Hemostasis Flashcards
Plasma vs. serum
Plasma - blood from which the cellular componenets have been removed
Serum - Plasma from which the clotting factors have been removed
Fibrinogen
Soluble protein converted to insoluble fibrin by thrombin
Vitamin K-dependent factors
Require vitamin K for the process of gamma-carboxylation that changes some Glu resides to Gla (γ-carboxy glutamate) with an extra negative charge necessary for the binding of Ca+2 and to phospholipid membrane surfaces
Nitric oxide
Inhibits the activation of platelets
NO stimulates the production of cGMP dependent protein kinase (G-kinase), which inhibits signaling and Ca²⁺ mobilization, preventing platelet activation.
Secreted by endothelial cells
Also a potent vasodilator
Prostacyclin (PGI2)
Prostaglandin member of the eicosanoid family of lipid molecules
Inhibits platelet activation similarly to NO
Secreted by endothelial cells
Also a potent vasodilator
Heparan sulfate
Glycosaminoglycan surface receptor on endothelial cells
Negatively charged and binds to anti-thrombin III (AT3)
Heparin is a stronger negatively charged glycosaminoglycan
Anti-thrombin III (AT3)
Degrades clotting factors II (aka thrombin), IX, and X
Binds to heparan sulfate and heparin
Heparin
Negatively charged, sulfated glycosaminoglycan, free-flowing in blood, produced by mast cells and basophils.
Binds to AT3 (like heparan), causing a conformational change, activating AT3 and inhibiting thrombin (factor II), factor X, and other factors.
Where is heparan sulfate located?
Cellular level
It is a glycosaminoglycan surface receptor on endothelial cells
Thrombomodulin
Endothelial membrane surface protein binds to thrombin (factor II), preventing thrombin from converting soluble fibrinogen to insoluble fibrin (part of the platelet plug).
While bound to thrombomodulin, thrombin activates protein C, which, along with protein S, degrades cofactors V and VII.
Steps of hemostasis
- Vascular spasm
- Platelet plug formation
- Coagulation
- Clot retraction and repair
- Fibrinolysis (clot busting)
Vascular spasm
- Endothelin release - endothelial cells secrete endothelin (21-AA peptide) which binds to receptors (various cells, including smooth muscle) leading to Ca2+ mobilization and muscle contraction causing vasoconstriction
- Myogenic mechanism - direct injury to smooth muscle cells triggers contraction by the myocyte itself, not external stimuli, helping maintain constant renal blood flow in response to BP changes
- Pro-inflammatory signals (i.e. histamine) are released subsequent to vessel/tissue damage along with endothelin - initially engage the nociceptor activation that leads to pain reflex and smooth muscle contraction
Proinflammatory signals also tends to lead to vasodilation over time
Platelet plug formation
Upon injury to vessel walls - platelets aggregate and adhere, forming a plug to prevent blood loss (primary hemostasis)
Von Willebrand factor and platelet plug formation.
vWF release - endothelial cells release vWF - large multimeric glycoprotein
vWF binds to factor VIII - prolonging its life
vWF naturally binds to exposed collagen - platelets passing by bind to vWF via their glycoprotein 1b receptors (Gp1b)
Once platelets become activated, what three signals do they release?
ADP - attracts more platelets
Thromboxane A2 (TxA2) - assists in platelet activation/aggregation, vasoconstriction, and degranulation
Serotonin - vasoconstrictor
Secondary hemostasis
The extrinsic and intrinsic pathways of the coagulation cascade
Both ex/in paths lead to fibrin formation - they converge
Intrinsic pathway is aka the contact activation pathway
Extrinsic pathway is aka the tissue factor pathway
What is the primary pathway for the initiation of coagulation?
The extrinsic pathway (tissue factor pathway)
What causes blood to coagulate in vitro?
The intrinsic pathway
Intrinsic pathway
Serine proteases become activated beginning w/ factor XII
Factor XII interacts w/ negative charges on platelets and becomes activated (factor XIIa)
Factor XIIa activated XI to XIa
XIa activates IX to IXa
With the help of cofactor VIII and Ca2+, IXa activates X to Xa of the common pathway
13-11-9(8)-10 (factor steps)
Takes 4-6 min. for Fx
Exrinsic pathway
Initiated by cell damage
Endothelial cells secrete tissue factor (aka factor III) when damaged
factor III acts as a cofactor for factor VII to VIIa - which then activates factor X of the common pathway
Quicker than intrinsic (~30s)
Common pathway
Factor Xa and V (and platelet factor) activate factor II (prothrombin) to factor IIa (thrombin)
Thrombin is a serine protease that polymerizes soluble fibrinogen molecules together to form insoluble fibrin
Thrombin also activates XIII to XIIIa - which crosslinks fibrin to create the fibrin mesh that holds the platelet plug down preventing it from getting loose and causing a distant thrombotic event
Clot retraction and repair
While in the plug formation platelets contract to close the injury and secrete PDGF to trigger mitosis in damaged smooth muscle cells, regenerating collagen fibers. They also produce VEGF to help regenerate the endothelial lining.
Fibrinolysis
Endothelial receptor tissue plasminogen activator (t-PA) - converts plasminogen in the plasma into plasmin
Plasmin - digest fibrin mesh into degradation products (D-dimer)
What regulator mechanisms keep platelet activation and coagulation cascade in check?
- Protein C
- Anti-thrombin
- Tissue factor pathway inhibitor
- Plasmin
- Prostacyclin
Protein C
Anticoagulant
Vitamin K-dependent serine protease - becomes activated by thrombin when bound to thrombomodulin on endothelium
Activated protein C (along with protein S) degrades cofactors V and VII
Deficiencies in proteins C or S may lead to thrombophilia (tendency to develop thrombosis)
Anti-thrombin
Serine protease inhibitor (serpin) that degrades serine proteases: thrombin, along with factors IXa, Xa, XIa, and XIIa
Tissue Factor Pathway Inhibitor (TFPI)
Limits the action of tusse factor (factor III) - in turn inhibits VIIa and Xa
Plasmin
Generated by proteolytic cleavage of plasminogen (plasma protein produced by liver) - catalyzed by tissue plasminogen activator (t-PA), which is produced by endothelium
Plasmin proteolytically cleaves fibrin into fibrin degradation products (including D-Dimers)
Prostacyclin
Is released by endothelium and it keeps platelets in an inactive state by preventing the Ca2+ mobilization w/i platelets
Prothrombin time (PT)
Measures extrinsic pathway
Primarily inhibited by warfarin
Warfarin blocks the gamma-carboxylation of the certain Glu’s
PT uses plasma rather than whole blood - does not measure platelet Fx
Partial thromboplastin time (PTT)
Measures intrinsic pathway
PT uses plasma rather than whole blood - does not measure platelet Fx
What activates antithrombin 3 and slows aPTT?
Heparin (primarily) and heparan
Blood clotting made simple…
Xarelto
Inhibits factor Xa
So does Eliquis
Pradaxa
Inhibits IIa
Endothelial cell contributions to thrombosis
Tissue factor (III) to initiate the extrinsic pathway
vWF that binds platelets to collagen at the injury site
vWF binds factor VII and protects VIII
Endothelial cell contributions to hemostasis
Surface heparan and plasma heparin (from mast cells) molecules activate anti-thrombin III (AT3) to prevent clotting
Thrombomodulin/throbmin complex activates protein C, which along w/ protein S (cofactor) degrades factors Va and VIIIa to slow clotting
Adenosine diphosphatase cleaves ADP; reducing platelet adhesion
Production of NO
Production of prostacyclin (PGI2); inhibits platelet aggregation
Vit K role in clotting
Vit K is needed for the post-translational modification of some of the clotting factors in the liver
Helps carboxylate 10-12 glu AA’s of factors II, VII, IX, and X to yield γ-carboxyglutamic acid (Gla)
The extra negative charges of Gla bind Ca2+ so the coagulation factors can stick to the phosphatidylserine on the sruface of activated platelets
Phosphatidylserine is normally in the cytoplasmic side of lipid bilayers, but it appears on the outside of activated platelets
Warfarin
Vit K antagonist - inhibits vit K epoxide reductase (VKOR) - the enzyme responsible for reduction of vit K to its active form
Without reduced vit K - Gla residues cannot be formed from the Glu residues of factors II, VII, IX, and X such that they are less negatively charged and less likely to form the Ca2+ bridge w/ phosphatidylserine of activated platelets
Von Willebrand Disease
Most common bleeding disorder (~1% of pop.)
If vWF is absent/reduced - 50% cases - prolonged aPTT due to factor VII not being protected - defect in intrinsic pathway
Hemophilia
A - factor 8 def
B - factor 9 def
Both A/B deficits in intrinsic path
Healthy endothelial cell
Endothelial injury
Unactivated platelets
Dense bodies - procoagulants thromboxane A2 (TXA2), ADP, and calcium
TXA2 and ADP bind to the TXA2 receptor (TXA2-R) and the ADP receptor, specifically P2Y12, respectively
The clotting cascade
How would defect in the common clotting pathway affect PT/aPTT
Elevation in both PT and PTT
In normal blood flow processes, endothelial cells secrete which of the following modulators that functions to keep vessels dilated?
A. Endothelin
B. Anti-thrombin III
C. Prostacyclin
D. Thrombomodulin
E. Thromboxane A2
C. Prostacyclin
A patient is started on warfarin for atrial fibrillation. Which of the following coagulation factors will be affected due to the inhibition of vitamin K?
Factors II, VII, IX, and X
In the extrinsic pathway of coagulation, which factor secreted by endothelial cells activates factor VII leading to the entry into the common pathway?
Tissue factor
Endothelial cells express the surface receptor thrombomodulin, which leads to the activation of which factor that degrades factors Va and VIIIa to slow clotting?
Protein C
A 42-year-old man is undergoing surgery and develops excessive bleeding. His prothrombin time (PT) is prolonged, but his aPTT is normal. Which pathway is most likely impaired?
Extrinsic
As part of the platelet plug formation, platelets bind to one another via which combination of receptor ligands?
Glycoprotein IIb/IIIa and fibrinogen
C. Thrombomodulin and thrombin
Heparan sulfate receptor is another modulator
B. GpIIb/IIIa (aka integrin)
Endothelial cell modulators
Hemophilia C
Deficiency of Factor XI
Autosomal recessive
