PLATELETS

Maturation sequence of megakaryoblast takes about 5 days. Platelets are produced directly from the mekaryocyte
cytoplasm. As the megakaryocyte matures, clusters of granules aggregate to form platelets.

1. Megakaryoblast
20-50 um diameter
Blue cytoplasm
N/C ratio is about 10:1
Multiple nucleoli
Fine chromatin

2. Promegakaryocyte
20-60 um diameter
Less basophilic cytoplasm
Chromatin becomes coarse
Irregularly shaped nucleus, may show show
slight lobulation
N/C ratio is 4:1 to 7:1

3. Granular megakaryocyte






4. Mature megakaryocyte
40-120 um diameter
Cytoplasm contains coarse clumps of granules
aggregating into little bundles, which bud off
from the periphery to become platelets
Multiple nuclei are present
No nucleoli is visible
N/C ratio is less than 1:1

5. Platelet/thrombocyte
1-4 um diameter
Light blue to purple, very granular
o Chromomere granular and located
centrally
o Hyalomere surrounds the chromomere,
nongranular and clear to light blue
MORPHOLOGIC DIFFERENTIATION OF MEGAKARYOCYTIC CELL SERIES

Maturation Stage Cytoplasmic
Granules
Cytoplasmic
Tags
Nuclear Features Thromocytes
Visible
Megakaryoblast Absent Present Single nucleus, fine
chromatin, nucleoli
No
Promegakaryocyte Few Present Double nucleus No
Megakaryocyte Numerous Usually absent Two or more nuclei No
Metamegakaryocyte Aggregated Absent Four or more nuclei Yes

PLATELET STRUCTURE

Composed of 60% protein, 30% lipid, 8% carbohydrate, various minerals, water and nucleotides
Divided anatomically into four areas: peripheral zone, sol-gel zone, organelle and membranous system

1. Peripheral zone
Glycocalyx
Plasma membrane
Submembranous area

2. Sol-gel zone
Microfilaments: actin and myosin
Microtubules

3. Organelle zone
Alpha, dense granules
Mitochondria, lysosomal granules

4. Membranous system
Dense tubular system
Open canalicular system
(surface connecting system)
SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN
HEMOSTASIS

ROLE IN
HEMOSTASIS
SUBSTANCE SOURCE COMMENTS ON PRINCIPAL
FUNCTION
Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation
pathway
Fibrinogen Alpha granules Converted to fibrin for clot formation
Factor V Alpha granules Cofactor in fibrin clot formation
Factor VIII:vWF Alpha granules Assists platelet adhesion to subendothelium
to provide coagulation surface

Promote aggregation ADP Dense bodies Promote platelet aggregation
Calcium Dense bodies Same
Platelet factor 4 Alpha granules Same
Thrombospondin Alpha granules Same
Promote
vasoconstriction
Serotonin Dense bodies Promotes vasoconstriction at injury site
Thromboaxane A
2

precursors
Membrane
phospholipids
Same
Promote vascular
repair
Platelet-derived growth
factor
Alpha granules Promotes smooth muscle growth for vessel
repair
Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vessel
repair
Other systems
affected
Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis

2
antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis
C1 esterase inhibitor Alpha granules Complement system inhibitor

HEMOSTASIS

Process that retains the blood within the vascular system during periods of injury, localizes the reactions involved
to the site of injury, and repairs and re-establishes blood flow through the injured vessel
A system in dynamic balance that when tipped by deficiencies (congenital or acquired) of the procoagulant portion
or excesses of the fibrinolytic portion, results in uncontrolled bleeding (hemorrhage); when tipped by deficiencies
(congenital or acquired) of the fibrinolytic portion or uncontrolled activation of the procoagulant portion, the result
is excessive clot formation or persistence of clot (thrombosis)

SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS
ROLE IN
HEMOSTASIS
SUBSTANCE SOURCE COMMENTS ON PRINCIPAL
FUNCTION
Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation
pathway
Fibrinogen Alpha granules Converted to fibrin for clot formation
Factor V Alpha granules Cofactor in fibrin clot formation
Factor VIII:vWF Alpha granules Assists platelet adhesion to subendothelium to
provide coagulation surface
Promote
aggregation
ADP Dense bodies Promote platelet aggregation
Calcium Dense bodies Same
Platelet factor 4 Alpha granules Same
Thrombospondin Alpha granules Same
Promote
vasoconstriction
Serotonin Dense bodies Promotes vasoconstriction at injury site
Thromboaxane A
2

precursors
Membrane
phospholipids
Same
Promote vascular
repair
Platelet-derived
growth factor
Alpha granules Promotes smooth muscle growth for vessel
repair
Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vessel
repair
Other systems
affected
Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis

2
antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis
C1 esterase inhibitor Alpha granules Complement system inhibitor


BASIC TERMINOLOGY FOR CLINICAL FINDINGS IN BLEEDING DISORDERS:
1. Petechiae purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary ability to withstand
normal blood pressure and trauma
2. Purpura hemorrhage of blood into small areas of skin, mucous membranes, and other tissues
3. Ecchymosis form of purpura in which blood escapes into large areas of skin and mucous membranes, but not
into deep tissues
4. Epistaxis nosebleed
5. Hemarthosis leakage of blood into joint cavities
6. Hematemesis vomiting of blood
7. Hematoma swelling or tumor in the tissues or a body cavity that contains clotted blood
8. Hematuria rbc in urine
9. Hemoglbinuria hb in urine
10. Melena stool containing dark red or black blood
11. Menorrhagia excessive menstrual bleeding

COAGULATION FACTORS
1. Coagulation factors are also known as enzyme precursors or zymogens. They are found in plasma along with
nonenzymatic cofactors and calcium,
2. Zymogens are substrates having no biologic activity until converted by enzymes to active forms called serine
proteases.
a. The zymogens include II, VII, IX, X, XI, XII and prekallikrein
b. The serine proteases are IIa, VIIa, IXa, Xa, XIa, XIIa and kallikrein
3. Cofactors assist in the assist in the activation of zymogens and include V, VIII, tissue factor, and HMWK
4. In its active form, factor XIII is a transglutamase.
5. Fibrinogen is the only substrate in the cascade that does not become an activated enzyme.

Blood factors are produced mostly in the LIVER and circulate in an inactive precursor form.

COAGULATION FACTOR NOMENCLATURE WITH PREFERRED NAMES AND SYNONYMS

NUMERAL PREFERRED NAME SYNONYMS
I Fibrinogen
II Prothrombin Prethrombin
III Tissue factor Tissue thromboplastin
IV Calcium
V Proaccelerin Labile factor
Accelerator globulin (aCg)
VII Proconvertin Stable factor
Serum prothrombin conversion accelerator (SPCA)
VIII:C Antihemophilic factor (AHF) Antihemophilic factor globulin (AHG)
Antihemophilic factor A
Platelet cofactor 1
IX Plasma thromboplastin component (PTC) Christmas factor
Antihemophilic factor B
Platelet cofactor 2
X Stuart-Prower factor Stuart factor
Prower factor
Autoprothrombin III
XI Plasma thromboplastin antecedent Antihemophilic factor C
XII Hageman factor Glass factor
Contact factor
XIII Fibrin stabilizing factor Laki-Lorand factor
Fibrinase
Plasma transglutaminase
Fibrinoligase
- Prekallikrein Fletcher factor
- High-molecular-weight kininogen Fitzgerald factor
Contact activation cofactor
Williams factor
Flaujeac factor



SCHEMATIC DIAGRAM OF
PHYSIOLOGIC HEMOSTASIS. Note that
the contact phase factors comprising
factor XII, HK (high molecular weight
kininogen) and PK (prekallikrein) are shown
grayed; although factor XI can be activated
by this route under artificial in vitro
conditions such as in the PTT test (see Fig.
38-8 ), this pathway is not believed to
contribute to normal physiologic
hemostasis. Similarly, whereas Tissue
factor/VIIa can directly activate X to Xa in
the in vitro PT test under conditions which
supra-physiological concentrations of
Tissue factor are employed, this reaction is
shown as grayed because it does not
contribute significantly to clot formation
under normal in vivo physiological
conditions. Normal clotting in vivo
accordingly is initiated when sufficient
Tissue factor/VIIa becomes available to
activate factor IX to IXa. Subsequently, IXa
in the presence of VIIIa activates X to Xa,
which in turn activates prothrombin to
thrombin in the presence of Va. Thrombin
not only then proceeds to clot fibrinogen
and to activate platelets (see Fig. 38-2 ),
but additionally exerts critically important
positive feedback by activating factors VIII
and V. Thrombin has further been shown
capable of activating factor XI, thereby
providing an additional pathway for the
activation of factor IX. PL (phosopholipid
present on the surface membranes of
platelets in vivo) and Ca
++
(calcium ions)
contribute to reactions as indicated.










CHARACTERISTICS OF COAGULATION FACTORS
Factor Active Form Pathway
Participation
Vitamin K
Dependent
Present in BaSO
4

Adsorbed Plasma
I
Fibrin clot Common No Yes
II
Serine protease Common Yes No
V
Cofactor Common No Yes
VII
Serine protease Extrinsic Yes No
VIII:C
Cofactor Intrinsic No Yes
IX
Serine protease Intrinsic Yes No
X
Serine protease Common Yes No
XI
Serine protease Intrinsic No Yes
XII
Serine protease Intrinsic No Yes
XIII
Transglutaminase Common No Yes
Prekallikrein
Serine protease Intrinsic No Yes
HMWK
Serine protease Intrinsic No Yes

INHIBITORS OF COAGULATION
Major site of inhibition: endothelium and platelet
1. Protein C degrades factor Va and VIIIa
2. Protein S degrades factor Va and VIIIa
3. Antithrombin III major inhibitor of thrombin, also inhibits factors IXa, Xa, XIa, XIIa, kallikrein and plasmin
4. Heparin cofactor II inhibit thrombin
5.
2
macroglobulin forms a complex with thrombin, kallikrein and plasmin, thus inhibiting their activities
6. Extrinsic pathway inhibitor (EPI)
Lipoprotein assoc. coagulation inhibitor (LACI) inhibits the VIIa-tissue factor complex
7. C1 inhibitor inactivator of factor XIIa and kallikrein, it also inhibits factor XIa and plasmin
8.
1
antitrypsin inhibitor of thrombin, Xa and XIa

DISORDERS OF COAGULATION CAUSING CLOTTING FACTOR DEFICIENCIES
FACTOR INHERITED COAGULOPATHIES ACQUIRED COAGULOPATHY
INHERITANCE
PATTERN
COAGULOPATHY
I Autosomal recessive

Autosomal dominant
Afibrinogenemia

Dysfibrinogenemia
Severe liver disease
Diffuse intravascular coagulation
Fibrinolysis
II Autosomal recessive Prothrombin deficiency Liver disease
Vit K deficiency
Anticoagulant therapy
V Autosomal recessive Factor V deficiency
(OWRENS dis, Labile factor
def)
Severe liver disease
Diffuse intravascular coagulation
Fibrinolysis
VII Autosomal recessive Factor VII deficiency Liver disease
Vit K deficiency
Anticoagulant therapy
VIII X-linked recessive

Autosomal dominant
Hemophilia A

vWD
Diffuse intravascular coagulation
Fibrinolysis
IX X-linked recessive Hemophilia B Liver disease
Vit K deficiency
Anticoagulant therapy
X Autosomal recessive Factor X deficiency Liver disease
Vit K deficiency
Anticoagulant therapy
XI Autosomal recessive Hemophilia C
(common in Eastern
European Jewish descent/
Ashkenazi Jews)
*
XII Autosomal recessive Factor XII deficiency *
XIII Autosomal recessive Factor XIII deficiency Liver disease
Diffuse intravascular coagulation
Fibrinolysis
Prekall Autosomal recessive Fletcher trait *
HMWK Autosomal recessive Fitzgerald trait *
* Unclear whether any acquired disorders cause factor XI or XII deficiencies or prekallikrein or HMK deficiency
CLASSIFICATION OF VON WILLEBRAND DISEASE
TYPE DESCRIPTION
1 Partial quantitative deficiency of von Willebrand factor (vWF)
2 Qualitative deficiency of vWF
2A Decreased platelet-dependent vWF function with selective deficiency of high-molecular-weight multimers
2B Increased affinity for platelet glycoprotein Ib
2M Decreased platelet-dependent vWF function with high-molecular-weight multimers present
2N Markedly decreased binding of factor VIII to vWF
3 Complete deficiency of vWF

Lee and White clotting time method
Equipment: water bath 37
o
C; glass test tubes 13 x 100 mm; stopwatch and plastic syringe (10 mL) and 20-
gauge needle. (Brown page 215)
From Brown
Prothrombin Time:
Test tubes, 13 x 100 mm 0.1 mL patients plasma 0.2 mL (200 L) thromboplastin-calcium
reagent
APTT:
13 x 100 mm tube 0. 2mL plasma 0.2 mL APTT reagent 0.2 mL
CaCl
2

From Steiniger
Prothrombin Time:
0.1 mL plasma 0.2 mL (200 L) PT reagent
APTT:
12 x 75 mm tube 0.1 mL PPP 0.1 mL APTT reagent 0.1 mL
CaCl
2



DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION SCREENING TESTS
Abnormal partial thromboplastin time (PTT) alone
Associated with bleeding: VIII, IX, XI defects
Not-associated with bleeding: XII, prekallikrein (PK), high-molecular-weight kininogen, lupus anticoagulants
Abnormal prothrombin time (PT) alone
Factor VII defects
Combined abnormal PTT and PT
Medical conditions: anticoagulants, DIC, liver disease, vitamin K deficiency, massive transfusion
Rarely dysfibrinogenemias, factors X, V, and II defects

FAMILIES OF COAGULATION PROTEINS
THROMBIN-SENSITIVE
COAGULATION PROTEINS
PROTHROMBIN FAMILY CONTACT FAMILY
I, V, VIII and XIII
Not vitamin K dependent
Consumed during coagulation
Absent in serum
Present in adsorbed plasma
II, VII, IX and X
Vitamin K dependent
Present in serum (except II)
Absent in adsorbed plasma
XII, XI, prekallikrein,
HMWK
Not vitamin K dependent
Present in serum
Present in adsorbed plasma

SUBSTITUTION STUDIES

DEF. PT APTT TT SUBSTITUTION STUDIES
Normal Plasma Adsorbed Plasma Aged Serum
I Abn Abn Abn C C NC
II Abn Abn N C NC NC
V Abn Abn N C C NC
VII Abn N N C NC C
VIII N Abn N C C NC
IX N Abn N C NC C
X Abn Abn N C NC C
XI or XII N Abn N C C C
N Normal NC NOT CORRECTED
Abn Abnormal C CORRECTED
PT Prothrombin Time
APTT Activated Partial Thromboplastin Time
TT Thrombin Time
CIRCULATING ANTICOAGULANTS
Prolonged APTT and PT not corrected
Inactivate an activated coagulation factor or block interaction between coagulation factors and platelets
Ex Lupus inhibitor
Nonsp anticoagulant
IgG, IgM and IgA which interfere with phospholipid portion of the complex:
Xa-Va-calcium-plt phospholipid
Platelet neutralization procedure
Dilute Russell Viper Venom time

INSTRUMENTATION FOR TESTS OF HEMOSTASIS
1. Visual detection of fibrin clot formation
Tilt tube method

2. Electromechanical detection of fibrin clot formation
Fibrin strand formation is detected using a wire
loop or hook; has been incorporated into a semi-
automated mechanical instrument
Instrument: FIBROMETER



3. Photo-optical detection of fibrin clot formation
Detection of fibrin clot formation depends on the
increase in light scattering associated with the
conversion of soluble fibrinogen molecules to the
insoluble polymerized fibrin clot
Semi-automated instruments: Electra 750 and
750A, Fibrintimer series, and FP 910 Coagulation
Analyzer
Automated instruments: Ortho Koagulab 16S and
40A, the Coag-A-Mate X2 and XC, and the MLA
Electra 700 and 800
FIBRINOLYSIS
Digestion of fibrin clot, keeps the vascular system free of deposited fibrin/fibrin clot
Occurs when plasminogen is converted to plasmin

PLASMINOGEN ACTIVATORS
1. Intrinsic activators
Factor XIIa
Kallikrein
HWK
2. Tissue type
Urokinase-like PA

3. Therapeutic activators
Treatment for thromboemboli
Streptokinase
Urokinase
Tissue-like PA

INHIBITORS OF FIBRINOLYSIS
1.
2
antiplasmin ________________
2.
2
macroglobulin
3. Thrombospondin
4. PA inhibitor 1 and 2


Degradation of cross-linked fibrin by plasmin Degradation of fibrinogen and non-crosslinked fibrin
by plasmin


PATHOLOGIC FIBRINOLYSIS
PRIMARY FIBRINOLYSIS
Excessive amounts of plasminogen activators from damaged cells/malignant cells
Converts plasminogen to plasmin in the absence of fibrin formation

SECONDARY FIBRINOLYSIS
DIC: uncontrolled, inappropriate formation of fibrin within the blood vessels
Infection; Neoplasm; Snake bite; HTR