COAGULATION AND PLATELETS

NORMAL HEMOSTASIS ENDOTHELIUM

- tightly regulated events w/c allow blood to be - Acts like a “switch”
kept in fluid state yet permit formation of clot ff - The balance between the anticoagukant and
vascular injury procoagulant activities of endothelium often
- prevent bleeding determines whether clot formation, propagation
or dissolution occurs
Components: - Endothelial cells are activated by trauma,
- Vascular wall (endothelium) infectious agents, hemodynamic forces, plasma
- Platelets mediators and cytokines
- Coagulation cascade
- Fibrinolytic system ANTI-THROMBOTIC PROPERTIES
A. Antiplatelet effects
PLATELETS o Intact endothelium, prostacyclin, NO
B. Anti-coagulant effects
o Thrombomodulin, Tissue Factor
Pathway Inhibitor
C. Fibrinolytic effects
o tPA (tissue plasminogen activator)

ENDOGENOUS ANTICOAGULANTS:
1. Antithrombin III
o Inhibits the activity of thrombin and
other serine proteases
o IXa, Xa, XIa, XIIa
o Activated by binding to heparin-like
molecules on endothelial cells
2. Protein C and Protein S
o Vitamin k-dependent proteins that act
in a complex to inactivate factors Va
and VIIIa
3. Tissue Factor Pathway Inhibitor
o Produced by endothelium that
inactivates tissue-factor VIIa complexes

PROTHROMBOTIC PROPERTIES
A. Platelet effects
o Interaction w/ ECM (vWF) after injury
B. Procoagulant effects
1. Platelet adhesion
o Tissue factor
- Von willebrand factor C. Anti-fibrinolytic effect
o Deficiency: Von Willebrand disease
o Secrete PAI (plasminogen activator
- GpIb protein
inhibitor)
o Deficiency: Bernard-Soulier Syndrome
- GpIIb-IIIa complex COAGULATION CASCADE
o Deficiency: Glanzmann thrombasthenia rd
- 3 arm in homeostatic process
2. Change in Shape
- series of enzymatic conversions
3. Granule release (Secretion)
- each step proteotically cleaves an inactive
proenzyme into an active enzyme, culminating in
Alpha granules Dense granules thrombin formation
Fibrinogen ADP
Fibronectin Calcium
Factor 5, 8 Serotonin
PDGF Epinephrine
TGF- β

4. Recruitment
5. Aggregation (hemostatic) plug
- ADP: IIb/IIIa
- Fibrinogen: bridges platelets → Plt aggregation

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COAGULATION AND PLATELETS

Surface-bound zymogens
“Contact System”
Factor 12: auto-activates when associated w/ a negatively
charged surface such as a glass tube and several
physiologic substance

Vitamin K-dependent zymogens

- phospholipid bound
- allows binding of there proteins to phospholipids
and cell membranes (activated)

Cofactors/substrates
- Act as receptor for coagulation proteins
- Accelerate the reactions in w/c they participate
- Functions as a substrate of one or more enzymes
that participate in their formation and
inactivation

PRIMARY HEMOSTASIS (PLATELET PLUG FORMATION)

1. Adhesion: platelet to non platelet interaction
2. Platelet shape change and secretion
3. Aggregation: platelet to platelet interaction

SECONDARY HEMOSTASIS (FIBRIN CLOT FORMATION)

1. Formation of Thrombin
2. Formation of Stable Fibrin Clot

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INHIBITORS OF FIBRINOLYSIS
1. Plasminogen activator inhibitor 1 (PAI-1)
o Rapidly inhibits tPA
2. α-2 Antiplasmin (α-1-AP)
o inactivates plasmin by forming
inhibitory complex w/ circulating
plasmin
3. Thrombin-activatable fibrinolysis inhibiot (TAFI)
o Cleaves the C-terminal lysin residues of
fibrin, preventing the coactivation of
plasminogen by fibrin

INCREASED CLEARANCE – Ag-Ab complexes are formed in

the circulations, spontaneously activate the clotting
factors
- Consumed in the process = deficient

FIBRINOLYTIC SYSTEM
FIBRINOLYSIS – process by w/c cross-linked fibrin is broken
down in order to avoid excessive thrombosis

Proteins involved in fibrinolysis:

1. Plasmin- directly degrades fibrin
2. Plasminogen- inactive protein (zymogen)
EVALUATION OF HEMOSTASIS
o made in endothelial cells
TEST OF VASCULAR PLATELET PHASE:
o found in: tissues, urine, plasma,
- Bleeding time
lysosomal granules & vascular
TESTS OF COAGULATION CASCADE:
endothelium
- Clotting Time
3. Tissue plasminogen activator
- Activated Partial Thromboplastin Time (APTT)
o From endothelial cells
- Prothrombin Time (PT)
o Converts plasminogen to its active
- Thrombin Time (TT)
form: Plasmin
a. Single chain urokinase
BLEEDING TIME
b. 2-chain urokinase plasminogen activators
- Measures the time taken for the blood vessel
- both are normal plasminogen activators of the
constriction and platelet plug formation
body
- Detects defective platelet function
- secreted after inflammation
- Screening tests for acquired and congenital
- cleave plasminogen into plasmin
platelet defects
- assess vascular platelet function problems

2 types:
1. DUKE METHOD- ear lobe
2. IVY METHOD- forearm; preferred method
- pressure & incision can be fairly well
standardized
- allows multiple testing
- easier control of bleeding if It becomes excessive

**Do not perform if platelet counts <75,000

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COAGULATION AND PLATELETS

CLOTTING TIME: limitations

DECREASED CT INCREASED CT
*Rough handling of blood *extreme increases in
specimen temperature
*Traumatic Venipuncture *pH variation
*Frequent tilting of tube *performance of test at
*unclean tubes room temperature

CLOTTING TIME: Interpretation

Reference range: 5-15 mins
increased:
- circulating anticoagulants
- severe clotting factor deficiencies (<1-2%)
- a/hypofibrinogenemia
INTERPRETATION
Reference range: 1-7 mins (Ivy) **Rough measure of coagulation, limited use due to poor
1-6 mins (Duke) sensitivity
- If prolonged: nearly always indicates a clinical
abnormality APTT- Assess: intrinsic & common pathway
- Increased in states w/ abnormal platelet PPP- Reagents:
function: Surface activator Kaolin, micronized silica,
o Thrombasthenia celite, ellagic acid
o Von Willebrand’s disease Phospholipid Cephalin- to replace
o Storage pool disease platelet phospholipid
o Bernard Soulier syndrome Calcium Calcium is required in
o Prolonged fibrinolytic states molar excess for
coagulation to occur
BLEEDING TIME: sources of error
FALSE POSITIVE FALSE NEGATIVE
*BP cuff maintained too *BP cuff maintained too
high low
*Incision to deep *Incision too shallow
*Disturbing clot w/ filter
paper
*Fibrinogen <100mg/dl
*Platelet<100,000/mm3
*Drug ingestion affecting
platelet function

CLOTTING TIME: Principle

- “Lee-White Method”
- rough measure of all intrinsic factors
- wide variations
- limited test sensitivity – not used anymore
- whole blood, when exposed to foreign surface,
will form solid clot
PROCEDURE:

Use: Spectrophotometer

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COAGULATION AND PLATELETS

PROTHROMBIN TIME INTERPRETATION:

Assess: Extrinsic & common pathway - The thrombin time will be prolonged when
Principle: Clot formation functional fibrinogen levels are <1.0 g/L
PPP- Reagents:
Tissue Factor w/ *Tissue factor binds to DECREASED FIBRINOGEN
phospholipid FV!! And initiates A. Congenital deficiencies of fibrinogen
coagulation - Afibrinogenaemia or hypofibrinogenaemia
*Exogenous phospholipid - Dysfibrogenaemia (dysfunction fibrinogen)
is used to replace platelet o May be present in normal or reduced
phospholipid amounts
Calcium Req’d for recalcification o Ex: hypo-dysfibrinogenaemia
B. Acquired deficiencies
INTERPRETATION - DIC
- Following thrombolytic therapy
- Liver disease
- Malignancy

TEST FOR FIBRINOLYTIC PATHWAY

1. Fibrin Degradation Products (fibrin split
products) +
2. D-dimer Fibrin Fragment +
3. Whole Blood Lysis
4. Euglobulin Clot Lysis
5. Plasminogen Assay
6. α 2-anti-plasmin Assay

APTT Alone PT alone APTT & PT

8, 9, 11 7 10, 5, 2

THROMBIN TIME
- Direct measure of: Fibrinogen function
-Principle: reflects the rate of thrombin-induced cleavage
of fibrinogen to fibrin monomers and polymerization of
hydrogen bonded fibrin polymer

METHOD:
- Human Thrombin (or bovine thrombin) is added
to platelet poor plasma at 37C
- Time taken for the formation of a fibrin clot
recorded
- Normal Value: 13-15sec
AFFECTED BY:
- Extremely low fibrinogen levels
- Abnormal fibrinogen thrombin inhibitors
- High concentrations of immunoglobulin

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