Primary Hemostasis: Platelet Form and Function

Cara Calvo, MS, MT(ASCP), SH(ASCP) UW, 2009.

Graphic adapted from URL http://www.med-ed.virginia.edu/courses/path/innes/nh/function.cfm, 2009.

Lab M 419 Clinical Coagulation Fall 2009

Learning Objectives When you have

finished the reading assignment, after careful study, and following this lecture, you will be able to:
1. Define and correctly use the key terms that appear in italic on pages 146 157, and 571 574 of the course text. Recall the differentiating features of primary and secondary hemostasis. Describe the ultrastructure of a resting platelet, noting how it changes once a platelet is activated. Relate platelet ultra structural features to specific hemostatic and other biological functions of PLTs. Discuss each of the platelet plasma membrane receptors identified in the course text in terms of the identity of their ligands and the actions resulting from receptor-ligand interaction. Explain the relationships among platelet function, von Willebrand factor, and fibrinogen and their impact on hemostasis. Discriminate the numerous functions of platelets in hemostasis. Summarize the steps in platelet activation leading to a platelet plug. Include a discussion of the biochemistry and morphological changes necessary for platelets to play their role in primary and secondary hemostasis. Compare and contrast the effects of the anti-platelet drugs on platelet function. Drugs of interest include aspirin and clopidogrel. Synthesize knowledge of platelet form and function to correctly respond to test questions and solve subject-matter related case studies.

2. 3.
4. 5. 6. 7. 8. 9. 10.

Lecture Outline

Overview Platelet Structure & Function Platelet Structure: Peripheral Zone

Glycocalyx Plasma membrane
Lipids Receptors SCCS

Overview Platelet Function PLT Function: Vascular Integrity PLT Plug Formation: Adhesion
Von Willebrand Factor Membrane Receptor GP1b Collagen Shape Change

DTS
Cytoskeleton
Microtubules Microfilaments

PLT Plug Formation: Aggregation

ATP Ionized Calcium Receptors GPIIb/IIIa Fibrinogen Phases

Platelet Structure: Sol-Gel Zone

Platelet Structure: Organelle Zone

Cytoplasmic Content PLT Granules
Alpha Dense Lysosomes

PLT Plug Formation: Secretion DRUGS and PLT Function Summary: PLT Form & Function

Overview: Platelet Structure & Function

Anucleate cytoplasmic pieces shed from megakaryocyte Metabolically active
Phospholipid source for clot formation Release chemicals that mediate physiological response to tissue damage leading to hemorrhage, thrombosis and tissue repair House receptors that facilitate PLT activation and PLT participation in primary and secondary hemostasis

1/3rd PLTs released from BM are sequestered by spleen PB Ref range: 150 450 x 109/L
Graphic accessed at URL http://hsc.virginia.edu/medicine/clinical/pathology/educ/innes/text/nh/platelets.html & http://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20megakaryocyte%20x50a.jpeg

Platelet Structure - Peripheral Zone

Glycocalyx Plasma Membrane Bilayered
Lipids Receptors Surface-connected canalicular system (SCCS)

Dense tubular System (DTS)

Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.

Glycocalyx
Amorphous exterior coat Adhesive Negatively charged Site of PLT functional environment
BLD Group specificity Tissue compatibility PLT antigenicity Adhesion receptors

Electron micrographs from a resting platelet (x10,000), or from an activated platelet showing pseudopodia emission (x5,000). (Pictures have been kindly provided by Dr. J. White).

Graphic accessed http://www.platelet-research.org/2/morph_rest.htm, 2009.

PLT Plasma Membrane: Lipids

Plasma layer
Phosphotidylcholine Sphingomyelin

Cytoplasmic layer
Phosphotidylinositol Phosphotidylethanolamine Phosphotidylserine

Sources of arachidonic acid

PLT activation
TXA2

Graphic accessed URL http://en.wikipedia.org/wiki/File:Eicosanoid_synthesis.svg 2008.

PLT Plasma Membrane: Receptors

Mediate adhesion & aggregation Facilitate secretion Categorized
Cellular adhesion molecules (CAMs)
Integrins Leucine-rich repeat Immunoglobulin gene Selectin Seven-transmembrane receptor (7-TMR)

Graphic accessed URL http://www.platelets.se/resource/Platelet%20function.jpg, 2009.

PLT Plasma Membrane: Receptors

Name GPIIb/IIIa GPIb/IX/V Category Integrin LRR Ligand Fibrinogen vWF vWF Thrombin Effect PLT aggregation PLT adhesion

PAR 1 PAR 2 P2Y1 P2Y12

GP VI P-selectin

7-TMR

Thrombin
ADP

PLT activation: aggregation

Ig Gene Lectin

Collagen PSGL

PLT activation: aggregation WBC tethering & binding

M&M graphic accessed/adapted URL http://www.tu-pc.com/fondos/media/2452.jpg. 2009.

PLT Plasma Membrane: SCCS

Tubular invagination of membrane
glycocalyx

Transport of substances released from PLT granules to exterior Adsorption/storage of hemostatic proteins

White, JG and Clawson, CC. The surface-connected canalicular system of blood platelets: a fenestrated membrane system (1980). Am Journal of Pathology, 101, 353-364, and Graphic accessed URL http://www.chelationtherapyonline.com/GarryGordon/images/Copy%20(3)%20of%20Slide1.GIF , 2009.

Dense Tubular System (DTS)

Endoplasmic reticulum remnant
From megakaryocyte

Calcium storage/release Prostaglandin & Thromboxane synthesis

Graphic accessed URL http://www.platelet-research.org/2/morph_rest.htm, 2009. Histochemistry (1982) 76:189-196 , Am J Pathol. 1976 May; 83(2): 283298 & Thromb Haemost. 1978 Oct 31;40(2):224-31

Platelet Structure: Sol-Gel Zone

PLT Cytoskeleton
Microtubules
Tubulin
PLT discoid shape Secretory process PLT pseudopod morphology

Microfilaments
Actin/Myosin
Contractile protein: PLT shape-change

Graphic accessed URL http://health.upenn.edu/News/News_Releases/platelet.jpg, 2009.

Platelet Structure: Organelle-Zone

Cytoplasmic content
Anucleate Few mitochondria Granules
Alpha Dense Lysosomes

Graphic accessed URL http://www.platelet-research.org/2/morph_rest.htm, 2009.

PLT Granules Content

Granule Alpha Thromboglobulin PF4 PDGF Fibrinogen, Factors V & VIII vWF Plasminogen a1-antiplasmin HMWK Fibronectin Dense ADP/ATP Calcium Serotonin Lysosomes Proteolytic, hydrolytic enzymes PLT agonist Regulates PLT activation Promotes vasoconstriction Digest vessel wall matrix and debris Inhibit heparin; vessel repair Inhibit heparin Vessel repair Fibrin formation PLT Adhesion Precursor of plasmin (fibrinolysis) Plasmin inhibitor Contact activation: intrinsic coagulation path Promotes PLT spreading Function

Primary hemostasis, Secondary hemostasis

Overview: Platelet Function

PRIMARY HEMOSTASIS
Form platelet plug: damaged endothelia Nurture endothelia

SECONDARY HEMOSTASIS
Reaction surface for coagulation
Graphic accessed at URL http://www.medicine.mcgill.ca/physio/209A/Blood/blood6a.htm, 2007.

PLT Function: Vascular Integrity

Inner layer - monolayer of endothelia
Glycocalyx coat PLT Adhesion WBC/PLT binding
P-selection

Middle layer smooth muscle, connective tissue

Vasoconstriction Binds/Activates PLTs Tissue factor source

Outer layer fibroblasts, collagen, mast cells

Tissue factor source
Graphic accessed at URL http://cellapplications.com/images/humanimages/HCtAEC2115-s2.jpg, 9/12/08.

PLT Function: Vascular Integrity

Endothelial Function Consequence

ANTICOAGULANT
Secrete Prostacyclin (PGI2) Smooth, continuous, negatively charged surface Vasodilation PLT aggregation inhibition PLT and Proteins repelled blood flows

Secrete nitrous oxide

Secrete heparan sulfate Secrete TFPI

Vasoconstriction neutralized
Anticoagulation Activation of tissue factor pathway inhibited

PROCOAGULANT
Secrete vWF Secrete adhesion molecules Exposure of Tissue Factor PLT Adhesion WBC and PLT binding Coagulation initiated

Platelet Plug Formation: Adhesion

Platelets bind to exposed adhesive subendothelial connective tissue
Collagen vWF Fibronectin

Mechanism components
vWF: links PLT to endothelial binding site PLT receptor GPIb Collagen fibers

Actin contracts & pseudopods form REVERSIBLE

Facilitates activation
M&M graphic accessed http://www.dicksons.com.au/images/body/products/prod_mm_guys.gif, 2009.

M&M graphic accessed URL http://www.jauntyquills.com/pics/WebPics/M&Mgroup.jpg, 2009

Platelet Plug Formation: Aggregation

Platelet-Platelet interaction Mechanism components
ATP Ionized calcium Fibrinogen PLT receptor GPIIb/IIIa

Initial aggregation REVERSIBLE Secondary aggregation IRREVERSIBLE = white clot, a.k.a platelet plug formed.

Graphic accessed URL http://www.medscape.com/content/2003/00/45/62/456257/art-jic456257.fig1.jpg, 2008.

Platelet Plug Formation: Secretion

Discharge of granules contents Markers of PLT activation
PF4 PDGF Thromboglobulin

Promote & Amplify PLT activities

Primary hemostasis Secondary hemostasis

Graphic accessed http://www.uptodate.com/online/content/images/hema_pix/Platelet_interactions.jpg, 2009.

Drugs and PLT Function

Aspirin
Acetylsalicyclic acid
Irreversibly inhibits Cyclooxygenase

Clopidogrel
Plavix
irreversibly inhibits P2Y12

Dipyridamole
inhibits Thromboxane synthase

Abciximab
ReoPro

Brinkman WT, Terramani TT, Najibi S, Chaikof EL. Platelets: is aspirin sufficient or must we know how to pronounce abciximab? Semin Vasc Surg. 2

(Pronounce: ab-SIKS-ih-mab)

inhibits GP IIb/IIIa

Graphic accessed URL http://www.scielo.br/img/revistas/abc/v85n3/en_26011f1.jpg, http://www.medscape.com/pi/editorial/cmecircle/2002/1000/figure-7.gif & http://www.australianprescriber.com/upload/issue_files/3004_f1_antiplatelet.gif ,2009.

Summary: PLT Form and Function

Platelets are derived from BM megakaryocytes. Circulating platelets are anucleate, but have a distinctive anatomy and physiology that enables them to play important roles in primary and secondary hemostasis. The PLT membrane houses numerous receptors that control PLT activation. PLT function can be inhibited by drugs which target key PLT activation enzymes and receptors. PLTs adhere to surfaces, aggregate with each other, and secrete their granules contents in order to plug damaged areas in endothelia, modulate coagulation, and aid tissue repair.

Quiz Time! (non-graded)

The Case
From - Ciesla, B. Hematology in Practice (2007). Philadelphia: FA Davis, p.241.

A 35-year-old female, took aspirin 16 hours ago because of a headache Gravid 1, Para 1 Mother has history of bleeding after tooth extraction Physician needs to determine if the patient has a bleeding disorder Pre-Op (scheduled to have ovarian cysts removed) lab tests
PLT count 320 (Ref. Range 150 435K/mL) Bleeding Time 11 minutes (Ref. Range 2 9 minutes) Coagulation studies were within normal limits

M&M graphic accessed and adapted from URL http://www.prnewswire.com/mnr/mars/31278/ , 2009.

Quiz Time Question

This patient has a bleeding disorder.
Interpret the lab data, correlate lab data with patient information & synthesize knowledge of platelet form and function to explain.

Graphic accessed URL http://www.expressionery.com/images/CheckDesign/NCMGR.jpg, 2009.

Answer
Bleeding time is ABN suggests disorder w/primary hemostasis
Consider PLT disorder
ACQUIRED? Patient took aspirin (PLT function inhibitor) within last 24 hours
Drug-mediated PLT aggregation problem

FAMILIAL? Patients mother has history of bleeding after tooth extraction

vWD?

Rest of the story factor VIII, vWF Ag and activity assays were done. All results WRR. PLT aggregometry was performed: only primary wave w/epinephrine and flat-line w/AA. BT was repeated 72 hours after discontinuing aspirin BT returned to normal.

Graphic Accessed URL http://drmagrann.com/Hematology/Unit%204/5%20Shape%20Change.jpg, 2009.

PLT Shape Change

Ig Super Family
The interaction between cell adhesion molecules can be considered to be identical to the binding of a ligand to its receptor. This is an important concept because often (if not always) the binding of two cells sets up intracellular events (signal transduction) in the adherent cells.
Professor Danton ODay,
http://www.erin.utoronto.ca/~w3bio315/lecture5.htm, 2005.

Graphics accessed URL http://www.mcld.co.uk/hiv/images/immunoglobulinSuperfamily.gif & http://www.erin.utoronto.ca/~w3bio315/picts/lectures/lecture5/CellAdhesionTypes1.jpg, 2009.

References
Rodak BF, Fritsma GA, and Doig K. (2007). Hematology Clinical Principles and Applications. St. Louis, Missouri. Saunders Elsevier. Pages 146-157, 571-574. Platelet Research Laboratory at URL http://www.platelet-research.org/ Lab Tests On-line Platelet Function Tests at URL http://labtestsonline.org/understanding/analytes/pl atelet_function/glance.html Hartwig, JH and Desisto, M. The Cytoskeleton of the Resting Human Blood Platelet: Structure of the Membrane Skeleton and Its Attachment to Actin Filaments. The Journal of Cell Biology: 112(3);407-425 University of Virginia Health System, Pathology, Normal Hemostasis, Platelet Function at URL http://www.meded.virginia.edu/courses/path/innes/nh/function.cfm Platelet at Wikipedia URL http://en.wikipedia.org/wiki/Platelet

Graphics accessed URL http://us.mms.com/us/images/char_spotlight_red.png, 2009.