Week 8-9 Flashcards
how do errors in hemostasis occur
-missing parts that are needed to produce a clot
-missing components needed to breakdown a clot or inhibit clotting
What three systems interact to provide hemostasis
vascular intima
-platelets
-plasma coagulation system
What is primary hemostasis
associated with BV and PLTs - short term response
-activated by small injuries to blood vessels and expose subendothelial collagen
-BV contract to seal wound and stop blood flow
-PLTs cling to site of injury , secrete granule contents and aggregate with other PLTS
What is secondary hemostasis
activated by large injuries to blood vessels and surround tissues
-involves platelets and coag system
-delayed, long term response
-TF (activator) is exposed on cell membranes activating zymogen to generate thrombin and thrombin converts Fibrinogen to Fibrin - forming a fibrin clot
What are the 3 layers of the BV and what do they include
-Vascular intima - Endothelial cells + subendothelial matrix which releases anticoagulant, procoagulant and fibrinolytic
-Vascular media
Vascular adventitia - outermost
What are the ANTIcoagulant properties of intact endothelium
anticoagulant glycosaminoglycan heparan sulfate
coagulation extrinsic pathway regulator tissue factor pathway inhibitor
maintains cell membrane thrombomodulin a protein C coagulation control system activator
What happens when then blood vessel is injured
the vessel’s properties switch from ANTI-coagulant to PRO-coagulant
What are the procoagulant properties of damaged vascular intima
induces vasoconstriction
-binds VWF and PLTs
-secretes adhesion molecules Pselectin, ICAMs, PECAMs
What is the role of PLATELETS at the time of injury
Adhesion, Aggregation and Secretion
What is the adhesion function of platelets
-PLTs adhere to nonPLT surfaces like subendothelial collagen via VWF through GP Ib/IX/V receptor
Reversible, closes endothelial gaps
-secretes growth factors
VWF is needed for adhesion in arterioles
What is the aggregation function of platelets
when PLTs stick each other
-PLTs activated and there is receptor change GP IIb/IIIa to allow for PLT binding to VWF and FIB
-irreversible , platelet plugs form and contents are secreted
need fibrinogen
What is the secretion function of PLTs
-when PLTs start to discharge granule contents
-irreversible
-happens during aggregation
-PRO coagulation factors are secreted and needed for coagulation
What do the alpha granules of plts contain
Fibrinogen
vWF
Factor V,VIII
Plasminogen
HMWK
Protein S
What do dense granules contain
ADP
ATP
Calcium
Serotonin
Epinephrine
Thromboxane
What is the Extrinsic pathway composed of
1). Exposed TF on subendothelial cells bind to Factor VII.
2). TF-VIIa activates Factors IX and X. IXa-VIIIa complex also activates Factor X
4). Xa-Va activates Factor II (Prothrombin) into Factor IIa (Thrombin)
5) Thrombin cleaves Fibrinogen and cross links Fibrin by activating Factor XIIIa
What is the intrinsic pathway composed of
1). Exposure to negatively charged surfaces activates contact factors (XII, Pre-K, HMWK)
2). Contact factors will activate XI which goes onto activate IXa
3). IX-Va will activate Factor X to continue on.
What are the Vitamin K dependent factors?
The Prothrombin Group:
FII (prothrombin), FVII, FIX and FX
Regulatory proteins Protein C and Protein S
What are the three coagulation complexes
Extrinsic Tenase
Intrinsic Tenase -more efficient than Extrinsic tenase at activating Prothrombinase Complex
Prothrombinase
Each complex involves a protease (enzyme activity) a co-factor (stabilizing and enhancing enzyme activity), Ca++ (to bind factors/cofactors), & phospholipid (negatively charged)
Cell-based Physiologic Coagulation
can be described through independent phases such as
Initiation
Amplification
Propagation
What is the initiation phase
triggered with formation of the Extrinsic tenase complex on the surface of TF-bearing cells
Complex activates low levels of Factors IX and X to generate a small amount of Thrombin
What is the amplification phase
T-hrombin feedback loop creates a cascade of activation
-Activates small amounts of Factors V, VIII, XI and activates more platelets
-Serves to amplify more
-Thrombin generation
-Begins Fibrin formation
What is the propagation phase
-Activation of cofactors V and VII by thrombin during amplification phase to bidn activated PLT membranes - to become receptors for X and IX
-IXa generated from initiation phase binds to VIIIa on PLT membrane forming Intrinsic tenase complex
-Thrombin activation of FXI in amplification phase also activates more FIX
-This process initiates Intrinsic pathway
How is a fibrin mesh formed
Exposed fibrin monomer alpha and beta chain ends (called E domains) have immediate affinities for D Domains of neighbouring fibrin monomers – Leads to polymerization
After activation by thrombin, XIII covalently crosslinks fibrin polymers (btw adjacent D domains) to form a stable urea-insoluble fibrin clot
Actions of Thrombin
-Converts fibrinogen to fibrin
-Procoagulant (Activates FV, FXIII & FXI > to generate more thrombin)
-Enhances activity of FV & VIII (for fibrin crosslinking)
-Platelet aggregation
-Involved in Factor Feedback Inhibition
Thrombin bound to Thrombomodulin activates Protein C pathway to suppress coagulation
What is the PT assay
when PT reagent is mixed with PLT poor plasma
the PT reagent has TF or thromboplastin in phospholipids and CaCL
activates the extrinsic and common pathways
The reagent mix activates fibrin polymerization =clotting and FVII
-FVII has the shortest half life (6 hours) and effects PT the greatest
-monitors Warfarin/Coumadin therapy
-Formation of clot is visually detectable
To distinguish between vitamin K deficiency and liver disease
the laboratory practitioner determines factor V and factor VII levels. Both factor V and factor VII are reduced in liver disease; only factor VII is reduced in vitamin K deficiency.
What is Activated Partial Thromboplastin Time or APTT
-looks at issues in the intrinsic and common pathway
-Used to monitor standard (unfractionated) Heparin anticoagulant therapy
-assay has two steps/reagents
1-Contact activator - negatively charged particulate activator like silica
-phospholipids ( partial thromboplastin because there is no TF or Ca)
2-CaCl
Thrombin Time
assess deficiencies or dysfunction of fibrinogen or the presence of an inhibitor to thrombin (FIIa)
-qualitative test
-add thrombin to plasma and time the clot formation
-The less fibrinogen available the longer it takes to form a clot
-bypasses all other factors and just measures FI- fibrinogen
What is the fibrinogen assay
-Add thrombin to PPP
-Produce Standard curve Time vs. Fibrinogen concentration
-Take patient’s Fib concentration from graph
If patient is fibrinogen deficient, we expect PROLONGED clotting time (therefore, lower concentration)
Regulation of the Coagulation Pathway
Regulated by:
Vascular intima
Regulatory proteins
prevent uncontrolled thrombosis
What is a serpin
Serine protease inhibitor to II, VII, IX, X
What is the tissue factor pathway inhibitor
regulates Extrinsic system
-inactivates FX and then both bind to FVIIa to inactivate. Once the intrinsic pathway is active , TFPI will shut down the extrinsic pathway and Xa
Xa + IXa production will shift to intrinsic pathway as propagation phase occurs
-amplification of Thrombin occurs through FIXa
What is what of the protein C pathway
The Protein C System changes Thrombin from a procoagulant to an anticoagulant
Thrombin bindsThrombomodulin
Thrombin becomes inactive and can no longer activate procoagulant factors or PLTs
Thrombin-Thrombomodulin/T-T complex activates Protein C System
Thrombin activates Protein C (Activated Protein C/APC)- serpin
APC dissociates from EPCR and binds cofactor Protein S (stabilizes APC)
APC-Protein S complex inactivates (by further cleaving or digesting) FVa and FVIIIa
Slows or blocks Thrombin generation and coagulation
what is the PrC pathway
-Thrombin-Thrombomodulin/T-T complex activates Protein C System
-Thrombin activates Protein C (Activated Protein C/APC)- serpin
-APC dissociates from EPCR and binds cofactor Protein S (stabilizes APC)
-APC-Protein S complex inactivates (by further cleaving or digesting) FVa and FVIIIa
-Slows or blocks Thrombin generation and coagulation
What is Antithrombin (AT)
Serpin
-slow acting , weak inhibitor. Wont interfere with normal clotting unless activated by heparin
-Needs heparin to bind and inhibit Thrombin
- Antithrombin’s activity is accelerated 2000-fold by binding to heparin
-circulating in plasma is Heparin cofactor II = Serpin that exclusively inactivates Thrombin
-binds and inactivates Thrombin (FIIa) and FIXa, FXa, FXIa, FXIIa, prekallikrein, and plasmin
-
Where is heparin from
Mast cell granules
endothelial cell heparan sulfate
through therapeutic heparin
What is heparin Cofactor 2
-needs heparin for anticoagulation
-inactivates thrombin in the presence of heparin
-prevents conversion of fibrinogen to Fibrin
SERPIN
like ANTITHROMBIN
What is z dependent protease inhibitor ZPI
SERPIN
ZPI + Protein Z = inhibits Xa
-can also inhibit FXIa in the presence of heparin
TFPI inactivates
FXa by binding the TF:VIIa complex
APC inactivates
FVa and VIIIa when thrombin binds thrombomodulin which activates Pro C to APC. APC needs to complex with free Pro S to function efficiently
What is Fibrinolysis
-break down of fibrin clot
-forms Fibrin degradation products
Fibrinolysis Pathway Proteins
Plasminogen
Tissue Plasminogen Activator (tPA)
& Urokinase
Plasminogen Activator Inhibitor PAI-1
α2-antiplasmin
What is plasminogen
circulating zymogen made in the liver
-TPA and UPA are activated (or released) and act to convert Plasminogen to Plasmin
What is plasmin
-serine protease that degrades fibrinogen
FV, VIII (both in circulation) and Fibrin polymer in clot
- free plasmin is regulated by α2-antiplasmin (SERPIN)
What is Tissue Plasminogen Activator (TPA)
Plasminogen activator
-secreted from endothelial cells
-primary activator and most potent
-converts plasminogen to plasmin
-higher affinity for fibrin than fibrinogen
-cleaves fibrin bound plasminogen
-used for therapeutic thrombolytic therapy - clot busting drug - recombinant TPA
-Circulating TPA is usually bound to an inhibitor and then cleared for circulation
What is urokinase
Plasminogen Activator
-secreted by kidney - urinary tract epithelial cells , monocytes and macrophages
-primary activator in the genitourinary system
-very specific
-not bound to fibrin
released mostly during
urological surgery and prostate cancer
What is streptokinase
-plasminogen activator
-cleaves plasminogen to plasmin
-bacterial endotoxin , can cause sepsis and is a product of B hemolytic streptococci
-pathogenic and can trigger DIC
-not a serine protease
-complexes with plasminogen
-antigenic
-when used therapeutically it can induce an immune response
Fibrin Degradation Products
plasmin after cleaving fibrin and fibrinogen produces
X, Y, D, E, &
D-Dimer:
Specific product of digestion of crosslinked fibrin only (marker of thrombosis and fibrinolysis important when trying to diagnose DIC)
Thrombin cleaves A & B fibrinopeptides
plasmin cleaves E & D fragments from fibrinogen
-fragments can inhibit hemostasis and contribute to bleeding by preventing PLT activation
Plasminogen Activator Inhibitor-1 (PAI-1)
Fibrinolysis Inhibitors
Principal inhibitor of plasminogen activation
Inactivates TPA & UPA
Prevents conversion of plasminogen to active plasmin
SERPIN
-produced by endothelial cells, monocytes, smooth muscle cells,
-platelets have a pool of PAI1
Plasmin Inhibitors -Irreversibly bind to free plasmin in circulation
2-anti-plasmin ** neutralize free plasmin
2-macroglobulin (secondary)
PLASMIN INHIBITORS:
1-antitrypsin
AT - especially in the presence of heparin
C 1 esterase inhibitor
all serpins
ALL bind and neutralize free plasmin»_space; Inhibits fibrinolysis
Thrombin
-made as a result of coag system activation
ACTS AS BOTH:
initiator of fibrinolysis
induces tPA release **
inhibitor of fibrinolysis
stimulates endothelial cell release of PAI-1
Thrombin Activatable Fibrinolysis Inhibitor (TAFI)
Synthesized in the liver
Activated by Thrombin-Thrombomodulin complex (same complex that activates Protein C pathway)
antifibrinolytic enzyme
Inhibits fibrinolysis:
Prevents the binding of TPA & plasminogen to fibrin
Blocks the formation of plasmin
Results is suppression of fibrinolysis
DIC
excessive clotting due to excess of thrombin present in the plasma and a weak plasmin response
-when normal mechanisms are overwhelmed
-results in overconsumption of coagulation factors and platelets and results in bleeding and shock
Triggering Mechanism of DIC
1.Activation of the extrinsic coagulation pathway by the release of TF
2.Direct activation of Factors X or II (prothrombin)
3.Activation of the intrinsic pathway
Activation of the extrinsic coagulation pathway by the release of TF
causes what
triggers DIC
primary initiator of DIC
Trauma / surgery
Obstetrical complications
Tumor / leukemia
Bacterial infection
Sepsis (activate Contact Factors (XII, HMWK)
Direct activation of Factors X or II
causes what
triggers DIC
Snake venom
Liver disease
Activation of Intrinsic System (uncommon)
triggers DIC
Liver disease
Heat stroke
Sepsis (activates both extrinsic and intrinsic)
Burns
Immune complex disease
What is Primary Fibrinolysis
presentation is similar to DIC
plasmin is formed in the absence of clot formation (unlike DIC the uncontrolled clotting doesnt occur)
Triggered by
enzymes in plasma capable of activating plasminogen
urological procedures
metastatic prostatic carcinoma
hepatic disorders
DIC coag report will show
prolonged PT and APTT
low fibrinogen
PLT and PBF- confirm thrombocytopenia and schitocytes
-high d dimer rules out DIC
What is the D dimer test
Detects active fibrinolysis - This indirectly implies the thrombosis
-Quantitative or semi-quantitative immunoassay
-uses monoclonal antibodies against D- dimers on microlatex particles
-specific marker for thrombosis – only formed if fibrin is broken down by plasmin
Lab tests for DIC
1). Protein C, Protein S, and Antithrombin (AT) assays – Decreased monitors plasma
2). Chromogenic plasminogen assay/ TPA assay/ PAI-1 assay – Decreased good for analyzing systemic fibrinolysis
serum FDP is replaced by quantitative D dimer
What will you see in primary fibrinolysis
increased
FDP, D DIMER AND clotting time
Low or Absent
Fibrinogen and Shisto
normal
PLT count and ATIII
Because overconsumption of coagulation factors doesn’t occur for this disease, Protein C, Protein S, and Antithrombin (AT) assays may be useful in diagnosis
