Week 6 Flashcards
What is hemostasis
what are the system properties
-physiologic process that keeps blood from circulating
-balances hemorrhage and coagulation
properties
procoagulant - initiate clotting if required
anticoagulant - prevent undesirable clotting
fibrinolytic/fibrinolysis - clot breakdown
what is the hemostasis cascade divided into
Primary hemostasis - platelet plug formation and vasconstriction
Secondary - fibrin clot formation and Coagulation Activation
fibrinolysis - fibrin clot breakdown
when is hemostasis initiated
-disruption of vascular endothelial lining or with cell turnover by trauma or disease
-blood is then exposed to subendothelial connective tissue
-blood vessels contract to seal wound - vasocontraction to reduce blood flow
-then the three processes start
what things need to interact to stop bleeding, allow for clotting and then clot breakdown
-blood vessels with tissue factor to help with vasocontriction
-platelets- for platelet adhesion, aggregation and secretion
-plasma components - zynogens, cofactors, control proteins and Fibrinogen
-fibrinolytic proteins - fibrin breakdown
-inhibitors - inhibit coagulation
what roles do the inner BV layer and subendothelium of the blood vessel play
Inner BV layer - Vascular Endothelium
-needs to be unbroken for best blood flow
-this layer releases:
Anticoagulant - when clot is formed or prevent spontaneous clotting
Procoagulant - when there is injury
Fibrinolytic - parttakes in clot dissociation
Sub endothelium
-Supports surface protein called Tissue factor -procoagulant -INITIATION of COAGULATION
-degree of vasoconstriction depends of BV type, size and muscle = SMOOTH CELLS CONTRACT
What role in hemostasis does RBC, WBC and PLT play
RBC: adds bulk and structural integrity to fibrin clot
WBC- wound healing
-monos and lymphs have tissue factor on their surface to trigger coagulation
Platelets-
PROCOAGULANT only - initiates and controls hemostasis
-PLT dont prevent or break down clots instead they adhere, aggregate and secret granule contents to help form a clot
What is primary hemostasis
What is it triggered by and what do the PLTs to when activated
-PLTs have alpha and epsilon granules
-triggered by injury to BV walls that expose subendothelial collagen
-PLTs can become activated and
Adhere to site of injury
Secrete granule contents
Aggregate with other PLTs
what is the short lived reponse that occurs in primary hemostasis
how is the bleeding reinforced in the long term
if there are defects in primary hemostasis what will it cause
-the short response to vessel damage is produced by vasoconstriction and platelet plug formation
-in order for bleeding to be controlled in the long term the platelet plug must be reinforced with fibrin
-Defects in primary hemostasis can cause chronic hemorrhages - can be fatal
eg- when someone has collagen abnormalities, thrombocytopenia, platelet disorders or vWf disease
What is secondary hemostasis
-plasma coagulation protein activation that results in fibrin clot formation
-involves PLT and coaug system
-delayed- long term response
-activated by big injuries to BV and surrounding tissues
-triggered by Tissue Factor TF (activator)
-a membrane protein found in subendothelial smooth muscle cells, fibroblasts released when endothelial cells are disrupted, monos and lymphs
What is the coagulation cascade that occurs in 2ndary hemostasis
-series of plasma protein interactions
-coagulation factors are serine proteases circulating as zymogens/inactive enzymes . During coagulation, they become activated through a domino effect to produce thrombin
After injury occurs
-PLTs will adhere and aggregate at site of injury until new endothelial cells grow
-the injury exposes tissue factor in subendothelial cells
-the coagulation system initiation occurs ON TF BEARING CELLS
-the coag reactions are then propagated on PLT surface
-GENERATING THROMBIN-FIBRIN-CLOT STABILIZED - FIBRINOLYSIS
What is the fibrin clot in 2ndary hemostasis
-the clot is a fibrillar protein that give the semi solid state to clot or thrombus
-the clot is created by breakdown of fibrinogen by thrombin (serine protease)
-fibrinogen has no binding sites but fibrin monomers are able to polymerize to site of trauma/injury
What are the components of Plasma * LISTEN TO RECORDING IF THESE NEED TO BE MEMORIZED
Zymogens and & co factors
-16 coag or clotting factors -PROCOAGULANTS
-working in a cascade to form fibrin clot
8 serine proteases
Co-factors
vWf (Factor 8 carrier and platelet adhesion)
Phospholipids
Calcium
Fibrinogen
Control proteins
-once initiated they control Coag (Anticoag)
Serine protease inhibitors
Co factors -
2-Prothrombin - serine protease
3,
5 (Labile factor),
7- Stable factor - serine protease
8 (antihemophilic),
ix (9) Christmas factor -SP
10 (X) - Stuart Prower Factor - SP
11 (XI) Plasma thromboplastin antecedent (PTA) -SP
12(XII) Hageman factor -SP
Prekallikrein - Fletcher factor , PK - SP
High molecular weight kininogen (fitzgerald HMWK)
What do the reactions in the coagulation cascade need
- Cellular base of negatively charged phospholipids (subendothelial cells or PLT surface)
2.Ca2+ to bind co/factors to those neg charged phospholipids ( coagulation only occurs at site of injury)
3.Plasma coag proteins : Serine proteases for enzyme activity and Co factors for stabilizing & enhancing enzyme activity
summary of cascade
Coag cascade + serine proteases & cofactors =thrombin (FIIa)
-Thrombin breaks down fibrinogen (FI) to form fibrin
What three in vivo complexes and cofactors does the coagulation cascade form **listen to lecture portion
VIIa & TF , phospholipid and Ca2 (extrinsic tenase) which then activate IX and X
IXa & VIIIa, phospholipid and Ca2 (intrinsic tenase) which activate X
Xa & Va, phospholipid and Ca2 (prothrombinase) which activate prothrombin
Contact factor pathway (XII, pre-K, and HMWK)
What are the 3 main pathways that are formed in the cascade and what do they consist of
Extrinsic pathway:
TF (FIII), VIIa + Common
- Intrinsic pathway:
XIIa*, XIa, IXa, VIIIa + Common - Common pathway:
Xa, Va, IIa (Thrombin), FI (Fibrinogen)
Describe what occurs in the Extrinsic pathway:
TF (FIII), VIIa + Common
TF + VIIa + Ca2+ + PL = Extrinsic tenase complex
‘Extrinsic’ because TF not found in blood
Initiates coagulation after tissue injury & TF exposure
-TF is exposed in subendothelium activating FVII forming the Extrinsic tenase complex TF:VIIa
-FX is activated by the TF:VIIa complex initiating the common pathway producing thrombin
-FIX is activated to IXa which initiates intrinsic pathway
Describe what occurs in the Intrinsic pathway:
Consists of factors found in plasma
True ‘in vivo’ intrinsic pathway starts at FIXa and FXIa
FIX is activated by Extrinsic path
FXI is activated by Thrombin-Contact Factor complex can also activate FXI
Extrinsic pathway activates small amount of FIX
FXIa further activates FIX to FIXa
FIXa joins cofactor VIIIa forming Intrinsic tenase complex
-Occurs on PLT surface
-Complex also activates FX , initiating the Common pathway
What is the contact factor complex that is found in the intrinsic pathway
-can activate FXI
-Contact factors’= HMWK, pre-K, XIIa
-activated through contact with neg charged foreign substances
In vivo examples, stents, valve prostheses, and
bacterial cell membranes
In vitro, siliconized glass, kaolin, or ellagic acid act
as negatively charged surfaces that activate FXII
-dont play a large role in invivo coag and trauma injuries
What is the common pathway consisting of and what does it activate
F-X is activated by both Extrinsic & Intrinsic pathways
-FXa “activates” FV - Va and forms the Prothrombinase complex (Xa & Va on PLT membrane in presence of Ca2+)
-Prothrombinase complex ‘activates’ Prothrombin (FII) into THROMBIN (FIIa)
-Thrombin then converts Fibrinogen (FI) to Fibrin (red clot formation)
-Thrombin activates FXIII to cross-link fibrin or stabilize clot
What type of feedback loop is created in common pathway
-Thrombin positive feedback loop
-Thrombin loops back into cascade to further activate V, VIII, XI, and PLTs until solid clot is formed
-Once ‘Intrinsic’ pathway is active ‘Extrinsic’ gets shut down
-Tissue factor inhibitors stop the ‘Extrinsic’ system (no more activation of X & IX)
-Antithrombin comes in to prevent further initiation of thrombin once clot is solid
What are the 3 phases of cell based coagulation
Can be described via interdependent phases:
Initiation
Amplification
Propagation
What is the initiation phase of cell based coag
-triggered with formation of Extrinsic tenase complex on surface of cells with TF
-this complex then activates small levels of FIX and FX to create a small amount of thrombin
What is the amplification phase of cell based coag
-when the thrombin feedback loop creates a activation cascade
-activates FV, FVIII, FXI (this initiates the intrinsic pathway) and more platelets
-helps to generate Thrombin generation
-starts fibrin formation
What is the propagation phase of cell based coag
-in amplification phase Thrombin activates Cofactors Va and VIIIa to bind PLT membranes becoming receptors for Xa and IXa
-the IXa binds to VIIIa on PLT membrane= Intrinsic tenase complex
-which activates FX 50-100 times higher than extrinsic complex
- FXa binds FVa to form Prothrombinase complex which activates prothrombin to create burst of thrombin
-the thrombin cleaves fibrinogen to fibrin forming a fibrin clot
-Thrombin also activates FXIII to help stabilize the clot via cross links (formation of red clot)
what role do procoag cofactors play in 2ndary hemostasis
Bind serine proteases to stabilize & enhance their reactivity
What role does Tissue Factor TFIII play in 2ndary hemostasis
Procoagulant Cofactor
-initiates coag cascade as transmembrane receptor for 7
-found on subendothelium of extravascular cells like SM cells, fibroblasts or on leuks (mono/lymph during inflammatory or septic conditions)
What role does FV and FVIII play in 2ndary hemostasis
Procoagulant Cofactor
-soluble plasma protein
-liable and degrade quick in plasma so they are used up during clotting
What role does HMWK play in 2ndary hemostasis
Procoagulant Cofactor
-circulating plasma protein that binds to FXIIa and Prekallikrein (preK) in intrinsic factor complex
What role does HMWK play in 2ndary hemostasis
Procoagulant Cofactor
-soluble glycoprotein that circulates in plasma and is stored in platelet alpha granules
-when platelets are activated they release/secret FV at injury site
-synthesized in BM megakaryocytes
-unstable in plasma so it is a labile factor
-acts as cofactor to FXa in prothrombinase complex
What role does FVIII play in 2ndary hemostasis * RECORDING OF SLIDE AFTER
Procoagulant Cofactor
-very unstable in plasma except during coag
-circulates bound to vWf which helps to increase its stability for about 12 hours
-produced by hepatocytes and microvascular endothelial cells
-known as Anti-hemophilic factor - X linked
-deficiency causes Hemophilia A
-when stored as whole blood or THAWED plasma, FVII level decreases 50% after 5 days
-cofactor to FIX which forms intrinsic tenase complex
What role do Serine Proteases play
IIa, VIIa, IXa, Xa, XIa, XIIa & Prekallikrein (pre-K)
-start as zymogens (inactive enzymes) that circulate in plasma
- become proteolytic enzymes which are activated by cleavage on the cell/platelet surface at site of injury
-coag factors are produced in the liver which is why when the liver fails the pt bleeds more
-factors II, VII, IX and X are Vitamin K - dependent
What role does Vitamin K play
-carboxylates factors in liver so they can bind Ca and phospholipids so they can participate in the coag pathway
-it gives factors a second caboxyl group needed to be effective factors
-if there is not Vit K then the factors will not be functional
-Vit K dependent factors- FII, VII, IX, and X, and regulatory control proteins, protein C, S, and Z
-Coumadin/Warfarin - the blood thinner works by blocking Vitamin K so factors cannot bind to Ca because they wont get that second carboxyl group. There will be no clotting
-Vitamin K antagonists are Epoxide reductase and Quinone reductase
What role does FIX play
and what if youre deficient
-serine protease known as Christmas factor
-dependent on Vitamin K
-its cofactor is FVIIIa and together they make the intrinsic tenase complex to activate FX
-deficiency causes Hemophilia B or Christmas Disease (X linked recessive factors and sever bleeding)
What role does FXI play and what does its deficiency cause
-serine protease but its not part of any complex
-activated by contact factor complex or thrombin which then activates FIX
-if deficient it leads to Hemophilia C or Rosenthal Syndrome
What is FXII
and what does its deficiency cause
-serine protease which forms part of the contact factor complex
-becomes activated by contact with negatively charged foreign surfaces
-FXIIa transforms pre K into active Kallikrein which then activates HWMK in Bradykinin and activates FXI
-deficiencies in contact factors dont cause bleeding disorders but it lengthens in vitro coagulation testing
What is Thrombin FIIa
-serine protease
-main enzyme of coag cascade
Triggers positive feedback loop to make more of itself
-activates FV and FVII - >Va and VIIIa
-activates FXI–>FXIa
-induces platelet aggregation and activation
-cleaves fibrinogen to fibrin FI to FIa PRIMARY FUNCTION
-activates FXIII–>FXIIIa (fibrin stabilizing factor
Thrombin regulates coagulation and fibrinolysis
FINAL SERINE PROTEASE
What is FI - fibrinogen
-glycoprotein produced by the liver
-absorbed, carried and released by PLTs (alpha granules)
-highest plasma concentration of procoagulants
-part of PLT aggregation because it joins activated PLTs together by their GP IIB/IIIa receptors
-primary substrate of Thrombin which digests it into fibrin monomers that polymerize forming fibrin polymer - a lattice fibrin network)
what is FXIII
-transglutaminase
-fibrin stabilizing factor which crosslinks adjacent D domains of fibrin polymers to form meshwork of fibrin polymers that are resistant to fibrinolysis or are insoluble
What is VWF
-plasma coagulant
-LARGE multimeric glycoprotein
-produced by megakaryocytes and endothelial cells. Stored in alpha granules and Weibel-Palade endothelial cells which are released on injury.
-people with type O blood have lower vWf
What is the primary function of vWf
-initiates primary hemostatsis by bridging PLTs and subendothelial collagen during PLT adhesion
-has a receptor site to support PLT aggregation
-Carrier protein for FvIII
-abnormalities in VWF structure and concentration can cause severe bleeding
What are some coagulation regulatory mechanisms
what are the main control proteins
-inhibitors and their cofactors regulates serine proteases in the coag cascade by using anticoag feedback loops
-this ensures that coag is localized and not systemic maintaining a balance by slowing procoagulant activation and suppressing thrombin production
Main control proteins/regulators:
-Tissue Factor Pathway Inhibitor (TFPI)
-Activated Protein C (APC) & Protein S
-Antithrombin (AT) & Heparin Cofactor II
-Protein Z & Protein Z-dependent -Protease Inhibitor (ZPI)
What is the Tissue Factor Pathway Inhibitor
-serine protease inhibitor
-made by endothelial cells and expressed on PLT membranes
-primary regulator of Extrinsic pathway because it inhibits the extrinsic tenase complex and FXa
-Protein S is its cofactor which enhances its reactions 10 fold
how does Tissue Factor Pathway Inhibitor inhibit coagulation
in two steps:
TFPI binds and inactivates FXa
TFPI:Xa complex binds and inactivates TF:VIIa on subendothelial membrane so no more Xa can be formed.
-after the intrinsic pathway is activated, TFPI shuts down the extrinsic tenase complex and Xa
- Xa + IXa production shifts to Intrinsic pathway
What is the Protein C Regulatory System
-changes thrombins function from procoagulant to anticoagulant
-thrombin binds to endothelial membrane protein called Thrombomodulin causing thrombin to become inactive so it cant activate procoag factors or PLTs
-Thrombin-Thrombomodulin/T-T complex activates Protein C System
-Thrombin activates Protein C (Activated Protein C/APC)- serpin
-APC binds Protein S to stabilize itself
-APC:ProtS complex inactivates FVa and FVIIIa
-this blocks Thrombin generation
What is the Protein S Regulatory System
-cofactor that binds and stablizes APC and TFPI
-enhances Xa inhibition by TFPI tenfold and increases action of T-T complex 5 fold
-made in the liver and circulates in cofactor free form and bound (unavailable) form
what is Antithrombin (AT)
-Serine protease inhibitor (SERPIN)
-prevents further activation of Thrombin in common pathway
-Binds and neutralizes serine proteases:
Thrombin (FIIa) and FIXa, FXa, FXIa, FXIIa, prekallikrein, and plasmin
-needs heparin for proper coag activity because AT activity is increased 2000 fold by binding to it
-this is what the pharma Heparin is based off of
-Heparin cofactor II is what circulates in plasma it exclusive inactivates Thrombin
What is Protein Z + Protein Z-dependent Protease Inhibitor (ZPI)
ZPI =Serine protease inhibitor (SERPIN)
Protein Z - nonproteolytic cofactor
Vitamin K-dep plasma glycoprotein synthesized in liver
Increases the ability of ZPI to inhibit factor Xa 2000-fold
ZPI+Protein Z/PL + Ca2+= serpin- potent inhibitor of FXa
ZPI also inhibits FXIa
-Separate reaction that does not require protein Z, phospholipid, and Ca2+
-The inhibition of FXIa is accelerated twofold by the presence of heparin
What is fibrinolysis
-clot digestion
-helps to restore normal blood flow
-starts a few hours after an insoluble fibrin clot is produced (fibrin polymerization and crosslinking)
-localized to site of injury needs plasminogen, Tissue plasminogen activator and Urokinase Plasminogen Activator
-once the clot is digest it leave behind fibrin degradation products
Fibrinolytic proteins vs Fibrinolysis Inhibitors
Fibrinolytic proteins -Dissolve clot to restore blood flow
Fibrinolysis Inhibitors -Prevents excessive or early fibrinolysis
how does fibrin degradation begin
Fibrinolytic proteins Plasminogen, TPA, and UPA are joined into fibrin as the clot forms
-once the clot forms because of inflammation and coag, TPA and UPA will be activated/released and will convert Plasminogen to Plasmin which is the primary serine protease of fibrinolytic system
What is TPA
Tissues plasminogen activator
-serine protease
-secreted by activated endothelial cells
-released when there is inflammation and coagulation, binds to fibrin clot covalently
-hydrolyzes plasminogen in fibrin and converts it to plasmin to initate degradation
-TPA in circulation is bound to fibrinolysis inhibitor -Plasminogen Activator Inhibitor-1 (PAI-1) which is neutralized and taken out
What is Urokinase Plasminogen Activator (UPA)
-serine protease
-secreted by urinary tract epithelium monocytes, and macrophages
-binds with plasminogen and TPA as clot is formed but does not bind fibrin tight
-converts plasminogen to plasmin which starts fibrin degradation so normal blood flow can be restored
What are plasminogen and plasmin
-both plasma zymogen serine proteases that are made by the liver
-abundant in plasma
-as fibrin clot polymerizes plasminogen binds it
-plasminogen gets converted to plasmin when it is cleaved by fibrin bound TPA/UPA
Bound plasmin -digests fibrin, restores flow and prevents SYSTEMIC clotting
FREE plasmin -digests plasma fibrinogen FI, FV, FVIII. Levels are controlled by alpha 2 antiplasmin
What is α2-Antiplasmin (AP)
fibrinolysis inhibitor
-Serine protease inhibitor (SERPIN)
-made in the liver
-inhibitor of free plasmin
-slows fibrinolysis because it competes with plasminogen binding with fibrin, binds to plasmin quickly and reversibly, inactivates plasmin
what is Plasminogen Activator Inhibitor-1 (PAI-1)
-Serine protease inhibitor (SERPIN)
-Produced by endothelial cells, megakaryocytes, smooth muscle cells, fibroblasts, monocytes, adipocytes, hepatocytes
-Principal inhibitor of plasminogen activation
-Inactivates TPA & UPA
-Prevents conversion of plasminogen to active plasmin
What is Thrombin Activatable Fibrinolysis Inhibitor (TAFI)
-Plasma procarboxypeptidase
-Synthesized in the liver
-regulates inflammation and healing
-Activated by Thrombin-Thrombomodulin complex (same complex that activates Protein C pathway)
Acts as an antifibrinolytic enzyme
Inhibits fibrinolysis:
Prevents binding of TPA & plasminogen to fibrin
Blocks formation of plasmin
Causes suppression of fibrinolysis
- if you have coagulopathies with factor deficiencies, like Hemophilia- decreased thrombin can reduce TAFI activation causing increased fibrinolysis and more bleeding
-In thrombotic disorders-
Increased thrombin can increase activation of TAFI causing decreased fibrinolysis making thrombosis worse
What are some Fibrin Degradation Products
-when plasmin cleaves fibrin and fibrinogen it produces fibrin fragments :X, Y, D, E, & D-Dimer
these fragments inhibit hemostasis
-prevent PLT activation by inhibiting hemorrhage
-prevent fibrin polymerization
how are Fragments X, Y, D, and E produced
-by systematic digestion of either fibrin or fibrinogen by plasmin
how are D-Dimer Fragments produced
-Specific product of digestion of crosslinked fibrin only
-Therefore, a marker of thrombosis and fibrinolysis
-can be detected by monoclonal AB for D dimer ag
What do you use a d dimer assay for
-Identify chronic and acute DIC
DIC- Disseminated Intravascular Coagulation (uncontrolled activation of thrombin & consumption of coag. factors, plts and fibrinolytic proteins)
-Rule out venous thromboembolism
Suspected deep venous thrombosis (DVT) or pulmonary embolism (PE)
