SEM423 Hema 2 Week 1  Dense granules- calcium, ADP, serotonin, ATP,

General Characteristics of platelets: Pyrophosphate

1.  Mitochondria- for energy production
2.
3. 4. Membranous system
4.  Dense tubular system- serves as the site of
5. arachidonic acid metabolism, functions as a calcium-
6. sequestering pump that maintains platelet
7. cytoplasmic calcium levels
 Open canalicular system/ Surface Connecting
Megakaryocytopoiesis/Thrombopoiesis- platelets System- release of granules, provides direct
are produced directly from the megakaryocyte cytoplasm. communication between intracellular and
extracellular compartments
*Thrombopoietin: primary hormone influencing
platelet maturation.
Hemostasis-process that retains blood within the vascular
*Endomitosis-nuclear division without system during periods of injury, localizes the reactions
cytoplasmic division, each megakaryocyte produces 2,000 to involved to the site of injury, and repairs and re-establishes
4,000 platelets. blood flow through injured vessel
- Stoppage of blood flow
Stages of Platelet Maturation: (Megakaryocytic - Involves the interaction of blood vessels, platelets,
system involves FOUR Stages) coagulation, fibrinolysis, and tissue repair.
Stage I Megakaryoblast- earliest recognizable stage,
round oval or maybe kidney shaped nucleus, deeply Steps in hemostatic response
basophilic (blue) color of cytoplasm; 20-50 um; NC Ratio: 10:1 1. Injury to endothelium or vasculature
2. Primary hemostasis-formation of primary hemostatic plug
3. Secondary hemostasis- formation of fibrin clot
Stage II Promegakaryocyte- irregular shape, horseshoe/
4. Fibrinolysis-removal of clots
irregularly- shaped nucleus, less basophilic cytoplasm; 20-
60um
Primary Hemostasis
- Vascular system: arteries, capillaries & veins
Stage III Granular Megakaryocyte- smaller in size
- Platelets
nucleus, pinkish cytoplasm; in this stage the granules become
 Vasoconstriction- first response to injured vessel,
prominent.
initiated by serotonin & thromboxane A2
Substances released by the Endothelial cells:
Stage IV Mature Megakaryocyte- largest cell in bone 1. Prostacyclin (PGI2)- inhibits platelet activation;
marrow, totally pink cytoplasm; gives rise to 2,000-4,000 stimulates vasodilation. Act as an anticoagulant
platelets per megakaryocyte. and reduces blood flow rate.
2. Adenosine- stimulates vasodilation. Reduces
Platelet/Thrombocyte- light blue to purple and very blood flow rate.
granular cytoplasm, life span: 8-11 days/9-12 days 3. Thrombomodulin- endothelial surface receptor
*_________________________: spleen for thrombin and enhances fibrinolytic activity
*_________________________: peripheral blood for Protein C. Act as an anticoagulant and for
*Platelet count is higher in patients without a spleen fibrinolytic activity.
(___________________) 4. Heparan Sulfate- coats endothelial cell surface
*Platelet count is lower in patients with splenomegaly and weakly enhances activity of anti-thrombin
(___________________) III. Act as an anticoagulant.
5. Tissue plasminogen activators- converts
Platelet Structure: plasminogen to plasmin. Fibrinolytic
1. Peripheral Zone 6. Von Willebrand factor (vWF)- secreted by
 Glycocalyx- outer space, provides surface for coagulation endothelium and are required for platelet
factors adhesion. Coagulation.
 Plasma membrane-consists of 30 or more glycoproteins,
phospholipids & platelet VIII-vWF  Formation of platelet plug
GpIb: receptor for vWF 1. Adhesion- exposed subendothelial surface
GpIIb/IIIa: receptor for fibrinogen (collagen in vivo, glass in vitro), occurs in the
GpVa: receptor for thrombin presence of vWF (receptor: GpIb)
 Submembranous area Von Willebrand Dse: No vWF
Bernard Soulier Syndrome (Giant platelet
2. Sol-Gel zone syndrome): Lacks GpIb
 Microfilaments
Actin and Myosin- responsible for clot retraction, aka 2. Activation- platelets undergo viscous
actomyosin or thrombosthenin (Contractile unit of metamorphosis/ pseudopod formation
platelet) - morphologic and functional changes
 Microtubules- responsible for maintaining shape in platelets, agonists: ADP, thromboxane A2, PAF,
collagen, thrombin
3. Organelle zone *arachidonic acid to thromboxane A 2 by
 Alpha granules- platelet factor, platelet-derived growth cyclooxygenase
factor, platelet fibrinogen, factor V, vWF, β-
thromboglobulin, thrombospondin, fibronectin and 3. Secretion- release of platelet granules
platelet albumin Alpha Granules (Gray Platelet syndrome)
 Dense Granules (Wiskott-Aldrich Syndrome, Platelet count 1: routine procedure, collection
Hermansky Pudlack Synbdrome, Chediak Higashi Platelet count 2: collected through glass bead
Syndrome) collecting system
Calcium- involved mostly in coagulation
ADP- stimulates platelet aggregation % PA= PC1-PC2 X100
Serotonin- vasoconstrictor PC1

4. Aggregation- platelet attachment to each other 5. Clot Retraction Time

through the receptor receptor: GpIIb-IIIa, Normal clot retraction: normal number of
fibrinogen and calcium. Exposure of functioning platelets, calcium, ATP, normal
phospholipids on the platelet surface, providing fibrinogen level and interaction with thrombin
a site for fibrin formation and thrombogenesis. a. Castor oil/Hirschboeck-N.V 15-45 mins,
formation of dimpling/ droplet like serum
Laboratory Tests on the surface of blood drop
1. Platelet count- N.V 150-400 x109/L, area: 1 mm2, b. Stefanini- 3-5 ml blood (37OC) 1/2/16/18/24
dilution: 1:100 hours
Direct Methods: Normal: appreciable within 1 hour,
A. Tokantin Method: complete with 18-24 hours
- Rees-Ecker-diluent c. McFarlane-5 ml blood 37OC (1 hour)
- Sodium Citrate- anticoagulant CR= vol.serum x100
- Formalin- Preservative TV
- Brilliant Cresyl Blue- stain N.V 44-67%
- Light microscope Abnormal: thrombocytopenia, low or
B. Brecker-Cronkite Method: abnormal fibrinogen, paraproteinemias and
- Uses 1% ammonium oxalate as Glanzmann’s thrombasthenia
diluting fluid
- REFERENCE METHOD for manual 6. Capillary Fragility Test/ Torniquet Test/ Rumpel
platelet count. Leede Test
C. Unopette method - Detects abnormality of the capillaries
- Uses tripotassium EDTA and due to a structural weakness in the
ammonium oxalate capillary walls or thrombocytopenia
- Principle: Hemolysis of red cells by - Abnormal: Hereditary telangiectasia,
hypotonicity and complete block of may be normal in hemophilia and Vit. K
platelet activity by chelating Mg def.
and Ca with EDTA and ammonium - positive test is found in
oxalate. thrombocytopenia, decreased fibrinogen
and in vascular purpura
2. Bleeding Time-primary screening test; the - inflate BP cuff to a point hallway
actual time it takes for a standard wound to stop between the systolic and diastolic
bleeding. pressures (never exceed 100 mmHg),
Detects: maintain pressure for 5 mins. Remove
A. Abnormalities of platelet number and BP cuff and wait for 5 to 10 minutes
function before proceeding, count petechiae.
B. Factor VIII:VWF deficiency 1+= few petechiae on the anterior part of the
C. Abnormalities of vessel wall structure forearm
Methods: 2+=many petechiae on the anterior part of the
*Duke Method: fingertip/earlobe, N.V 2-4 mins forearm
3+= multiple petechiae over the whole arm and
*Ivy Method: blood pressure cuff was inflated to back of the hand
40 mmHg, result wound was blotted every 30 4+= confluent petechiae on the arm and back of
seconds with filter paper until bleeding stopped, the hand
N.V 3-6 mins 7. Platelet Estimation on PBS
- 7-21/ 8-20 platelets/OIF
Platelet Bleeding Probable disease or cause - Count 10 OIF
Count Time Reporting of Platelet Estimate:
Normal Prolonged Qualitative platelet defect Estimate Reporting
VWD 0-49,000 /uL Markedly decrease
Vessel wall structure abnormality 50,000-99,000/ uL Moderately decreased
Low Normal Autoimmune thrombocytopenia 100,000- 149,000/ uL Slightly Decreased
Low Very Qualitative and Quantitative 150,000- 199,000/ uL Low Normal
Prolonged Platelet Deficiency 200,000-400,000/ uL Normal
401,000- 599,000/ uL Slightly Increased
3. Platelet aggregation- in vitro test to determine 600,000- 800,000/ uL Moderately Increased
the ability of platelets to aggregate with certain  800,000/ uL Markedly Increased
agonist: Epinephrine, Collagen, ADP, Ristocetin,
PRP + agonist -> O.D monitored DISORDERS OF HEMOSTASIS
A. BASIC TERMINOLOGIES
4. Platelet Adhesiveness (Salzmann)- measure 1. Petechiae – purplish, red, pinpoint hemorrhagic spots in
ability of platelets to adhere in glass surfaces, the skin caused by loss of capillary ability to withstand normal
N.V 26-60% blood pressure and trauma
 2. Purpura – hemorrhage of blood into small areas of skin,  Autoimmune Vascular Purpura
mucous membranes and other tissues; appears first as red  Drug-induced purpura – quinine, procaine, penicillin,
but later on turns purple and finally brownish yellow color aspirin, sulfonamides, sedatives, coumarins
3. Ecchymosis – form of purpura in which in which blood  Allergic purpura – Henoch’s purpura
escapes into large areas of skin or mucous membranes, but (gastrointestinal), Schonlein purpura (joints)
not into deep tissues  Infectious purpura – purpura may form for several
4. Epistaxis – nosebleed reasons:
5. Hemarthrosis – leakage of blood into a joint or cavity 1. Result of an inflammatory response to infection
6. Hematemesis – vomiting of blood 2. An autoimmune response
7. Hematoma – a swelling or tumor in the tissues or body 3. Bacterial products
cavity that contains blood 4. Toxins or direct injury caused by infectious
8. Hematuria – presence of intact RBCs in the urine agent
9. Hemoptysis – expectoration of blood secondary to
hemorrhage in the larynx, trachea, bronchi or lungs C. QUANTATIVE PLATELET DISORDERS
10. Melena – a stool containing dark red or black blood 1. Thrombocytopenia
11. Menorrhagia – excessive menstrual bleeding  Decreased production
a. Aplastic anemia – generalized bone marrow
B. BLEEDING DISORDERS DUE TO VASCULAR EFFECTS suppression leading to a decrease on all cell
1. Hereditary Connective Tissue Defects types
 Ehlers-Danlos Syndrome b. Selective suppression of the megakaryocyte by
– The individual has hyperextensible joints and chlorothiazide (diuretic)
hyperplastic skin c. Thrombocytopenia with absent radius (TAR) –
– Skin, vasculature, and bones of affected individuals absent or decreased and abnormal bone
lack structural support marrow megakaryocytes, congenital deformities
– The defect may lie in a peptidase enzyme deficiency of the arm
that converts collagen to procollagen d. Myelophthisic process – a space occupying a
 Pseudoxanthoma Elasticum lesion in the bone marrow such as metastatic
– Connective tissue elastic fibers in small arteries are tumor, fibrosis or leukemia
calcified and structurally abnormal  Dilutional Loss- extensive blood transfusion often is
– Subarachnoid and gastrointestinal bledding are the accompanied bythrombocytopenia, the degree of which
most common causes of death is directly proportional to the number of units
transfused.
2. Acquired Connective Tissue Defects  Nonimmune Destruction
 Scurvy (Vitamin C deficiency) a. Artificial Surfaces- induce platelet adherence as
– Acquired disorder of dietary deficiency of vitamin C well as formation of platelet microaggregates.
– Ascorbic acid is required for the formation on intact b. Immune Platelet Destruction- platelet
structure of the vascular basement membrane destruction by immune mechanisms, associated
– Without vitamin C, hydroxylation of the amino acids with increased levels of IgG or complement on
proline and lysine cannot take place and collagen will the platelet surface.
not be formed c. Disseminated Intravascular Coagulation-
 Senile purpura prolonged all coagulation factors with
– The aging process that brings about a degeneration decreased platelets.
of collagen, elastin and subcutaneous fat d. Hemolytic Uremic Syndrome and TTP
e. Increased platelet sequestration by spleen
3. Hereditary Alterations of Vessel Wall Structure
 Hereditary Hemorrhagic Telangiectasia 2.Thrombocytosis
– Characterized by vascular malformations and surface a. Primary- uncontrolled proliferation of
skin lesions called telangiectasias platelets.
– The small blood vessels are focally disorganized and b. Secondary (Reactive)- a broad spectrum of
dilated throughout the body, their wall support is acute and chronic illnesses illicit an increase
poor, and their ability to contract is diminished in platelet production
 Congenital Hemangioma (Kasabach-Meritt Syndrome) D. QUALITATIVE PLATELET DISORDERS
– A disorder associated with tumors composed of 1. Adhesion Defects
vessels that commonly swell and bleed at the surface A. Bernard Soulier Syndrome
B. Von Willebrand Disease
4. Acquired Alterations of Vessel Wall Structure 2. Aggregation Defects
 Diabetes Mellitus A. Glanzmann’s thrombasthenia
– The large vessels may become atherosclerotic, and B. Afibrinogenemia
the capillary basement membrane may thicken, thus 3. Storage Pool Defects
blocking the normal flow of blood A. Gray Platelet Syndrome
– Most often affected are the glomerulus and retina B. Wiskott-Aldrich Syndrome
 Amyloidosis C. Hermansky-pudlak Syndrome
– It can involve and obstruct the function of many D. Chediak Higashi Syndrome
organs, including the vascular system, in which there 4. Acquired Defects
is deposition of the fibrillar protein called amyloid A. Drugs-aspirin, carbenicillin
B. Diet
5. Endothelial Damage – damaged caused by C. Disease:
autoimmune of infectious agents to the endothelial - Myeloproliferative disease
lining of the blood vessels may lead to - Uremia
hypercoagulation or hypocoagulation - DIC
- Paraproteinemia