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Hemostasis/Coagula+on
• Hemostasis • Coagula+on
• Components – Blood vessels – Platelets – Coagula+on proteins – Fibrinolysis – Serine protease inhibitors
– Kinin system – Complement system
• Hemostasis – Primary
• Forma+on of the “platelet plug” • Ini$ally halts the loss of blood
– Secondary • Reinforcement of the platelet plug by fibrin forma/on
Balance between bleeding and cloEng
Major components
Minor components
3
Secondary Hemostasis • Purpose—to reinforce the primary platelet plug (primary hemostasis) • Products of secondary hemostasis
1. Thrombin 2. Fibrin
• Defects lead to 1. Decreased fibrin produc+on 2. Reduces the stability of the formed clot
• Mediated by proteins—coagula+on factors • Coagula+on factors are produced in the liver
– Factor VIII and vWF probably excep+ons • Coagula+on cascade describes the ac+vity of the coagula+on proteins • Consists of:
1. Substrates—substances acted upon by an enzyme 2. Cofactors—components that takes part in a catalyzed reac+on 3. Enzymes (mostly serine proteases)—components that speeds up a chemical
reac+on
7
Coagula+on Cascade
• Paul Morawitz—1905
8
Tissue damage
Thrombokinase
Prothrombin + Ca2+
FIBRIN CLOT
Thrombin
Fibrinogen
1. MacFarlaine—cascade concept 2. Davie and Ratnoff—waterfall concept 3. Modern Concepts
9
Surface, XII Prekallikrein HMWK
Simplified Coagula+on Cascade
IX IXa
Tissue Factor VII(VIIa)
VIII VIIIa
Xa X
II Thrombin V Va
Fibrinogen Fibrin
PC
aPC
Platelet Aggregation
Stable Clot
XIII
XIIIa
Central Player • Converts Fibrinogen to Fibrin • Platelet ac+va+on • FV, VIII, XI • FXIII XI XIa
Coagula+
on Factors
Factor Name Pathway I Fibrinogen Common
II Prothrombin Common
III Tissue Factor Extrinsic
V Labile Factor Common
VII Proconver+n (Stabile Factor) Extrinsic
VIII An+hemophilic Factor Intrinsic
IX Plasma thromboplas+c component (PTC)
Intrinsic
X Stuart-‐Prower factor Common
XI Plasma thromboplas+n antecedent (PTA)
Intrinsic
XII Hageman Factor Intrinsic
XIII Fibrin-‐stabilizing factor ?Common
PK Prekallikrein (Fletcher) Intrinsic
HMWK High molecular weight kininogen (Fitzgerald)
Intrinsic
IV Ionized calcium
VI Not assigned
10
Extrinsic Pathway • Endothelial damage occurs à allows TF to enter the
vessel lumen
• FVII floa+ng by in the circula+on binds to TF [+ (Ca2+)] forming a TF:VIIa complex
• TF:VIIa complex converts FX to FXa
• Therefore the [TF:VIIa +Ca + PF3] complex is also known as the “EXTRINSIC TENASE”
• FXa + Va converts prothrombin (FII) to thrombin (IIa)
– This results in the ini+al burst of thrombin (IIa)
• Thrombin feeds back up to ac+vate FV, FVIII, FXI, FXIII
• TF:VIIa complex also converts FIXα to FIXa
• FIXa + FVIIIa also converts FX to FXa – [FIXa + FVIIIa + Ca2+ + PF3] is also known as
the “INTRINSIC TENASE”
• Fig 24-‐14
11
Intrinsic Pathway • Consist of
1. PK – prekallikrein (Fletcher) 2. HMWK – high molecular weight kininogen (Fitzgerald) 3. FXII – Hageman factor 4. FXI – plasma thromboplas+n antecedent
• Characteris+cs
1. Ini+ated in vivo by exposure to subendothelial collagen which has a nega+ve charge 2. In the in vitro by glass, kaolin, celite, ellagic acid, silica 3. Ini+ated under pathologic condi+ons by exposure to lipopolysaccharide (bacteria) 4. PK, HMWK, FXII do NOT play a significant role in in vivo hemostasis
a. Pa+ents with PK, HMWK, FXII à do NOT bleed (but tend to thrombose) b. Increased interac+on of these factors with
– Complement system – Kinin system – Fibrinoly+c system
12
Intrinsic Pathway 1. Exposure to nega+vely charged collagen
(subendothelium or phospholipids)
2. Ini+ally FXII is auto-‐ac+vated to XIIa and binds to high molecular weight kininogen (HMWK)
– Forms a complex that binds to the subendothelial collagen layer
3. FXIIa in the presence of prekallikrein (PK) and HMWK converts FXI to FXIa
4. FXIa + Ca2+ converts FIX to FIXa
5. [FIXa + FVIIIa + Ca2+ + PF3] complex à “INTRINSIC TENASE complex” converts FX to FXa
6. FVIIIa – a cofactor orients FIXa and FXa in the proper orienta+on to alach to the platelet phospholipid surface (PF3)
• Figure 24-‐15
13
Complexes
Figure 24-‐20 Intrinsic and Extrinsic Tenase Complexes
Figure 24-‐21
Prothrombinase Complex
14
Common Pathway
• [FXa + FVa + PF3 + Ca2+] – prothrombinase complex – converts prothrombin (II) to thrombin (IIa)
• FVa – a cofactor posi+ons the tenase complex (extrinsic or intrinsic) to interact with FII on the platelet phospholipid surface
• Prothrombin à THROMBIN (IIa)
• IIa à circula+ng fibrinogen (FI) to soluble fibrin monomers
• IIa à FXIII to FXIIIa
• FXIIIa cross-‐links the fibrin monomers into an insoluble fibrin polymer (CLOT)
15
Figure 24-‐16
Addi+onal Fact about the Common Pathway
• Intrinsic and extrinsic pathways converge on the common pathway • Involves the intrinsic and/or extrinsic tenase complex • Prothrombinase complex à Prothrombin to THROMBIN
• Thrombin has mul$ple func$ons 1. Converts fibrinogen to fibrin 2. Ac+vates factor XIII 3. Ac+vates platelets 4. Ac+vates FVIII 5. Ac+vates FV 6. Ac+vates FXI
7. Ac+vates Protein C 8. Binds to Thrombomodulin 9. Mitogen for fibroblasts
Prothrombinase Complex
Xa
Va
Platelet (PF3)
Vitamin K-‐Dependent Factors
• II—Common pathway • VII—Extrinsic pathway • IX—Intrinsic pathway • X—Common pathway
• C—Protein C pathway, inhibitor • S—Protein C pathway, inhibitor • Z—Probably a cofactor
– Protein Z-‐related inhibitor (ZPI)—inhibits factor Xa and XIa
17
prothrombo+c
an+thrombo+c
Mechanism of Ac+on of VKDF’s • Contain glutamic acid residue
– Terminal GLA located at the COOH terminal end
– γ-‐carboxyglutamic acid
• Allows calcium to alach the VKDF’s to the phospholipid surface of the platelet
• Absence of vitamin K à to reduced func+on of the VKDF’s
– PIVKA’s—”proteins induced by vitamin K antagonists”
• Func+on of the GLA domains 1. Binds the complex to PF3 using
Ca2+ as a bridge 2. Folds the protein into the proper
configura+on for complex forma+on
18
Ac+on of Thrombin on Fibrinogen
• Fibrinogen molecule consists of 6 chains – Aα, Bβ, γγ chains
• Thrombin proteoly+cally cleaves a. Fibrinopep+de A from the Aα-‐
chain b. Fibrinopep+de B from the Bβ-‐
chain Ø Leads to a forma+on of a
soluble fibrin monomer (from the remaining chains)
• Fibrin monomers polymerize “end-‐to-‐end” and “side-‐to-‐side”
• Factor XIIIa catalyzes the cross-‐linking of glutamine and lysine bonds à polymeriza/on of soluble fibrin monomers à to insoluble fibrin meshwork
19
Forma+on of a Fibrin Clot
1. Thrombin proteolyzes fibrinopep+des A and B from fibrinogen to yield soluble fibrin monomer
2. Soluble fibrin monomer then associates side-‐to-‐side and end-‐to-‐end to form fibrin polymers
3. Thrombin-‐ac+vated FXIIIa covalently cross-‐links the fibrin polymers into an increasingly complex structure and an ul+mately insoluble clot
• D = peripheral domains • E = central domain
20
Fibrinolysis • Presence of fibrin triggers the ac+va+on of plasminogen to plasmin
• Plasminogen is the target substrate of the ac+va+on systems and ater cleavage becomes the ac+ve enzyme plasmin
• Plasmin then splits fibrin and fibrinogen into fragments – Interfere with thrombin ac+vity, platelet func+on, and fibrin polymeriza+on, leading to clot
dissolu+on
21
Func+on of Fibrinolysis • Fibrinolysis and Repair
1. Fibrinolysis 1. Physiologic process of removing unwanted fibrin deposits 2. Gradual progressive enzyma+c cleavage of fibrin to soluble
fragments 3. Fragments are removed by RES 4. Re-‐establishes blood flow in vessels—maintains patency 5. Involves proteoly+c diges+on of fibrinogen and fibrin by plasmin 6. In+mately related to fibrin forma+on
2. Repair • Involves release of PDGF
Key Player in Fibrinolysis • Plasminogen
– Becomes bound to fibrin at the +me of fibrin polymeriza+on – Bound plasminogen is converted to a two-‐chain ac+ve plasmin molecule
• Plasmin – Serine protease that systema+cally digests fibrin
• Also digests FV, FVIII, Fibrinogen – Binding to fibrin clot via lysine bonds prevents systemic ac+vity – Plasmin is the main enzyme in fibrinoly+c system
23
Ac+vators and Inhibitors of Fibrinolysis
Ac/vators
1. Tissue plasminogen ac+vator (t-‐PA) – Plasmin acts primarily on fibrin, but is a
nonspecific destroyer • Hydrolyzes plasminogen bound to
the fibrin clot à ini+ates fibrinolysis
• 2 forms of tPA – Synthe+c recombinant TPA
used in thromboly+c therapy – Secreted from endothelial
cells
2. Urokinase – Secreted from urinary tract epithelial
cells—intrinsic plasminogen ac+vator – Does not bind firmly to fibrin, so it is not
extremely effec+ve
3. Streptokinase – Extrinsic, therapeu+c ac+vator
Ac+vators and Inhibitors of Fibrinolysis
Inhibitors
1. Plasminogen Ac+vator Inhibitor (PAI-‐1) – Prevents t-‐PA and urokinase from ac+va+ng free fluid-‐phase plasminogen
2. α2-‐An+plasmin
– α2AP rapidly inhibits free plasma
– Produced in the liver
PAI-‐1
Naturally Occurring Inhibitors (Regulators) of the Coagula+on Cascade
1. An+thrombin (AT)—major regulator of coagula+on cascade a. Inhibits IIa, Xa, and the other serine
proteases b. Serves as a cofactor for heparin and other
heparinoids
2. Ac+vate Protein C (aPC)—inhibits Va and VIIIa a. PC—when ac+vated becomes APC b. PS—serves as a cofactor to PC
3. Heparin Cofactor II (HCII)—Similar to AT, but only inhibits IIa
4. Tissue Factor Pathway Inhibitor (TFPI)—inhibits extrinsic tenase
5. Thrombin Ac+vatable Fibrinolysis Inhibitor (TAFI)—thrombin ac+vatable fibrinlysis inhibitor
6. Thrombomodulin (Tm)—thrombomodulin—regulates thrombin—alenuates thrombin’s procoagulant ac+vity conver+ng it into an an+coagulant
26
Kallikrein-‐Kinin System
• PK and HMWK needed to enhance or amplify the contact factors – XIIa + HMWK converts PK to
kallikrein – Kallikrein feeds back to
accelerate XII à XIIa – Speeds up the intrinsic ac+va+on
• Ac+va+on of FXIIa acts as the common link between A. Fibrinoly+c system B. Kinin system C. Complement system D. Coagula+on system
28
Kallikrein-‐Kinin System
• System of proteins that when ac+vated à to the release of vasoac+ve kinins
• Kinins are ac+vated by Kallikrein à release both HMWK and LMWK • Kinins are involved in physiological and pathological processes
– Regula+on of blood pressure and flow (via modula+on of the renin-‐angiotensin pathway – Blood coagula+on – Cellular prolifera+on and growth, Angiogenesis, Apoptosis, and Inflamma+on
• Kinin ac+on on endothelial cells à 1. Vasodila+on 2. Increased vascular permeability 3. Release of tPA 4. Produc+on of nitric oxide (NO) 5. Mobiliza+on of arachidonic acid à PGI2 produc+on 6. Chemotaxis
• In hemostasis –bradykinin is released from HMWK
29
Cell-‐based Model of Hemostasis
• Proposed to replace the tradi+onal cascade
• Specific cellular surface receptors for coagula+on proteins promote hemostasis
• Occurs in 3 phases 1. Ini/a/on 2. Amplifica/on 3. Propaga/on
31
The Role of Platelets in Hemostasis
Collagen Other factors TF
Thrombin
Platelet
Platelet Platelet
Platelet
Platelet
Activated platelet
Activated platelet
Activated platelet
Platelet Platelet
Platelet Platelet
Adhesion
Aggregation
Contraction
Secretion
Primary Haemostasis
=
Activated platelet
Activated platelet
Activated platelet
Activated platelet
This plug of activated platelets, localised to the site of injury, provides the phospholipid surface upon which Secondary Haemostasis takes place
Initiation, Amplification, Propagation
VIIIa IXa
Hoffman M & Munroe DM. A cell-based model of hemostasis. Thromb Haemost 2001; 85: 958-965
+ activates various factors
Initiation
Amplification
Propagation
Hemostasis
Ø Vasoconstric+on Ø Platelet plug forma+on
l Ac+va+on l Adherence l Aggrega+on
Ø Stabiliza+on of platelet plug l Coagula+on cascade l Fibrin
XII
XIIa
XI XIa
IX
X X
VII
IIa II
Fibrin Fibrinogen
VIIa TF
TF
IXa VIIIa
Xa Va
Intrinsic
Common
Extrinsic
Plt
Xa IIa VIIa TF
Xa Va
IIa Plt
Xa
IXa VIIIa
Xa Va
IIa
Plt
Plt
Fg
Fb
vWF
Fb
Plt
Plt Fb
Fb
Fb
vWF
Tissue factor bearing cell
Subendothelial collagen
Plt
IIa
Ini+a+on
Amplifica+on
Propaga+on
Platelet activation
Ø Triggered by l Sub-‐endothelial collagen l IIa l ADP l TXA2 l 5HT l Epinephrine
Platelet activation
• Results in; – Greatly increased surface area
– Dense granules release Ca2+, 5HT, TXA2, ADP
– Alpha granules release factor V, fibrin, VWF
Platelet activation
• Platelet phospholipid bilayer is actively controlled
• Resting internal surface is procoagulant • Bilayer everts during activation, exposing
procoagulant surface