MTAP2-S4 – Hematology 2 ■ Prolonged results:

RW INTERNS EDITION © ● Presence of clots

Rene Jesus Alfredo R. Dinglasan, RMT ○ Clotting factors have already been
consumed
HEMOSTASIS ○ HOW TO AVOID CLOTS: Properly
● Maintenance of blood flow within the vascular system mix the blood and the anticoagulant
● Important considerations in blood collection for hemostasis inside
testing: ● Increased anticoagulant concentration
○ If the patient has many bruises or mentions a tendency (AC)
to bleed (a reason to expect excessive bleeding), the ○ Caused by:
phlebotomist should extend the time for observing the ■ Short draw (underfilled tube)
venipuncture site from 1 to 5 minutes and should apply ● Lead to prolonged APTT
a pressure bandage before dismissing the patient. ■ Elevated hematocrit (>55%)
○ In the routine venipuncture, we leave the patient. If the ● Adjust the volume of
patient has a hemostatic problem we need to observe anticoagulant inside the
for a longer period of time. Light Blue top so that the
○ Factors V and VIII volume of plasma is just
■ Considered as labile factors (easily enough
destroyed) ● No need to adjust if the
○ Cold temperature (1 to 6 C) storage patient has low hematocrit
■ Refrigeration can cause interferences in the
coagulation testing
■ causes precipitation of: von Willebrand factor Note:
■ activation of: Factor VII ● Phlebotomists must provide tubes with relatively
■ destruction of: platelets decreased anticoagulant volumes for collection of blood
■ Hemostasis sample must be stored at Room from a patient whose hematocrit is high.
temperature only (18-24degC) ● The following formula may be used for the adjustment:
○ 0.105 to 0.109 M (3.2%) buffered Sodium Citrate C = (1.85 X 10-3) (100-H) V
■ Preferred anticoagulant in hemostasis ● where C is the volume of sodium citrate in
■ Found in LIGHT BLUE TOP tubes milliliters, V is volume of whole blood-sodium
● 3-4 tube inversions citrate solution in milliliters, and H is the
● No over inversions because it activates hematocrit in percent
the platelet ● There is NO evidence suggesting a need for increasing
● 9:1 blood:anticoagulant ratio the volume of anticoagulant for specimens from patients
■ May increase the stability of Factors 5 & 8 with anemia, even when the hematocrit is < 20%.
● EDTA and Heparin cannot increase the ● Sample problem:
stability of Factors 5 & 8 ● To collect 5 mL of blood and anticoagulant
● Also other tubes requires 8 inversions mixture from a patient whose hematocrit is 65%,
■ Sometimes Light blue top contains CTAD calculate the volume of sodium citrate as follows:
instead of Na citrate ● C = (1.85 X 10-3) (100-H) V
○ CTAD (Citrate, Theophylline, Adenosine, ● C = (0.00185)(100-65) x 5.0 mL
Dipyridamole) ● C = (0.00185)(35) x 5.0 mL
■ Found in the light blue top ● C = 0.32mL of 3.2% sodium citrate should
■ For PF4 (Platelet Factor 4) and β-TG remain in the tube
(Thromboglobulin) assays
■ Also used in coagulation test
■ ASSUMPTION: 3-4 inversions Needles for Hemostasis Specimens
○ Factors that may affect coagulation test results (PT
and/or APTT): Application Preferred Needle Gauge
■ Shortened test results: and Length
● Hemolysis
● Excessive agitation Adult with good veins, 20 or 21 gauge, thin-walled,
○ Proper tube inversions to avoid specimen < 25 mL 1.0 or 1.25 inches long
platelet activation
● Prolonged tourniquet application Adult with good veins, 19 gauge, 1.0 or 1.25 inches
○ Apply tourniquet 3-4 inches above specimens > 25 mL long
the venipuncture site in less than 1
minute Child or adult with small, 23 gauge, winged-needle set;
○ Leads to hemoconcentration → friable, or hardened apply minimal negative
Shortening of PT and APTT due to veins pressure
entry of tissue thromboplastin
● Excessive needle manipulation Transfer of blood from syringe 19 gauge, slowly inject
○ As we insert the needle, Factor 3 to tube through tube closure
has already entered the syringe
thus, leading to shortening of PT Syringe with winged-needle 20, 21, or 23 gauge,
and APTT set thin-walled; use only for
● Platelet contamination Small, friable, or hardened
○ Make sure to centrifuge the veins or specialized
specimen and aspirate the plasma coagulation testing
properly
○ PLASMA is used
 ● composed of fibrous connective tissue that
Hemostasis Specimen Storage Times and Temperatures contains autonomic nerve endings and the vasa
vasorum (small networks of blood vessels that
Application Temperature (RT) Time supply nutrients to the tissues of the wall).

PT with no 18 to 24 C 24 hours
unfractionated
heparin (UFH) in
specimen

PTT with no 18 to 24 C 4 hours

unfractionated
heparin (UFH) in
specimen

PTT for monitoring 18 to 24 C Separate within 1

UFH therapy hour, test
within 4 hours

PT when UFH is 18 to 24 C Separate within 1

present in hour, test
specimen within 4 hours B. Types of Blood Vessels
1. Arteries (size: 4 mm)
● distributing blood vessels that leave the heart
○ Arteries = Away from the heart
Two Stages of Hemostasis: ● have the thickest walls of the vascular system
● Arterioles (30 μm): microscopic continuation
PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS (smallest version) of arteries that give off
branches called METARTERIOLES, which in
Involves the following: Involves the following: turn join the capillaries
● Constriction of ● Formation of fibrin 2. Veins (5 mm) - LARGEST
damaged blood meshwork through ● collecting blood vessels that return to the
vessels activated coagulation heart
○ Purpose of factors ○ Veins = Vavalik sa puso moh <3
hemostasis: To ○ Platelet plug will ● larger, have a more irregular lumen than
keep the blood be carried away by arteries (the largest among the three types of
inside your body. the plasma flow blood vessels)
● Formation of platelet thus, fibrin ● Venules (size: 20 μm) – microscopically sized
plugs meshwork is veins; connect the capillaries to the veins
○ Platelet plugs are important because 3. Capillaries (8 μm) - SMALLEST
formed following it serves as the ● blood passes from the arterial to the venous
the many blanket of the system via the capillaries
different platelet plug ● capillaries are the thinnest walled and most
processes: ● Inhibition of activated numerous of the blood vessels.
■ ADHESION coagulation factors
■ AGGREGATION ● composed of ONLY ONE CELL LAYER of
■ SECRETION ○ To prevent the simple squamous epithelium (unlike the vessels
**Platelet plug : Seal that further activation of the arterial and venous systems), which
prevents the further outflow of of clotting factors permits a more rapid rate of transport of
the blood from the blood materials between blood and tissue.
vessels ● SINUSOIDS – specialized types of capillaries
found in particular locations such as in the bone
marrow, spleen and liver.
PRIMARY HEMOSTASIS
BLOOD VESSELS Some of the SUBSTANCES released from or found on the
A. 3 coats (tunics) composing the tissue in a blood vessel wall: surface of intact endothelial cells:
1. TUNICA INTIMA
SUBSTANCE ACTION/S
● also known as tunica interna; Innermost layer
● Line by the endothelium – made of simple
squamous epithelium lining the blood vessels PROSTACYCLIN ● a.k.a.: Prostaglandin I2 (PGI2)
● forms the smooth glistening surface of ● Inhibits platelet activation
endothelium that lines the inner tubular cavity ● produced by the EICOSANOID
(lumen) PGI - inhibits SYNTHESIS PATHWAY of
● Endothelial cells: Responsible for secreting the TXA - activates endothelial cells
substances of the blood vessels participating in ● penetrates the platelet and binds at
the hemostasis its IP receptor
2. TUNICA MEDIA
● composed of smooth muscle and elastic fibers EICOSANOID SYNTHESIS PATHWAYS
● Second layer; Thickest coat ● Found in the blood vessels →
3. TUNICA ADVENTITIA Prostaglandin I2 (PGI2) →
● also known as tunica externa; outermost layer inhibits platelet activation
● Also found inside the platelet →
 Thromboxane (TXA2) which
activates platelets ● TTP - Thrombotic Thrombocytopenic Purpura
○ caused by deficiency of ADAMTS-13
Adenosine ● stimulates vasodilation ○ Clogging of blood cells; leads to
(metabolic ○ Vasodilation = widening of blood thrombocytopenia
product of ATP vessel lumen
and ADP) ○ Vasoconstriction = narrowing of
PLATELETS
blood vessel lumen
● aka: THROMBOCYTES
● arise from a bone marrow cell called megakaryocytes
Thrombomodulin ● a thrombin cofactor ● important in both primary and secondary hemostasis
(TM) ● binds thrombin and reduces its ability ○ Majority of the functions are in the primary
to participate in the clotting process hemostasis
● thrombomodulin + thrombin activate ● described as cells with granular cytoplasm but no nuclear
= material
○ PROTEIN C ● platelets cluster with the RBCs near the center of the blood
■ Activated protein C vessel
(APC) - inactivates ● platelets move back and forth with the WBCs from venules
factors Va and VIIIa into the white pulp of the spleen
○ TAFI (Thrombin Activatable ● LIFE SPAN: 9 TO 10 DAYS
Fibrinolysis Inhibitor) ● On a Wright-stained PBS:
■ An antifibrinolytic ○ platelets are spread throughout the RBC monolayer
enzyme (7 to 21 cells per 100x field)
■ Controls the ○ platelets have an average diameter of 2.5 μm (or, 2
overclotting to 4 μm)
**Thrombin →. An enzyme that activates ● 3 major functions:
the clotting factors : 1, 5, 8 and 13 1. To form an aggregate plug of platelets that can
However, if the thrombin is already slow down or stop blood loss
captured and bound with thrombomodulin 2. To participate in plasma coagulation
it will have reduced capacity to activate 3. To preserve the endothelial lining of the blood
clotting factors vessels
● Reticulated Platelets
Heparan sulfate ● weakly enhances activity of ○ aka: STRESS PLATELETS
ANTITHROMBIN (previously called ○ appear in compensation for
Antithrombin III) - natural thrombocytopenia
anticoagulant ○ newly released from megakaryocytes and still
contain RNA
TPA (tissue ● major plasminogen activator ○ markedly larger than the usual platelets
plasminogen ● TPA activates plasminogen (diameter in PBS: exceeds 6 μm [MPV reaches
activator) ● PLASMINOGEN → PLASMIN 12 to 14 fL])
○ clinical use:
Plasmin - AKA Fibrinolysin; an enzyme ■ can help differentiate bone marrow
that dissolves clot failure from peripheral destruction in
thrombocytopenia
VWF (von ● Major functions: ■ early predictor of bone marrow
Willebrand ○ Aids in platelet adhesion recovery after chemotherapy and
Factor) (platelets adhere to the foreign transplantation
surface) ● Reticulated Platelets - potentially prothrombotic (may be
○ Acts as a carrier protein for associated with increased risk of cardiovascular disease)
Factor VIII
■ Thus, vWF deficiency SIZE OF THE PLATELETS
also affects Factor VIII ● Normal: 2.5 μm (average)
● Sites of synthesis: ● MEAN PLATELET VOLUME (MPV)
○ Endothelial cells ○ Reference Range: 6.8 to 10.2 fL
○ Megakaryocytes - largest cells ○ Counterpart of MCV
in the bone marrow ○ Tells the size of platelets
● Sites of storage: ○ Pertains to the average volume of individual platelets
○ Weibel-Palade bodies (found in a specimen
in the blood vessels) ○ EDTA causes swelling of platelets (causes
○ Alpha granules (found in PLTs) approximately 20% increase in MPV during the first
hour).
○ Should be based on EDTA specimens that are
ADAMTS 13 between 1 to 4 hours old
● “a disintegrin and metalloproteinase with a ■ After 1 hour, the size has stabilized
thrombospondin type 1 motif, member 13” ■ Also, do not measure beyond 4 hours
● aka: VWF- Cleaving Protease ○ increased: LARGE; decreased: SMALL
● a plasma enzyme secreted by the: LIVER
● regulates the size of circulating VWF by cleaving
ultra-long VWF multimers (ULVWF) into shorter
segments (have less hemostatic potential)
 CYTOPLASM
● On a Wright-stained PBS, platelets
appear lavender and granular.
● two general parts:
○ Chromomere (aka
Granulomere)
■ Centrally located
■ Granular
Examples of disorders characterized by: ○ Hyalomere
■ Peripherally-located
Small Platelets Large Platelets ■ Non-granular

WAS (Wiskott-Aldrich BSS (Bernard-Soulier MEGAKARYOCYTOPOIESIS

Syndrome) Syndrome) ● aka: MEGAKARYOPOIESIS
● Inheritance: X-Linked ● Inheritance: autosomal ● process by which megakaryocytes develop from
recessive recessive hematopoietic stem cell (HSC)
● Some of the ● Some of the ● THROMBOPOIETIN (TPO)
characteristics: characteristics: ○ Major regulator of platelet production
○ Small platelets ○ Giant platelets ○ Produced primarily by the: LIVER
○ Eczema ○ Thrombocytope ○ 70, 000 Dalton molecule
○ Thrombocytopenia nia ○ Possesses 23% homology with erythropoietin
○ Dense granules ○ GP1B/IX/V (EPO)
deficiency complex ○ MPL – TPO receptor site present at all
deficiency maturation stages (from BFU-Meg to PLTs)
*small platelet is an important ● Cause problem in ○ TPO concentration in the plasma is inversely
diagnostic feature of this platelet adhesion proportional to platelet and megakaryocyte
disease mass, suggesting that membrane binding and
consequent removal of TPO by thrombocytes is
TORCH infections Gray Platelet Syndrome the primary platelet count control mechanism.
Toxoplasma (GPS) ○ Some of the functions of TPO:
Other agents ● Inheritance: autosomal ■ Stimulates megakaryocytopoiesis
Rubella virus recessive (Some of the cytokines that function
Cytomegalovirus ● Some of the with it to stimulate this process include
Herpesvirus characteristics: IL-3, IL-6, and IL-11.)
○ Large, gray ■ Induces the proliferation and
● The patient shows platelets maturation of megakaryocytes
similar S/S → small ○ Mild bleeding ■ Induces thrombocytopoiesis
platelets that they tendencies
manifest ○ Thrombocytope ● Megakaryocyte (general characteristics):
nia ○ largest cells in the bone marrow (size: 30 to 50
○ Fibrosis of the um)
marrow ○ has a multilobulated nucleus and abundant
○ Alpha granule granular cytoplasm
deficiency ○ <0.5% of all bone marrow cells
○ Bone marrow smear: 2-4 megakaryocytes per
MYH9 gene mutations 10x low-power field

● May-Hegglin Anomaly ● Megakaryocyte Progenitors

○ Thrombocytopenia ○ Progenitors: immature hematopoietic cells that
○ Large platelets cannot be recognized under the light
○ Inclusion bodies microscope
○ Dohle body-like ○ Precursors: immature hematopoietic cells that
inclusions seen in can be recognized under the light microscope
Neutrophil, ○ 3 megakaryocyte lineage-committed
Eosinophil, progenitor stages:
Basophil and ■ Burst-forming unit (BFU-Meg) –
Monocyte) least mature
○ Leukopenia ● participate in normal mitosis
● Sebastian Syndrome ○ Cytoplasmic and
● Fechtner Syndrome nuclear division
● Epstein Syndrome ■ Colony-forming unit (CFU-Meg)
● participate in normal mitosis
○ Cytoplasmic and
*Large and Giant platelets are different in size ranges nuclear division
■ Light-density CFU (LD-CFU-Meg) –
SHAPE OF THE PLATELETS most mature
● Resting and circulating platelets = ● loses its capacity to divide
Biconvex or disk-shaped ● performs endomitosis
● Activated platelets = Spherical with ○ Nuclear division w/o
pseudopods cytoplasmic division
 ❖ All of theses progenitors resemble The megakaryocyte progenitor that undergoes endometriosis
LYMPHOCYTES is:
A. MK-I
● Megakaryocyte Precursors (Terminal Megakaryocyte B. BFU-Meg
Differentiation Stages) C. CFU-Meg
○ stages wherein the observers are able to D. LD-CFU-Meg
recognize the unique Wright-stained
morphology of the cells in bone marrow smears PLATELET ULTRASTRUCTURE
or H and E-stained bone marrow biopsy ● Ultrastructure of the platelets - studied using scanning
sections and transmission electron microscopy, flow cytometry,
○ MK-I stage (megakaryoblast) and molecular sequencing
■ least differentiated
■ cannot be reliably distinguished from I. Platelet Plasma Membrane
myeloblasts or pronormoblasts (light ● Selectively permeable
microscopy) ● Provides phospholipids that support platelet
■ begins to develop MOST of its activation internally and plasma coagulation
cytoplasmic ultrastructure (including externally
α-granules, dense granules, and the ● Anchored within the membrane are glycoproteins and
demarcation system (DMS)) proteoglycans
○ MK-II stage (promegakaryocyte) ● PHOSPHOLIPIDS
■ identified by the appearance of nuclear ○ Neutral phospholipids:
lobularity ■ Phosphatidylcholine and sphingomyelin
○ MK-III stage (megakaryocyte) ■ Found in the plasma layer
■ most abundant ○ Anionic or polar phospholipids
■ easily recognized at 10X magnification ■ Phosphatidylinositol (support platelet
(basis is size: 30 to 50 μm) activation by supplying arachidonic acid)
■ At the full maturation of the cell, ■ Phosphatidylethanolamine, and
platelet shedding proceeds. ■ Phosphatidylserine (flips to the outer
■ One megakaryocyte may shed 2,000 surface upon activation and is the
to 4,000 platelets. charged phospholipid surface on which 2
■ Platelet shedding – aka: coagulation pathway complexes
thrombopoiesis, assemble)
thrombocytopoiesis ● These 2 complexes are:
■ Thrombopoietin - major regulator of ○ Tenase complex
platelet production ○ Prothrombinase complex
■ Found in the inner cytoplasmic layer
MK-I MK-II MK-III ● GLYCOCALYX
○ The platelet membrane surface
○ Absorbs albumin, fibrinogen, and other plasma
Nucleus Round Indented Multilobed
proteins through endocytosis
Nucleoli 2 to 6 Variable Not visible II. Platelet Receptors
○ Receptors: receive ligands
Chromatin Homogeneo Moderately Deeply and
us condense variably
condensed Glycoprotein Platelet Membrane Receptors

N:C ratio 3:1 1:2 1:4 Electrophoresis Current Ligand Remarks

Nomenclature Nomenclature
Mitosis Absent Absent Absent
GP Ia/IIa Integrin : α2β1 Collagen None
Endomitosis Present Ends Absent
Integrin : αvβ1 Vitronectin
Cytoplasm Basophilic Basophilic Azurophilic
and and Integrin : α5β1 Laminin
granular granular
Integrin : α6β1 Fibronectin
Demarcation Present Present Present
System (DMS)
Part of
cytoplasm that GP VI CAM of the Collagen Key collagen
delineates immunoglobulin -have more receptor,
individual plt gene family
during
significant triggers
thrombocytopoi effect than platelet
esis GP Ia/IIa activation,
release of
TXA2 and
Quiz: ADP that
increase the
 ○ P-selectin or CD62 quantification by flow
avidity of cytometry is a means for measuring in vivo
integrins platelet activation
α2β1 (GP
Ia/IIa) and III. PLATELET CYTOSKELETON
αIIbβ3(GPIIb/II ● microtubules, actin microfilaments, and intermediate
Ia) microfilaments control platelet shape change,
extension of pseudopods, and secretion of granule
contents
○ MICROTUBULES
GP Ib/IX/V CAM of the VWF and ◦GPIbα - ■ Formed by Tubulins
leucine-rich thrombin vWF-specific ■ Thick, circumferential bundle of these
repeat family bind GPIbα ; site maintains platelet’s discoid shape
thrombin reside just within, although NOT
cleaves a ◦Bernard-Sou touching, the plasma membrane
site on lier syndrome ■ Platelets become round when
GP V - deficiency of microtubules disassemble in the cold.
GPIb/IX/V However, upon warming to 37◦C, they
recover their original discoid shape.
■ Aside from maintaining the platelet’s
discoid shape, microtubules also move
GP IIb/IIIa Integrin : Fibrinogen, ◦Key inward on activation to enable
αIIbβ3 VWF fibrinogen expression of α-granule contents.
receptor ■ During platelet shape change,
◦Glanzmann’s microtubules also reassemble in long
thrombasthe parallel bundles to provide rigidity to
nia - deficiency pseudopods.
of GPIIb/IIIa ■ Resting: Disk shape
■ Active: Spherical shape with
Note: pseudopods
▫Aggregation - when platelets adhere to other platelets ○ MICROFILAMENTS
▫Adhesion - platelets adhere to foreign surface ■ Location: between the microtubules
▫Ligand – a molecule that binds to another molecule (used and the membrane
especially to refer to a small molecule that ■ Formed by ACTINS
specifically binds to a larger molecule) ■ Actin:
▫CAM – cell adhesion molecule ● anchors the plasma membrane
glycoproteins and
proteoglycans
● also present throughout the
Platelet STR (“Seven-Transmembrane Repeat”) platelet cytoplasm
● globular and amorphous (in
Receptors
resting platelets)
● As cytoplasmic calcium
RECEPTOR LIGAND concentration increases, actin
becomes filamentous and
PAR 1 Thrombin contractile
● NOTE: The cytoplasm also contains intermediate
PAR 4 Thrombin filaments (Desmin and Vimentin) - connect with actin and
the tubules, maintaining the platelet shape.
P2Y1 ADP
Activate the IV. PLATELET GRANULES
P2Y12 ADP platelets ● α-granules contents and lysosomes’ contents
flow through the SCCS
TPα and TPβ TXA2 ● dense granules travel to the plasma membrane

α2-adrenergic Epinephrine a.k.a. ○ ALPHA GRANULES

ADRENALINE ■ 50 to 80 granules in each platelet
■ Stain medium-gray (osmium-dye transmission
electron microscopy preparations)
IP PGI2 Inhibit plt
■ Most of the α-granule contents are proteins that
activation
participate in secondary hemostasis (called
coagulation).
Additional Platelet Membrane Receptors ■ As the platelets becomes activated:
● FcγIIA (CD32) – a low-affinity receptor for the ● α-granule membranes fuse with the SCCS
immunoglobulin Fc portion (has a role in a perilous condition ● contents of the α-granule flow to the nearby
known as heparin-induced thrombocytopenia) environment
● P-selectin (CD 62) – an integrin that helps platelet binding ● In the nearby environment, the contents of
to endothelial cells, WBCs, and one another the α-granule:
○ found on the α-granule membranes of the ○ participate in platelet adhesion
resting platelet but travels via the SCCS to the ○ participate in platelet aggregation
surface of activated platelets ○ support plasma coagulation
 ■ Some of the platelet α-granule proteins include:
Serotonin (5-HT) Vasoconstrictor that binds endothelial
cells and platelet membranes
LOCATION PROTEINS PRESENT 5-hydroxytryptamine

α-granules (but ◦β-thromboglobulin Histamine

not in the ◦PF-4
cytoplasm) ● These two can be measured Calcium (Ca2+) and Divalent cations support platelet
using CTAD anticoagulant Magnesium (Mg2+) activation and coagulation
● Able to inhibit heparin →
induced coagulation Epinephrine Adrenaline
◦HMWK
◦PAI-1 (plasminogen activator
inhibitor-1) LYSOSOMES
◦Plasminogen ● Few in number
◦Protein C inhibitor ● Stain positive for arylsulfatase, β-glucuronidase, acid
◦EGF (endothelial growth factor) phosphatase, and catalase
◦PDGF (platelet-derived growth factor) ● Lysosomes’ contents flow through the SCCS
◦TGF-β (transforming growth factor-β) ● Lysosomes probably digest vessel wall matrix
● Support mitosis of vascular components during in vivo aggregation and may also
fibroblasts and smooth muscle digest autophagic debris
cells
● Promote wound healing
V. SCCS (Surface-Connected Canalicular System)
α-granules and ◦Fibrinogen
○ The plasma membrane invades platelet interior,
platelet cytoplasm ◦Fibronectin
producing SCCS.
◦Albumin
○ Glycocalyx is less developed in the SCCS and lacks
◦Immunoglobulins
some of the glycoprotein receptors present on the
◦VWF
platelet surface.
◦Thrombospondin
○ SCCS is the route for:
◦Factor V
■ endocytosis
■ secretion of α-granule contents
■ secretion of lysosome contents
α-granule ◦P-selectin
membrane
VI. DTS (Dense Tubular System)
○ Parallel and closely aligned to the SCCS
α-granule ◦GP IIb/IIIa ○ A condensed remnant of the rough endoplasmic
membrane and ◦GP IV reticulum
plasma membrane ◦GP Ib/IX/V ○ Sequesters calcium and bears a series of enzymes that
support platelet activation
○ These enzymes include:
○ DENSE GRANULES (MPCHASE) ■ phospholipase A2, cyclooxygenase, and
● 2 to 7 dense granules per platelet thromboxane synthetase (support the eicosanoid
● Aka: DENSE BODIES synthesis pathway that produces thromboxane A2)
● stain black (opaque) when treated with osmium in ■ phospholipase C (supports production of inositol
transmission electron microscopy triphosphate (IP3) and diacylglycerol (DAG))
● They migrate to the plasma membrane and release their ○ “CONTROL CENTER” for platelet activation
contents directly into the plasma upon platelet activation. ■ Contains the necessary materials needed for the
● The dense granule contents are vasoconstrictors activation of platelets
and platelet agonists that intensify primary hemostasis.
● AGONIST or ACTIVATORS - a substance which initiates PLATELET ACTIVATION
a response when combined with a receptor (ex. ligands) ● Platelet Activation Pathways:
● Dense granules are important in the primary hemostasis ○ G proteins
● Some of the components of dense granules include: ■ aka: GUANINE NUCLEOTIDE-BINDING
MPCHASE (Magnesium, Phosphate, Calcium, PROTEINS
Histamine, APD/ATP, Serotonin/5-hydroxytryptamine, ■ control cellular activation for all cells (not
Epinephrine/Adrenaline) just platelets) at the inner membrane
surface
■ Stimulate the two other platelet
Components Remarks pathways: EICOSANOID SYNTHESIS
PATHWAY and IP3-DAG PATHWAY
ADP Supports neighboring platelet
aggregation by binding to P2Y1 and ● Eicosanoid synthesis pathway
P2Y12 ○ aka: PROSTAGLANDIN, CYCLOOXYGENASE,
OR THROMBOXANE PATHWAY
ATP Function unknown, but ATP release is ○ Eicosanoid: a mediator derived from the
detectable upon platelet 20-carbon atom arachidonic acid (20 in Greek is
activation "eicosa") or a similar fatty acid
○ examples of eicosanoids: prostacyclin,
Phosphate thromboxane, etc.
 ○ Collagen (receptor: GPC), ADP (receptors: P2Y1 ● VWF unrolls (similar to a carpet) and adheres to the
& P2Y12), Thrombin (receptors: PAR1 and PAR4), injured site. (NOTE: VWF circulates as a globular
and Epinephrine (receptor: Alpha 2 adrenergic protein. However, it becomes fibrillar as it unrolls.)
receptor) bind their respective membrane ● VWF exposes sites that partially bind the GPIb-α
receptors (The combination activates portion of the platelet GP Ib/IX/V → This is a reversible
phospholipase A2 found in DTS.) – not sure pa binding process that “tethers” or decelerates the platelet.
○ Phospholipase A2 → releases arachidonic acid ○ Platelets tangled in the specific area
(also called 5,8,11,14-eicosatetraenoic acid) from ● Platelet and VWF interactions remain localized by
membrane phosphatidylinositol (polar ADAMTS-13 that digests “unused” VWF.
phospholipids) ● The VWF-GP Ibα tethering reaction is temporary, and
○ Arachidonic acid is acted upon by the platelet rolls along the surface unless GP VI comes
cyclooxygenase, peroxidase, and thromboxane into contact with the exposed collagen.
synthase to produce TXA2 → which activates ● When collagen binds platelet GP VI, it activates internal
the platelet. platelet activation pathways, releasing:
○ TXA2 suppresses adenylate cyclase and ● TXA2 and ADP (agonists)
reduces cyclic AMP concentration. This allows the ○ TXA2 binds to TPα and TPβ → platelet STR
release of ionic calcium from the DTS. Ionic receptors
calcium supports the contraction of actin ○ ADP binds to P2Y1 and P2Y12
microfilaments, activating the platelet. ■ trigger a reaction that increases the
○ REMEMBER: affinity of α2β1 (GP Ia/IIa) for collagen
■ The eicosanoid pathway in the endothelial (foreign surface for the platelets)
cells is almost the same EXCEPT that ■ GP VI and GP Ia/IIa → receptor of
prostacyclin synthase replaces collagen; GP VI is the major receptor
thromboxane synthase. (thromboxane ● The combined effect of GP Ib/IX/V, GP VI, and α2β1
synthetase in platelets; prostacyclin (GP Ia/IIa) causes the platelet to become firmly attached
synthase in endothelial cells) to the damaged surface, where it subsequently loses its
■ In endothelial cells, the eicosanoid discoid shape and spreads.
pathway result is prostaglandin I2 (aka: ● TXA2 and ADP:
prostacyclin) ○ also secreted from the platelet to the
■ Prostacyclin penetrates the platelet and microenvironment, there they activate adjacent
binds to its IP receptor. platelets through their respective receptors
■ Prostacyclin accelerates adenylate ○ provide inside-out activation of αIIbβ3 (GP
cyclase, increasing cyclic AMP, and IIb/IIIa), which assists in platelet aggregation.
sequesters ionic calcium to the DTS.
■ In intact blood vessels, the endothelial cell B. AGGREGATION
eicosanoid pathway suppresses platelet ● Platelets adhere to other platelets
activation through the mechanisms stated ● Blood vessel injury releases tissue factor (TF) from
above. endothelial cells.
● TF triggers generation of thrombin which reacts with
● INOSITOL TRIPHOSPHATE-DIACYLGLYCEROL platelet PAR1 and PAR4. This further activation
(IP3-DAG) PATHWAY generates the COAT platelet (“collagen and thrombin
○ Related to Eicosanoid synthesis pathway activated”) platelet (integral to the cell-based coagulation
○ G-protein activates phospholipase C model).
○ Phospholipase C cleaves membrane ● Meanwhile, αIIbβ3 (GP IIb/IIIa) assembles from its
phosphatidylinositol 4,5-bisphosphate to form resting membrane units αIIb and β3.
IP3 and DAG. ● αIIbβ3 (GP IIb/IIIa) binds fibrinogen and VWF and
○ IP3 promotes release of ionic calcium from the supports platelet-to-platelet aggregation → seal the
DTS, which activates actin microfilament wounds to stop bleeding
contraction. ● Thrombocytes lose their shape and extend pseudopods.
○ IP3 may also activate phospholipase A2 ● Phosphatidylserine flips to the outer layer (establishing a
○ DAG activates a multistep process: activation of surface for the assembly of coagulation factor
phosphokinase C (which triggers phosphorylation complexes).
of the protein pleckstrin (which regulates actin ● As platelet aggregation goes on, membrane integrity is
microfilament contraction). lost, and a syncytium of platelet cytoplasm forms as they
use internal energy sources.
Processes Involved in Platelet Activation: C. SECRETION
● Blood vessel injury: ● The activation of the platelets trigger actin microfilament
○ Exposes collagen contraction.
○ Releases VWF ● Intermediate filaments also contract (moves the
○ Releases tissue factor circumferential microtubules inward and compressing the
granules).
**”This processes may occur simultaneous” - Rodaks ● Phosphatidylserine is the phospholipid on which two
coagulation pathway complexes assemble: tenase and
A. ADHESION prothrombinase (both supported by ionic calcium)
● Platelets adhere to a foreign surface ● The alpha granule contents fibrinogen, Factor V and
● initiated by exposure of the subendothelial collagen Factor VIII, and VWF (which binds and stabilizes Factor
● damaged endothelial cells release VWF from VIII) are secreted and increase the localized
cytoplasmic storage organelles → Weibel-Palade concentrations of these clotting factors. Their presence
bodies (WPb) further supports the action of tenase and
prothrombinase.
 ● α-granules contents and lysosomes’ contents flow
through the SCCS trigger a reaction that increases the from highly refractile dirt and debris, one must
affinity of α2β1 remember the platelets’ characteristics when they are
● Dense granules travel to the plasma membrane seen using a phase-contrast microscope.
● Most of the α-granule contents are proteins that The platelets have the following characteristics:
participate in secondary hemostasis (called coagulation). ● Diameter: 2 to 4 μm
● The dense granule contents are vasoconstrictors and ● Shape: round or oval
platelet agonists that intensify primary hemostasis. ● Color: light purple sheen
● Lysosomes probably digest vessel wall matrix ○ (NOTE: “Ghost” erythrocytes are
components during in vivo aggregation and may also frequently seen in the background.)
digest autophagic debris 5. Platelets are counted in the 25 small squares of the
CENTRAL LARGE SQUARE (1 MM2) of the
COMMON TESTS FOR PRIMARY HEMOSTASIS hemocytometer on both sides. On the other side of
A. PLATELET COUNT the counting chamber, repeat the counting process.
● Reasons why platelets hard to count: Compare the counted cells of both squares.
○ Platelets adhere to foreign surfaces (like skin
and dried walls of pipets). IMPORTANT:
○ Platelets easily disintegrate. ● difference between the total cells counted on
○ They are hard to differentiate from debris. each side should be < 10%.
○ Platelets are unevenly distributed in the blood ● greater difference could indicate uneven
because they tend to clump. distribution (requires that the procedure be
● Normal Value (general): 150 to 450 x 109/L (SI) repeated)
● *platelet count decreases after 65 years old to 122 to ● formula for percentage difference:
350 X 109/L in men and 140 to 379 X 109/L in women.
○ In normal individuals: PERCENTAGE DIFFERENCE:
■ 2/3 of platelet mass = CIRCULATION
■ 1/3 of platelet mass = SPLEEN
● Platelet count of < 100,000/μL (100 x 109/L) is the most
common cause of clinically important bleeding.
● If the patient develop splenomegaly → decreased
platelet count as the spleen traps more and more
platelets; Splenectomy → thrombocytosis WHERE,
● V1 - larger number
PHASE-CONTRAST MICROSCOPE METHOD ● V2 - smaller number
● Reference method (described by Brecher and
Cronkite) 6. Average the number of platelets counted on the two
● EDTA whole blood is diluted 1:100 with 1% sides. Using the average, compute the PLT count
ammonium oxalate to lyse the non nucleated using the following formula:
RBCs. *(Reminder: This is the general formula used for
○ ammonium oxalate is the diluent for manual cell counts and can be used to compute any
the manual platelet count type of cell count.)
● Platelets are counted in the 25 small squares of
the central large square (1 mm2) of the
hemocytometer.
● The accuracy of the manual thrombocyte count
must be verified by performing a platelet count
estimate on a Wrightstained Area (mm2) - size of squares = 1mm2
● PBS made from the same specimen.
● Alternatively, a light microscope can also be
used. However, visualizing the platelets may be
more difficult.

PROCEDURE:
1. In a small test tube, prepare a 1:100 dilution by
placing 20 μL of blood (well-mixed) into 1,980 μL of
1% ammonium oxalate
2. Mix the dilution properly and charge the counting
chamber. (NOTE: A special thin, flat-bottomed SAMPLE PROBLEM:
hemocytometer is used for phase-microscopy platelet ◦ platelets counted (one side): 208 (V1)
counts.) ◦ platelets counted (another side): 200 (V2)
3. The charged hemocytometer is then placed in a moist ◦Dilution: 1:100 = 100
chamber for 15 minutes to allow the platelets to
settle. (MOIST CHAMBER – may be prepared by
placing a piece of damp filter paper in the bottom of a
Petri dish. An applicator stick broken in half can serve
as a support for the hemocytometer.)
a. WBC count - 10 minutes
b. Platelet count - 15 minutes
4. Platelets are counted using the 40x objective lens
(400x total magnification). To distinguish the platelets
 ○ N0 level of functioning platelets
○ Calcium
○ Adenosine Triphosphate/ATP

1. HIRSCHBOECK/CASTOR OIL
● Evaluates clot retraction time
● Procedure:
a. Place a few drops of castor oil into a clean
test tube.
b. Place 1 drop of fresh blood (without
anticoagulant) into the castor oil then start
the timer.
c. Observe for extrusion of droplet-like serum
(“dimpling”) on top portion of the drop of
blood
d. Stop the timer as soon as “dimpling” of
serum is detected.
PLATELET COUNT ESTIMATION ● N.V.: 15 to 45 minutes
● To determine the approximate number of platelets per ● Reminders:
field, examine the thin area of the slide (where only a few ○ Abnormal characteristic of extruded serum:
red blood cells slightly overlap) using the oil immersion ■ Dark yellow - jaundice
objective. ■ Cloudy - plasma cell myeloma
● A normal (wedge) blood smear should demonstrate ■ Milky - diabetes, leukemia, after meals
approximately 7 to 21 cells per 100x field
● For the estimation of the platelet count: 2. MAC FARLANE METHOD
○ Scan ten (10) oil immersion fields for the ● Uses a calibrated tube
number of platelets ● Evaluates the degree of clot retraction
○ Average number of platelets/OIF X 20,000 = ● Procedure:
estimated plt. ct. per μL a. Place 5 mL of fresh venous blood into a clean
○ In occasions of significant anemia or calibrated tube.
erythrocytosis, use the following formula for the b. Insert a glass rod into the tube with the
platelet estimate: expansion immersed in the column of blood.
c. Fit a cork into the glass rod by boring a hole
at the center of the cork and allowing the
projecting tip of the glass rod pass through
● *200 – average number of RBCs per oil
the hole. This will cover the tube.
immersion field in the optimal assessment area
d. Place the tube in a 37oC water bath and
● Not accurate, only estimation
observe every 5 to 10 minutes for
coagulation.
Estimates may be reported using the following table: e. One hour after firm clotting, allow the tube to
stand at room temperature for retraction to
PLATELET ESTIMATE OF: REPORT AS: occur. (Retraction has taken place if the clot
has shrunk and has attached itself onto the
0 to 49,000/uL Marked decrease glass rod).
f. After retraction has taken place, remove the
cork.
50,000 to 99,000/uL Moderate decrease
g. Remove the glass rod and attached clot.
h. Measure the amount of expressed serum
100,000 to 149,000/uL Slight decrease directly against the calibration of the tube.
i. Computation:
150,000 to 199,000/uL Low normal

200,000 to 400,000/uL Normal

● Normal Value: 44 to 67%
401,000 to 599,000/uL Slight increase ● REMINDERS:
○ Interaction of platelets with fibrinogen and fibrin
600,000 to 800,000/uL Moderate increase must also be normal for clot retraction to occur
○ Abnormal Clot Retraction observed in:
Above 800,000/uL Marked increase ■ Thrombocytopenia
■ Low or abnormal fibrinogens
■ Paraproteinemias (multiple myeloma,
B. CLOT RETRACTION (CR) macroglobulinemia) that interfere with
● Thrombosthenin - a contractile protein important for CR fibrin formation
● Principle: ■ Glanzmann’s thrombasthenia
○ Normal blood clot contracts (in doing so, it ● Platelet is incapable of
expresses serum) interacting with fibrin
○ Degree of clot retraction: measured on the
amount of expressed serum C. PLATELET AGGREGOMETRY
● Normal clot retraction requires:
○ N0 level of fibrinogen LIGHT-TRANSMITTANCE (OPTICAL) PLATELET
AGGREGOMETRY
 ● Designed to test platelet-rich plasma (plasma with a
platelet count of 200,000 to 300, 000/uL) Hereditary Hemorrhagic ● Most common inherited
● To prepare PRP: Telangiectasia (HHT) vascular bleeding disorder
○ Sodium citrate-anticoagulated blood is checked a.k.a. Osler-Weber-Rendu ● PT & APTT: NORMAL
visually for clots Disease) ● Inherited as: Autosomal
○ Then it is centrifuged at 50 x g for 30 minutes dominant (AD)
with the stopper in place to maintain the pH. ● Characterized by localized
○ Supernatant PRP is transferred by a plastic dilation of capillary walls
pipette to a clean plastic tube (skin & mucous membrane)
○ The tube is then sealed and stored at 18 to 24
C (ambient temperature) until the test is begun. Ehlers-Danlos Syndrome ● Characterized by
● PRP-based light-transmittance aggregometry is initiated (a.k.a. Cutis hyperextensible joints,
NO LESS THAN 30 minutes after the specimen is Hyperelastica) hyperplastic skin
centrifuged and completed within 4 hours of the time of ● Inherited as: Autosomal
collection. *hyperplastic/elastic skin* dominant (AD)
● To produce adequate PRP, the original specimen must
measure 9 to 12 mL of whole blood. Light Transmittance Marfan’s Syndrome ● Characterized by: skeletal
aggregometry is UNRELIABLE when the patient’s defects (long extremities),
whole-blood platelet count is < 100, 000/uL. **The thumb and wrist ● Arachnodactyly (“spider
signs are part and parcel of fingers)
the diagnostic criteria for ● Inherited as: Autosomal
PLATELET AGGREGATES STUDIES Marfan syndrome dominant (AD)

Examples of EXAMPLES OF DISORDERS WITH: Senile Purpura ● characterized by the

Aggregating presence of bruised areas
Reagents Normal response Abnormal response (common in ELDERLY on the forearms of elderly
person) persons (due to
Adenosine Bernard-Soulier Glanzmann’s degeneration of collagen)
diphosphate, Syndrome (BSS) thrombasthenia
Collagen, Henoch-Schonlein ● most commonly seen in
Epinephrine Von Willebrand Disease Purpura CHILDREN
(ACE) (VWD) ● vascular abnormalities:
(a.k.a.: Allergic Purpura or most probably caused by
Ristocetin Glanzmann’s Bernard-Soulier Nonthrombocytopenic immunologic damage to the
thrombasthenia Syndrome (BSS) Purpura) endothelial cells
● characterized by
Von Willebrand Plt count: NORMAL gastrointestinal hemorrhage
Disease (VWD) and joint swelling
**In blood vessel disorders
normal platelet count but
PLATELET LUMIAGGREGOMETRY bleeding is present
● measures platelet aggregation and ATP release (from
dense granules) Waterhouse-Friderichsen ● characterized by severe
● ATP release is measured by addition of LUCIFERIN to a Syndrome capillary damage following
blood sample. meningococcal septicemia
● performed on: WHOLE BLOOD DILUTED WITH
SALINE
Kasabach-Meritt ● associated with tumors
Syndrome composed of vessels that
DISORDERS OF PRIMARY HEMOSTASIS commonly swell and bleed
(a.k.a. Congenital at the surface
VASCULAR DISORDERS Hemangiomata)
**General laboratory test results:
● NORMAL RESULTS in these tests = Platelet count, ● characterized by defects in
Platelet function tests, Coagulation tests
Scurvy
(Ascorbic acid/Vitamin C) the synthesis of collagen
● ABNORMAL RESULTS in these tests = and hyaluronic acid
deficiency)
○ Bleeding time ● One of the earliest
■ integrity of blood vessel manifestations: BLEEDING
Vitamin C - important in
○ Rumple-Leede Test GUMS
maintaining the integrity of
■ tourniquet test evaluating the integrity ● PT and APTT: NORMAL
blood vessel walls
of blood vessel and check for
presence of Petechiae

VASCULAR
COMMENTS
DISORDERS
PLATELET DISORDERS
Cirrhosis of the liver
A. QUANTITATIVE Portal hypertension
● Thrombocytopenia (decreased platelet count)
Massive Blood Transfusions
Examples of conditions with: Impaired or Decreased
Platelet Production
● Thrombocytosis (increased platelet count)
BERNARD-SOULIER SYNDROME GROUPS EXAMPLES OF ASSOCIATED CASES
○ Autosomal recessive (AR)
○ Decrease platelet production in bone marrow A.) Reactive Thrombocytosis ● Recovery from
○ Platelet receptor deficiency of GP Ib-IX-V ● aka: SECONDARY splenectomy
■ Receptor for VWF THROMBOCYTOSIS ● Acute blood loss
○ Presence of GIANT PLATELETS ● characterized by: ● Major surgery
Moderately increased
Fanconi anemia platelet count
○ Bone marrow disorder
○ Characterized by Aplastic Anemia B.) Autonomous ● Primary Myelofibrosis
TAR syndrome Thrombocytosis (PMF)
○ “Thrombocytopenia with Absent Radii” ● aka: ● Chronic Myelogenous
○ Low platelet count Primary Leukemia (CML)
thrombocytosis ● Essential
Viral infections ● characterized by: Thrombocythemia (ET)
Leukemia Markedly increased ● Polycythemia Vera (PV)
platelet count
Megaloblastic anemias
WAS (Wiskott-Aldrich syndrome)
○ X-linked recessive B. QUALITATIVE
○ Small platelets Examples of conditions with: PLATELET ADHESION
○ Eczema and dense granules deficiency DISORDER
MYH9 gene mutations
○ May-Hegglin Anomaly ● Bernard Soulier Syndrome (BSS)
■ Platelet and WBC disorder ○ Deficiency of the GP Ib/IX/V
■ Autosomal dominant (AD) ○ Inherited as: AR
○ Associated with:
■ Some of the characteristics: ■ Decreased platelet count
● Leukopenia ■ Giant platelet
● Thrombocytopenia ■ Normal aggregation in: ADP, Collagen,
● Dohle body-like inclusions (seen Epinephrine (A, C, E)
in Neutrophil, Eosinophil, ■ Abnormal aggregation in: Ristocetin
Basophils, and Monocytes) ● VWD
○ Sebastian Syndrome, Fechtner Syndrome, ○ Deficiency of VWF
Epstein Syndrome ○ Inherited as: Autosomal Dominant (AD)
○ GIANT platelets ○ Associated with
■ Decreased Factor VIII
■ Normal PT, Prolonged APTT
Examples of conditions with: INCREASED PLATELET ■ Normal aggregation: A, C, E
DESTRUCTION ■ Abnormal aggregation: Ristocetin

HUS (hemolytic uremic syndrome) Examples of conditions with: PLATELET AGGREGATION

○ occurs as a complication of a diarrheal DISORDER
infection from E. coli O157:H7 infection
● Glanzmann’s thrombasthenia
DIC (disseminated intravascular coagulation) ○ Inherited as: Autosomal recessive (AR)
ITP (immune thrombocytopenic purpura) ○ Deficiency of GP IIb/IIIa
TTP (thrombotic thrombocytopenic purpura) ○ Characterized by:
■ Very prolonged BT
○ ADAMTS-13 deficiency →
■ Abnormal clot retraction
HIT (heparin-induced thrombocytopenia) ■ Abnormal aggregation response in ACE
■ Normal aggregation response in Ristocetin
Examples of conditions with: Increased Splenic ++++++ need additional information 2:48PM
Sequestration ● Hereditary afibrinogenemia

Gaucher disease Examples of conditions with: PLATELET SECRETION

DISORDER
Hodgkin’s disease
Sarcoidosis
Lymphoma
 ● Thromboxane Pathway Disorders concentration: be prolonged.
● Cyclooxygenase or thromboxane synthetase 200-400 mg/dL
deficiency Increases
approximately 10mg/dL
● Storage pool diseases per decade in the elderly
○ ALPHA GRANULE DEFICIENCY
■ Gray platelet syndrome Essential for platelet
● Large platelets aggregation (links
● Mild bleeding tendency activated platelets
○ DENSE GRANULE DEFICIENCIES through their GP IIb/IIIa
■ Hermansky-Pudlak syndrome platelet fibrinogen
■ Chediak-Higashi syndrome receptor
■ TAR syndrome
■ Wiskott-Aldrich syndrome Platelet alpha granules
absorb, transport, and
release abundant
DISORDER INHERITANC ALBINISM PLATELET fibrinogen
E PATTERN COUNT
____
Hermansky- AR Oculocutane Normal
Pudlak ous albinism II Prothrombin
● Mol. weight (Daltons):
71, 600
Chediak- AR Partial Low
● Half-life(hours): 60
Higashi Albinism
● Mean plasma conc: 10
mg/dL
Wiskott- X-linked No Albinism Low
Aldrich recessive
III Tissue factor/Tissue Factor III is not ordinarily
thromboplastin/Thromboki present in the plasma
Thrombocyto AR No Albinism Normal
nase
penia with
● Mol. weight (Daltons): No wounds = No Factor
absent radius
44,000 III
(TAR)
● Half-life(hours):
insoluble Factor III is ALWAYS
TAR Syndrome ● Mean plasma conc: present in the tissues →
● Platelet counts are usually 10,000 to 30,000/uL in none there is no such thing as
infancy but overtime normal levels will be achieved Factor III deficiency
within 1 year of birth
IV Calcium ions
SECONDARY HEMOSTASIS ● Mol.weight (Daltons):
Coagulation Factors 40
● Coagulation factors are produced primarily in the LIVER ● Half-life(hours): N/A
(except for Factors III and IV) ● Mean plasma conc: 8
● Factor VII - produced in a number of tissues (however, to 10 mg/dL
the major production site is the liver)
● The vWF portion of VIII: vWF - made by V Proaccelerin/Labile Factor V Leiden -
megakaryocytes and endothelial cells factor/Thrombogen mutant factor V (Dutch
● Increased in liver disease are Factors I and VIII ● Mol.weight (Daltons): investigators from the
● Factor VII has the shortest half-life (6 hours). With acute 330,000 city of Leiden first
liver dysfunction, levels of factor VII are reduced early. ● Half-life (hours): 24 described this mutation)
PT is a very good test to assess liver function in the ● Mean plasma
acute setting because Factor VII is needed for the concentration: 1 Remember that factors V
extrinsic pathway. mg/dL and VIII are inactivated
● All deficiencies of coagulation factors are transmitted as by protein C-protein S
autosomal recessive with the exceptions of Factor VIII complex. However,
deficiency and Factor IX deficiency, which are **Factor VI → it’s just an Factor V Leiden
transmitted as X-linked recessive. activated factor V (mutated factor V) is
NOT activated and leads
Characteristics of Clotting Factors: to excessive clot
formation

Factor Name/s Important Notes Factor V deficiency =

“Owren’s disease” or
I Fibrinogen Most concentrated of all “Parahemophilia”
● Mol.weight (Daltons): the plasma
340,000 procoagulants VII Proconvertin/Stable Factor Shortest half-life (6
● Half-life (hours): 100 ● Mol.weight (Daltons): hours)
to 150 If fibrinogen level is <100 50,000
● Mean plasma mg/dL, PT and aPTT will ● Half-life(hours): 6 Used in liver function
 ● Mean plasma conc: tests 58,800
0.05 mg/dL ● Half-life(hours): 48 to
First to be affected by 52
Warfarin therapy ● Mean plasma conc: 1
mg/dL
VIII Antihemophilic Factor A Free FVIII is unstable in
(AHF-A)/Antihemophilic plasma (it circulates XI Antihemophilic Factor C Factor XI deficiency is:
globulin (AHG) bound to vWF). During (AHF-C)/ Plasma “Hemophilia C” or
● Mol.weight (Daltons): coagulation, thrombin Thromboplastin “Rosenthal
330,000 cleaves factor VIII from Antecedent (PTA) Syndrome”
● Half-life(hours): 12 vWF and activates it ● Mol.weight (Daltons):
● Mean plasma conc: 143,000 >50% of cases = Seen in
0.01 mg/dL Factor VIII deficiency = ● Half-life(hours): 48 to Ashkenazi Jews
“Hemophilia A” or 84
“Classic Hemophilia” ● Mean plasma conc:
0.5 mg/dL
*Factor VIII circulates in the blood bound to vWF (“Factor VIII
complex) XII Hageman factor/ Glass ● Factor XII
*Factor VIII complex may be symbolized as follows (based on its Factor/ Contact Factor deficiency: has
characteristics): ● Mol.weight (Daltons): NO bleeding
(1) Factor VIII, Factor VIIIC, Factor VIII:C 84,000 tendency; has
- Refers to procoagulant portion ● Half-life(hours): 48 to thrombotic
- Measured by standard Factor VIII assays and 70 tendency
APTT ● Mean plasma conc: 3
- Markedly decreased in Hemophilia A mg/dL
(2) Factor VIII:Ag
- Refers to the antigenic properties XIII Fibrin Stabilizing Factor/ To detect Factor XIII
- Measured by immunoassays Fibrinase/ Laki-Lorand deficiency, one may
(3) Factor VIIIR:RCo Factor use the 5M Urea
- Refers to the portion responsible for platelet ● Mol.weight (Daltons): Clot Solubility Test
aggregation in the presence of ristocetin 320,000 (aka Duckert’s Test)
- Termed as ristocetin cofactor ● Half-life(hours): 150 [Results:
(4) Factor VIII:vWF ● Mean plasma conc: 2 - Clot is insoluble
- Also termed the von Willebrand Factor mg/dL in 5M UREA =
- Required for normal platelet adhesion FACT. XIII is
(5) vWF:Ag present
- Antigenic portion of the von Willebrand factor - *Clot is soluble
- Was previously termed as Factor VIII related in 5M UREA =
antigen (VIIIR:Ag)
- Measured by immunoassays PK Prekallikrein Aka: Fletcher Factor
● Mol.weight (Daltons):
VWF Von Willebrand Factor ● Largest molecule in 85,000
● Mol.weight (Daltons): the human plasma ● Half-life(hours): 35
600,000 to 20,000,000 ● VWF has receptor ● Mean plasma conc: 35
● Half-life(hours): 24 sites for BOTH to 50 mg/dL
● Mean plasma conc: 1 platelets and
mg/dL collagen (helps to HMWK High-molecular weight
bind platelets to Kininogen/ Reid Factor/
exposed Williams Factor/ Fitzgerald
subendothelial Factor/ Flaujeac Factor
collagen during ● Mol.weight (Daltons):
platelet adhesion) 120,000
● GP Ib/IX/V - primary ● Half-life(hours): 156
platelet surface ● Mean plasma conc: 5
receptor for VWF mg/dL

IX Christmas Factor/ Factor IX deficiency = Important considerations:

Antihemophilic Factor B “Hemophilia B” or ● Platelet phospholipids (NOT in the list), particularly
(AHF-B)/ Plasma “Christmas Disease” phosphatidylserine, are considered coagulation factors
thromboplastin also. They were once called collectively as platelet factor
Component (PTC) First patient to be 3 (PF-3)
● Mol.weight (Daltons): diagnosed with ● Eight coagulation factors are enzymes that circulate in
57,000 Factor IX deficiency: an active form zymogens. These are:
● Half-life(hours): 24 Stephen Christmas ○ Prothrombin (II)
● Mean plasma conc: ○ VII
0.3 mg/dL ○ IX
○ X
X Stuart-Prower Factor ○ XI
● Mol.weight (Daltons): ○ XII
○ Prekallikrein
 ■ these 7 enzyme become serine
proteases when activated Notes:
○ XIII - becomes transglutaminase ● This concept of coagulation process is used
● The coagulation factors that functions as cofactors are: extensively to interpret in vitro laboratory tests and to
○ Tissue Factor (III) identify factor deficiencies; however, this does NOT
○ Factor V sufficiently describe the complex interdependent
○ Factor VIII reactions that occur in vivo
○ HMWK ● Thrombin: activates factors 1,5,8, and 13
● Tenase complex: includes 9a, platelet factor-3
Groups of Clotting Factors: (PF-3), Calcium, and 5a
● Important when dealing with substitution ● Calcium - involved in all phases of coagulation, except
in the contact phase
I. According to Coagulation Pathway ● Stabilized fibrin clot - NOT soluble to 5M urea

INTRINSIC EXTRINSIC COMMON CELL-BASED (IN VIVO, PHYSIOLOGIC) COAGULATION:

COAGULATION COAGULATION COAGULATION ● Aside from the procoagulant and anticoagulant plasma
PATHWAY PATHWAY PATHWAY proteins, normal physiologic coagulation needs the
FACTORS FACTORS FACTORS presence of two cell types for formation of coagulation
complexes
○ Tissue factor-bearing cells (usually
XII, XI, IX, VIII III and VII X, V, II, I
extravascular)
(12, 11, 9, 8) (3 and 7) (10, 5, 2, 1)
○ Platelets (intravascular)
Remember: There is no such thing as Factor 3 deficiency ● Coagulation in vivo can be described as occurring in two
phases:
II. According to Properties ○ INITIATION - occurs on tissue factor-bearing
cells and generates 3% to 5% of total thrombin
FIBRINOGEN PROTHROMBIN CONTACT produced
GROUP GROUP GROUP ○ PROPAGATION - occurs on platelets; produces
≥ 95% of the total bilirubin
I, V, VIII, XIII II, VII, IX, X XII, XI, PK, HMWK ● Coagulation is initiated, in vivo, on tissue factor-bearing
(1,5,8,13) (2, 7, 9, 10) (12, 11, cells. TF:VIIa activates factors IX and X, producing
Prekallikrein, enough thrombin to activate platelets and factors V, VIII,
Aka: Present in High-mol.-weight and XI. coagulation proceeds on activated platelet
THROMBIN serum except kininogen) phospholipid membranes with the formation of IXa:VIIIa
-SENSITIVE for factor II and Xa:Va complexes, which produces a burst of
GROUP thrombin that cleaves fibrinogen to fibrin

Other information COMMON TESTS FOR SECONDARY HEMOSTASIS:

I. APTT/aPTT
● Absent in ● Adsorbed ● Vitamin K-
serum from the independent ● “Partial” - because the reagent consists only of the PL
plasma using (phospholipid) portion of the tissue thromboplastin
● Vitamin K EITHER: ● Calcium- (prepared from brain or plant phospholipids)
independent and ○ Barium independent ● “Activated” - because there are negatively charged
Calcium sulfate activators to be added [solid particulate materials such as
dependent ○ Aluminu silica, kaolin, celite, and bentonite; or soluble activator,
m e.g. ellagic acid]
● Liver can ● APTT - an improvement of the PTT in that the variable of
synthesize these ● Only group contact activation is removed by the addition of an
factors without vit that is activator to obtain complete contact activation (results in
K and calcium Vitamin shortening of the PTT and narrowing of the normal
K-dependent range). Different activators have been recommended:
● Increased in P I solid particulate materials such as silica, kaolin, celite,
SO ● Calcium- and bentonite; or soluble activator, e.g. ellagic acid. It
Pregnancy dependent must NOT be assumed that all activators give
Inflammatio comparable results.
n (Acute ● Reference range: 35 to 45 seconds (generally)
Phase ● An example of a clot-based coagulation screening test
Reactants) ● Specimen requirements:
Stress ○ CITRATED platelet-poor plasma (prepared by
Oral specimen centrifugation for 15 minutes at 2500 x
Contracepti G; plasma platelet count should be <15,000/uL)
ves Intake ○ Aspirate the topmost part of the plasma
● Materials and reagents:
○ APTT reagent = two components:
PLASMA-BASED (IN VITRO) COAGULATION (INTRINSIC, ■ Platelet substitute (phospholipid) -
EXTRINSIC, and COMMON PATHWAYS): supplied to substitute for PF3
■ Activator - added to achieve maximum
activation of the contact factors
○ CaCl₂
 ○ Controls ○ To be computed ONLY for samples from patients
○ Test tubes (12 x 775-mm glass tubes) who have achieved a stable anticoagulation
○ Pipette response with Coumadin. During the first week
○ 37 C water bath of Coumadin therapy, the physician must
● Uses: interpret PT results in seconds, comparing them
○ To monitor patients on heparin therapy with the reference interval.
■ Unfractionated Heparin (UFH) - refers ○ A target INR range of 2.0 to 3.0 is
also to the Unfractionated form of recommended for most indications. The
heparin physician adjusts the Coumadin dosage to
○ Used to detect coagulation factor deficiencies in the achieve the desired INR of 2.0 to 3.0 (or 2.5 to
common and extrinsic pathways (10, 5, 2, 1 and 7) 3.5 if the patient has a mechanical heart valve).
■ Factor 3 deficiency is not included ○ Introduced to minimize the differences in PT
■ Remember: There is no such thing as results due to different reagent-instrument
Factor 3 deficiency combinations
● Procedure: ○ Formula:
○ 0.1 mL of PPP is added to 0.1 mL of APTT
reagent
○ Mixture is then incubated at 37 C (for the period
of time specified by the reagent manufacturer
[approx. 3 to 5 mins])
○ Following incubation, 0.1 mL of warmed CaCl₂
is added
○ The time for clotting to occur is then recorded
● Reporting APTT results ○ ISI stands for International Sensitivity Index
○ APTT is reported in seconds (to the nearest ○ Theoretically, the more sensitive
tenth) along with the reference range thromboplastin has ISI < 1.0, and less
II. PT - Protime or Prothrombin Time sensitive reagents have an index that is > 1.0
● Reference range: 10-13 seconds (generally) (the more sensitive the thromboplastin reagent,
● An example of a clot-based coagulation screening test the longer the resulting PT; the less sensitive
● Specimen requirements: the reagent, the shorter the resulting PT)
○ Citrated platelet-poor plasma (prepared by
specimen centrifugation for 15 minutes at 2500 RELATED TOPICS TO PT AND APTT:
X g; plasma platelet count should be Therapeutic Anticoagulants
<15,000/uL) I. Oral Anticoagulant (coumarin drugs)
● Materials and reagents: ● Examples: Warfarin - most commonly used
○ Thromboplastin-Calcium Chloride (CaCl2) ○ (Warf - “Wisconsin Alumni Research
reagent (PT reagent; Simplastin) Foundation”)
○ Controls ○ Inhibits the synthesis certain clotting
○ Test tubes (12 X 75-mm glass tubes) factors: 2, 7, 9, and 10
○ Pipette ○ A vitamin K antagonist
○ 37 C water bath ○ In cases of overdosage, administer:
● Uses: ■ Vitamin K supplement,
○ To monitor patients in Warfarin therapy (oral ■ Coagulation factor
anticoagulant) concentrates, or
○ To detect coagulation factor deficiencies in the ■ Fresh frozen plasma.
COMMON and EXTRINSIC PATHWAY
● Procedure: II. Intravenous Anticoagulant
○ Aliquot of patient plasma and control are ● Example: Heparin (Unfractionated Heparin) -
warmed according to the method being used. most commonly used
○ Warm PT thromboplastin reagent by incubating ○ In case of overdosage, administer:
it (37 C for 3 to 5 mins.). Protamine sulfate
○ 0.2 mL of PT reagent is added to 0.1 mL of ● Heparin has no anticoagulant activity of its own
plasma (patient or control). (acts as an anticoagulant by accelerating the
○ The time for clotting to occur is then recorded. binding of antithrombin to target enzymes (e.g..
● Reporting PT results: Thrombin and factor Xa))
Prothrombin time may be reported in several ● UFH (Unfractionated heparin) - used routinely
ways: in cardiac surgery. UFH therapy requires
○ Patient time with the control time (in seconds) monitoring with partial thromboplastin time
○ Patient time (in seconds) with the reference (PTT) or activated clotting time (ACT) assay. It
range is administered intravenously.
○ Prothrombin ratio (which is the PT of the patient ● ACT (Activated coagulation time)
divided by the mean of the reference range and ○ An example of a clot-based
multiplied by 100) coagulation screening test
● INR (International Normalized Ratio) ○ A point of case assay that is used in
○ Standardized way of reporting PT (NOT a new clinics, at the inpatient’s bedside, in
laboratory test) the cardiac catheterization laboratory,
○ It is merely a mathematical calculation (corrects or in the surgical suite, and it is
for the variability in PT results caused by specifically useful at the high UFH
variable sensitivities of the thromboplastin dosages.
agents used by laboratories).
 ○ In this test, a particulate activator is ○ Contains all coagulation factors
added to blood, the mixture is rocked, except Factor III
and the interval to clotting is recorded 2. Aged plasma
○ Contains all coagulation factors
● Low-Molecular-Weight-Heparin (LMWH) except Factor V and VIII (labile)
● Produced by the controlled fragmentation of heparin 3. Aged serum
● React with the regulatory protein antithrombin III to ○ Contains all coagulation factors
inhibit activated factor X (factor Xa) but not thrombin except Factors I, V, VIII, XIII, and II
(factor IIa) 4. Adsorbed plasma
● administered by subcutaneous injection ○ Contains all coagulation factors
● Some of the advantages of using LMWH include: EXCEPT factor 2, 7, 9, and 10.
○ Lowered incidence of heparin-induced
thrombocytopenia compared with POSSIBLE CASES:
unfractionated heparin ● CASE 1:
○ Lowered incidence of hemorrhage and ○ PT = Prolonged
osteoporosis ○ APTT = Prolonged
● Some of the disadvantages of using LMWH include: ❖ COMMON PATHWAY (10, 5, 2, or 1)
○ Increased cost compared with traditional
(unfractionated) heparin ● CASE 2:
○ More complicated monitoring of LMWH than ○ PT: Normal
traditional heparin ○ APTT: Prolonged
❖ INTRINSIC PATHWAY (12, 11, 9, or 8)
Coagulation Instrumentation
- Instrument methodologies used for coagulation testing ● CASE 3:
are classified into five groups based on the end-point ○ PT: Prolonged
detection principle: ○ APTT: Normal
1. Mechanical/ Electromechanical ❖ EXTRINSIC PATHWAY (7)
● Examples:
● BBL Fibrometer (uses electromechanical clot
detection system that measures a change in
conductivity between two metal electrodes in SAMPLE PROBLEMS:
plasma) ● PROBLEM 1:
● Diagnostica Stago analyzers (In these ○ PT = Normal
analyzers, an electromagnetic field detects the ○ APTT = Prolonged
oscillation of a steel ball within the 1. APTT + Fresh plasma = Corrected
plasma-reagent solution. Viscosity starts to 2. APTT + Aged plasma = Not Corrected
increase as fibrin strands form, slowing the 3. APTT + Aged serum = Not Corrected
movement. When the oscillation decreases to a 4. APTT + Adsorbed plasma = Corrected
predefined rate, the timer stops, indicating the ❖ FACTOR VIII DEFICIENCY
clotting time of the plasma.)
2. Photo-optical (Turbidimetric)
● Coagulometers that use this principle detect a III. Stypven time (Russel’s Viper Venom Time)
change in plasma optical density during clotting. ● used to detect deficiencies in fibrinogen, prothrombin,
3. Nephelometric and factors V and X
● A modification of photo-optical end-point ● differs from the PT in that deficiencies in factor VII are
detection NOT detected
● Here, 90-degree or forward-angle light scatter, ● uses the coagulant properties of Russel’s viper venom,
rather than OD, is measured obtained from the snake Vipera russeli
4. Chromogenic (amidolytic) ● venom is capable of bypassing the action of Factor VII
● Uses a synthetic oligopeptide substrate and directly activating Factor X to Xa.
conjugated to a chromophore, usually ● may help differentiating Factor VII and Factor X
para-nitroaniline (pNA) deficiencies
● A means for measuring specific coagulation ● Normal Value: 20 to 25 seconds
factor activity because it uses the factor’s **Dilute Russel’s Viper Venom Time (dRVVT) - used to detect
enzymatic (protease) properties lupus anticoagulants
● Useful to evaluate specimens from patients who - Normal value: 30 to 35 seconds
have circulating inhibitors or who are on
anticoagulant treatment because the inhibitors IV. Thrombin Time
do not interfere in the chromogenic assay ● Prolonged thrombin times are found when the fibrinogen
5. Immunologic level is low, when function of fibrinogen is impaired, and
● Based on antigen-antibody reactions similar to in the presence of heparin, fibrin(ogen) degradation
those used in nephelometry products and streptokinase
Substitution Test ● TT is a sensitive test in detecting heparin inhibition
- may be used to identify specific factor deficiency by ● Normal Value: 15 to 18 seconds
mixing correction reagents with patient’s plasma
and then performing the PT and/or APTT V. Reptilase time
- may also be used to detect lupus anticoagulant ● Reptilase is an enzyme found in the
and specific factor inhibitors venom of the Bothrops atrox snake and
- uses the following: is capable of converting fibrinogen to
1. Fresh plasma fibrin.
 ● Reptilase is unaffected by heparin disease
● Normal Value: 10 to 15 seconds Circulating
Thrombin Times and Reptilase Times in Different Conditions anticoagulant (e.g.
lupus)

Conditions Thrombin Time Reptilase Time Vit K deficiency

Oral anticoagulants
Prolonged Prolonged N N
Hypofibrinogenemia Prolonged Prolonged Factor V, X, II
deficiency
Immunologic Prolonged Normal
Heparin (large
antithrombins
amount)
Prolonged Prolonged Prolonged N Liver disease
Heparin therapy Prolonged Normal Fibrinogen
deficiency/disorder

VI. Clotting Time N N N Low Thrombocytopenia

● Slide or Drop Method
○ Perform proper skin puncture procedure and Disseminated
wipe off the first drop of blood. Prolonged Prolonged Prolonged Low intravascular
○ Start timer as soon as the second drop of blood coagulation
appears.
○ Transfer the second drop of blood onto the **Adapted from: Dacie and Lewis - Practical Hematology, by:
center of the slide. Bain, Bates, Laffan, and Lewis
○ Pass the tip of a needle through the drop of
blood every 30 secs. and note the formation of EXAMPLES OF DRUGS AFFECTING THE COAGULATION
fibrin strands. SYSTEM:
○ Stop timer as soon as fibrin strands are seen
clinging at the tip of the needle. DRUG EFFECTS
○ N.V.= 2 to 4 mins
Penicillin G - Weakens platelet function, platelet
● Lee and White (Whole Blood Clotting Time) Method (carbenicillin and aggregation, and interaction of vWF with
○ Place three test tubes (labeled as tube 1, 2 and ticarcillin) platelets
3) in a 37 C water bath. - Increases bleeding time
○ Place 1 mL blood into each of the three tubes,
starting with tube 1. DDAVP - Increases plasma levels of VIII:C by
○ Start timer as soon as blood enters tube 1. (1-desamino-8-D releasing endogenous protein from
○ Gently tilt tube 3 every 30 secs. and observe for -arginine body storage sites
clotting. vasopressin) - Can shorten the APTT
○ After clotting is observed in tube 3, observe
tube 2. When clotting is observed in tube 2,
Isoniazid (INH) -Can produce thrombocytopenia
observe tube 1.
-Increases bleeding time
○ When no flowing of blood is
observed upon tilting tube 1,
Aspirin products -Prolongs bleeding time by inhibiting
stop the timer. Clotting time is
thromboxane A 2 synthesis in the platelets
the time it takes for blood to
coagulate in tube 1.
○ N.V. = 7 to 15 mins Phenothiazines -Stimulates the production of a “lupus
anticoagulant” in circulation
SAMPLE RESULTS FOR SOME HEMOSTATIC TESTS -Prolongs the APTT

Phenylbutazone Weakens platelet function but enhances

TESTS the effect of warfarin (may increase the PT
RELATED in a patient on both drugs)
Prothromb Plt CONDITIONS -May prolong the bleeding time
APTT TT
in time count
Metronidazole -Improves the effect of warfarin in a
Normal hemostasis
patient on both drugs
Disorder of platelet
function -Can prolong the PT
Factor XIII
N N N N deficiency Streptomycin -Stimulates formation of inhibitors to
Disorder of vascular Factor V
hemostasis -Can prolong the PT and possibly the
Disorder of APTT
fibrinolysis

Prolonged N N N Factor VII deficiency CIRCULATING INHIBITORS (CIRC. ANTICOAGULANTS):

● Acquired inhibitors of coagulation
Factor VIII, IX, XI, ● Usually detected by the prolongation of the PT or APTT
XII, Prekallikrein, which is NOT corrected by the addition of normal (fresh)
N Prolonged N N
HMWK deficiency plasma
Von Willebrand’s ● Two groups of inhibitors include:
 Plasminogen ● a plasma zymogen produced by the
Specific Inhibitors Non-specific inhibitors liver
● stored and transported in eosinophils
● Antibodies against ● Not directed against specific ● Fibrin-bound plasminogen become
specific coagulation coagulation factors converted into active plasmin
factors Example: ● plasmin is a serine protease (bound
● Associated with 1. Lupus anticoagulant plasmin digests clot and restores blood
bleeding ● IgG, IgM, IgA vessel patency)
● Examples: Antibodies ● Found in 10% of SLE ● free plasmin is capable of digesting
to factors VIII and IX patients plasma fibrinogen, factor V, factor VIII,
(MOST COMMON) ● Normal or slightly and fibronectin, causing a potentially
increased PT and deadly primary fibrinolysis (however,
increased APTT plasma α2-antiplasmin rapidly binds
● Directed against and inactivates any free plasmin in the
phospholipids and are circulation)
generally not associated
with bleeding Tissue ● Serine protease secreted by activated
● Tests: plasminogen endothelium, activates plasminogen
○ Platelet activator (TPA) ● Circulating TPA is bound to inhibitors
neutralization test such as PAI-1 and is cleared from
○ Dilute RVVT plasma
○ Substitution test
2. Paraproteins Urokinase ● Another intrinsic plasminogen activator
3. Fibrinogen degradation Plasminogen ● Secreted by urinary tract epithelial
products Activator (UPA) cells, monocytes
● UPA does NOT bind firmly to fibrin,
and has a relatively minor physiologic
NATURALLY OCCURRING INHIBITORS OF COAGULATION
effect
● TWO MAJOR ANTICOAGULANT SYSTEMS IN THE
BODY:
1. PROTEIN C/PROTEIN S SYSTEM Plasminogen ● PAI-1 is the principal inhibitor of
■ Protein C = activated by thrombin Activator plasminogen activation
bound to the thrombomodulin Inhibitor 1 ● Inhibits both TPA and UPA and thus
■ Activated Protein C = INACTIVATES (PAI-1) preventing them from converting
the factors Va and VIIIa) plasminogen to plasmin
■ Protein S = Cofactor to protein-C
α2-antiplasmin ● inhibits plasmin, synthesized in the
2. PLASMA SERINE PROTEASE INHIBITOR liver
SYSTEM (serpins) ● primary inhibitor of free plasmin
■ Antithrombin (formerly antithrombin III) ● ε-aminocaproic acid and tranexamic
is the main serine protease inhibitor acid = antifibrinolytic (both inhibit the
■ Antithrombin inhibits: IIa, VIIa, IXa, Xa, proteolytic activity of plasmin)
XIa, prekallikrein, XIIa
Thrombin-Activa ● synthesized in the liver
table Fibrinolysis ● becomes activated by the
Inhibitor (TAFI) thrombin-thrombomodulin complex
FIBRINOLYSIS (the same complex that activates the
● Final stage of coagulation and is the dissolution of the protein C pathway; however, the two
formed clot functions are independent)
● Starts a few hours after fibrin polymerization and ● activated TAFI functions as an
cross-linking antifibrinolytic enzyme
● TPA and UPA - two activators of fibrinolysis that are TESTS FOR FIBRINOLYSIS
released in response to inflammation and coagulation A. D-dimer test
● Fibrinolytic proteins accumulate on fibrin during clotting. ● Plasmin lyses the fibrin and produces FDP that
Plasminogen, plasmin, TPA, UPA, and PAI-1 become contain the portion called D-dimer
incorporated into the fibrin clot ● Plasmin will also lyse fibrinogen , but will NOT
● TPA and UPA activate fibrin-bound plasminogen several produce the D-dimer
hours after clot formation, degrading fibrin and ● The presence of D-dimer indicates that a stable
reestablishing normal blood flow during vascular repair fibrin clot has been lysed.
● FSP/FDP (+) = DIC or pathological fibrinolysis
○ FDP(+), D-dimer (-) = Pathological
PROTEINS OF THE FIBRINOLYSIS PATHWAY fibrinolysis
○ FDP(+), D-dimer (+) = DIC
Name Comments
B. Euglobulin Lysis Time
● Euglobulins -proteins that precipitate when
plasma is diluted with water and acidified (1%
acetic acid)
 ○ include plasminogen, plasmin,
fibrinogen, and plasminogen activators
● PROCEDURE:
○ patient’s citrated PPP is diluted w/
water, acidified and refrigerated
○ euglobulins will precipitate, which is
then redissolved and clotted with
thrombin
○ if plasminogen (in the euglobulin
fraction) is converted to plasmin, it will
lyse the clot
○ time needed for complete lysis (at 37
C) is the euglobulin lysis time
● Euglobulin lysis time is typically longer than 2
hours.
● Lysis in less than 2 hours = increased
fibrinolytic activity

“Sunrise, show my weary heart, that

A new day will soon arrive”

🔅🔆 🔅
- Sunrise; Ben&Ben

Good luck and God Bless!