Day 12, Lecture 2: Proteins: Hemostasis and Coagulation Flashcards
1
Q
Do cells adhere to the endothelial cells or subendothelial cells
A
- Endothelial cells line the interior of blood vessels; when these cells are intact, inhibits coagulation, enhances clot digestion, and inactivates platelets
- Subendothelial cells are normally not exposed to blood until injury. When exposed by injury, platelets adhere to subendothelial cells
2
Q
if platelets encounter subendothelial cells then they
A
bind and activate it
3
Q
What types of medical treatments could lead to coagulation problems due to acquired platelet deficiencies
A
Chemotherapy, Radiation, Immuno-suppressants
4
Q
Clotting factors
A
- soluble plasma proteins made in the healthy liver that circulate in inactive form
- cascades on the activated platelet surface turns on thrombin, a serine protease, that converts soluble fibrinogen into insoluble fibrin that glues platelets and wound together
5
Q
Platelet adhesion
A
- a platelet receptor protein recognizes collagen of subendothelial cells via von Willebrand factor protein intermediate (if deficient, von Willebrand disease- one of the most common genetic bleeding disorders) thus leading to platelet adhesion
6
Q
Platelet activation
A
- Fibrinogen receptor appears on platelet surface
- Fibrinogen helps glue the platelets together (platelet aggregation) to form a platelet plug (within a few minutes)
- This plug can slow bleeding, but fragile and it may re-bleed
- Also phosphatidyl serine in platelet cell membrane flip-flops form the inner to outside surface of the bilayer. This new surface is essential for proper coagulation; clotting factors in plasma now can localize together on the activated platelet
7
Q
Clotting cascade
A
- Wound
- Platelet adhesion
- receptor protein recognizes collagen of subendothelial cells via von Willebrand factor
- Platelet activation
- Fibrinogen receptor appears on platelet surface. Platelet aggregation to form a platelet plug (within a few minutes)
- also phosphatidyl serine in platelet cell membrane flip-flops from the inner to outside surface of the bilayer. this new surface is essential for proper coagulation; clotting factors in plasma now can localize together on the activated platelet
- Clot initiation
- Tissue factor is released from exposed cells and starts a coagulation cascade to turn on thrombin
- this cascade is though the central pathway by activating the dormant zymogen Factor X to become Factor Xa
- Factor Xa is an active protease that cuts and thus turns on thrombin to start making fibrin glue
- Tissue factor is released from exposed cells and starts a coagulation cascade to turn on thrombin
- Clot formation
- Thrombin converts soluble fibrinogen into insoluble fibrin strands. This clot is much more effective at stopping bleeding than the platelet plug alone.
- Clot stabilization
- Transglutaminase crosslinks the glutamine and lysines of fibrin (within 8-10 min)
- Clot retraction
- Platelets tighten clot by contracting a smooth muslce protein, called thrombosthenin (about 1 hour)
8
Q
Factor Xa production cascade
A
- Two ways to make active Factor Xa form X zymogen:
- extrinsic pathway
- needs tissue factor
- main clot initiator
- Factor VII and Subendothelial membrane protein, tissue factor, combine to form a complex that converts X to Xa
- (note: normally extrinsic pathway is sufficient for hemophiliacs (missing VIIIa of intrinsic pathway) to combat spontaneous hemorrhages except in tissue factor-poor areas like joints and muslces (these continual bleeds lead to arthritis)
- intrinsic pathway
- needs RNA from damaged cells (contact phase)
- supportive
- Factor XII binds to this surface and becomes 105- fold more active
- proteolytically clips more XII (autoactivates) and activates various complexes of [kallikrein, high molecular weight kininogen and XI] by cleavage. XIa then cleaves Factor IX
- IXa subsequently cleaves X; a cascade that makes Xa
- VIIIa is a helper factor that stimulates IXa 105 fold (deficient in hemophiliacs), but it is not a protease itself
- extrinsic pathway
9
Q
Severe Hemophilia A
A
- result of arginines at positions 1689 or 372 of Factor VIII is mutated to another residue that cannot be cleaved by the protease thrombin; therefore VIII is never transformed into the useful active form (1 in 10,000 occurence in males)
10
Q
Hemophilia B
A
- missing factors IX
11
Q
Hemophilia C
A
missing factor XI
12
Q
Thrombin Production
A
- Step 1
- upon wounding and platelet activation, prothrombin is localized to the activated platelet surface
- A unique modification, the addition of gamma-carboxyglutamate residues (Vitamin K-cofactor dependent carboxylation fo glutamate in the liver), allows the zymogen to bind
- Step 2
- On the platelet surface, factor Xa, a serine protease, cleaves prothrombin in 2 places.
- Active thrombin falls off platelet (lipid binding domain cut off) and gets enmeshed in fibrinogen/fibrin network
- Thrombin now starts cleaving fibrinogen to form insoluble fibrin clot. (note that thrombin still has disulfide bonds to hold the thrombin together)
13
Q
Negative control of thrombin
A
- antithrombin and heparin inhibit thrombin when it strays too far from clot site. These inhibitors prevent clotting from wandering away from the wound site with activated platelets
14
Q
Amplification of Thrombin
A
- Thrombin can also activate soem of the early parts of cascade by triggering zymogens and factors (Factor V, VIII) for a burst of clotting- feedback amplification for even higher amounts of thrombin production
15
Q
Fibrin Clot formation
A
- Structure of Fibrinogen
- 3 domains
- DED
- Flexible triple chain rods
- 6 polypeptides
- very soluble
- 6 polypeptides
- 3 domains
- Aggregation
- platelets bind fibrinogen D domains and form crosslinks between platelets
- Proteolysis and Activation
- Thrombin cleaves off A and B fibrinopeptides > exposes binding domain E which interacts with D domains of other fibrin molecules. (fibrinopeptides have a high negative charge and keep fibrinogen from interacting)
- Polymerization
- Formation of staggered array of monomers to form fibrous aggregates
- Crosslinking
- Thrombin activates transglutaminase, an enzyme that forms bridges between D/D and D/E domains> stabilizes the clot
16
Q
Fibrin clot removal
A
- Plasmin
- Protease cleaves fibrin at flexible triple chain rods between the D and the E domains
- Formation of active plasmin
- Tissue plasminogen activator (tPA) and plasminogen zymogen bind to the clot network and form a complex
- plasminogen is then proteolytically clipped by tPA to form active plasmin
- The plasmin cleaves fibrin
- Antiplasmin
- in the plasma inhibits any plasmin released from the clot
17
Q
How do you restrict the clot to damaged tissue
A
- Thrombin that escapes the clot is inactivated by antithrombin III inhibitor that is bound to heparin on endothelial cells of intact blood vessels
- Thrombin escapes the clot binds to thrombomodulin of intact enothelial cell surface. This regulatory protein changes thrombin’s proteolytic specificity so now thrombin activates protein C in the plasma.
- activated protein C and protein S together degrade Factors Va, VIIIa of the clotting cascade
- Thromboxane (TxA2) from activated platelets stimulates:
- other platelets to activate (for faster clotting!)
- Healthy intact endothelial cells to produce the small molecule prostaglandin I2 (PGI2) that inhibits platelet activation
- These small molecules TxA2 and PGI2 have a short half-life (about 1 minute) therefore the effects are very local and fine-tuned
