I. Atomic force microscopy (AFM) can be used to characterize different platelet types and their propensity for aggregation by imaging their morphology. II. Measuring the strength of interactions between platelet surface receptors and ligands like fibronectin and fibrinogen with techniques like bio-membrane force probing could quantify differences in interaction strength between platelet types. III. Studying how various activation factors affect interaction strength between different platelet morphologies would improve understanding of platelet aggregation.

1. Dr. Mudasir A Lone

2. 09/02/2016 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)

3. 09/02/2016 3
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

4. 09/02/2016 4
C
D
AA
BA
A
A
C
D
B
C
D
B
B
B
C
C
1. Different possibilities For Interactions

5. 09/02/2016 5
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.

6. 09/02/2016 6
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.

7. 09/02/2016 7
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)

8. 09/02/2016 8
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

9. 09/02/2016 9
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.

10. (a) Specific, (b) non-specific, (c) no adhesion (d)
multiple force displacement curves of the interaction
Plot of adhesion force or binding interaction
strength.

11. 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

12. 09/02/2016 12
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

13. 09/02/2016 13
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

14. 09/02/2016 14
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 CAmorphous

15. 09/02/2016 15
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

16. 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.