11 07 2014 Hemostasis Flashcards
What is the primary initiating event of hemostasis?
hemostasis: bleeding and clotting
Damage to a blood vessel/ endothelium injury
- blood exposed to tissue factor, von willebrand factor, collagen
What is a platelet and where does it come from
produced in bone marrow– bleb off of megakaryocytic cytoplasm. They contain granules full of important chemicals.
Life span is 7-10 days
Primary Hemostasis
Formation of platelet plug
- Activation:
When blood vessel is damaged, sub endothelial cell matrix is exposed ( collagen* and vWF and Tissue Factor)
vWF is released from damaged endothelium and binds to exposed collagen
- Adhesion
Platelets attach to vWF via GPIb receptor - Aggregation
Binding of platelets to vWF activates platelet cell surface receptors : GPIIb/IIIa
- binds fibrinogen
= 3D platelet-fibrinogen mesh = Platelet plug
Changes shape of platelet (pseudopods and platelet granulation)
- Secretion:
Degranulation – release of granules (alpha granules and dense bodies- increase aggregation and platelet activation) activates more platelets = bind to fibrinogen and make a thicker platelet plug
What are the receptors on platelets crucial for hemostasis?
GPIIb/ GPIIIa
GPIb
Absence of above = Glanzmann’s thrombasthenia, bernard Soulier syndrome
Facter V and III receptors ( and maybe VIIa receptor)
receptors for neutrophils, monocytes
- components of dead neutrophils can activate platelets and other clotting events
Von Willebrand Factor (vWF)
- describe protein
- who processes it and why?
- Where is it usually found?
- What are the two important functions of vWF?
protein - multimer
Rapidly processed by ADAMTS13 metalloprotease
- if not processed = bind and activate platelets in an unregulated manner
Found in alpha granules of platelets OR in vascular endothelium cells
- facilitates platelet adhesion to the vessel wall by linking platelet membrane receptors to the subendothelial matrix
“ the glue” - plasma carrier for factor VIII
- extends half-life of Factor VIII from 8-15min to 13 hours
Details about the process of Secretion in primary hemostasis. Explain why aspirin/ NSAIDs interfere with platelet function
Granule secretion is a phospholipid dependent event
Exposure of the platelet to collagen initiates a series of events in which arachidonic acid (lipid bilayer of the platelet) is converted to thromboxane A-2 by cyclo-oxygenase 1 (Cox-1)
Presence of thromboxane A-2 = secretion
- it is also a potent vasoconstrictor (enhancing blood stasis
Aspirin blocks COX-1
What exactly do dense bodies do and what is the name of the syndrome when they are not present?
Dense bodies are stored in the granules of platelets. They increase platelet activation
Syndrome: Hermansky-Pudlak syndrome
Severe Thrombocytopenia
- causes
- signs/symptoms
decreased platelet numbers.
- not being made
- being destroyed
- sequestered – Check spleen
Bleeding in mouth and gums
Bruising
nosebleeds
petechia– pinpoint red dots
Von Willebrand disease
Autosomal Dominant
deficiency of VWF = can’t stop a bleed after an injury quick enough.
Recall that vWF carries factor 8!! So deficiency in VWF = have an effect on factor 8 (may be less because of short half life when vwf is not around)
What causes a prolonged bleeding time
- vascular fragility
- Collagen abnormality (Marfan syndrome)
- Low platelet count
- low or abnormal vWF
- Abnl platelet function
PT test
Prothrombin time
Evaluates the extrinsic coagulation system down to the formation of the fibrin clot
* VII, X, V, II, I (separate test)
Normal reference is 11 to 15 seconds
PTT test
Partial Thromboplastin time
Evaluates the intrinsic coagulation system down to formation of a fibrin clot
* VII, XI, IX, VII, X, V, II, I
Normal reference is 25- 40 secs
What would you suspect if PT is prolonged?
Fact VII deficiency
- life threatening bleeds
What would you think of if a patient has a prolonged PTT?
deficiency: 8, 9, 11, 12
Factor 12 deficiency has no bleeding problems
PT and PTT are both prolonged
Factor II deficiency, V, 10
Platelet Function Analyzer –PFA- 100 test
measures time for platelets under shear stress to clot off aperture
- glass tube coated with collagen and epinephrin or ADP (platelet activators)
specific for vWD and platelet dysfunction but NOT sensitive to common late let defects
- this is not a screening test, it is a diagnostic test
PFA- 100 test
measures time for platelets under shear stress to clot off aperture
specific for vWD, not sensitive to common late let defects
- this is not a screening test, it is a diagnostic test
Platelet flow cytometry
Platelets are labelled with antibodies directed against surface membrane glycoproteins
CD41— GpIIB
CD61 – GPIIIa
CD42b — GPIb : inhibits ristocetin – depndent binding of vWF
Platelet flow cytometry
Platelets are labelled with antibodies directed against surface membrane glycoproteins
CD41— GpIIB
CD61 – GPIIIa
CD42b — GPIb : inhibits ristocetin – depndent binding of vWF
Ristocetin
antibiotic that causes clotting
— the platelets clot in presence of von Willebrand factor when exposed to this antibiotic
Aspirin
irreversibly acetylates and inhibits platelet COX-1
Clopidogrel
blocks ADP activation of GPIIb/ GPIIIa receptor complex
- block binding to fibrinogen = block the 3D platelet plug
ReoPro or Abciximab
Anti- integrin monoclonal antibody that binds GP IIb/ IIIa
- block binding to fibrinogen = block the 3D platelet plug
ReoPro
Anti- integrin monoclonal antibody that binds GP IIb/ IIIa
- block binding to fibrinogen = block the 3D platelet plug
Is the platelet plug strong? Will it stay put?
no, one can brush it away very easily and therefore keep bleeding. We need to do something else to strengthen the platelet plug
Platelet Count
normal : 150,000 to 400,000 cells/mm^3
Bleeding time test
Evaluates platelet function up to the formation of temporary platelet thrombus
Normal interval is 2-7min
Steps of the intrinsic pathway of secondary hemostasis
Factor 7 is activated
Activates 11
Activates 9 (with the help of factor 8)
Activates 10 –> common pathway
Steps of the intrinsic pathway of secondary hemostasis
Factor 7 is activated
Activates 11
Activates 9 (with the help of factor 8)
Activates 10
Steps of the extrinsic pathway of secondary hemostasis
Factor 7 is activated via tissue factors
Activates factor 10 —> common pathway
What is the new Cell based model of hemostasis/ “physiological pathway”
- Tissue injury = TF is exposed (non enzymatic lipoprotein). TF binds Factor 7 = active complex
- TF- 7a complex catalyzes the activation of X –> Xa and 9 –> 9a
- 10a in the presence of factor 5a activates prothrombin (II) to thrombin
- Thrombin activates 5, 8, 9, 10 —> further platelet aggregation
- 9a in the presence of 8a activates 10 –> 10a (on platelet surface)
- 10a in the presence of 5a catalyzes prothrombin to thrombin
- 7a bound to platelet surface acti gates 10
- 11 activated by thrombin. Then 11a activates factor 9
- Thrombin IIa catalyzes the conversion of fibrinogen to fibrin
What is the new Cell based model of hemostasis/ “physiological pathway”
- Tissue injury = TF is exposed (non enzymatic lipoprotein). TF binds Factor 7 = active complex
- TF- 7a complex catalyzes the activation of X –> Xa and 9 –> 9a
- 10a in the presence of factor 5a activates prothrombin (II) to thrombin
- Thrombin activates 5, 8, 9, 10 —> further platelet aggregation
- 9a in the presence of 8a activates 10 –> 10a (on platelet surface)
- 10a in the presence of 5a catalyzes prothrombin to thrombin
- 7a bound to platelet surface acti gates 10
- 11 activated by thrombin. Then 11a activates factor 9
Factors V and VIII
co-factors with no enzymatic activity of their own.
Va —> activates 10 —> Prothrombin
VIIIa –> activates 9 –> activates 10
People missing factor V or VIII make clots, but not as fast.
- similar clinical appearance between factor 8 and 9 deficiencies
Who are the factors that require vitamin K?
Factors 2, 7, 9, and 10
- they are gamma - carboxylated proteins that require vitamin K to become carboxylated
- carboxylation takes place in LIVER
Can’t give liver patient with low thrombotic factors vit. K
- bad liver failure all thrombotic factors EXCEPT 8 will be low. – made by endothelial cells too along with vWF
and Protein C and S - anti-coagulation
Hemorrhagic Disease of the newborn
Give infants vitamin K shots to prevent hemorrhaging
Factor 13
fibrin stabilizing factor; activated by thrombin to covalently cross-link the fibrin polymers
- provides clot strength
- Without factor 13, clots dissolve more rapidly
Comes in after fibrin is made therefore it is not picked up by PT or PTT test.
- D-Dimer!!!
Factor 13
fibrin stabilizing factor; activated by thrombin to covalently cross-link the fibrin polymers
- provides clot strength
- Without factor 13, clots dissolve more rapidly
Comes in after fibrin is made therefore it is not picked up by PT or PTT test.
Thrombin time
clinical test that measure the amount of time it takes to convert fibrinogen to fibrin
low levels of fibrinogen or dysfibrinogenemia will show prolonged thrombin times.
- usually have normal PT or PTT
Heparan Sulfate and Thromodulin
normal endothelial cells express heparan sulfate and Thrombomodulin on their surface.
Both play an active role in turning off thrombin
When endothelium is damaged, that localized area now lost protective mechanism that prevented a blood clot
How does heparan sulfate work?
binds to Antithrombin (AT) protein synthesize by liver AND endothelial cells.
Makes AT-Heparan complex – directly turns off thrombin and inactivates 10a
How does heparan sulfate work?
binds to Antithrombin (AT) protein synthesize by liver AND endothelial cells. It helps turn off thrombin
AT deficiency
rare, autosomal dominant thrombotic disorder
Do not respond well to heparin (medication that mimics Heparan)
Homozygous AT deficiency is lethal in utero
How does thrombomodulin work
Thrombomodulin- thrombin complex!
Activates Protein C.
Protein C and S function?
Protein C turns of 5a and 8a by cleavage at specific sites
Protein S co-enzyme of Protein C and is necessary for full activation of Protein C.
Protein C and S deficiencies?
- type of inheritance
Autosomal dominant thrombotic disorders
Factor V Leiden (FVL)
point mutation on gene for factor V = conformational change in the binding site for PC.
Va is resistant to cleavage
-Activated protein C resistance
Factor V Leiden (FVL)
point mutation on gene for factor V = conformational change in the binding site for PC.
Va is resistant to clotting
Fibrinolysis (in general)
process of breaking down the clot
- measure fibrin split products (FSP)
High levels of FSP =increased fibrinolysis
If fibrinogen or fibrin and FSP are low = liver failure
Fibrinolysis (in general)
process of breaking down the clot
Tissue plasminogen activator (tPA) and urokinase (to a lesser degree) roles?
both are serene protease which convert plasminogen to plasmin. Plasmin then breaks down the clots (recall that plasmin is incorporated into the clot via attachment to fibrinogen and fibrin)
= fibrin split products (FSP) are debris from this process
used to open occluded blood vessels
- Pulmonary Embolism
- Stroke
- heart attack
Tissue plasminogen activator (tPA) and urokinase (to a lesser degree) roles?
both are serene protease which convert plasminogen to plasmin
D-dimer
specific FSP – covalent cross linking is resistant to fibrinolysis
D-dimer are specific to fibrinolysis of an actual clot.
Plasminogen activator inhibitor-1 (PAI-1)
down regulation of tPA.
Deficiency = bleeding disorder since it leaves tPA unopposed.
alpha2 antiplasmin
circulating plasma protein that scavenges up free circulation plasmin. Deficiency = bleeding disorder since plasmin wandering around the body would actively break down every clot in the body.
- alpha 2 k.o in mice does not bleed but is resistant to injury induced pulmonary embolism
Anti-thrombin
made in liver and endothelium
-most important inhibitor of thrombin and 10s
Directly binds and blocks thrombin, 9a, 10a, and 11a
Action is enhanced by heparan sulfate
Function of heparin?
keeps clot from growing but it doesn’t break it down.
blood thinner
Dysfibrinogenemia
A variety of disorders resulting from mutations which may render a clot:
- resistant to fibrinolysis
- most sensitive to lysis
- resistant to cross linking
some result in both bleeding and clotting disorder
What are some inherited thrombophilia (endothelial cell)
- endothelial protein C recpetor mutations
- Kruppel-like factor KLF2 polymorephisms
- Endothelial apoptosis
- Variable expression of ADAMTS-13
