Dato’ Dr Yasmin Ayob

� Blood vessels

� Platelet

� Coagulation factors

Phase I

� Vascular constriction

� Platelet adhesion � aggregation

Phase II

� Activation of clotting factors

� clot formation

Phase III

� Fibrinolysis

Vessel injury

vasocontriction

Reduced

Blood flow

Collogen

exposure

Platelet

Release reaction

Primary

Haemostatic plug

Platelet

fusion

STABLE

HAEMOSTATIC

PLUG

Thrombin

Fibrin

Tissue Factor

Blood Coagulation

� Monolayer of cells, inner surface of blood vessel

� Uninterrupted: necc for survival

� Rests on the subenthothelial matrix

� Secrete collagen, elastin, mucopoly-saccharides laminin, fibronectin, vWF, vitronection, thrombospandin

� Maintain blood flow by

- influencing the vascular tone, blood pressure

� - achieved by secretion of rennin, endothelium, ED RF, nitrous oxide adenosine, prostacyclin

� Anti platelet & anticoagulant properties

� Prevent platelet adhesion

� Expression of thrombomodulin and tissue factor inhibitor (TFPI)

Coagulant properties� Production of tissue factor

� Binds factor IX, X, V, HMWK

� Contain FXIII activity

� Produce TFPI

� Fibrinolytic Properties

Secrete plasminogen activators & plasminogen activator inhibitor

Repair properties of endothelium

� Migration of adjacent

� Enhance smooth muscle migration & fibroblast function

� Interacts with leukocytes

- inflammation

- adhesion molecules

� On escaping from damaged vessels at high shear rates, platelets adhere via Gp 1b-IX-V to vWF and collagen to the subendothelial vessel wall

� This activates platelets exposing Gp IIb/IIIa, fibrinogen receptor

� Exposing other receptors: P2Y1 and P2Y12 for ADP,

and thrombin and thromboxane receptors.

� � platelet aggregation & activation

� �provide receptors for other clotting factors for coagulation

� Activation of blood clotting factors

� Fluid blood ���� insoluble fibrin clot

� Biological amplification system

(l mol XIa � 2x108 mol of fibrin)

� Circulating precursor

� Proteolysis

� Activation

� Fibrinogen � fibrin

(form stable clot with platelets)

VIIa

TF-BEARING

CELL

ACTIVATED PLATELET

HEMOSTASIS

THROMBIN

FIBRIN HEMOSTATIC PLUG

INITIATION

OF HEMOSTASIS

PROPAGATION/AMPLIFICATIONOF HEMOSTASIS

TF

� Initiation: Tissue damage � endothelial cells are activated which expresses TF on the surface which is a receptor for FVII

� TF – FVII Complex

?

TF – FVIIa

Not clear what activate FVII but once coagulation is activated other proteins can activate FVII including Xa and VIIa.

- Direct activation of X to Xa

- Stimulation of amplification system

- To a lesser extend activate XI to XIa on the platelet surface

X Xa

Prothrombin Thrombin

� Small amount of thrombin generated activated FVIII, FV, platelets

� initiates amplification phase

Va Ca++

TF-VIIa

� Produce ‘thrombin burst’ where larger amount of thrombin is generated

� Thrombin generation

- on platelet surface, XI �XIa

- XIa binds to GP Ib and activates IX

- IXa with VIIIa as cofactor activates X

� Thrombin

- driven by direct activation of FIX by TF:VIIa

� activate FX (FVIII, Ca++)

� Thrombin �

TF

X

Xa Va

II

IIaTF-bearingcell

VIII/vWF VIIIa

V Va

platelet

activated platelet

VIIa

VIIaIX

IXa

VIIIa VaIXa

X

Xa

II

IIa

TF

XIa

IX

XI XIa

UHe/AC

NORMAL HEMOSTASIS

TFPI

� Explosive generation of thrombin converts large amount of soluble fibrinogen into fibrin

� This is stabilized by XIIIa ( XIII �XIIIa by thrombin)

� Fibrin enmeshes the platelet aggregate �stable hemostatic plug

Fibrinopeptide

releaseFibrinogen

Fibrin monomer

Fibrin polymer

H-bonding

Cross-linked fibrin

Transamides bonding

Thrombin

XIII

XIIIa

Inactivate FVIIIa and FVa

� stop thrombin burst

When binds to thrombomodulin activate protein C

Activated protein C in the presence of protein S inactivate VIIIa & Va

Thrombin

Thrombomodulin

Complex

Protein C Protein CaProt. S

PL

Ca++

FV/FVIII inactivation complex

inactive

� TF – FVIIa – responsible for generation of FXa and thrombin

� These are sufficient to activate FV, FVIII and platelet aggregation locally

� Inhibition by TFPI – FXa cannot maintain coagulation

� FXa generation amplification by IXa & VIIIa

� Explain why FVIII, FIX deficiency cause bleeding

� FIX deficiency is less serious

� Explain why FVIIa & activated PCC works in the presence of FVIII inhibitors

� Plasma contain proteins that prevent excessive intravascular coaulation

� Raised levels does not cause bleeding but reduce amount will cause thrombosis

� i) Antithrombin – potent inhibitor of Thrombin, Xa & XIa, and the TF-VIIa complex

ii) Protein C & S

Protein Z binds to Z-protease inhibitor & the complex inactivtes Xa

� TFPI is a single-chain polypeptide which can reversibly inhibit Factor Xa (Xa) and Thrombin(Factor IIa).

� While Xa is inhibited, the Xa-TFPI complex can subsequently also inhibit the FVIIa-TFcomplex.

� TFPI contributes significantly to the inhibition of Xa in vivo.

BLEEDING MAY BE CAUSED BY

1. DEFECTIVE PROCOAG. ACTIV. OR

2. HYPERACTIVE F-LYSIS OR

3. IMBALANCED SYSTEMS

PROCOAGULANT ACTIVITIES• coagulation proteins• TF exposure• phospholipid expression• on thrombin activated

platelets• TAFI

FIBRINOLYTIC

ACTIVITIES.

• activators (tPA)• plasminogen binding to fibrin

- Extrinsic & intrinsic pathway

- Able to test the ‘2 pathways’

- Able to understand coagulation

- Fail to represent the true picture

� No clinical problem with deficiency of FXII, prekallikrein or HMWR ( APTT)

� FXI deficiency – don’t always present with bleeding

� FVIII deficiency � bleeding

(even though ‘extrisnic pathway’ is intact)

� FVII activate FIX, FX

� TF is a natural constituent of many nonvascular cells

� Identification of TFPI

� Takes place while clotting is taking place

� Plasminogen converted to plasmin by tissue plasmin activator

� Plasmin digest fibrin (fibrinogen, FV, FVIII)

� split (degradation) products

� Fragments D & E detected in DIC

� Inactivation of plasmin by plasminogen activator inhibitor (PAI-1)

� PAI-2 – synthesized by placenta especially at the end of pregnancy

� Thrombin activatable fibrinolytic inhibitor (TAFI) - cleaves lysine from fibrin preventing plasminogen binding to fibrin

COAGULATION & FIBRINOLYSIS

� Agents inhibiting platelet function (aspirin, thienopyridines, Gp IIb/IIIa inhibitors)

� Heparins ( Unfractinated , LMWH, pentasaccharide)

� Coumarin derivatives (warfarin, acenocoumarol)

� Direct thrombin inhibitors (argatroban, bivalirudin, lepirudin,ximelagatran)

� Thrombolytic agent (streptokinase, urokinase, tPA)

� Aspirin & other NSAIDs –inhibit cyclooxygenase-1 & -2

� Thienopyridine (ticlopidine 7 clopidogrel)derivative inhibit binding of ADP to platelet receptor P2Y12

� Gp IIb/IIIa inhibitors impairs binding to fibrinogen – preventing aggregation

COUMARIN

� Inhibits effective synthesis of Factors II, VII, IX & X; protein C, S & Z, osteocalcin ( not involved in clotting)

� These factors require carboxylation of their glutamic acid residues to allow them to bind to phospholipid surfaces on the vascular endothelium.

� This process is linked to the oxidation of vitamin K to form vitamin K epoxide which is recycled back to its reduced form, requiring enzyme vitamin K epoxide reductase

� Coumarin inhibits the epoxide reductase – diminishing vitamin K stores – inhibiting synthesis of these factors

� Production of PIVKA – proteins induced by vitamin K absence.

� It may promote clot formation temporarily –causes the decline in protein C & S in the first 36 hrs, reduced degradation of FVa & FVIIIa

� Initial prolongation in INR after loading dose of 5 mg due to reduced FVII – full antithrombotic effect does not take place until FII drops a few days later

� The haemostasis system becomes temporarily biased towards thrombosis – it is beneficial to co-administer heparin that acts on FII which helps to reduce the risk of thrombosis for 4-5 days until full effect of warfarin is achieved

� A rare but serious complication resulting from treatment with warfarin is warfarin necrosis, which occurs more frequently shortly after commencing treatment in patients with a deficiencyof protein C.

� Patients when starting on warfarin, the initial reduction of protein C levels faster than the coagulation factors, increase the blood's tendency to coagulate when treatment is first begun (many patients when starting on warfarin are given heparin in parallel to combat this), leading to massive thrombosis with skin necrosisand gangrene of limbs.

� Its natural counterpart, purpura fulminans, occurs in children who are homozygous for certain protein C mutations.

� During the first trimester—particularly between the sixth and ninth weeks of pregnancy—a constellation of birth defects known variously as fetal warfarin syndrome (FWS), warfarin embryopathy, or coumarin embryopathy can occur.

� characterized mainly by skeletal abnormalities, which include nasal hypoplasia, a depressed or narrowed nasal bridge, scoliosis, and calcifications in the vertebral column, femur, and heel bone which show a peculiar stippled appearance on X-rays. Limb abnormalities, such as brachydactyly (unusually short fingers and toes) or underdeveloped extremities, can also occur.

� Common non-skeletal features of FWS include low birth weight and developmental disabilities.[

� Usually, warfarin is avoided in the first trimester, and a low molecular weight heparin is substituted. With heparin, risk of maternal hemorrhage and other complications is still increased, but heparins do not cross the placental barrier and therefore do not cause birth defects.

� Various solutions exist for the time around delivery.

� Warfarin administration in the second and third trimesters is much less commonly associated with birth defects - when they do occur, are considerably different from fetal warfarin syndrome.

� The most common congenital abnormalities associated with warfarin use in late pregnancy are central nervous systemdisorders, including spasticity and seizures, and eye defects.Because of such later pregnancy birth defects, anticoagulation with warfarin poses a problem in pregnant women requiring warfarin for vital indications, such as strokeprevention in those with artificial heart valves.

� Is a naturally-occurring anticoagulant produced by basophils and mast cells –High MW (3-30 kDa), stored in the mast cell granules & released in the vasculature at the site of injury.

� Unfractionated heparin (MW-12-15 kDa) produced commercially is derived from mucosal tissues of pocine intestine & bovine lung

� LMWHs are derived from heparin by chemical or enzymatic depolymerization, yielding fragments approximately one third the size of heparin - mean molecular weight of 4,500 to 5,000 d, with a distribution of 1,000 to 10,000 d.

� Pentasaccharide (fondaparinux)

� Only about one third of an administered dose of heparin binds to AT, and this fraction is responsible for most of its anticoagulant effect.

� The remaining two thirds has minimal anticoagulant activity at therapeutic concentrations, but at concentrations greater than usually obtained clinically, both high-affinity and low-affinity heparin catalyze the AT effect of a second plasma protein, heparin cofactor II

� The heparin-AT complex inactivates a number of coagulation enzymes, including thrombin factor (IIa), factors Xa, IXa, XIa, and XIIa.

� Of these, thrombin and factor Xa are most responsive to inhibition, and human thrombin is about 10-fold more sensitive to inhibition by the heparin-AT complex than factor Xa

� To inhibit thrombin, heparin must bind to both the coagulation enzyme and AT, but binding to the enzyme is less important for the inhibition of activated factor X (Xa)

� Molecules of heparin containing < 18 saccharides do not bind simultaneously to thrombin and AT and are therefore unable to catalyze thrombin inhibition.

� In contrast, very small heparin fragments containing the high-affinity pentasaccharide sequence catalyze inhibition of factor Xa by AT.

� By inactivating thrombin, heparin not only prevents fibrin formation but also inhibits thrombin-induced activation of factor V and factor VIII.

� Unfractionated heparin (UFH) and LMWH also induce secretion of tissue factor pathway inhibitor by vascular endothelial cells that reduce procoagulant activity of tissue factor VIIa complex, and this could contribute to the antithrombotic action of heparin and LMWH.

� Heparin resistance has been associated with AT deficiency, increased heparin clearance, elevations in heparin binding proteins, and elevations of factor VIII, fibrinogen, and platelet factor 4 (PF4).

� Aprotinin has been reported to cause drug-induced heparin resistance.

� The association with nitroglycerin is controversial.

� Factor VIII or fibrinogen levels are elevated in response to acute illness or pregnancy.

� Elevation of the levels of factor VIII alters the response of the APTT to heparin without diminishing the antithrombotic effect, as the anticoagulant effect of heparin (measured by the APTT) and the antithrombotic effect measured by anti-Xa activity become dissociated.

� For patients who require > 35,000 U of UFH per 24 h, the dose should be adjusted to maintain anti-Xa heparin levels of 0.35 to 0.70 IU/mL

� Substitution of LMWH may be inadvisable in such patients due to its long half-life and the lack of an effective neutralizing agent.

� LMWHs are defined as heparin salts having an average molecular weight of less than 8000 Da and for which at least 60% of all chains have a molecular weight less than 8000 Da. These are obtained by various methods of fractionation or depolymerisation of polymeric heparin.

� Low-molecular-weight heparins and and fondaparinux target anti-factor Xa activity rather than anti-thrombin (IIa) activity, with the aim of facilitating a more subtle regulation of coagulation and an improved therapeutic index.

� With LMWH and fondaparinux , there is a reduced risk of osteoporosisand heparin-induced thrombocytopenia (HIT).

� Monitoring of the APTT is also not required and indeed does not reflect the anticoagulant effect, as APTT is insensitive to alterations in factor Xa.

� LMWH therapy is monitored by the anti-factor Xa assay, measuring anti-factor Xa activity. The best time to perform an anti-Xa assay (if indicated in renal failure or morbid obesity) is 4 h after sc injection of a weight-adjusted dose of LMWH.

� Average molecular weight: heparin is about 15 kDa and LMWH is about 4.5 kDa.[7]

� Once-daily dosing by subcutaneous injection, rather than a continuous infusion of unfractionated heparin.[8]

� No need for monitoring of the APTT coagulation parameter.[9]

� Possibly a smaller risk of bleeding.

� Smaller risk of osteoporosis in long-term use.

� Smaller risk of heparin-induced thrombocytopenia, a feared side effect of heparin.

� The anticoagulant effects of heparin are typically reversible with protamine sulfate, while protamine's effect on LMWH is limited.

� Has less of an effect on thrombin compared to heparin, but maintains the same effect on Factor Xa.

� A serious side-effect of heparin is heparin-induced thrombocytopenia (HIT). HIT is caused by an immunological reaction that makes platelets a target of immunological response, resulting in the degradation of platelets. This is what causes thrombocytopenia. This condition is usually reversed on discontinuation, and can generally be avoided with the use of synthetic heparins.

� There is also a benign form of thrombocytopenia associated with early heparin use, which resolves without stopping heparin.

� Elevation of serum aminotransferase levels, which has been reported in as many as 80% of patients receiving heparin. This abnormality is not associated with liver dysfunction, and it disappears after the drug is discontinued.

� Hyperkalemia, occurs in 5 to 10% of patients receiving heparin, and is the result of heparin-induced aldosterone suppression.