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What are the main processes?
Ø clot formation or thrombus formation
Ø can have removal by fibrinolysis
processes occur at surface

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Who are the main players?
Ø surface
Ø platelets
coagulation proteins
1.1 Components of Blood
A. What is blood made of?
Ø cells
Ø red blood cells
Ø white blood cells (important in inflammation)
Ø neutrophils
Ø monocytes
Ø eosinophils
Ø lymphocytes
Ø basophils
Ø platelets
Ø proteins
Ø > 200 known

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Figure 22-25. White blood cells. (A-D) Electron micrographs showing,
respectively, a neutrophil, a basophil, an eosinophil, and a monocyte. Electron
micrographs of lymphocytes are shown in Figure 23-4. Each of the cell types shown
here has a different function, which is reflected in the distinctive types of secretory
granules and lysosomes it contains. There is only one nucleus per cell, but it has an
irregular lobed shape, and in (B), (C), and (D) the connections between the lobes are
out of the plane of section. (E) Light micrograph of a blood smear stained with the
Romanowsky stain, which colors the white blood cells strongly. (A-D, courtesy of
Dorothy Bainton; E, courtesy of David Mason.)

Alberts, Bray, etc. from Pubmed Books

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Who are the main players?
Ø surface
Ø platelets
coagulation proteins
1.1 Components of Blood
A. What is blood made of?
Ø cells
Ø red blood cells
Ø white blood cells (important in inflammation)
Ø neutrophils
Ø monocytes
Ø eosinophils
Ø lymphocytes
Ø basophils
Ø platelets
Ø proteins
Ø > 200 known

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http://www.bendigo.latrobe.edu.au/biolsc/bradly/images/megakaryocyte.jpg

B. Platelets
Ø non-nucleated
Ø disk-shaped
Ø diameter 3-4 um
Ø average volume 10 x 10-9 mm3
Ø produced in bone marrow
Ø average circulation concentration 250,000 cells/ul (red cells: 5 * 106 cells/ul)
Ø 0.3% of total blood volume (red cells: 40-50% of total blood volume)
Ø Function:
Ø initially arrest bleeding through formation of platelet plugs
Ø stabilize platelet plugs by catalyzing coagulation reactions
Ø leads to formation of fibrin

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1.2 Platelet structure Fig 1
Two states: nonstimulated and activated state
Ø Ø activated:
Ø extremely sensitive and respond to minimal stimulation
Ø activation causes platelets to become sticky
Ø change shape to irregular spheres with spiny pseudopods
Ø internal contraction
Ø extrusion of storage granule contents into extracellular environment
Ø secreted platelet products stimulate other platelets
Ø irreversible platelet aggregation
formation of fused platelet thrombus Fig 2

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1.2 Platelet structure Fig 1
Two states: nonstimulated and activated state
Ø discoid shape is maintained by circumferential bundle of microtubules (part of
cytoskeleton): cytoskeleton – actin, microtubules, intermediate filaments
Ø external surface coat of platelet: membrane-bound receptors (glycoproteins (b
and IIb/IIIa) – involved in platelet adhesion (platelet-surface) and platelet-platelet
aggregation
Ø membrane: phospholipid surface (interactions with phospholipids help
accelerate coagulation reactions)
Ø membrane is a spongy, canal-like (canalicular) open network – reactive surface
to which plasma factors can selectively adsorb
Ø substantial muscle protein (actin, myosin) – internal contraction when platelets
are activated
Ø 3 types of cytoplasmic storage granules
Ø α-granules (contain platelet factor 4, β-thromboglobulin, proteins found in
plasma (fibrinogen, albumin, fibronectin, coagulation factors V and VIII)
Ø dense granules (contain ADP, calcium ions, and serotonin)
lysosomal granules (contain enzymes such as acid hydrolases

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http://www.akh-wien.ac.at/biomed-research/htx/anatomy.htm

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http://expmed.bwh.harvard.edu/projects/signaling/
http://www.indstate.edu/thcme/mwking/blood-coagulation.html
The initial activation of platelets is induced by thrombin binding to specific receptors on the
surface of platelets, thereby initiating a signal transduction cascade. The thrombin receptor is
coupled to a G-protein that, in turn, activates phospholipase C-γ (PLC-γ). PLC-γ hydrolyzes
phosphatidylinositol-4,5-bisphosphate (PIP2) leading to the formation of inositol
trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular Ca2+
stores, and DAG activates protein kinase C (PKC). The collagen to which platelets adhere
as well as the release of intracellular Ca2+ leads to the activation of phospholipase A2
(PLA2), which then hydrolyzes membrane phospholipids, leading to liberation of
arachidonic acid. The arachidonic acid release leads to an increase in the production and
subsequent release of thromboxane A2 (TXA2). This is another platelet activator that
functions through the PLC-γ pathway. Another enzyme activated by the released
intracellular Ca2+ stores is myosin light chain kinase (MLCK). Activated MLCK
phosphorylates the light chain of myosin which then interacts with actin, resulting in
altered platelet morphology and motility. One of the many effects of PKC is the
phosphorylation and activation of a specific 47,000-Dalton platelet protein. This activated
protein induces the release of platelet granule contents; one of which is ADP. ADP further
stimulates platelets increasing the overall activation cascade; it also modifies the platelet
membrane in such a way as to allow fibrinogen to adhere to two platelet surface
glycoproteins, GPIIb and GPIIIa, resulting in fibrinogen-induced platelet aggregation.
Activation of platelets is required for their consequent aggregation to a platelet plug.
However, equally significant is the role of activated platelet surface phospholipids in the
activation of the coagulation cascade.

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1.5 Coagulation
There are many proteins involved Fig 3
Ø discovered by putting proteins in a test tube and seeing if they make a clot
Ø designation of Roman numerals are in order of discovery
Ø absence of any one factor can lead to abnormalities in clotting (bleed to death);
famous hemophiliac of royalty in Great Britain
Ø intrinsic and extrinsic (surface reactions and bulk i.e. factors derived from
tissues)
Ø end result: insoluble fibrin gel when thrombin acts on fibrinogen
Ø cascade of reactions (enzymatic reactions; proteolytic cleavages); enzymes can
activate many substrate molecules à reactions are quickly amplified and significant
amounts of thrombin are produced à leads to platelet activation, fibrin formation,
and arrest of bleeding

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http://hsc.virginia.edu/medicine/clinical/pathology/educ/innes/text/nh/function.html

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http://hsc.virginia.edu/medicine/clinical/pathology/educ/innes/text/nh/
function.html

• Normal platelets in the blood stream have a discoid shape and have
little or no interaction with other platelets or endothelium.

• Endothelial damage, specifically exposure of underlying collagen as

shown at left, triggers platelet adhesion. First, von Willebrand's
factor(vWF) binds to subendothelial collagen. This results in
conformational changes in vWF allowing vWF to bind to the GP Ib
receptor on platelets.

• Exposure of the basement membrane and release of tissue factor from

damaged endothelialcells serves to activate coagulation pathways.

Here is a strategy that you could think about blocking the collagen-
vWF-platelet receptor interaction. Design drug. Alpha2beta1 integrin
(Erin showed crystal structure from PDB file).

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Following adhesion, proteins and soluble products interact with platelet membrane receptors to cause
activation. At time of activation, platelets become spherical and develop cytoplasmic hair-like
filipodia. The GP IIb/IIIa receptor complex Ds conformation allowing binding of fibrinogen. The
contents of the alpha and dense granules are secreted. All of the above lead to platelet aggregation.

1.3 Platelet adhesion

Platelets adhere to artificial surface and injured blood vessels
Ø Interaction: platelet glycoprotein Ib (GP Ib)and connective tissue elements that have become
exposed (collagen); requires plasma von Willebrand factor (vWF) as an essential cofactor
Ø ~ 15,000 molecules of GP Ib per platelet acts as surface receptor for vWF
Ø hereditary absence of GP Ib or vWF è defective platelet adhesion and serious abnormal
bleeding
Artifical surface:
Ø platelet adhesion to artificial surface mediated through
Ø platelet glycoprotein IIb/IIIa (40,000 copies per platelet)
Ø receptor for adhesive plasma proteins that support cell attachment
Ø fibrinogen, vWF, fibronectin, vitronectin
Ø resting platelets do not bind these proteins
Ø platelet activation causes a conformational change in GP IIb/IIIa
Ø fibrinogen or other proteins could adsorb onto an artificial surface (not normally found on

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http://www.akh-wien.ac.at/biomed-research/htx/anatomy.htm

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http://jama.ama-assn.org/issues/v281n15/fig_tab/jcc71004_f1.html

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At left you see vWF bound to collagen; vWF bound to platelet GP Ib, and fibrinogen bound to
activated platelet GP IIb/IIIa. The fibrinogen acts as a glue binding platelets together.
The contents of platelet granules are discharged, exponentially magnifying the accumulation of
platelets and fibrin at the site of injury. This process is known as platelet aggregation, the end result
of which is formation of a platelet-fibrin plug or thrombus.

1.4 Platelet aggregation (after adhesion)

Complex series of reactions initiated to
Ø release of dense granule containing ADP
Ø formation of thrombin
Ø generation of thromboxane A2
=> these together act in concert to recruit platelets in a growing platelet aggregate Fig 2
Platelet stimulation by these agonists causes
Ø expression on platelet surface of activated GP IIb/IIIa which lead to...
Ø binding of plasma proteins that support platelet aggregation
Ø fibrinogen is most important protein supporting platelet aggregation
Ø platelet-platelet interaction involves Ca2+-dependent bridging of adjacent platelets by
fibrinogen molecules
Ø Note: platelets will not aggregate in the absence of fibrinogen, GP IIb/IIIa or Ca2+
Thrombin binds directly to platelets
Ø Activates platelets which then catalyze the production of more thrombin
Ø Stimulate ADP release and thromboxane A2 formation

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The fibrin gives the platelet mass strength allowing it to function as a secure patch
and a protected base for repair and healing.
Note that many of the platelets appear as empty sacks having discharged their
granules.

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web.vet.cornell.edu/.../CPmodules/ heme1/images/pltfibr.jpg

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