11/15 - Secondary Hemostasis

Front 1

Pathway activated by tissue injury?

Back 1

Extrinsic pathway

Front 2

Pathway activated by contact?

Back 2

Intrinsic pathway

Front 3

Common pathway

Back 3

Front 4

What converts fibrin to CLF? What is CLF?

Back 4

Fibrin --> CLF (cross-linked fibrin) via XIIIa

Cross-linking stabilizes fibrin (otherwise transient)

Front 5

Intrinsic vs extrinsic pathway

Back 5

Extrinsic pathway is initial activation

Intrinsic pathway is an amplification

Front 6

Factor II

Back 6

Prothrombin (converted to IIa - thrombin)

Front 7

Clotting cascade feedback

Back 7

Positive feedback

Front 8

Reactions in clotting cascade w/out cofactors?

Back 8

Slow, but they do proceed

Front 9

Most factors are activated or inactivated by...

Back 9

proteolytic cleavage

Front 10

Factors II, VII, IX, X (and protein C and S) have ____ domains which are ____ charged and attach to _____

Back 10

Gla domains

Negatively charged

Attach to platelet surface

Front 11

Factors II, VII, IX, X are _____ dependent factors

Back 11

Vitamin K

Front 12

Conversion of X --> Xa requires...

Back 12

IXa w/ cofactor VIIIa

VIIa w/ cofactor Tissue Factor

Front 13

Platelets bind to collagen with the help of ____

Tiny amount of factor ____ is in form of _____

Injury exposes _____. ____ and ____ bind to ____.

Back 13

Platelets bind to collagen with help of vWF

Tiny amount of VII is in form of VIIa

Injury exposes TF, VII and VIIabinds to TF (extrinsic pathway)

Front 14

TF-VIIa is called _____

TF-VIIa converts a little ____ to ____ and ____ to ____

Back 14

Extrinsic tenase (Xase)

IX --> IXa (intrinsic pathway)

X --> Xa (common pathway)

Front 15

Xa feedback higher up

Back 15

Main action is to convert II --> IIa

Also feeds back to convert V--> Va and VIII --> VIIIa

Front 16

Complex of Xa/Va called....convert ___ to ___

Back 16

prothrombinase (prothrombin --> thrombin)

Front 17

VIIIa and IXa called _____ and convert more ____ to ____

Back 17

Intrinsic tenase or Xase - convert more X to Xa

Front 18

Once thrombin is active, it activates _____

Back 18

lots of factors (V, VII, VIII) this is the amplification

Also converts XI to XIa - amplifying the intrinsic pathway (converts more IX to IXa)

Front 19

Thrombin effect on platelets

Back 19

Thrombin activates platelets (via binding to PAR 1, 3, 4) - cause secretion of granules (vWF, fibrinogen, Factor Va)

Front 20

Cross-linker for platelets?

Back 20

Fibrinogen

Front 21

What converts fibrinogen to fibrin? What also needs to happen to help cross-link fibrin?

Back 21

IIa (thrombin) converts fibrinogen to fibrin

IIa also converts XIII to XIIIa which is needed to cross-link fibrin

Front 22

Extrinsic pathway

Back 22

• TF exposed upon exposure of subendothelium

• Binding of TF to VIIa allows activation of small amount of both X and IX

• VIIa is present in small amounts in the plasma and cannot initiate hemostasis w/out TF

Front 23

Extrinsic pathway - thrombin production?

Back 23

•The extrinsic pathway produces only asmall amount of thrombin, which is insufficient (in isolation) for hemostasisin most situations

Front 24

Complete deficiencies of VII/TF?

Back 24

Not compatible with life

Front 25

Tissue factor

Back 25

-Cell bound initiator of homeostasis, some soluble

-Transmembrane protein

1) Extracellular portion interacts w/ VIIa

2) Serves as cofactor for VIIa

3) Has no intrinsic protease activity

4) Found on fibroblasts, smooth muscle, microparticles

5) Expressed in endothelial cells in inflammation and other diseases (atherosclerosis)

-Necessary to maintain vascular integrity

Front 26

Factor VII

Back 26

-Extrinsic pathway

-Shortest half-life of any hemostatic factor

-Small amount of circulating VIIa that is available to bind with TF

-TF does not activate VII

-Complete loss of VII incompatible w/ life

-Thrombin can activate VII --> VIIa

Front 27

Intrinsic pathway - function

Back 27

-Feedback/amplification loop

-Significantly increases the production of thrombin

-Required for hemostasis in most situation

Front 28

"Intrinsic" factors

Back 28

XII, XI, IX, VIII

Front 29

Complete deficiencies of intrinsic factors?

Back 29

•Complete deficiencies of intrinsicfactors are compatible withlife,unlike extrinsicandcommon pathway factors

Front 30

Factor IXa activates _____. Activation process?

Back 30

•Factor IXaactivates factor X

–IXa is a slow activator of X withoutits cofactor: VIIIa

–Remember: X is activated byboth factor VIIIa/IXa andthe TF/VIIa complex

Front 31

Factor VIII is activated to Factor VIIIa (cofactor for...?) by ______.

Factor VIII is normally bound by ____ which extends the half-life of VIII. Activation _______.

Back 31

•FactorVIII is activated (VIIIa) by thrombin

VIIIa is the cofactor for IXa activation of X)

•FactorVIII bound by vWF, which extends the ½ life of VIII. VIII --> VIIIa dissociates it from vWF

Front 32

Common pathway - why is it called common, important factors/substrate, common pathway deficiencies

Back 32

•Called “common” as it is the portion ofthe cascade that is common to both the intrinsic and extrinsic pathways

•Factors X, V, prothrombin, and fibrinogen

•Complete deficiencies of X, V, orprothrombin are not compatible with life

Front 33

Factor X is activated to Xa by ______.

Xa activates ______. Cofactor?

Xa is a therapeutic target for _____ because of ______.

Back 33

•Factor X is activated to Xa byeither the extrinsic “Xase” complex (TF/VIIa) orthe intrinsic “Xase” complex

•Xa activatesprothrombin (to thrombin) relatively slowly until factor V is activated bythrombin (Va is acofactor)

•Xa is a therapeutic target foranticoagulation because of its central role in hemostasisC

Front 34

Common pathway: prothrombin + thrombin

Back 34

-Activation of prothrombin --> thrombin via Xa with cofactor Va

-IIa is considered master effector and regulator of hemostatic cascade

-Thrombin upregulates its own production (XI, VIII, V)

-Thrombin downregulates its own production (activation of protein C when associated w/ thrombomodulin)

Front 35

Thrombin interacts with/cleaves many proteins

Back 35

–Fibrinogen (I), V, VII, VIII, XI

–Factor XIII (stabilizes fibrin)

–Thrombomodulin

–Thrombin activatablefibrinolysis inhibitor (TAFI)

–PARs 1, 3, 4 (on platelets)

–C5 and Osteopontin (Th 1cytokine) for lymphocyte activation

Front 36

Almost all coagulation is initiated by the _____ with a positive feedback loop from the _____

Back 36

•Almostall coagulation is initiated by the “extrinsic” cascade(TF/VIIa)with apositive feedback loop from the “intrinsic” cascade

Front 37

Factors important for contact activation (on an anionic surface) include:

Back 37

–Prekallikrein(enzyme, activates XII)

–High Molecular Weight Kininogen(co-factor)

–Factor XII (Enzyme, activates factor XI)

Front 38

Deficiencies of contact pathway?

Back 38

do NOT result in a bleeding disorder

Front 39

In vitro vs in vivo contact pathway

Back 39

•Initiates fibrin formation invitro (i.e.aPTT)

•In vivo, may be involved in production ofbradykinin(vasodilator) and fibrinolysis onendothelial cells

Front 40

Structure of fibrinogen + cleavage by thrombin

Back 40

•Dimer consisting of three pairs ofchains: Aα, Bβ, andγ

•Thrombin cleaves fibrinopeptides Aand B from the Aα and Bβchains, respectively

•Cleavage of fibrinopeptide A isall that is required for formation of the fibrin polymer

•Fibrin polymers formed from thisthrombin-mediated process are unstable and easily lysed

Front 41

Factor XIII

Back 41

•Plasma transglutaminase

•Cross-links fibrin polymers via acovalent bond between the glutamine to lysine residues of the γ-chainof fibrin

•Also slowly crosslinks the α-chain

•Will also crosslink inhibitors offibrinolysis (α2-antiplasmin)into the fibrin clot, this stabilizes the clot

•Activated by thrombin

Front 42

Vitamin K dependent factors

Back 42

-Coagulant factors: II, VII, IX, X

-Anticoagulant proteins: C and S

-Glutamyl carboxylase - microsomal enzyme, requires Vit K as cofactor, converts Glutamate residues in the "Gla domain" on precursor proteins to y-carboxyglutamyl (Gla) residues via addition of CO2

-Gla domain: Ca2+ dependent binding of phospholipids

Front 43

____ is added to glutamate to create gamma-carboxy-glutamate

Back 43

CO2

Front 44

Recycling of Vitamin K - drug significance and enzyme significance

Back 44

• Vitamin K must be converted back toreduced form by epoxide reductase torecycle
  
• Warfarin (coumadin) blocks epoxide reductase
  
• Also note many isoforms of epoxide reductase withdifferent activities, so Warfarin mustbe titrated and monitored, a PitA for both patient and physician
  

Front 45

Calcium - clotting factor ____; role in hemostasis; anticoagulants that target Ca2+

Back 45

•Originally described as “clotting factorIV”

•Absolutely required for hemostasis

•Citrate or EDTA (calcium chelators) arepotent anticoagulants

•Vitamin K-dependent factors require Ca2+ binding to function

•Three Ca2+ bindingsites on fibrinogen that are important for maintaining its stability/structure

Front 46

Most of the clotting factors are synthesized in the ______

Back 46

liver - liver diseases affect clotting

Front 47

What can cure VIII deficiency?

Back 47

Liver transplant (cure hemophilia A)

Front 48

Inhibitors of coagulation

Back 48

• The coagulation cascade is tightly controlled to prevent pathologic thrombus formation
• Multiple inhibitors of coagulation
• Coagulation is inhibited primarily at three points:
• 1) Activation of X by TF/VIIa complex (TFPI)
• 2) Activity of thrombin (thrombomodulin, antithrombin)
• 3) Cofactor activity of VIIIa and Va (proteins S and C)

Front 49

TFPI

Back 49

-Tissue factor pathway inhibitor

-Exists as both soluble and membrane bound forms (membrane bound is predominant in humans, mainly on endothelial cells)

-TFPI binds to complex of TF, VIIa, and Xa

-Inhibits extrinsic pathway of coagulation, especially in areas adjacent to clot where endothelium is intact

Front 50

Thrombomodulin

Back 50

-Cell-associated factor (mostly endothelial cells) that binds thrombin and alters its activity

-Main role is to prevent clot/thrombus on endothelial cells

-Decreases activity against/affinity for: Fibrinogen, factors V, VIII, and XI, PARs (platelet receptors)

-Increases (significantly) activity for: protein C, thrombin activatable fibrinolysis inhibitor (TAFI)

Front 51

Proteins C and S

Back 51

•Protein C is activated by the thrombin-thrombomodulincomplex, binds to endothelial protein C receptor (EPCR)

•Protein S is a cofactor for protein C

•Activated protein C inactivates factors Va and VIIIa,especially on endothelial surfaces

•Vitamin K dependent proteins. ½ life of protein C is short (6-10 hours) so Warfarincauses paradoxical clotting, so give heparin for first 4 days

•APC (activated protein C) is also anti-inflammatory–Can be used for the treatment of severesepsis

Front 52

Antithrombin

Back 52

-SerPIn (serine protease inhibitor) attached to endothelial surface

-Inhibits activity of both Xa and thrombin (IIa)

-Activity of antithrombin is greatly increased by heparins

Front 53

Factor V Leidan

Back 53

Factor V Leiden is mutation infactor V that is resistant to protein C inactivation

Front 54

Heparin

Back 54

endogenous sulfated glycosaminoglycan

produced by mast cells in mammals

enhances activity of antithrombin

Front 55

Antithrombin and ____ Combine together to inhibit _____

Back 55

heparin

Xa (prevents conversion of II to IIa)

Front 56

Unfractionated heparin blocks...

Back 56

Thrombin AND Xa

Front 57

Fibrin is degraded by...

Back 57

plasmin - cleaves fibrin and other proteins at lysine-arginige bonds

Front 58

Plasminogen is primarily activated by plasmin by...

Back 58

tissue plasminogen activator (tPA) - from endothelial cells or uPA - urokinase-type plasminogen activator (from kidneys, bind endothelial cells via uPAR)

Front 59

Both ____ and ____ bind to fibrin, increasing the fibrinolytic potential of both enzymes

Back 59

plasmin

tPA

Front 60

What on endothelial cells binds tPA an dplasminogen?

Back 60

Annexin A2

Front 61

Fibrinolytic system (w/out inhibitors)

Back 61

Front 62

D-dimers

Back 62

Not from dissolution of fibrin polymer - from dissolution of cross-linked fibrin (end pieces) - measure of clot formation

Front 63

Inhibition of fibrinolysis

Back 63

-a2-antiplasmin directly inhibits plasmin

-plasminogen activator inhibitor 1 (PAI-1) directly inhibits plasminogen activity

-fibrinolysis is indirectly inhibited by thrombin/thrombomodulin activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

-TAFI cleaves the C-terminal Lys residues from fibrin - these are the sites of binding for plasmin and tPA

Front 64

Fibrinolytic system with inhibitors? (draw)

What do deficiencies in these inhibitors cause?

Back 64

Front 65

Deficiencies of VIII or IX are known as ____

Back 65

Hemophilias

Hemophilia A - factor VIII deficiency

Hemophlia B - factor IX deficiency

Front 66

Extrinsic vs intrinsic Xase

Back 66