Coagulation

Question 1

Primary hemostasis is accomplished by ______

Secondary hemostasis is accomplished by _____

Answer 1

Platelets

Fibrin

Question 2

Dissolution of a clot is driven by _________

Answer 2

Fibrinolysis

Question 3

Do primary and secondary hemostasis happen in isolation of one another?

Answer 3

No - there is significant cross-talk between the two

Question 4

How long do platelets survive in the blood?

Answer 4

10 days

Question 5

What is a good estimation of how many platelets can be made by a megakaryocyte?

Answer 5

10,000

Question 6

When platelets are acitvated, they begin to develop _______, which are cytoplasmic projections that aid in adherence.

Answer 6

Filopodia

Question 7

The inner leaflet of the plasma membrane contains _________ phospholipids, and this balance is maintained by _______

Answer 7

negatively charged

Enzymes: floppases, flippases, scramblases

Question 8

Microtubules below the surface of the platelet aid in platelet _______ and _______

Answer 8

shape change

granule secretion

Question 9

When quiescent, platelets have a highly folded canalicular system that represents extensive invagination of the surface membrane. What is the purpose of this structure?

Answer 9

1) upon platelet activation this surface can evaginate quickly increase the surface area available for platelet-matrix or platelet-ligand interactions
2) granules can fuse with the surface connected canannicular system discharging their contents and resulting in rapid secretion of these contents to the exterior of the cell.

Question 10

The dense tubular system is the analogue of the ________ in muscle cells and is a major storage site for what ion? Why is this significant?

Answer 10

Sarcoplasmic reticulum

Major storage site for calcium which is released intra-cellularly upon platelet activation –> Ca2+ important for activation of PLA2 which is used to convert arachidonic acid to prostaglandins, including thromboxane which is essential for platelet activation.

Question 11

What do alpha granules contain?

What do dense granules contain?

Answer 11

von Willebrand factor, fibrinogen, and other proteins important for platelet recruitment and function

dense on electron microscopy because of high calcium content also contain ADP, ATP, serotonin, and histamine

Question 12

von Willebrand Factor

• Where is it synthesized?
• When is it necessary for platelet adhesion?
• Describe its synthesis, secretion and processing

Answer 12

• Endothelial cells
• Under conditions of high shear stress
• vWF is an exceptionally large protien, it dimerizes in the ER and the multimerizes in the golgi where it is secreted in a large endosome. Upon fusion with the membrane, ADAMTS13 cleaves the large vWF into smaller multimers that are able to diffuse in the blood and bind to subendothelial collagen at the site of injury

Question 13

Fibrinogen is made in the _______ and secreted and it is important in platelet aggregation.

Answer 13

Liver

Question 14

Platelet Receptors

• Glycoprotein Ib binds to ______ and is (always/occassionally) present on the surface of the platelet
• Glycoprotein IIb IIIa binds to _____ and ______ and is present on the surface in what form?
• GPCRs on platelet surface bind to _____ and ______
• P2Y1 is a G protein is bound by ______ and is (stimulatory or inhibitory)
• P2Y12 is a G protein that is bound by ______ and is (stimulatory or inhibitory)
• What are the receptors for collagen on the platelet?

Answer 14

• vWF, constitutively
• Fibrinogen and vWF, present in form that is inaccessible to ligands and requires that the platelet be activated before it can bind to ligands
• Thomboxane A2 and thrombin-proteases (thrombin molecules that act as proteases to cleave the N-terminal extracellular side of the receptor)
• ADP and prostacyclin (PGI₂), stimulatory
• ADP, inhibitory
• Glycoprotein VI and Glycoprotein alpha 2 beta 1

Question 16

Formation of thromboxane is inhibited by _______

Answer 16

Aspirin (NSAIDs)

Question 17

Describe the process of platelet activation in general.

Answer 17

Resting platelets in the circulation do not interact with each other or the endothelial surface. Upon exposure of sub-endothelial collagen after an injury, platelets bind to the collagen in a process known as adhesion. The binding of platelets to the collagen initiates a process of platelet activation which is amplified by binding of other ligands to cell surface receptors (see next few slides). In the process of platelet activation the platelets change shape (disc to sphere), release their granule contents, synthesize and release Thromboxane A2, expose negatively charged phospholipids on their surface to provide a surface for coagulation protein reactions and alter the conformation of the integrin alpha 2b, beta 3a (GP IIB, IIIA) so it can interact with its ligands (fibrinogen and others) and mediate platelet aggregation (platelets sticking to one another)

Question 18

What is the difference between vWF when it is in the blood vs. bound to collagen?

Answer 18

When in the blood, it is tightly coiled, hiding its binding sites. When it binds to collagen, its shape changes and causes elongation of the vWF into the long arm structure that we know.

Question 19

Describe the structure of fibrinogen.

Answer 19

This molecule is composed of 6 chains (3 identical polypeptide pairs (α, β,and γ) linked by di-sulfide bonds

Question 20

Why is it necessary to sequester negatively charged membrane phospholipids on the inner leaflet prior to activation?

Answer 20

Because the negative charge facilitiates platelet adhesion / activation, so these negatively charged phospholipids are moved to the outer leaflet only when needed for these tasks.

Question 21

All coagulation proteins are synthesized in the _______

Answer 21

Liver

Question 22

What are the vitamin k dependent enzymes?

Answer 22

2, 7, 9, 10

Question 23

What is the initiator of blood clotting?

Answer 23

Tissue factor - this is present in high amounts as integral membrane protein in cells that surround the vasculatorebut is not normally exposed to the blood

Question 24

What does the little “a” indicate when talking about the coagulation cascade proteins?

Answer 24

Either:

1) Active enzyme that has been converted from zymogen to active form
2) Protein has taken on a different configuration that is more active and better able to serve its function

Question 25

How does in vitro coagulation differ from in vivo?

Answer 25

In vitro, HMWK binds to PK, which converts 12 to 12a

12a converts 11 to 11a

11 a converts 9 to 9a

9a converts 10 to 10a

10a converts 2 to 2a

2a converts:

• Fibrinogen to fibrin
• 11 to 11a
• 13 to 13a

13a converts fibrin to cross-linked fibrin

cross-linked fibrin forms scaffold for platelet clot

Question 26

Vitamin K dependent enzymes are converted from _____ to ______ in the ______ before secretion

Answer 26

zymogens –> active enzyme

ER

Question 27

What is the redox state of vitamin K that is used for vitK dependent enzymes?

What reactions does it mediate?

Why is this important for coagulation cascade?

Answer 27

Reduced form

Carboxylations

Adds COO- group so that the enzyme can bind to Ca2+ in the blood which helps the enzyme “sit down” on the surface of the plasma membrane of cells that express tissue factor –> also allows for more surface area for reactions to take place

Question 28

Describe tissue factor.

Answer 28

It is a membrane lipoprotein that is not functional in the absence of lipid. It has no enzymatic activity but rather binds to the enzyme 7a and functions as a major initiator of coagulation. It is located in extravascular cells and small microparticles present in the blood (in both cases it is in the inactive form until bound by 7a).

Question 29

Describe Factor 7.

Answer 29

It is a single glycoprotein that is made in the liver. When it is activated, it has 2 chains that are conneted via disulfide bond. There is very little 7a that circulates in the blood, most is as 7.

Question 30

Why is it important to have mechanisms to inhibit clotting?

Answer 30

To ensure the clot is limited to the site of injury

To ensure that the blood clot does not become pathologic under normal circumstances

Question 31

What is Antithrombin?

Answer 31

A serine protease inhibitor that is produced in the liver and circulates in the blood. When it comes in contact with endothelial cells, it can bind to GAGs present on their surface. This enhances its ability to bind to serine proteases, like thrombin, so it is able to bind to thrombin and remove it from circulation.

Question 32

Describe the action of protein C.

Answer 32

Protein C is produced in the liver and circulates in the blood. When it comes in contact with endothelial cells it can bind to EPCR on their surface. This receptor is kept in close proximity to thrombomodulin, so when thrombin binds to thrombomodulin and protein C binds to EPCR, the thrombin can cleave the protein C to its active form. The active protein C then combines with protein S as a cofactor and is able to inactivate factors 5a and 8a, which inhibits formation of important coagulation cascade proteins.

Question 33

What are the many actions of thrombin?

Answer 33

Cleaves:

• Fibrinogen –> fibrin
• Factor 13 –> Factor 13 a
• Factor 5 –> Factor 5a
• Factor 8 –> Factor 8a
• Protein C –> active protein c

Question 34

Describe tissue factor pathway inhibition.

Answer 34

A molecule called tissue factor pathway inhibitor is produced in endothelial cells and then binds to those same endothelial cells. It inhibits tissue factor by binding to factor 10a and then binding to the TF-7a complex. This all occurs on the endothelial cell surface.

Question 35

Describe how plasminogen works to prevent clotting.

Answer 35

Tissue plasminogen activator (tPA) is released from endothelial cells. It contains lysine residues and binds to other lysine residues on the surface of fibrin on the surface of a clot. Plasminogen also contains lysine residues and binds to the surface of fibrin via lysines on fibrin surface. When in proximity, tPA converts circulating plasminogen (produced in liver) to plasmin –> plasmin cleaves firbin into smaller peptides. These peptides are called D-dimers if they are prouduced from degradation of cross-linked fibrin or fibrin degradation products if they are produced from degradation of fibrinogen.

Question 36

What are 2 important regulators of plasmin formation?

Answer 36

Plasmin Activator Inhibitor –> inhibits tissue plasminogen activator

Alpha 2 antiplasmin –> inhibits plasmin directly

Question 37

The endothelial cell produces Nitric Oxide, PGI₂ and ADPase to inhibit clotting. What do each of these molecules do?

Answer 37

NO - Secretion from endothelial cell results in production of cGMP which inhibits platelets

PGI₂ (prostacyclin) –> Inhibits Ca2+ release from dense tubules which reduces the production of thromboxane –> reduces platelet activation

ADPase –> degrades ADP that is secreted by adhering platelets to prevent activation of other platelets

Question 38

Although we learned that tissue factor is largely found on peripheral vascular cells, some tissue factor is stored in microvesicles that circulate. What is this tissue factor used for?

Answer 38

It is stored in microvesicles in an inactive form. When it is secreted from the microvesciles, it can bind directly to receptors on platelets and cause platelets to form new layers of blockage for the platelet plug. This is important because the initial platelets that occlude the injury will see tissue factor in the peripheral vascular cells, but later platelets will not so it would be difficult to form a large and effective plug without this circulating TF.