Hemostasis

Question 1

Describe the role of platelets in the hemostatic process and the relationship between platelet structure and function.

Answer 1

Platelets = responsible for formation of primary hemostatic plug
o Plug hole at site of vascular injury

Structure:
Small, anucleate cell fragments
• Appear as granular bluish cell fragments
Derived from bone marrow megakaryocytes
• Production mediated by cytokine Thrombopoietin (TPO)
• Survive 7-10 days in circulation
• Removed by spleen
• Note: about 30% platelets are sequestered by spleen
• Released in response to epinephrine
• Large circulating platelets in thrombocytopenic patient = peripheral destruction/consumption of platelets
Normal value: 150-450 k/ul blood

Activated by interacting with subendothelial matrix and/or soluble agonists
o Shape change
o Conformational activation of GpIIb/IIIa complex
o Granule release
o End result = thrombus contraction

Question 2

Explain what endothelial cells do so platelets do not adhere to blood vessels under normal circumstances.

Answer 2

Normal circumstances = healthy endothelial cells have multiple mechanisms for inhibiting coagulation:
1) Barrier function = sequesters active Tissue factor
2) Expresses:
Prostacycin (PGI2)
• Made via arachidonic acid pathway
• Opposes platelets thromboxane A2 activity
• Causes vasodilation and platelet inhibition
Endothelial nitric oxide synthase (eNOS)
• Generates NO → vasodilation
ectoADPase (CD39)
• Degrades ADP and ATP
• Inhibits platelet activation and recruitment
Heparin sulfate
• Binds AT3 → accelerates protease inhibition
Thrombomodulin
• At low thrombin levels = enhances activated protein C formation

Question 3

Platelet plug formation: adhesion

Answer 3

• Folded vWF factor binds to exposed ECM
• Blood flow → high shear → unfolding of vWF
• Exposes binding sites for platelet GpIb
• Platelet binds to vWF

Question 4

Platelet plug formation: activation

Answer 4

From variety of agonists:
• Thrombin from tissue factor exposure at injury site
• Collagen from ECM
• Thromboxane A2 from platelet arachidonic acid (via COX-1 pathway)
• Others: epinephrine, ADP, serotonin

Stimulates:
• Platelets shape change and spreading
• Conformational activation of GpIIb/IIIa
• Binding sites for coagulation proteins = negative charges (acidic phospholipids) flipped to outside = nidus for soluble coagulation factors
o Vitamin K dependent factors → gamma-carboxy glutamic acid (high density of negative charge)

Platelet procoagulant activity
• Activated platelets provide a procoagulant surface
• Exposes phosphatidylserine (PS) on outer membrane
• Accelerates local thrombin generation

Question 5

Platelet plug formation: aggregation

Answer 5

• GpIIb/IIIa conformational change allows receptor to bind fibrinogen
• Result = cross-linking of activated platelets

Question 6

Platelet plug formation: secretion

Answer 6

Platelet α-granules and dense granules → substances recruit/activate additional platelets
1) α-granules:
o Adhesive proteins (fibrinogen, vWF)
o Factor V = enhances coagulation
2) Dense granules:
o ADP/ATP, serotonin = activate more platelets
3) Growth factor and chemokines = recruit inflammatory cells and initiate wound healing

Locally amplifies coagulation, vasoconstriction, and wound repair

Question 7

Describe the production and functions of von Willebrand factor (vWF)

Answer 7

Production:
• Synthesized and stored in endothelial cells, and megakaryocytes
• Also found in subendothelial CT
• vWF in circulation = normally folded so doesn’t bind platelets

Functions:
Major carrier protein for Factor VIII
• Prolongs ½ life of Factor VIII
• If decreased vWF = decreased VIII
Required for platelet tethering and adhesion to exposed subendothelial matrix under high shear flow conditions
• Binds surface → flow causes it to unfold
• Exposes its GpIb binding sites

Question 8

Describe the role of desmopressin

Answer 8

o Desmopressin acetate (DDAVP) stimulates release of vWF

o Use desmopressin to treat von Willebrand disease

Question 9

Describe the function of ADAMTS13

Answer 9

o vWF secreted in a range of multimer sizes
o Ultra-large molecule = very sticky and unfold spontaneously
o The protease ADAMTS13 cleaves these extra-large factors= Down-regulates vWF activity

If inherited or acquired ADAMTS13 defect → Thrombotic Thrombocytopenic purpura (TTP)
• Formation of large vWF-platelet aggregates
• Deposit in microvasculature → tissue injury

Overall: size matters for vWF function

Question 10

Describe the Initiation Phase of Hemostasis

Answer 10

Threshold-mediated

Vascular injury → Tissue factor (TP) is exposed → binds VIIa

TP-VIIa complex activates VII to VIIa
• Also activates small amounts of IX and X
• Inhibited by tissue factor pathway inhibitor

Xa converts prothrombin to thrombin

Thrombin activates Cofactors, V, VIII, and XI
• Cofactor activation important for forming membrane-bound complexes
• Xia activates more IX

Question 11

Describe the Propagation Phase of Hemostasis

Answer 11

Exponential burst of thrombin → fibrin clot formation

Cofactor (VIII and V) activation forms membrane bound complexes:
IXa/VIIIa
• Required for explosive generation of thrombin
Xa/Va

Thrombin:
1) Cleaves fibrinogen to fibrin → Polymerizes into insoluble thrombus
2) Activates XIII → XIIIa
• XIIIa crosslinks alpha and gamma chains of fibrin
• Stabilizes fibrin clot
3) Binds thrombomodulin = accelerates Protein C activation
4) Activates surface receptors on other cell types
• Includes: endothelium, monocytes, smooth muscle cells, fibroblasts
• Modulates migration of inflammatory cells, wound healing, other processes

Question 12

Describe the role of vitamin K in coagulation.

Answer 12

Vit K dependent: Protein C, Factor VIIa, Factor IXa, factor Xa, and Prothrombin (factor II)

Proteins have 2 ends:
1) Gamma-carboxylation end
• Synthesized as zymogens by liver
• From post-translational processing (Vit K dependent reaction)
• Mediates Ca2+ dependent binding to membrane surface = mediated proper conformation for enzymatic activity to occur (Ca2+ acts as bridge)
2) Serine protease end
• Enzymatic activity

Warfarin = inhibits Vit K dependent gamma-carboxylation

Question 13

Describe the co-factors involved in the clotting system

Answer 13

Co-Factors also involved = holds complex (protease and substrate) together to work even more efficiently
o Helps localize pro- and anti-coagulant activity

Include:
Protein S
• Vit K dependent
• Cofactor for activated protein C
Tissue factor
• Integral membrane protein normally expressed by extravascular cells
• Abundant on adventitial cells
• Exposure triggers clotting
• Binds VIIa to form a complex that activates X
Factor Va
• Requires proteolytic activation from thrombin
• Stable cofactor
• Binds Xa to form a complex that activates prothrombin
Factor VIIIa:
• Require proteolytic activation from thrombin
• Unstable (degrades over time)
• Binds factor IXa to form a complex that activates factor X

Question 14

Describe Thrombomodulin (TM)

Answer 14

On endothelial surface

Binds thrombin at low concentrations → changes substrate specificity of thrombin
• Goes from pro- to anti-coagulant
• Accelerates rate of Protein C activation

Question 15

Describe the coagulation inhibitors

Answer 15

Tissue factor pathway inhibitor (TFPI)
•	Complexes with Xa → TFPI-Xa
•	Thus = amount of Xa available is limited for thrombin generation 
•	Complex inhibits TF-VIIa complex
•	Regulates initiation phase 

Antithrombin III (AT3)
•	Protease inhibitor of IXa, Xa, and thrombin
Action accelerated by:
•	Heparin (drug)
•	Heparan sulfate (on cell surface)

Activated Protein C (APC)
• Vit K dependent zymogen
• Uses Protein C as a cofactor (Protein C = activated by thrombin bound to thrombomodulin)
• Inactivates Va and VIIIa via proteolysis → terminates propagation phase

Factor V Leiden = Protein C-resistant form of Va (from mutated APC cleavage site) → increased risk of thrombosis

Question 16

List the enzymes involved in fibrinolysis; describe how fibrinolysis is regulated, and how the activation of plasminogen is localized to the fibrin clot.

Answer 16

• Fibrinolytic pathway = serine protease cascade
o Creates plasmin = breaks fibrin clots
o Regulated by protease inhibitors, localization to fibrin, and endothelial surface receptors

Components:
1) Plasminogen/plasmin:
• Plasminogen secreted by liver
• Converted to plasmin by plasminogen activators
• Digests cross-linked fibrin → degradation product D-dimer
• Can use D-dimer to diagnosis DIC and venous thromboembolism
2) Plasminogen activators = secreted by endothelial cells
Tissue plasminogen activator (tPA)
• Binds fibrin before activating plasminogen
Urokinase
• Low affinity for fibrin so activates plasminogen with or without fibrin
Note: cellular receptors present for both plasminogen and plasminogen activators
• Help maintain pro-fibrinolytic endothelial surface
• Removes fibrin in places where not needed

Fibrinolysis inhibitors:
α2-antiplasmin (α2-PI)
• Made in liver
• Inhibits circulating plasmin by forming 1:1 complex
• Less efficient at inhibiting plasmin bound to fibrin clot
• Result = keeps plasmin activity localized to clot

Plasminogen activator inhibitor-1 (PAI-1)
• Released by endothelial cells, monocytes/macrophages, hepatocytes, adipocytes, platelets
• Stabilizes clots by delaying onset of fibrinolysis

Question 17

List some laboratory tests used to evaluate the hemostatic and fibrinolytic systems and describe how each test is used clinically to evaluate bleeding disorders.

Answer 17

PT and aPTT (below)

Mixing tests
o If prolonged clotting time
o Repeat test with 50/50 mix of patient plasma and normal plasma
o If clotting time restored → due to single coagulation factor deficiency
o If not corrected → due to coagulation inhibitor

Specific factor activity
o Identifies missing/deficient factor
o Mix patient plasma with plasma deficient in specific clotting factors
o Able to quantify degree of deficiency by measuring clotting times in serial dilutions

Platelet function testing
Bleeding time
• Does not predict surgical bleeding
PFA-100 (platelet function analyzer-100) screen
• Whole blood passed though small glass tube
• Measure time until occlusion (closure time)
• Does not predict surgical bleeding
• Abnormally long closure times could be from:
1) Abnormal platelet function (inherited or acquired)
2) Low platelet count
3) Low plasma vWF activity
Platelet aggregation testing
• Assess platelet response to specific platelet agonists
• Used in suspected patients (not as a screening test)

vWF evaluation
vWF level
Factor VIII activity (normally parallels vWF level)
vWF activity
• Ristocetin cofactor activity = antibiotic that induces conformational change in vWF
• Triggers binding to platelet GpIb → agglutination
Multimer analysis
• Evaluate size distribution of vWF multimers

Fibrinolysis
FDP (fibrin or fibrinogen degradation products)
• Now use more specific D-dimer test
D-dimer
• Specific product from plasmin digestion of cross-linked fibrin
• Demonstrates both thrombin and plasmin activity
Fibrinogen level
• May be low due to congenital deficiency (rare) or liver disease or DIC
α2-Antiplasmin level
• Rare = low due to genetic deficiency
• More common = liver disease, inhibitor consumption associated with circulating plasmin activity

Other tests:
Thrombin Time (TT)
• Add thrombin to plasma = measure clotting time
• Sensitive to low fibrinogen and dysfunctional fibrinogen; also to heparin
Factor XIII activity
• Screening test = urea clot solubility
• Non-cross-linked clot rapidly dissolves in 5 M of urea
• Screens for congenital factor XIII deficiency (rare disorder)
• Confirmed by more specific assays

Question 18

Be able to generate a differential diagnosis for abnormal results o the prothrombin time/INR and the activated partial thromboplastin time (aPTT).

Answer 18

Prothrombin time (PT)
o Measures extrinsic pathway
o Expressed in international normalized ration units (INR)
o Sensitive to factors VII, X, V, prothrombin, and fibrinogen
o Selective Prolongation = factor VII deficiency

Activated partial thromboplastin time (aPTT)
o Measures intrinsic pathway
o Sensitive to prekallikrein, high molecular weight kiningren, XII, factors XI, IX, VIII, X, V, prothrombin, and fibrinogen
o Prolongation but NOT cause bleeding: prekallikrein, high molecular weight kiningren, XII deficiencies
o Selective Prolongation = contact factors, XI, IX, VIII deficiency