keyword(s):  
Epithelial Cells ◽  
Fluorescence Intensity ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Living Cells ◽  
Variable Intensity ◽  
Macromolecular Assemblies ◽  
Dynamic Assembly ◽  
Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

scholarly journals WAKTU PENYIMPANAN TROMBOSIT TERKAIT JUMLAH DI KONSENTRAT TROMBOSIT

2016 ◽  
Vol 20 (3) ◽  
pp. 224
Author(s):  
Raehana Samad ◽  
Agus Alim Abdullah ◽  
Kusriny A.P. ◽  
Mansyur Arif
Keyword(s):  
Platelet Count ◽  
Storage Time ◽  
Blood Donors ◽  
Impedance Method ◽  
Storage Process ◽  
Series Design ◽  
The Stability ◽  
The Impact

The transfusion of thrombocyte concentrate is one of the important medical approaches to make the platelet count raised in patients with thrombocytopenia. The TC could change during the storage process, so the in vitro storaging should be considered to minimalize the alterations on the platelet count. The study was aimed to know the impact of storage time to maintain the platelet count stability in TC of transfusion blood by evaluating it. A study with Time Series design was performed in 30 samples of TC of transfusion blood collected from 30 blood donors. The samples was collected from the bag tube of TC and the test is performed as soon as possible by using haematological analyzer (Sysmex KX-21) with an impedance method, the rest of the samples was stored in an agitator on 22±2°C to perform a repeated count on day 5th and 7th. The collected data was analyzed with “Paired T Test”. The results of this study showed that the platelet count in TC of transfusion blood was decreased in the fifth day compared to the first day, but the decreased matter is insignificant, statistically (p=0.13). While after seventh (7) day, the platelet count is decreased significantly (p=0.00). The researchers concluded that the storage of TC for seven (7) days could not maintain the stability of the platelet count of the transfusion blood. There fore is suggested, a further study to evaluate the impact of storage time on platelets viability. So the quality of platelets in TC of transfusion blood can be evaluated.

FcγRIIa Enhances Thrombus Growth in Vitro and in Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 191-191
Author(s):  
Huiying Zhi ◽  
Lubica Rauova ◽  
Vincent M Hayes ◽  
Jimmy Crockett ◽  
Cunji Gao ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Function ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Wild Type ◽  
Integrin Αiibβ3 ◽  
Platelet Thrombus

Abstract Abstract 191 Outside-in signal transduction is one of several autocrine amplification loops that platelets employ to stabilize and consolidate a platelet thrombus following their adhesion to each other or to components of the extracellular matrix. Binding of soluble fibrinogen to activated integrin αIIbβ3 on the platelet surface, or binding of αIIbβ3 to platelet-immobilized fibrinogen, initiates an outside-in signaling cascade that results in the activation of integrin β3-associated Src family kinases, which in turn phosphorylate tyrosine residues within the cytoplasmic domain of the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, FcγRIIa. “Activation” of FcγRIIa sets off a cascade of events that result in the assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2(PLCγ2), through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. We have previously shown that either antibody-mediated or genetic disruption of the functional interaction between integrin αIIbβ3 and FcγRIIa blocks tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2, and inhibits platelet spreading on immobilized fibrinogen. The physiological significance of FcγRIIa in supporting platelet thrombus formation, however, remains unknown. To further explore the importance of FcγRIIa in platelet function, we compared the relative ability of wild-type FcγRIIa-negative and transgenic FcγRIIa-positive (FcγRIIaTGN) murine platelets to support thrombosis and hemostasis in a number of well-accepted models of platelet function. FcγRIIaTGN platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen. FcγRIIaTGN platelets also retracted a fibrin clot faster than did wild-type FcγRIIa-negative platelets. When anti-coagulated whole blood was perfused over a collagen-coated flow chamber under conditions of arterial shear, the rate and extent of adhesion, aggregation, and thrombus formation was significantly increased for FcγRIIaTGN platelets compared to their wild-type murine counterparts. Addition of Fab fragments specific for FcγRIIa to whole blood derived from either humans or FcγRIIaTGN mice strongly inhibited thrombus formation in the arterial in vitro flow chamber assay. Finally, to examine the in vivo relevance of FcγRIIa, mice were subjected to two models of vascular injury: electrolytic injury of the femoral vein and laser injury of cremaster arterioles. In both in vivo models, FcγRIIaTGN mice displayed increased thrombus formation compared with their wild-type, FcγRIIa-negative counterparts. Taken together, these data establish FcγRIIa as a physiologically-important functional conduit for αIIbβ3–mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis. Disclosures: No relevant conflicts of interest to declare.

The Effect of High Storage Temperature on the Stability and Efficacy of Lyophilized Tenecteplase

2020 ◽  
Vol 35 (5) ◽  
pp. 501-507
Author(s):  
Emily Henkel ◽  
Rebecca Vella ◽  
Andrew Fenning
Keyword(s):  
Critical Care ◽  
Storage Temperature ◽  
Short Exposure ◽  
Protein Drug ◽  
Single Chain ◽  
Study Objective ◽  
The Stability ◽  
The Impact ◽  
D Dimer

AbstractIntroduction:Tenecteplase is a thrombolytic protein drug used by paramedics, emergency responders, and critical care medical personnel for the prehospital treatment of blood clotting diseases. Minimizing the time between symptom onset and the initiation of thrombolytic treatment is important for reducing mortality and improving patient outcomes. However, the structure of protein drug molecules makes them susceptible to physical and chemical degradation that could potentially result in considerable adverse effects. In locations that experience extreme temperatures, lyophilized tenecteplase transported in emergency service vehicles (ESVs) may be subjected to conditions that exceed the manufacturer’s recommendations, particularly when access to the ambulance station is limited.Study Objective:This study evaluated the impact of heat exposure (based on temperatures experienced in an emergency vehicle during summer in a regional Australian city) on the stability and efficacy of lyophilized tenecteplase.Methods:Vials containing 50mg lyophilized tenecteplase were stored at 4.0°C (39.2°F), 35.5°C (95.9°F), or 44.9°C (112.8°F) for a continuous period of eight hours prior to reconstitution. Stability and efficacy were determined through assessment of: optical clarity and pH; analyte concentration using UV spectrometry; percent protein monomer and single chain protein using size-exclusion chromatography; and in vitro bioactivity using whole blood clot weight and fibrin degradation product (D-dimer) development.Results:Heat treatment, particularly at 44.9°C, was found to have the greatest impact on tenecteplase solubility; the amount of protein monomer and single chain protein lost (suggesting structural vulnerability); and the capacity for clot lysis in the form of decreased D-dimer production. Meanwhile, storage at 4.0°C preserved tenecteplase stability and in vitro bioactivity.Conclusion:The findings indicate that, in its lyophilized form, even relatively short exposure to high temperature can negatively affect tenecteplase stability and pharmacological efficacy. It is therefore important that measures are implemented to ensure the storage temperature is kept below 30.0°C (86.0°F), as recommended by manufacturers, and that repeated refrigeration-heat cycling is avoided. This will ensure drug administration provides more replicable thrombolysis upon reaching critical care facilities.

scholarly journals Sphingosine kinase 1 (Sphk1) negatively regulates platelet activation and thrombus formation

2014 ◽  
Vol 307 (10) ◽  
pp. C920-C927 ◽  
Author(s):  
Patrick Münzer ◽  
Evi Schmid ◽  
Britta Walker ◽  
Anna Fotinos ◽  
Madhumita Chatterjee ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Sphingosine Kinase ◽  
Flow Chamber ◽  
Negative Regulator ◽  
Sphingosine Kinase 1 ◽  
Shear Rates ◽  
Dense Granules ◽  
Platelet Formation

Sphingosine 1-phosphate (S1P) is a powerful regulator of platelet formation. Enzymes generating S1P include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Activation-dependent platelet integrin αIIbβ3activation and secretion of platelets lacking functional sphingosine kinase 1 ( sphk1−/−) and of wild-type platelets ( sphk1+/+) were determined utilizing flow cytometry and chronolume luciferin assay. Cytosolic Ca2+activity ([Ca2+]i) and aggregation were measured using fura-2 fluorescence and aggregometry, respectively. In vitro platelet adhesion and thrombus formation were evaluated using a flow chamber with shear rates of 1,700 s−1. Activation-dependent increase of [Ca2+]i, degranulation (release of alpha and dense granules), integrin αIIbβ3activation, and aggregation were all significantly increased in sphk1−/−platelets compared with sphk1+/+platelets. Moreover, while platelet adhesion and thrombus formation under arterial shear rates were significantly augmented in Sphk1-deficient platelets, bleeding time and blood count were unaffected in sphk1−/−mice. In conclusion, sphingosine kinase 1 is a powerful negative regulator of platelet function counteracting degranulation, aggregation, and thrombus formation.

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