Week 5: Primary Hemostasis

Hemostasis
Platelet function
Platelet anatomy
Megakaryocyte
Platelet kinetics
Platelet count
Aggregation studies
Von Willebrands
Bernard-Soulier
Aspirin (salicylate)

Vascular problems
Allergy
Viral infection
Collagen disorders
Vitamin C deficiency
Ehlers-Danlos
Render-Osler-Weber
telangiectasia
Petechiae, ecchymoses
Bleeding time
Vascular Injury
Serotonin and thrombaxane A
2
(TxA
2
) for
vasoconstriction
Prostacyclin PGI-2 for arteriole relaxation
to increase blood flow
Exposure of basement membrane and
collagen (negatively charged surface)
Inside of a vessel SEM x 2,500
Process of Hemostasis
Vascular injury
Platelet adhesion and activation
Platelet aggregation (1
o
hemostatic plug)
Fibrin formation via cascade (2
o

hemostasis)
Clot retraction (thrombasthenin)
Fibrinolysis and healing
Role of Platelets
Surveillance for
vascular integrity
Formation of 1
o

hemostatic plug
Activation of 2
o

hemostasis
Healing
Platelet Formation
Megakaryoblast undergoes endomitosis
Intermiediate stage promegakaryocyte
without granules
Megakaryocyte (2N to 64N) with over
100 diameter
IL3, GM-CSF, thrombopoietin
20% of platelet stored in spleen
Platelet
2 - 4 diameter
Round or oval
Hyalomere - clear peripheral zone
Granulomere - highly stained area with
granules
Platelet Anatomy
Peripheral zone with glycoprotein
receptors
Structural zone with contractile
microtubules (thrombasthenin)
Organelle zone with granules
Membrane with open cananicular and
tubule systems for increased surface area
and rapid release
Electron micrograph of a platelet x 25,000

Longitudinal peripheral microtubule (brown), endoplasmic reticulum
(blue), mitochondria (green), glycogen (black)
Platelet Function
Adhesion to basement membrane or collagen with
vWF and GP-Ib
Activation
Shape change from discoid to distorted
Exposure of GP-IIb/IIIa and other receptors
TxA
2
synthesis (cyclo-oxygenase dependent)

Membrane phospholipid Arachidonic acid
Arachidonic acid TxA
2
and prostaglandins
Platelet Function:
Glycoprotein Group or Gene Families
Integrins
Leucine rich glycoprotein family
Selectin family
Quadraspanin family
Immunglobulin supergene family
Integrins
Integral to membrane
Ca
++
dependent GPIIb/IIIa most abundant
Cell-cell or cell-substrata interaction
Receptor to Fib, vWF, vitronectin,
fibronectin

Leucine Rich GP Family
Adhesion to subendothelial collagen (COL)
mediated by vWF by transmembrane
complex GPIb/IX
Stabilizes PLT membrane by interaction
with cytoskeleton
Selectin Family
GMP-140 mediates adhesion of neutrophils
and monocytes to PLT
Quadraspanin Family
Plasma membrane protein p24/CD9
interacts with GPIIb/IIIa, modulating
adhesion molecules
Leads to Ca
++
increase and subsequent PLT
activation and aggregation
Immunoglobulin Supergene Family
Functional role unclear
Has role in cellular interactions
Platelet Activation
1
o
aggregation with agonists: ADP, epinephrin,
serotonin, PF4 (anti-platelet)
Release or secretion facilitated by TxA2
Dense body for 2
o
aggregation and
vasoconstriction (ADP, Ca
++
, serotonin)
-granule for heparin neutralization and clot
Platelet derived growth factor (PDGF) for
healing
Retraction
Signs and Symptoms of 1
o

Hemostasis Problems
Ecchymoses
Petechiae
Mucus membrane bleeding
Hematoma
Prolonged bleeding after minor surgery
Hereditary Vascular Problems
Hereditary (spider) telangiectasis (Osler-
Rendu-Weber): dilated superficial
capillaries
Ehlers-Danlos: collagen disorder
Marfan syndrome: connective tissue
Osteogenesis imperfecta
Acquired Vascular Problems
Senile purpura (Batemans): altered
connective tissue support
Cushing syndrome: metabolic
Scurvy: abnormal collagen
Allergy: vascular inflammation
Viral infection
Bleeding Time
For vascular and platelet functions
Duke (1910) on earlobes
Ivy (1941) on arm with 1mm x 3mm
incision
Mielke (1969) with 1mm x 10mm template
1980s: disposable devices (e.g., Simplate,
Surgicutt)
Bleeding Time
Quantitative Platelet Disorders
Thrombocytopenia
<100,000/ml BT prolonged
10,000 Bleeding in trauma or OR
<10,000 Spontaneous, CNS bleeding
Thrombocytopenia due to destruction
ITP (acute in children, chronic in young women)
with anti-glycoprotein
Drug reaction
Heparin induced thrombocytopenia
DIC and TTP

About Thrombotic Thrombocytopeneic
Purpura (TTP)
Disorder of systemic platelet aggregation in
microvasculature
Stimulus: unusually large vWf
In children: likely to be deficiency in vWf
metalloproteinase to break down vWf
In adults: vWf metalloproteinase inhibited by
autoantibodies
Low PLT count, intravascular hemolysis, RBC
fragmentation, high LDH

Quantitative Platelet Disorders
Thrombocytopenia due to decreased production
Aplastic anemia (e.g., Fanconis)
Fibrosis
Acute leukemia
Megaloblastic anemia
Hereditary (e.g., May-Hegglin, Wiscott-Aldrich,
Bernard-Soulier)
Splenic sequestration
HELLP syndrome (hemolysis, elevated liver enzyme,
low PLT) in pre-eclampsia
Dilution (massive transfusion)
Platelet Satellitosis in EDTA
Quantitative Platelet Disorders
Thrombocytosis
Primary with dysfunctions (e.g., CML,
ET)
Post splenectomy: also see HJ, etc.
Hemolytic anemia
Acute hemorrhage and surgery
Pseudo Thrombocytosis
Red cell abnormalities
HJ bodies
Clumped Pappenheimer bodies
nRBC
Malaria
Microspherocytes and schistocytes
White cell abnormalities
Unlysed WBC
WBC fragments and necrobiotic cells
Platelet Count
Rees-Ecker with brilliant cresyl blue
Brecker-Cronkite with ammonium oxalate
Unopette: similar to BC
Electronic counters
Qualitative Platelet Disorders
Berhard-Soulier: GP-Ib deficiency, adhesion
problem
Von Willebrands: vWF deficiency, adhesion
problem
Glanzmanns thrombasthenia: GP-IIb/IIIa
deficiency, aggregation problem -- cannot bind
vWF and Fib
Storage pool disease: dense body defect, secretion
problem
Qualitative Platelet Problems
Aspirin: inhibits cyclo-oxygenase (COX),
secretion problem, no TxA
2
Plavix (Clopidogrel) inhibits ADP receptor
Other medications affect GPIIa/IIIb
interaction with Fib
Uremia, secretion problem
Gray platelet syndrome: -granule defect
Hypofibrinogenemia
Aggregation Studies
ADP
reversible 1
o
wave
if ADP is released, then 2
o
wave
abnormal with aggregation and release problems
Epinephrin
similar to ADP
Collagen
direct release so only one wave of aggregation
Ristocetin
antibiotic
aggregation only with vWF and GP-Ib
Platelet Aggregometry
Platelet

Aggregation