Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

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Ultrastructural Characteristics of Platelets Separated from Plasma by ADP-Induced Aggregation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090804 ◽

1976 ◽

Vol 34 ◽

pp. 164-165

Author(s):

B.A. Shinoda ◽

M.D. Hardison ◽

S.F. Mohammad ◽

H.Y.K. Chuang ◽

R.G. Mason

Keyword(s):

Blood Platelets ◽

Gel Filtration ◽

Differential Centrifugation ◽

Ultrastructural Characteristics ◽

Induced Aggregation

The utilization of blood platelets in experimentation frequently requires their separation from blood and subsequent resuspension in media of known composition. Several methods are available for preparation of isolated platelets (1-3) by differential centrifugation or gel filtration, but most methods are tedious and time consuming. Often platelets obtained by use of such methods are in a state different functionally and ultrastructurally from that of platelets in plasma (4).Recently Mohammad, Reddick, and Mason (5) reported a method in which platelets were separated from plasma by ADP-induced aggregation, washed several times, and then incubated in a carefully selected medium that resulted in deaggregation of platelets.

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Thrombus formation on artificial surfaces: Correlative microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016176x ◽

1990 ◽

Vol 48 (3) ◽

pp. 840-841

Author(s):

Quintin J. Lai ◽

Stuart L. Cooper ◽

Ralph M. Albrecht

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Low Voltage ◽

Thrombus Formation ◽

Blood Platelets ◽

Polymer Surfaces ◽

Flow Conditions ◽

Transmission Electron ◽

Adhesion Of Platelets ◽

Microscopic Techniques

Thrombus formation and embolization are significant problems for blood-contacting biomedical devices. Two major components of thrombi are blood platelets and the plasma protein, fibrinogen. Previous studies have examined interactions of platelets with polymer surfaces, fibrinogen with platelets, and platelets in suspension with spreading platelets attached to surfaces. Correlative microscopic techniques permit light microscopic observations of labeled living platelets, under static or flow conditions, followed by the observation of identical platelets by electron microscopy. Videoenhanced, differential interference contrast (DIC) light microscopy permits high-resolution, real-time imaging of live platelets and their interactions with surfaces. Interference reflection microscopy (IRM) provides information on the focal adhesion of platelets on surfaces. High voltage, transmission electron microscopy (HVEM) allows observation of platelet cytoskeletal structure of whole mount preparations. Low-voltage, high resolution, scanning electron microscopy allows observation of fine surface detail of platelets. Colloidal gold-labeled fibrinogen, used to identify the Gp Ilb/IIIa membrane receptor for fibrinogen, can be detected in all the above microscopies.

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Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647055 ◽

1988 ◽

Vol 60 (02) ◽

pp. 328-333 ◽

Cited By ~ 44

Author(s):

N J de Fouw ◽

Y F de Jong ◽

F Haverkate ◽

R M Bertina

Keyword(s):

Plasminogen Activator ◽

Protein C ◽

Blood Platelets ◽

Plasminogen Activator Inhibitor ◽

Protein S ◽

Tissue Type ◽

Clot Lysis ◽

Activator Inhibitor ◽

Pai 1

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

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Decreased Platelet-Free Dopamine and Unchanged Noradrenaline and Adrenaline in Essential Hypertension

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647040 ◽

1988 ◽

Vol 60 (02) ◽

pp. 251-254 ◽

Cited By ~ 4

Author(s):

S E Kjeldsen ◽

K Gjesdal ◽

P Leren ◽

I K Eide

Keyword(s):

Body Weight ◽

Essential Hypertension ◽

Blood Platelets ◽

Plasma Catecholamine ◽

Catecholamine Synthesis ◽

Hypertensive Group ◽

Plasma Catecholamine Concentrations ◽

Noradrenaline And Adrenaline ◽

Over Time ◽

Normotensive Control

SummaryThe content of free-catecholamines in blood platelets is much higher than in plasma and platelet catecholamines must be taken up from plasma, since platelets lack the enzymes for catecholamine synthesis. There is some evidence that platelet catecholamine content under certain circumstances may be an integrated measure of plasma catecholamine concentrations over time. Platelet-free catecholamines were therefore assayed in 18 untreated patients with essential hypertension and in 16 normotensive control subjects. Mean platelet-free dopamine in the hypertensive group was 3.7 ± 0.4 pg/mg platelet weight, i.e. significantly less than the 6.5 ± 0.9 pg/mg found in the normotensive (p <0.005). Platelet contents of noradrenaline and adrenaline did not differ. Decreased platelet-free dopamine and unchanged platelet noradrenaline and adrenaline persisted after adjustment for increased body weight in the hypertensive group. Although the reasons for decreased platelet-free dopamine in the hypertensive group remain unknown, this finding may add to previous result showing facilitated release of granular contents from blood platelets in patients with essential hypertension. Our data do not support platelet levels of free-catecholamines to be a marker of increased sympathetic tone in essential hypertension.

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Platelet Adhesion to Native Collagens Involves Proteoglycans and May Be a Two-Step Process

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645970 ◽

1987 ◽

Vol 58 (02) ◽

pp. 786-789 ◽

Cited By ~ 5

Author(s):

O Behnke

Keyword(s):

Electron Microscope ◽

Platelet Adhesion ◽

Close Contact ◽

Blood Platelets ◽

Collagen Fibrils ◽

Platelet Membrane ◽

Step Process ◽

Rat Blood ◽

Wide Gap ◽

Rat Tail

SummaryAdhesion of rat blood platelets to native rat tail collagen fibrils was studied in the electron microscope under conditions that preserved collagen-associated proteoglycans (CAPG). The CAPG molecules were aligned in chain-like configurations that encircled the fibrils with a 65 nm period; they appeared to coat the fibrils completely and extended 60-100 nm away from the fibril. The initial platelet-fibril contact occurred between the platelet glycocalyx and the CAPG of the fibrils i.e. between two surfaces with net-negative charges. When close contact was established between the fibril surface proper and the platelet membrane, CAPG were not identified in the area of contact, and the collagen-platelet distance was reduced to a ~10-12 nm wide gap traversed by delicate links in register with fibril periodicities.

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Storage of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647709 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 405-416 ◽

Cited By ~ 3

Author(s):

M. R Hardeman ◽

Carina J L. Heynens

Keyword(s):

Storage Temperature ◽

Platelet Rich Plasma ◽

Blood Platelets ◽

Storage Period ◽

Serotonin Uptake ◽

Hypotonic Shock ◽

Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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The Effect of Mitomycin C on Platelet Aggregation and Adenosine 3′, 5′-Monophosphate Metabolism

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646667 ◽

1978 ◽

Vol 39 (01) ◽

pp. 177-185 ◽

Cited By ~ 3

Author(s):

Shuichi Hashimoto ◽

Sachiko Shibata ◽

Bonro Kobayashi

Keyword(s):

Platelet Aggregation ◽

Cyclic Amp ◽

Mitomycin C ◽

Blood Platelets ◽

Adenosine Diphosphate ◽

Cellular Membrane ◽

Adenyl Cyclase ◽

Cyclic Amp Phosphodiesterase ◽

Membrane Structure And Function ◽

And Function

SummaryThe effect of Mitomycin C on aggregation, adenosine 3′, 5′-monophosphate (cyclic AMP) metabolism and reactions induced by thrombin was studied in rabbit platelets. Mitomycin C inhibited the platelet aggregation induced by adenosine diphosphate or thrombin. The level of radioactive cyclic AMP derived from 8-14C adenine or 8-14C adenosine increased after incubating intact platelets with Mitomycin G. Formation of radioactive adenosine triphosphate also increased though mitochondrial oxidation was not stimulated. Similar effect was observed also in rabbit liver. Mitomycin C failed to stimulate platelet adenyl cyclase but inhibited cyclic AMP phosphodiesterase in the absence of theophylline. In the platelets preincubated with Mitomycin C, thrombin-induced inhibition of adenyl cyclase, stimulation of membrane-bound cyclic AMP phosphodiesterase, and release of 250,000 dalton protein from platelet membranes were prevented. These results suggest that Mitomycin C will affect cellular membrane structure and function, and this extranuclear effect of Mitomycin C will lead to inhibition of aggregation in blood platelets.

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Platelet and Shear Rate Promote Tumor Cell Adhesion to Human Endothelial Extracellular Matrix - Absence of a Role for Platelet Cyclooxygenase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646619 ◽

1989 ◽

Vol 61 (03) ◽

pp. 485-489 ◽

Cited By ~ 5

Author(s):

Eva Bastida ◽

Lourdes Almirall ◽

Antonio Ordinas

Keyword(s):

Cell Adhesion ◽

Shear Rate ◽

Tumor Cell ◽

Umbilical Vein ◽

Blood Platelets ◽

Flow Conditions ◽

Tumor Cell Adhesion ◽

Rate Dependent ◽

Static Conditions

SummaryBlood platelets are thought to be involved in certain aspects of malignant dissemination. To study the role of platelets in tumor cell adherence to vascular endothelium we performed studies under static and flow conditions, measuring tumor cell adhesion in the absence or presence of platelets. We used highly metastatic human adenocarcinoma cells of the lung, cultured human umbilical vein endothelial cells (ECs) and extracellular matrices (ECM) prepared from confluent EC monolayers. Our results indicated that under static conditions platelets do not significantly increase tumor cell adhesion to either intact ECs or to exposed ECM. Conversely, the studies performed under flow conditions using the flat chamber perfusion system indicated that the presence of 2 × 105 pl/μl in the perfusate significantly increased the number of tumor cells adhered to ECM, and that this effect was shear rate dependent. The maximal values of tumor cell adhesion were obtained, in presence of platelets, at a shear rate of 1,300 sec-1. Furthermore, our results with ASA-treated platelets suggest that the role of platelets in enhancing tumor cell adhesion to ECM is independent of the activation of the platelet cyclooxygenase pathway.

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