Blood coagulation 2

Question 1

What are coumarins used for?

Answer 1

Management of DVT
May also be used for accidental poisoning and deliberate poisoning (rat killer).

Question 2

Describe the origin of warfarin related to its chemical structure.

Answer 2

Coumarin in sweet clover is converted to dicoumarol when cloves is spoily - causes fatal bleeding in catal - is a anti-coagulant
Warfarin is a structural modification of dicoumarol (an analogue) so mimics its anti-coagulant effects.

Question 3

What makes up the Gla domain on a coagulation factor?

Answer 3

Glutamate in protein structure undergoes post-translational modification
Converted by Vitamin K dependent gamma-carboxylase to a 10 y-carboxyglutamate residue.

Question 4

Why are Gla domains important functionally in some coagulation factors?

Answer 4

Gla domains are highly negatively charged
So bind strongly to Ca2+
Ca2+ then also binds to negatively charged phosphatidylserine on platelet membrane/ other membrane
Enables closer interaction with other coagulation factors. (particular other factors that are also membrane bound)

Question 5

How is L glutamate converted to a y-carboxyglutamate?

Answer 5

By post translational modification
Vitamin K dependent Gamma-carboxylase catalyses the addition of the carboxy group to the gamma/4th carbon, loss of a hydrogen
This carboxy group comes from a CO2 molecule
O2 is also required, will donate one oxygen to hydrogens produced to form water
Other oxygen atom is donated to Vitamin K. This oxidised vitamin K.

Question 6

What is the importance of the state of vitamin K in the conversion of L-glutamate to 4-carboxy-glutamate?

Answer 6

Reduced vitamin K can act as a catalyst in this reaction
Over the course of the reaction vitamin K is oxidised - this form is known as Vitamin K1 epoxide - this is an inactive enzyme

Question 7

How is Vitamin K recycled to ensure continued production of 4-carboxyglutmater from L-glutamate?

Answer 7

End of reaction inactive VK in oxidised state (VK1 epoxide)
Acted on by Vitamin K epoxide reducase - converted back to VK (reduced form)
Can once against act as a catalyst in the reaction.

Question 8

What is the main target of Warfarin?

Answer 8

Vitamin K epoxide reductase.

Question 9

What is the clinical use of Warfarin?

Answer 9

Prophylaxis of DVT/PE

Question 10

Explain the use of Warfarin as prophylaxis for DVT/PE

Answer 10

Class - VitK dependent oral anti-coagulant
Chemistry - structural analogue of VitK
Pharmacology - is a competitive inhibitor of VitK epoxide reductase
Physiology: less reduced (active) VitK, increased oxidised (inactive) VitK.
Leads to decreased conversion of L-glutamater to gamma-carboxyglutamate
This prevents Gla domain formation on coagulation factors. Therefore disrupts coagulation by reducing the likelihood of coagulation factors interacting with each other.
Note this has a delayed effect - only affects newly produced coagulation factors, pre-existing factors must be used up first

Question 11

What coagulation factors are Vitamin K depdenent factors?
What is common to these?

Answer 11

Factor II or thrombin (common pathway)
Factor X and Xa (common pathway)
Factor VIIa and VII in the intrinsic pathway
Factor IX and IXa (extrinis pathway)

Question 12

How is platelet activation important for VitK dependent coagulation factors?

Answer 12

Platelet activation causes phosphatidylserine to flip from the inside to the outside plasma membrane
Enables interaction with VK dependent coagulation factors.

Question 13

Suggest why prothrombin contains a Gla domain but thrombin does not.

Answer 13

Prothrombin contains Gla domain to interact with platelet membrane, enables contact with factor Xa which also contains a Gla domain
When converted to thrombin the Gla domain is cleaved away.
Thrombin able to move away from the platelet membrane to interact with fibrinogen.

Question 14

What are the different coagulation factors affected by warfarin?
What are the consequences of this?

Answer 14

Interfers with factor:
A. IX and IXa - prevent conversion of X to Xa
B. VIIa - prevent conversion of X to Xa
C. Xa - prevent conversion of prothrombin to thrombin.

Question 15

What are the different proteins affects by warfarin and the consequences of this?

Answer 15

Interacts with Protein C - prevent degradation of FVa and FVIIIa
Interacts with Protein S - prevents activation of protein C

This has some counter interutitive effects by encouraging coagulation but is to a lesser extent that warfarins anti-coagulant effects.

Question 16

Describe how resistance to warfarin may originate>

Answer 16

Mutation in VitK epoxide reductase
Confromational change still enables enzyme to interact with VK (so promotes coagulation) but prevents an interaction with warfarin (loss of effect of anti-coagulant drugs)
Risk of coagulation.

Question 17

What are the different structures that thrombin activates/cleaves?

Answer 17

Fibrinogen
FV and FVIII
FVII
FXIII
Protein C
Binds to Proteinase-activated-receptors to activate platelets.

Question 18

What is factor XIII?

Answer 18

Is an inactive enzyme
Is a plasma glycoprotein
Is activated to FXIIIa by thrombin
Acts as a transglutaminase cross linking glutamine to lysine in fibrin
Action requires Ca2+ as a co-factor.
This stabilises the platelet plug.

Question 19

What is fibrinolysis?

Answer 19

When plasmin starts to dissolve a thrombus

Question 20

What is plasminogen?

Answer 20

A zymogen (inactive enzyme)
Is a plasma glycoprotein
Binds to lysine residues in fibrin

Question 21

How is plasmin formed?

Answer 21

Plasminogen binds to lysine residues on fibrin,
Adherent structures urokinase or tissue plasminogen activator, cleave plasminogen to plasmin.

Question 22

What is the role of plasmin?

Answer 22

Cleaves fibrin
Produces fibrin degradation products - including D-dimers.

Question 23

What is a D-dimer?

Answer 23

Produces by fibrin degradation

Fibrin chains are cross-linked by FXIIIa
Then cleaves by plasmin
Remaining fibrin product is a D-dimer

Question 24

What is the clinical use of Alteplase?

Answer 24

Is a fibronolytic
So is indicated after an acute myocardial infarction, pulmonary embolism, ischaemic stroke.

Question 25

Describe how alteplase acts as a fibronolytic.

Answer 25

Chemistry: is a recombinant version of human tPA (tissue plasminogen activator)
Pharmacology: enzymatic activation of plasminogen
Physiology: activates endogenous fibronloytic system, by converting plasminogen to plasmin which then cleaves fibrin
This leads to thrombus dissolution.

Question 26

What are some in vitro coagulants?

Answer 26

Glass surfaces (old fashioned blood bottles) causes blood to clot easily.
Results in clotted blood in the bottom of the tube and serum or cell free plasma collects at the top.
Actives the intrinsic pathway of blood clotting (FXII to FXIIa)

Question 27

What can be done to prevent in vitro coagulation?

Answer 27

Use citrate or EDTA
Acts as a chelator to reduce the amount of free calcium
Reaction occurs on an equilibrium, the strength of binding to Ca2+ hence inhibitory effect varies.
This is mainly concentration dependent - hence adding more Ca2+ would enable blood to clot again.
Prevents the activation of Ca2+ dependent coagulation factors such as V,VIII, XIII and VK dependent factors.

Question 28

What are the three different drugs that may be used to prevent in vitro blood clotting?

Answer 28

EDTA (stronger effect than citrate)
Trisodium citrate
Heparin

Question 29

What are the two different tests used to assess coagulation?

Answer 29

Prothrombin time
Activated partial thromboplastin time.

Question 30

How does a Prothrombin time test work?

Answer 30

Performed on a citrated blood sample
Measures the time taken for thromboplastin (TF, PL and Ca2+) to trigger the extrinsic pathway and eventually activate fibrin forming a clot
Defects anywhere along the extrinsic or common pathway can result in a prolonged pro-thrombin test time.

Question 31

What are some example conditions that could cause a prolonged prothrombin test time?

Answer 31

FVII deficiency
Warfarin induced deficiency (affects the extrinisic common more than the intrinsic common)

Question 32

What is the activated partial thromboplastin time?

Answer 32

Measures the time taken for a blood clot to form.
Performed on a citrated blood sample
Triggered by an activator e.g kaolin activating FXII (a), and a partial thromboplastin (just PS and Ca2=) required for later steps
Measures the timining of the intrinsic pathway and the common pathway.
A defect in the common or intrinsic pathway could prolong activated partial thromboplastine time.

Question 33

What are some example conditions/scenarios that may cause an elevated activated partial thromboplastin time (aPTT)?

Answer 33

A deficiency in the intrinsic or common pathway
Includes FVIII or FIX deficiency - as seen in haemophilia A & B.
Heparin - induced.

Question 34

What is the pro-coagulant antidotes used after rivaroxaban?

Answer 34

Andexanet alfa
is a recombinant of human FXa, binds to rivaroxaban, prevents binding to Fx, results in FXa being able to cleave prothrombin to thrombin
Can prevent severe bleeding from rivaroxaban overdose

Question 35

What is the pro-coagulant used after dabigatran?

Answer 35

Idarucizumab
Recombinant Fab
Prevents Dabigatran from binding to prothrombin, so prothrombin is not cleaved to thrombin.

Question 36

What is the use of 4-factor Prothrombin Complex Concentrate?

Answer 36

Is a combination medication made of FII, VII, IX and X.
Replaces inactive or deficient factors
Or acts as competitive substrates for administered drugs.

Question 36

Give an example of an anti fibrinolytic.

Answer 36

Tranexamic acid
6 aminocaproic acid

Question 36

What is the use of phytomednadione?

Answer 36

Is a pro-coagulant/antidote to warfarin toxicity.
Is a VK supplement, increased amount of reduced Vitamin K so VitK dependent coagulation factors are activated and the coagulation cascade can occur.

Question 37

Have do anti-fibrinolytics work?

Answer 37

Chemistry - are lysine mimetics
Pharmacology - target plasmin(ogen), kringle domains, prevent surface interaction with fibrin.
Competes with C-terminal lysines in fibrin to bind to these sites.
Physiology -
Blocks the plasmin(ogen): fibrin interaction
This decreases the conversion of plasminogen to plasmin, so lower plasmin concentration
This decreases fibrin clot dissolution.
Clinical - haemostatic, to decrease haemorrhage such as menorrhagia.

Question 38

What is the clinical use of desmopressin?

Answer 38

Used to increase blood volume and blood pressure
Used to treat diabetes insipidus (by acting as replacement for ADH)
Used to treat Haemophilia A and von Willebrand disease

Question 39

How is desmopressin useful as a drug?

Answer 39

Chem: is a cyclic decapeptide
Pharm: targets vasopressin receptors (GPCR), is an agonist.
Physiology: increased H2O reabsorption in the DCT/CD, acts as a vasocontrictor, and induces the release of stored factors from endothelial cells this increases circulating FVIII and vWF levels.