WK06L2 - Hemostasis II (Ben)

Question 1

What is the main difference in the general characteristics of the extrinsic vs. intrinsic pathways of blood coagulation?

Answer 1

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

Question 2

What is the main plasma factor involved in the intrinsic pathway of coagulation?

Where is it made and what is its general structure?

Answer 2

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

Question 3

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?

Answer 3

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)

Question 4

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?

Answer 4

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)

Question 5

What cleaves factor XII to activate it?

Answer 5

Kallikrein

Question 6

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?

Answer 6

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)

Question 7

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?

Answer 7

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.

Question 8

Described the sequence of events that results in factor XII activation.

Answer 8

1. DNA (from NETs) or polyphosphate (from bacteria/platelets) binds to factor XII
2. Bound factor XII then complexes with high molecular weight kininogen (HK) and prekallikrein (PK)
3. HK + DNA allow factor XII to activate PK into kallikrein (Kal)
4. Kallikrein then cleaves XII to convert it to α-XIIa
5. α-XIIa can cleave another α-XIIa to form β-XIIa

(some of this detail is not shown in the pic)

Question 9

Once activated, what happens with factor XII to initiate clotting?

Answer 9

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)

Question 10

Why is the DNA/polyphosphate-triggered XII activation pathway to coagulation important in infection?

Answer 10

Because the clotting that results can restrict pathogen movement from the site of infection.

Question 11

What is the relationship between polyphosphate length and and factor XII activation?

Answer 11

Longer polyphosphates from bacteria (rather than shorter ones from platelets) are better activators of factor XII.

(Shown in top 2 graphs)

Question 12

In what two situations is platelet polyphosphate a better activator of coagulation than bacterial polyphosphate?

Answer 12

1. Blocking of TFPI
2. Factor V activation

Question 13

How does platelet polyphosphate contribute to the intrinsic pathway of blood coagulation?

Answer 13

By increasing factor XI activation by thrombin.

(bottom right)

Question 14

How is factor XII directed towards either coagulation or inflammation processes?

Answer 14

• 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

Question 15

How is thrombin kept from causing clotting in areas away from the injury/inflammation where it is needed?

Answer 15

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).

Question 16

What is the concentration of antithrombin in the blood?

At what local concentration does thrombin overcome antithrombin and initiate clot formation?

Answer 16

3.4 μM

at only several nM, thrombin can overcome antithrombin

Question 17

What happens to thrombin concentration after it reaches levels high enough to overcome antithrombin and form a clot?

Why?

Answer 17

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.).

Question 18

What other clotting factors does antithrombin act on?

But what limitations are there to antithrombin’s activity on thrombin and these factors?

(And what other clotting inhibitor does not have these limitations?)

Answer 18

Antithrombin also acts on factor Xa and factor IXa.

BUT, it can only act on FREE unbound thrombin and factors.

Factors in complexes can only be inhibited by TFPI.

Question 19

What class of molecules does antithrombin belong to?

(Named based on their action.)

What are 2 other members of this class?

Answer 19

SERPINs

Serine Protease Inhibitors

• heparin cofactor II and α₁-protease inhibitor are both SERPINs

Question 20

Generally, how do SERPINs inhibit proteases (including thrombin)?

Give the steps.

Answer 20

1. Exposed polypeptide loop known as reactive site (red blob in pic) on SERPIN attracts its target protease (teal protein in pic)
2. Target protease binds to SERPIN with its active site on a peptide bond that it would normally cleave
3. As the protease cleaves the loop, the previously strained loop is relieved.
4. 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.

Question 21

What must happen for the reactive sites of the SERPINs antithrombin and heparin cofactor II to be exposed?

(and thus for these SERPINs to be active inhibitors of proteases such as thrombin)

Answer 21

They must bind a polysaccharide such as heparin (used clinically) or heparin sulfate (present on endothelial cells).

(This is not true for α₁-protease inhibitor, whose reactive site is normally exposed.)

Question 22

How does heparin affect antithrombin or other heparin-depdendent SERPINs?

Mention how they interact and what happens.

Answer 22

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.

Question 23

What is the second way in which heparin affects the activity of antithrombin on thrombin?

(Other than conformational activation of AT’s reactive site loop.)

Answer 23

Heparin binds exosite II of thrombin, placing it in its most susceptible conformation for inactivation by antithrombin.

In this way it bridges proteases (ie thrombin) with their inhibitors (ie AT).

Question 24

What happens to heparin once it aids in the irreversible complex formation of protease + protease inhibitor?

Answer 24

Heparin then detaches and can initiate another complex formation.

In this sense it is a catalyst rather than a reactant.

Question 25

What is the difference between therapeutically-used heparins and naturally occuring ones? What difference does this make? How does it affect their action?

Answer 25

Therapeutical heparins are shorter polysaccharides known as **low molecular weight heparins**. Because they are shorter they _bind fewer proteins_, _are cleared from blood more slowly_ and have _more stable/predictable pharmacokinetics_. Longer **LMWHs** have the same bridging/conformational change effects but shorter **pentasaccharides** can be used to induce _only_ the conformational change.

Question 26

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?

Answer 26

**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)

Question 27

In what two ways does **alpha2-macroglobulin** inhibit hemostatic proteases?

Answer 27

1. **Forming a "cage"** **around them** - conformational change results in trapping of the protease in A2-MG's central cavity 2. **Covalent Bonding** - _irreversible_ bonding of the protease to certain parts of A2-MG

Question 28

What is the mechanism for **alpha2-macroglobulin**'s "trapping" of proteases within its central cavity?

Answer 28

1. Protease _enters central cavity_ via one of the "open doors" existing on A2-MG in its normal secreted form. 2. Protease _cleaves a peptide bond_ on a substrate sequence of A2-MG. 3. A _conformational change_ occurs to A2-MG's structure "closing the doors" and trapping protease in its cavity.

Question 29

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?

Answer 29

1. A **surface Lys residue** on the protease contacts reactive, macroergic **thioester** bonds **between Glu + Cys** residues of A2-MG (purple loops) 2. **Thioester rearranges** spontaneously so that its **carboxyl group** **attaches to Lys** of the protease 3. 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.

Question 30

What molecules can **alpha2-macroglobulin** _inhibit_? What other molecules can it _interact with_ and how?

Answer 30

Can inhibit **plasmin**, **kallikrein**, or **thrombin** as well as **extracellular matrix proteases** and **metalloproteases**. ## Footnote (And any other proteases which will cleave the right bonds, I think, but these were mentioned in Wiki and lecture.) - Can also interact with _growth factors_ and _cytokines_ such as **insulin**, **TGF-B**, **IL-1B** etc. sometimes acting as a "carrier" of these proteins in blood.

Question 31

_Antithrombin_ and _alpha-2macroglobulin_ act on clotting-related proteases in what form? What _two_ other mechanisms can act on these proteases when they are in a _different form_? (And what is the shortcoming of one of these mechanisms?)

Answer 31

**AT** and **A2-MG** act on **_free_** proteases only. **TFPI** and **Activated Protein C (APC)** can act on **_protease complexes_**. (BUT... TFPI only acts in the _initial stages_ of clotting + can not block clotting once _prothrombinase_ forms)

Question 32

What are **tenase complexes**? What are the two different types of tenase complex and their components?

Answer 32

**Tenase complexes** are multi-factor complexes which cleave and activate **factor X**. 1. **Intrinsic Tenase** - complex of _IXa_ and _VIIIa_ * IXa cleaves X and VIIIa acts as "co-factor" to speed this up, intrinsic because not TF-dependent 2. **Extrinsic Tenase** - complex of _VIIa_ and _TF_ * VIIa cleaves X and TF is the co-factor/accelerator

Question 33

Which protein can **cleave** and **inactivate** prothrombotic complex _co-factors_ with the help of endothelial cells? What is its origin and general structure? Which co-factors does it inactivate?

Answer 33

**Protein C** (AKA factor XIV ... but not mentioned this way in lecture) - made in the **liver** and has a **Gla domain** for membrane binding - inactivates **factors Va** and **VIIIa** (of the prothrombinase and intrinsic tenase complexes)

Question 34

What two endothelial cell membrane proteins contribute to the _activation of Protein C_?

Answer 34

1. **Thrombomodulin (TM)** 2. **Endothelial Protein C Receptor (EPCR)**

Question 35

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)

Answer 35

1. Membrane-bound **thrombomodulin** **(TM)** binds to **free thrombin (T)** via its **exosite I**. 2. TM increases T's affinity for **protein C** **(PC)** 3. PC binds to **Endothelial Protein C Receptor (EPCR)** co-factor, which aids in the interaction of PC and T. 4. Thrombin cleaves and activates PC to **activated protein C (APC)**

Question 36

What 2 effects does **thrombomodulin** binding to **thrombin** have on thrombin activity? (HINT: Increases one thing and decreases two others.)

Answer 36

**TM** is _competitive_ with **fibrin** for binding of **exosite I**. If TM binds... 1. Thrombin affinity for **protein C** _increases_ 2. Thrombin affinity for **factor XIII** and **fibrinogen** _decreases_

Question 37

Describe the action of **activated protein C** after thrombin has activated it.

Answer 37

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...)

Question 38

What _two_ enyzmes can cleave and _activate_ **factor V**? What part do they cleave?

Answer 38

Both **thrombin** and **Xa** cleave the **B domain** of **factor V** to make **Va**.

Question 39

How exactly is **factor Va** inactivated? Include molecules involved and exact bonds cleaved. How does this differ in free solution and membrane-bound form?

Answer 39

**Activated Protein C** with the help of **protein S** cleaves **R306** (Arg) on Va. - In free solution, first must cleave **R**_5_**06** and then **R306** to inactivate. (Only R306 must be cleaved when membrane-bound)

Question 40

What is **factor V Leiden**?

Answer 40

A mutation of factor V that replaces **R506** (Arg) with a **glutamine** resulting in inability of **APC** to cleave factor Va in free solution. (Present in ~10% of population and leads to increased thrombosis)

Question 41

What happens to **factor V** (specifically V... _not_ Va) when **APC** cleaves it? What effects does the product of this cleavage have?

Answer 41

If **activated protein C** acts on the not-yet-activated **factor V** it can form **factor Vac**, an _anti-coagulant_ active form of factor V. ## Footnote (APC cleaves same R506 bond in V that it cleaves in Va) **Factor Vac** then acts a _co-factor_ of **APC + Protein S** in the _inactivation_ of **factor VIIIa** (part of the _intrinsic tenase complex_)