WK06L2 - Hemostasis II (Ben) Flashcards
What is the main difference in the general characteristics of the extrinsic vs. intrinsic pathways of blood coagulation?
Extrinsic - requires extravascular tissue factor
Intrinsic - uses coagulation factors present in the blood, is not a “hemostatic” pathway, but rather is important in inflammation + atherosclerosis
What is the main plasma factor involved in the intrinsic pathway of coagulation?
Where is it made and what is its general structure?
Factor XII
(AKA Hageman factor)
- made by the liver
- synthesized in an inactive single-chain form containing a heavy chain and a catalytic domain held together by a disulfide bridge
What 3 amino acids are important in the active site of factor XII?
What changes about them in the single-chain uncleaved form of the factor vs. the cleaved/activated form?
Serine, Histidine, Aspartate
- they are not exposed in single chain form, but cleavage of the single chain exposes them
(spread out in lower right of the image, marked in black… probably very hard to see on this card sajnos)
Factor XII has two forms.
What are they and what do they consist of?
Which is more active in the intrinsic pathway of blood clotting?
Factor XII alpha - two chain form, has been cleaved to split the heavy chain and light chain (catalytic domain), but they remain attached via the disulfide bridge
Factor XII beta - single chain form, more active in clotting, made by a further cleavage that removes the heavy chain, leaving only the catalytic domain and another small peptide
(arrows show cleavage points)
What cleaves factor XII to activate it?
Kallikrein
Part of factor XII is very similar to other molecules involved in hemostasis.
What part and what is it similar to?
How is it similar?
What does this mean for its function?
The heavy chain is similar to fibrinolytic proteases (such as tPA).
- contains kringle (K) and finger (F) domains, like tPA
- this indicates its importance in fibrin dissolution (in addition to clotting)
(patients without XII actually don’t have bleeding problems, but DO have thrombosis issues)
What are the two molecular triggers for factor XII activation?
Where do they come from?
What general characteristic of these molecules makes them suitable triggers for XII activation?
DNA - from NETs (Neutrophil Extracellular Traps) made up of DNA expelled from WBCs
Polyphosphate - from bacteria (stored energy form) and platelets (stored in “delta granules”)
They are long polymers with negative charge, and thus are good triggers of XII activation.
Described the sequence of events that results in factor XII activation.
- DNA (from NETs) or polyphosphate (from bacteria/platelets) binds to factor XII
- Bound factor XII then complexes with high molecular weight kininogen (HK) and prekallikrein (PK)
- HK + DNA allow factor XII to activate PK into kallikrein (Kal)
- Kallikrein then cleaves XII to convert it to α-XIIa
- α-XIIa can cleave another α-XIIa to form β-XIIa
(some of this detail is not shown in the pic)
Once activated, what happens with factor XII to initiate clotting?
β-XIIa is released from DNA and can activate factor XI which activates factor IX which (in complex with factor VIII) activates factor X which (in “prothrombinase complex” with factor Va) will activate thrombin.
(sorry)
Why is the DNA/polyphosphate-triggered XII activation pathway to coagulation important in infection?
Because the clotting that results can restrict pathogen movement from the site of infection.
What is the relationship between polyphosphate length and and factor XII activation?
Longer polyphosphates from bacteria (rather than shorter ones from platelets) are better activators of factor XII.
(Shown in top 2 graphs)
In what two situations is platelet polyphosphate a better activator of coagulation than bacterial polyphosphate?
- Blocking of TFPI
- Factor V activation
How does platelet polyphosphate contribute to the intrinsic pathway of blood coagulation?
By increasing factor XI activation by thrombin.
(bottom right)
How is factor XII directed towards either coagulation or inflammation processes?
-
coagulation
- when heavy chain is cleaved from alpha-XIIa, the newly formed beta-XIIa diffuses away from DNA/polypeptide to activate more kallikrein
- kallikrein can make more XIIa and activate XI, all leading to more fibrin generation
-
inflammation
- if alpha-XIIa remains on DNA it will act on complement proteins which attract new inflammatory cells
- denatured proteins resulting from inflammation (ex: amyloid plaques) attract XIIa and then XIIa perpetuates the inflammation via complement interaction
How is thrombin kept from causing clotting in areas away from the injury/inflammation where it is needed?
Antithrombin is made by the liver and released into the blood.
It acts as a pseudosubstrate for thrombin, blocking its activity in areas where thrombin concentration is low (away from injuries).
What is the concentration of antithrombin in the blood?
At what local concentration does thrombin overcome antithrombin and initiate clot formation?
3.4 μM
at only several nM, thrombin can overcome antithrombin
What happens to thrombin concentration after it reaches levels high enough to overcome antithrombin and form a clot?
Why?
Thrombin continues to increase in the blood near the injury until it is ~100x its clot-forming concentration (to almost 300 nm).
This occurs because of the high increases in the rate of thrombin formation which result from the membrane-bound complex formation mentioned before (prothrombinase, etc.).
Generally, how do SERPINs inhibit proteases (including thrombin)?
Give the steps.
- Exposed polypeptide loop known as reactive site (red blob in pic) on SERPIN attracts its target protease (teal protein in pic)
- Target protease binds to SERPIN with its active site on a peptide bond that it would normally cleave
- As the protease cleaves the loop, the previously strained loop is relieved.
- Carboxy terminal of broken peptide bond on SERPIN is attached to Ser residue on protease active site + as strain is relieved, SERPIN swings protease to its other pole (bottom right), breaking protease’s active site irreversibly.
How does heparin affect antithrombin or other heparin-depdendent SERPINs?
Mention how they interact and what happens.
Heparin has many negative charges which bind to the positively-charged heparin binding site (HBS) on antithrombin (AT) molecules.
This binding results in so-called conformational activation by causing AT’s reactive site loop (RSL) to become more exposed.
What is the large plasma protein that plays a role in inhibiting both coagulation and fibrinolysis?
Where is it made and what is its general structure?
Alpha-2 Macroglobulin
- made in the liver
- is a homotetramer of large size (~720 kDa) with a central cavity containing thioester bonds between Glu and Cys residues
- its central cavity is large enough to accomodate 2 protease molecules (seen in img)
In what two ways does alpha2-macroglobulin inhibit hemostatic proteases?
- Forming a “cage” around them - conformational change results in trapping of the protease in A2-MG’s central cavity
- Covalent Bonding - irreversible bonding of the protease to certain parts of A2-MG
What is the mechanism for alpha2-macroglobulin’s “trapping” of proteases within its central cavity?
- Protease enters central cavity via one of the “open doors” existing on A2-MG in its normal secreted form.
- Protease cleaves a peptide bond on a substrate sequence of A2-MG.
- A conformational change occurs to A2-MG’s structure “closing the doors” and trapping protease in its cavity.
What is the mechanism for alpha2-macroglobulin’s covalent binding of the proteases that it inhibits?
What is special about the requirements for this type of inhibition by A2-MG and what kind of inhibition is it?
- A surface Lys residue on the protease contacts reactive, macroergic thioester bonds between Glu + Cys residues of A2-MG (purple loops)
- Thioester rearranges spontaneously so that its carboxyl group attaches to Lys of the protease
- This forms a covalent isopeptide bond, inhibiting the protease.
- No enzymatic activity by the protease is necessary for this inhibition, only close proximity of the Lys + Glu residues.
- Results in irreversible inhibition by conformational change of protease.
What two endothelial cell membrane proteins contribute to the activation of Protein C?
- Thrombomodulin (TM)
- Endothelial Protein C Receptor (EPCR)
Describe the steps which result in activation of protein C on the surface of endothelial cells.
(Only activation… not protein C’s action after it is activated)
- Membrane-bound thrombomodulin (TM) binds to free thrombin (T) via its exosite I.
- TM increases T’s affinity for protein C (PC)
- PC binds to Endothelial Protein C Receptor (EPCR) co-factor, which aids in the interaction of PC and T.
- Thrombin cleaves and activates PC to activated protein C (APC)
Describe the action of activated protein C after thrombin has activated it.
With the help of co-factors EPCR and Protein S…
… APC can cleave + inactivate factor Va of the prothrombinase complex, converting it to factor Vi
(also acts on factor VIIIa, which comes up in a later card…)
