scholarly journals Platelet aggregation in flowing blood at a site of injury to an endothelial cell monolayer: quantitation and real-time imaging with the TAB monoclonal antibody

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 390-398
Author(s):  
EF Grabowski
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cell ◽  
Real Time ◽  
Platelet Adhesion ◽  
Cell Monolayer ◽  
Thromboxane B2 ◽  
Endothelial Cell Monolayer ◽  
Lysine Acetylsalicylate ◽  
Flowing Blood ◽  
A Site

Epifluorescence videomicroscopy permits real-time imaging of platelet adhesion/aggregation to a defined microinjury of a monolayer of endothelial cells exposed to flowing blood. The fluorescent label is the TAB murine monoclonal antibody directed against human platelet GP IIB, together with a fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The combination assures specificity for platelet membranes, yet leaves platelet function intact. TAB is first added to gently mixed, citrated human blood; the second antibody is added 1 hour after the first, mixing continuing for a second hour. Bovine aortic endothelial cell monolayers (ECMs), grown on rectangular cover glasses precoated with microfibrillar collagen, comprise one wall of a flow chamber mounted on a vertical microscope stage. A loop of 6–0 sterile suture is drawn across the ECM in order to create microinjuries of width 70 +/- 15 microns (mean +/- SD) oriented in a direction transverse to flow. Platelet adhesion/aggregation is virtually absent on intact and confluent regions of the monolayer. On micro-injury sites and at shear rates of 60 to 1,080 sec-1, however, computer-enhanced images obtained by means of videomicroscopy show arrival and adherence of single platelets resulting in the formation of platelet aggregates elongated in the flow direction. When the monolayers are pretreated with 1.0 mmol/L lysine acetylsalicylate, the mean aggregate thickness increases (2P less than .05) to 260 +/- 58% (mean +/- SE, N = 6) of control, aggregates are regularly shed downstream, and the surface area of the injury site covered by platelets is augmented (2P less than .05) from 14.8 +/- 3.9% to 49.2 +/- 4.7% (mean +/- SE, N = 6). Donor ingestion of aspirin, on the other hand, leads to an increase (2P less than .01) in percent surface coverage to 42.7 +/- 8.5 without a concomitant increase in mean aggregate thickness. In parallel with the above, outflow levels of serum thromboxane and prostacyclin are measured by radioimmunoassays (RIAs) for thromboxane B2 and 6-Keto-PGF1 alpha, respectively. Thromboxane B2 is increased (2P less than .01) by monolayer pretreatment with lysine acetylsalicylate from 5.08 +/- 1.47 to 9.35 +/- 2.42, but decreased (2P less than .05) after oral aspirin to 1.21 +/- 0.38 ng/mL (mean +/- SE, N = 6). Levels of 6-Keto-PGF1 alpha were reduced (2P less than .05) by monolayer pretreatment from 0.48 +/- 0.046 to 0.36 +/- 0.016 ng/mL. Platelet adhesion/aggregation at a site of injury to an endothelial cell monolayer, therefore, can be imaged in flowing blood in real time using a monoclonal antibody approach.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Platelet aggregation in flowing blood at a site of injury to an endothelial cell monolayer: quantitation and real-time imaging with the TAB monoclonal antibody

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 390-398 ◽  
Author(s):  
EF Grabowski
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cell ◽  
Real Time ◽  
Platelet Adhesion ◽  
Cell Monolayer ◽  
Thromboxane B2 ◽  
Endothelial Cell Monolayer ◽  
Lysine Acetylsalicylate ◽  
Flowing Blood ◽  
A Site

Abstract Epifluorescence videomicroscopy permits real-time imaging of platelet adhesion/aggregation to a defined microinjury of a monolayer of endothelial cells exposed to flowing blood. The fluorescent label is the TAB murine monoclonal antibody directed against human platelet GP IIB, together with a fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The combination assures specificity for platelet membranes, yet leaves platelet function intact. TAB is first added to gently mixed, citrated human blood; the second antibody is added 1 hour after the first, mixing continuing for a second hour. Bovine aortic endothelial cell monolayers (ECMs), grown on rectangular cover glasses precoated with microfibrillar collagen, comprise one wall of a flow chamber mounted on a vertical microscope stage. A loop of 6–0 sterile suture is drawn across the ECM in order to create microinjuries of width 70 +/- 15 microns (mean +/- SD) oriented in a direction transverse to flow. Platelet adhesion/aggregation is virtually absent on intact and confluent regions of the monolayer. On micro-injury sites and at shear rates of 60 to 1,080 sec-1, however, computer-enhanced images obtained by means of videomicroscopy show arrival and adherence of single platelets resulting in the formation of platelet aggregates elongated in the flow direction. When the monolayers are pretreated with 1.0 mmol/L lysine acetylsalicylate, the mean aggregate thickness increases (2P less than .05) to 260 +/- 58% (mean +/- SE, N = 6) of control, aggregates are regularly shed downstream, and the surface area of the injury site covered by platelets is augmented (2P less than .05) from 14.8 +/- 3.9% to 49.2 +/- 4.7% (mean +/- SE, N = 6). Donor ingestion of aspirin, on the other hand, leads to an increase (2P less than .01) in percent surface coverage to 42.7 +/- 8.5 without a concomitant increase in mean aggregate thickness. In parallel with the above, outflow levels of serum thromboxane and prostacyclin are measured by radioimmunoassays (RIAs) for thromboxane B2 and 6-Keto-PGF1 alpha, respectively. Thromboxane B2 is increased (2P less than .01) by monolayer pretreatment with lysine acetylsalicylate from 5.08 +/- 1.47 to 9.35 +/- 2.42, but decreased (2P less than .05) after oral aspirin to 1.21 +/- 0.38 ng/mL (mean +/- SE, N = 6). Levels of 6-Keto-PGF1 alpha were reduced (2P less than .05) by monolayer pretreatment from 0.48 +/- 0.046 to 0.36 +/- 0.016 ng/mL. Platelet adhesion/aggregation at a site of injury to an endothelial cell monolayer, therefore, can be imaged in flowing blood in real time using a monoclonal antibody approach.(ABSTRACT TRUNCATED AT 400 WORDS)

RHEOLOGY AND PRIMARY HEMOSTASIS

1987 ◽  
Author(s):  
F E Grabowski
Keyword(s):  
Endothelial Cell ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Cell Monolayer ◽  
Shear Rates ◽  
Primary Hemostasis ◽  
Flowing Blood ◽  
A Site ◽  
Wall Injury ◽  
Oral Aspirin

Overview The adhesion-aggregation of platelets to a site of vessel wall injury is a quintessential blood flow phenomenon. Firstly, platelets are driven to the vicinity of the vessel wall by a form of convective diffusion in which red cells both mechanically augment the effective platelet diffusivity (Turitto et al., Ind. Eng. Chem. Fund. 11:216-223, 1972; Grabowski et al., Ind. Eng. Chem. Fund. 11:224-232, 1972) and enhance the near-wall piatelet concentration (Ti11es and Eckstein, Microvasc Res., In press, 1987). Secondly, red cells subjected to physiologic shear forces are capable of secreting sufficient adenine nucleotides to induce primary platelet aggregation without themselves undergoing frank lysis (Reimers et al, Blood 64:1200-1206, 1984). This "humoral" effect of erythrocytes is likely to contribute to primary hemostasis in a shear stress-dependent manner. Thirdly, endothelial cells are able to modulate platelet aggregation at a site of vessel injury by producing prostacyclin (and perhaps other antithrombotic substances) in a manner which increases with vessel shear rate (Grabowski et al, Blood 62:301a, 1983); production for a large range of arterial shear rates appears to be limited by plasma-borne substrate (arachidonate). This manner of production ensures a concentration of prostacyclin in the near-wall region which remains relatively independent of shear rate.Imaging primary hemostasis. In our work, epi-fluorescence videomicroscopy has allowed real time imaging of platelet adhesion-aggregation to a simulated vessel wall injury. The injury model is an endothelial cell monolayer (ECM) across which, prior to ECM exposure to flowing blood, a 6-0 sterile suture is drawn in a direction transverse to flow. Microinjuries result which measure 70 ± 15μm (Mean ± SD) in width. The fluorescent label is the TAB murine monoclonal antibody (courtesy of Dr. R.P. McEver) directed against human platelet GPIIB, together with a fluorescein-conjugated goat F(ab')2 against murine inmunoglobulin. The injured ECM's, grown to confluence on rectangular cover glasses precoated with microfibrillar collagen, comprise one wall of a flow chamber mounted on a vertical microscope stage. On microinjury sites and at shear rates of 100 to 700 sec-1, computer-enhanced video images show adherence, remodelling and growth of chains of platelet aggregates. Aligned with the flow direction, these chains have a spacing of approximately 30)im, a length similar to the average endothelial cell diameter. One may speculate that such chains provide a scaffold for wound healing insofar as they are likely rich in agents chemotactic for leukocytes and in platelet-derived growth factor.Modulatory role of endothelium. When the ECM's are pre treated with 1.0 mM FC lysine acetyl sal icy late (LA), aggregate length increases (P<0.001) up totwo-fold, outflow levels by RIA of serum thromboxane B2 increase (8 of 8 paired runs), and outflow levels of prostacyclin by RIA for 6-Keto PGFiot decrease (5 of 7 paired runs). The Table gives data for one of four similar experiments at 270 sec-1 and following five minutes of flow. These data imply that products of ECM which are inhibitable by aspirin modulate local adhesion-aggregation; their inhibition, as by vasculitis or drugs, may give rise to thrombotic states.Bleeding disorders. Aggregate length is reduced in von Willebrand's disease (4 patients), Hermansky-Pudlak syndrome (2 patients), and after 300 mg oral aspirin (Tablet 4 donors). The reduction in the first two, however, is greater (P<0.01) than that for oral aspirin. With oral aspirin, further, there is a paradoxic increase in the percent platelet coverage of the injury area. Summary. Rheology has profound effects on the rate, structure, and modulation of primary hemostasis. Many of these effects can be studied via real-time, epi-fluorescence videomicroscopy of platelet adhesion-aggregation to a site of injury to an endothelial cell monolayer exposed to flowing blood. The model described has application to the study of thrombotic and hemostatic disorders and unstable angina.

Effect of Shear Stress on Platelet Adhesion to Expanded Polytetrafluoroethylene, a Silicone Sheet, and an Endothelial Cell Monolayer

ASAIO Journal ◽  
2000 ◽  
Vol 46 (6) ◽  
pp. 696-701 ◽  
Author(s):  
Katsuko Sakai Furukawa ◽  
Takashi Ushida ◽  
Hirohito Sugano ◽  
Tamotsu Tamaki ◽  
Norio Ohshima ◽  
...  
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Platelet Adhesion ◽  
Cell Monolayer ◽  
Endothelial Cell Monolayer ◽  
Expanded Polytetrafluoroethylene ◽  
Silicone Sheet

CHARACTERIZATION OF DISORDERS OF PLATELET-VESSEL WALL INTERACTION IN AN AGGREGOMETER INCORPORATING BLOOD FLOW PAST AN ENDOTHELIAL CELL MONOLAYER

1987 ◽  
Author(s):  
E F Grabowski ◽  
K McKenny
Keyword(s):  
Endothelial Cell ◽  
Cell Monolayer ◽  
Fold Increase ◽  
Flow Chamber ◽  
Fluorescent Label ◽  
Endothelial Cell Monolayer ◽  
Flowing Blood ◽  
Normal States ◽  
Wall Interaction

Epi-fluorescence videomicroscopy permits real-time imaging of platelet (plt) adhesion-aggregation to a defined microinjury site of an endothelial cell monolayer (ECM) exposed to flowing blood. The fluorescent label is the TAB murine monoclonal antibody (courtesy of Dr. R.P. McEver) directed against human pit cp HB, together with a fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The combination assures specificity for pit membranes, yet leaves pit function intact. Bovine aortic ECM, grown on rectangular cover glasses, comprise one wall of a flow chamber mounted on a vertical microscope stage. A 6-0 sterile suture, drawn across the ECM in a direction transverse to flow, creates microinjuries of width 70 ± 15 (mean ± SD). Pit deposition is virtually absent upon intact and confluent regions of the ECM. On microinjury sites and at a shear rate of 270 sec-1, however, computer-enhanced images show pit adherence, aggregation, and embolization. Pretreatment of the ECM with 1.0 mMFC lysine acetyl salicylate, further, leads to a three-fold increase in aggregate length. ECM products inhibitable by aspirin, therefore, modulate adhesion-aggregation in disease and normal states under physiologic flow conditions. The Table shows that nercent coverage of the injury area, and mean aggregate length readily discriminate normal, post-aspirin, and von Willebrand's (vWD's) bloods. Aggregate length is reduced in vWD's blood to a greater degree (p<0.01) than by oral aspirin, while the latter is associated with a paradoxic increase (p<0.01) in single plt adhesion.

scholarly journals ATP increases the migration of microglia across the brain endothelial cell monolayer

2016 ◽  
Vol 36 (2) ◽  
Author(s):  
Tomoji Maeda ◽  
Manato Inagaki ◽  
Yu Fujita ◽  
Takehiro Kimoto ◽  
Chiaki Tanabe-Fujimura ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Matrix Metalloproteinases ◽  
Real Time ◽  
Culture System ◽  
Cell Monolayer ◽  
Brain Endothelial Cell ◽  
Endothelial Cell Monolayer ◽  
Microglial Migration ◽  
The Brain

To elucidate the mechanism of microglial migration across the blood–brain barrier (BBB), we developed an in vitro co-culture system and analysed real-time BBB integrity during transmigration. We show that ATP promotes microglia transmigration via a mechanism involving microglial matrix metalloproteinases (MMPs).

Arthrospira platensis accelerates the formation of an endothelial cell monolayer and protects against endothelial cell detachment after bacterial contamination

2021 ◽  
pp. 1-11
Author(s):  
A. Krüger-Genge ◽  
S. Steinbrecht ◽  
C.G.H. Jung ◽  
Sophia Westphal ◽  
Stefanie Klöpzig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Bacterial Contamination ◽  
Cell Monolayer ◽  
Arthrospira Platensis ◽  
Bacterial Toxin ◽  
Cell Detachment ◽  
Endothelial Cell Monolayer ◽  
Positive Effects ◽  
Anti Oxidant

Within the last years a comprehensive number of scientific studies demonstrated beneficial effect of Arthropira platensis (AP) as dietary supplement due to a high content of proteins, minerals and vitamins. Positive effects like promoting the immune system, reducing inflammation and an anti-oxidant capacity are reported. In this study, the effect of an aqueous AP extract on primary human venous endothelial cells (HUVEC) was investigated. In addition, the effect of AP on HUVEC treated with a bacterial toxin (lipopolysaccharide, LPA), inducing an activation of HUVEC and cellular detachment, was analyzed. Depending on the concentration of AP extract a significantly accelerated formation of an endothelial cell monolayer was observed. Furthermore, the detachment of HUVEC after LPA addition was dramatically reduced by AP. In conclusion, the data are promising and indicatory for an application of Arthrospira platensis in the clinical field.

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