Dr. Mudasir A Lone

01/05/2023 2

I. The clumping together of two or more than two activated platelets.

II. Activation Factors:

a. Exposure to collagen due to injury in the endothelial layer.

b. vWF and tissue factor

c. Thrombin (formed with the help of tissue factor)

d. Negatively charged surfaces (e.g; Glass)

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III. Effects of activation results in:

a. Change in shape (Morphology)

b. Granule secretion (dense and alpha) into the blood.

c. Thromboxane A2 Synthesis, which activates other platelets.

Cytoskeletal remodelling

Inactivated Platelet Activated Platelets

A B C D

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C

D

AA

BA

A

A

C

D

B

C

D

B

B

B

C

C

1. Different possibilities For Interactions

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I. What your group has been working on ?

a. Effect of different agonists on the aggregation of platelets.

b. There is no emphasis on which cells are more prone to

aggregation ( and why?), role of tethers, membrane characteristics,

strength of the interaction forces that lead to aggregation?

II. Where is the room to improve our understanding?

a. Characterization, quantification and identification of

morphologically different platelets (activated) that

participate in aggregation (with and without agonists).

b. Measurement and strength of the inter-platelet interaction forces

that exist between morphologically different platelets (activated).

c. Influence of different activation factors on shape and strength of forces.

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I. Morphological Characterization of

Different Cell Types With Atomic Force

Microscopy.

Instead of using an incident beam to visualize

a sample, as would be the case in classical

Microscopy, AFM senses the small forces

(in the piconewton range, ,10-12

N) that act

on the sample surface.

a. Cells have been imaged with AFM.

b. Can provide high-resolution images of cell

surfaces under physiological conditions.

II. ?

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This image, taken with atomic force

microscopy, shows E. coli (2-6µm) bacteria

after they have been exposed to the

antimicrobial peptide CM15. The peptides have

begun destroying the bacteria’s cell walls.

(http://web.mit.edu/newsoffice/2010/micropeptides-

0315.html)

Topographical rearrangements shown by AFM images of myoblasts fusing due to

cytoskeletal dynamics during myogenesis.

(http://www.mechano-biology.ethz.ch)

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I. Characterization of Activated Cells On The Basis of Their Binding Strength With Bio-membrane Force Probe (BFP).

A B C D

Aggregation receptor is glycoprotein IIb/IIIa (gpIIb/IIIa); calcium-dependent

for fibrinogen. Other receptors areGPIb-V-IX complex (vWF) and GPVI

(collagen)

Connecting agent such as fibrinogen, fibronectin, vitronectin,thrombospondin, and vWF

(von Willebrand factor)

A

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The principle of atomic force microscopy. (A) The cantilever attached with a sharpened tip is scanned over the sample as the piezo transducer moves the sample in X–Y–Z

directions, using the feedback control to keep the amplitude of cantilever deflection (measured by an optical beam deflection system with a laser and photodiode detector)

constant. (B) Typical force curve and cantilever behavior on living cells. From position 1–2, the tip is approaching the cell surface. At position 3, the cantilever bends until the

force reaches the specified limit. The tip is then withdrawn towards positions 4 and 5. At position 5, with further retraction, the tip detaches from the surface. Between

positions 5 and 6, the cantilever return to its resting position and is ready for another measurement cycle.

(a) Specific, (b) non-specific, (c) no adhesion (d) multiple force

displacement curves of the interaction

Plot of adhesion force or binding interaction

strength.

AFM Study of the Effect of Multiple freeze drying on IgG

- AFM images reveal both crystalline and amorphous features. Globular, protein like features can also be observed.

- The size of the smaller globular features is similar to the size of monomeric IgG (~15-20nm) reported in previous AFM studies

(Ultramicroscopy 105:103-110). The larger globular features likely represent aggregates of IgG

250nm 250nm 250nm

Cycle 1 Cycle 2 Cycle 3

AFM images of freeze dried IgG samples, prepared without excipients (starting concentration 2mg/ml in 0.01M PBS)

Globular features

Crystalline

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AFM study of IgG Freeze Dried with 20mM Sucrose

- AFM images predominantly show amorphous regions with associated

globular features, again most likely due to IgG monomers and aggregates.

Crystalline features were rarely observed.

250nm 250nm 250nm

250nm

A B C

D

Amorhous

Crystalline

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AFM study of IgG Freeze Dried with 20mM Mannitol

- AFM images show both amorphous and crystalline features.

- Globular features appear to be associated with distinct regions on the sample

surface e.g. some crystalline features do not appear to be coated with a protein like

layer.

250nm250nm 250nm

A B C

150 nm

D

Crystalline

Amorphous

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IgG Freeze Dried with Sucrose and Mannitol in Combination [2]

- Images of once freeze dried IgG with Sucrose (40mM) and Mannitol (20mM) in combination.

- An increase in the molar concentration of sucrose predominantly reveals amorphous features

- Again globular features are associated with these regions.

250 nm 250 nm 250 nm

150 nm

A B C Amorphous

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IgG Freeze Dried with Sucrose and Mannitol in Combination [1]

- Images of once freeze dried IgG with Sucrose (20mM) and Mannitol (40mM) in

combination

- An increase in the molar concentration of mannitol reveals distinct crystalline

features in some areas.

- The globular features of IgG profoundly appear to be associated with the amorphous

material.

250nm 250nm 250nm

A B C

150nm

D

Crystalline

Amorhous

I. Characterization of different cell types and those more prone to aggregation is possible with

AFM imaging.

II. Measurement of ligand (fibronectin/fibrinogen) – cell surface receptor interactions would be

ideal to determine the strength of platelet interactions that contribute maximum to the

aggregation, and hence, would be useful for the quantification of cells showing high

propensity to aggregate.

III. Finally, effect of different agonists on different cell types can also be verified by the approach.

Thanks for listening

And Your Special Questions !!!