Week 8 part 2 - Haemostasis

Question 1

Haemostasis definition

Answer 1

the stopping of blood loss from damaged vessels and protect against haemorrhage. A highly complex, regulated process
(basically a plug formed)

Question 2

Mechanisms involved in haemostasis following a wound include:

Answer 2

• Vasoconstriction
• Platelet adhesion to the exposed tissue
• Platelet activation to form a haemostatic plug
• Reinforcement of plug by fibrin

Question 3

There are times we want to promote haemostasis

Answer 3

• Haemophilia
• Haemorrhage (after surgery or trauma etc)

Question 4

There are more times though that we want to inhibit
haemostasis (thrombotic disease)

Answer 4

• Myocardial infarct (heart-attack), stroke (arterial thrombosis-emboli)
• Deep vein thrombosis (venous thrombosis)

Question 5

facts and terms about blot clots

Answer 5

• Myocardial infarction = heart attack where blood flow is stopped to the heart musclke
• stroke = clot that blocks blood flow to the brain
• deep vein thrombosis = a clot that forms in one of the deep veins of the body which is usually in the leg (calf/thigh).
• pulmonary embelism = when a clot (usually from a dvt) breaks free and could travel in the blood stream to block an important organ e.g. heart

Question 6

what is haemostasis?

Answer 6

• Body’s response to blood vessel injury and bleeding.
• Involves a coordinated effort between platelets and numerous blood clotting proteins (or factors).
• Results in the formation of a blood clot and subsequent stopping of the bleed.
• Interactions between platelet activation and the coagulation cascade
• Thrombin is a critical mediator

Question 7

Thrombosis

Answer 7

‘haemostasis in the wrong place’; the formation of a haemostatic plug within the vasculature in the absence
of bleeding

Question 8

Predisposing factors (Virchow’s triad) include:

Answer 8

• Injury to the vessel wall (eg rupture of atherosclerotic plaque)
• Altered blood flow (eg veins in legs during prolonged sitting, turbulence)
• Increased coagulability of the blood (thrombophilia)

Question 9

Drugs affecting haemostasis/thrombosis

Answer 9

antiplatelet agents

anticoagulants (heparin, LMWHs, hirudin, warfarin)

fibrinolytic agents (anistreplase, alteplase, reteplase, streptokinase, urokinase)

Question 10

Haemostasis: formation of a platelet plug

Answer 10

1. platelets adhere to and are activated by exposed collagen at the site of vessel injury
2. activated platelets release ADP and thromboxane A2.
3. These chemical messengers work together to activate other platelets passing by
4. Newly activated platelets aggregate onto growing platelet plug and release even more platelet-attracting chemicals
5. normal(uninjured) endothelium releases prostacyclin and nitric-oxide, which inhibit platelet aggregation, so platelet plug is confined to site of injury.

Question 11

Platelet activation

Answer 11

Platelets are activated through an enzyme called cyclooxygenase-1 (COX-1). When blood vessel injury occurs, COX-1 produces thromboxane A2, which promotes platelet aggregation and blood clot formation. Inhibiting COX-1, such as with aspirin, reduces thromboxane A2 production, preventing excessive platelet activation and clotting.

Question 12

Antiplatelet agents

Answer 12

Antiplatelet agents reduce platelet aggregation

Used for prophylaxis(to treat disease) of arterial thrombosis (platelet-rich clots), including acute MI; pts at high risk of MI; after cardiac surgery. Reduce the risk of vascular events

Combining different classes of antiplatelet agent often leads to a synergistic effect, but increases the risk of bleeding

Question 13

Arachidonic acid metabolism

Answer 13

Arachidonic acid is released from membrane phospholipids by phospholipase A2

AA can then be catalytically converted into a series of biologically active metabolites (eicosanoids).

The three major pathways for eicosanoid production involve the actions of cyclooxygenases, lipoxygenases and epoxygenases

Question 14

PGs as mediators of platelet function

Answer 14

Arachidonate
> (Cyclooxygenase) (COX) >
Endoperoxides (PGG2, PGH2)
> [1] PGI2 (prostacyclin)
= INHIBITS AGGREGATION
> [2] TXA2 (thromboxane A2)
= PROMOTES AGGREGATION

Question 15

Antiplatelet drugs: aspirin

Answer 15

• Aspirin irreversibly acetylates the active site of COX-1, preventing the formation of endoperoxides.
• Major products downstream of COX include prostacyclin (inhibits aggregation) and thromboxane A2 (promotes aggregation)
• intereferes with haemostasis by inhibiting the production of thromboxane A2 (which promotes platelet aggregation and vasoconstriction)
• suppressing thromboxane A2 = impedes platelet aggregation and reduce clot formation
• thins the blood less likely to clot
• If aspirin reduces prostacyclin production, how does it act as an antiplatelet drug?
• Aspirin irreversibly inhibits COX1, thus reducing TXA2 synthesis for the
  lifespan of the platelet
• PERMANENT inactivation of platelet COX-1 (in circulation) and reduction in clotting mechanisms (TXA2 from platelets no longer available). Platelets do not have a nucleus- so cannot make new COX-1
• In contrast, the inhibition of prostacyclin formation is reversible,
  because the endothelium is capable of re-synthesising cyclooxygenase
  (have a nucleus)
• So, aspirin produces a very long coagulation time (clotting time)
• A single (low) dose of aspirin doubles clotting time, and this effect lasts for a week- not coincidentally, the time it takes platelets to regenerate
• Low dosing allows for reduced gastrointestinal side-effects of blocking PGs

Question 16

Antiplatelet drugs: aspirin MOA

Answer 16

• COX-1 activated when platelets become activated –> generates TXA2.
• TXA2 binds to TP receptors on platelets thereby increasing expression of GPIIb/IIIa on platelet cell membranes (see later slides)
• Aspirin: inhibits TXA2 production
  permanently from platelets –> chronically reduces platelet aggregation

Question 18

Aspirin Side Effects

Answer 18

AEs include:

Increased bleeding time (common);

GI irritation, haemorrhage/ulcers (see above) - these are rare, due to low dosing frequency – once/twice weekly, low dosages (e.g. 100-600 mg), and enteric coatings)

blood thinning!

Question 19

Antiplatelet drugs: purinergic antagonists

Answer 19

• ADP induces platelet aggregation by
  activating P2Y12 receptors
• Thienopyridines irreversibly inhibit P2Y12 receptors to inhibit platelet aggregation
• Clopidogrel is well absorbed orally; prodrug is converted to active metabolite by CYP enzymes (including CYP2C19) in the liver
• Variant alleles or CYP2C19 inhibitors may decrease effectiveness (25 SNPs identified to date, many of which are associated with alterations in activity)
• Used for prevention of ischaemic events in patients with symptomatic atherosclerosis; can be used in combination with aspirin
• AEs include diarrhoea, GI ulcer, increased risk of bleeding

Question 20

Antiplatelet agents (2)

Answer 20

GP IIb/IIIa inhibitors (eg abiciximab, Arofiban) prevent fibrinogen crosslinking of platelets

Question 21

Haemostasis: the role of thrombin

Answer 21

thrombin is a component of the clotting cascade and plays multiple roles in haemostasis

• stimulated conversion of fibrinogen to fibrin
• activates factor stabilising fibrin meshwork of clot
• enhances activation of more prothrombin into thrombin through positive feedback
• enhances platelet aggregation

through positive feedback, aggregated platelets secrete PF3, which stimulates clotting cascade that results in thrombin activation

Question 22

Blood coagulation

Answer 22

Fibrinogen (soluble) > (thrombin)> fibrin (insoluble)

• Activation initiates the coagulation cascade, amplifying the signal
• Thrombin (factor IIa) cleaves fibrinogen=fibrin; also activates factor XIII fibrinoligase, induces platelet aggregation and affects VSM tone
• Cascade requires control by endogenous inhibitors: eg antithrombin inhibits critical clotting factors (IXa, Xa, XIa, XIIa, thrombin).

Question 23

anticoagulants

Answer 23

Drugs affecting haemostasis/thrombosis
- heparin
- LMWHs
- hirudin
- warfarin

Question 24

Anticoagulants: direct thrombin inhibitors

Answer 24

• Hirudin was one of the first anticoagulants, and is a direct thrombin inhibitor
• Direct thrombin inhibitors do not rely on endogenous anticoagulant systems (compare with heparin and LMWH)
• Dabigatran is an orally-available DTI that reversibly inhibits thrombin (free and clot-bound) and thrombin-mediated platelet aggregation.
• Fixed dose produces predictable anticoagulant effect (promising alternative to warfarin)
• Rivaroxaban is an orally-available drug that inhibits factor Xa to reduce thrombin production. Similar indications/usage as dabigatran.

Question 25

Anticoagulants: heparins

Answer 25

• Heparin is family of large sulfated polysaccharides; not absorbed from the gut (charge, high Mw), administered IV or SC.
• Indirectly inhibits thrombin by enhancing anti-thrombin (AT) mediated inactivation of factor Xa and thrombin (increases affinity of AT for clotting factors)
• Inactivation of thrombin requires long polysaccharide chains that can complex with both AT and thrombin; these are absent in LMWH (eg enoxaparin).
• Limited efficacy on platelet/fibrin bound factors
• Main risk is haemorrhage: can be treated with protamine sulphate
  (complexes with and inactivates heparin). AEs include bruising,
  thrombosis, osteoporosis (long-term), hypersensitivity

Question 26

Anticoagulants: heparin vs LMWH

Answer 26

?

Question 27

Anticoagulants: vitamin K antagonists

Answer 27

• Vitamin K is a fat-soluble vitamin that is required for synthesis of factors II, VII, IX and X.
• Obtained from plants and from gut microflora; given routinely at birth in Australia
• Acts as a co-factor for carboxylase

Question 28

Anticoagulants: warfarin

Answer 28

• Warfarin is the most important oral anticoagulant*, however there are several issues associated with its use
• It competes with vitamin K for binding at vitamin K reductase (VKORC1)
• Orally acIve and rapidly absorbed
• Small distribution volume, strongly albumin-bound
• Peak concentration in blood within a few hours, yet peak pharmacological effects takes ~ 48h to develop
• The response of individual patients to warfarin can vary dramatically
• The VKORC1 gene is polymorphic
• Warfarin is metabolized by CYP2C9 (also polymorphic)
• Low margin of safety; requires
  frequent monitoring and dose
  individualization
• Multiple drug interactions (other
  albumin-bound drugs; antibiotics;
  P450)
• Crosses placenta

Question 29

Fibrinolysis

Answer 29

?

Question 30

Drugs affecting Fibrinolysis

Answer 30

?

Question 31

Fibrinolytics

Answer 31

• Endogenous plasminogen activators (serine proteases) present in blood
• At a thrombus, they cleave plasminogen (deposited on fibrin strands) to release plasmin
• Plasmin digests fibrin, fibrinogen, ECM proteins and some clotting factors
• Action is localized to the clot as plasminogen activators are mainly effective on plasminogen that is bound to fibrin; escaping plasmin is inactivated by circulating
  plasmin inhibitors

*Fibrinolytic drugs are used to dissolve clots and reopen occluded arteries in patients with acute MI within 12 h of onset. Small window of opportunity for administration (time is muscle/brain)
* Initial fibrinolytic drugs derived from haemolytic streptococci;
current versions utilise recombinant DNA technology.
* Alteplase is a recombinant tissue plasminogen activator. Greater activity on plasminogen bound to fibrin, which increases ‘clot selectivity’. Non-antigenic. Modified version of tPA including tenecteplase.
* Administered IV, major risk of bleeding (including GI
haemorrhage and haemorrhagic stroke).