PLATELETS (THROMBOCYTES)

Lecture by GK Mbassa

Purpose of knowing structure, biochemistry and functions of platelets

Understand qualitative platelet abnormalities

Gain knowledge on hemostasis for treatment of diseases

Know platelets role in tumor metastases, atherosclerosis and inflammation resulting from cytoplasmic fragments of megakaryocytes, e.g. arachidonic acid.

Morphology of platelets

Heterogenous in blood smears; discoid, spheroid, elongated, flat

Granular organelles distributed in cytoplasm.

Some organelles in centre (granulomere) Platelet cytoplasm is hyalomere, which is

clear Platelet is bounded by thin membrane,

smooth or having fine projections

EDTA minimizes platelet clumping Platelets clump to other cells

(erythrocytes and neutrophils), called satellitism.

Platelet volume in dog, pig, man is 7.6 – 8.3 fl, in cattle, equine, sheep, rat, guinea pig, mouse it is 3.2. – 5.4 fl, while in the cat it is 15.1 fl,

Platelet counts vary (1- 10 x 1011/l)

Larger platelets are metabolically and functionally more active than small platelets.

Scanning electron microscope show platelets to have discoid or lentiform shape, with smooth surfaces, slightly biconcave surface, has shallow indentations at external openings of the open canalicular system

Surface projections represent protractions of platelets granules

Surface features of platelets are similar in most species.

Platelets diameter length is 1.3 – 4.7 µm in dog, cat, equines, cow, sheep and goat.

Platelet thickness is 0.5 µm Transformed platelets acquire

pseudopods or projections, found also in normal blood Surface projections occur very fast when blood is taken out of vessel, vary in number and sizes between species

Ultrastructural features of the platelet Unit membrane covered with amorphous

material (external or exterior coat) Bundles of microtubules in matrix beneath

membrane Internal structure comprises of heterogenous

granules (alpha-granules) Clycogen particles Dense granules Mitochondria Lysosomes Peroxisomes

Poorly developed Golgi complex Endoplasmic reticulum (rarely) Spongy like channels, called open

canalicular system) Open canalicular system communicate

with substance of platelet, open to surface at invaginations.

Open canalicular system is lined by unit membrane, covered by external coat

Another system of platelet channels is the dense tubular system.

Dense tubular system occurs under marginal band of microtubules and appears to open to surface, but does not open on the platelet surface.

Platelets of many animals have similar morphology.

Platelets have two types of granules, (1) alpha-granules, and (2) dense granules.

Dense and alpha granules are homogeneously distributed, but vary in electron density, number and size.

Functional organization of the platelet

Platelet divided into 4 structural regions

(1) Peripheral zone (2) Sol-gel zone (3) Organelle zone (4) Membrane system

Peripheral zone Composed of external (exterior) coat, unit

membrane, sub-membraneous area Functions, maintain platelet integrity,

receive and transmit stimuli triggering platelet responses (adhesions, aggregations)

Exterior coat has glycoproteins (glycocalyx) contains mucopolysaccharides and Mg2+ dependent AT Pase, plasma proteins (fibrinogen, IgG, IgM), coagulation factors (vitamin K-dependent factors, factors V and VIII)

Glycoproteins have receptors for platelet activation and aggregation.

Seven glycoproteins recognised, including glycoprotein 1b (reaction site for von Willebrand factor, a component of coagulation factor VIII) necessary platelet adhesion to endothelium on injured blood vessel

Platelet membrane; maintains platelet integrity, rich in phospholipids.

Platelet phospholipids function in blood coagulation (eg

Sol-gel zone Represented by matrix of platelet

cytoplasm, contains microfilaments and microtubules, which function as cytoskeletal elements.

Microfilaments and microtubules maintain discoid platelet shape, form contractile system for shape change, pseudopod formation, internal contractions and granule secretion.

Microfilaments also function in clot retraction.

Microfilaments are also associated with thrombosthenin, a contractile protein (has actin-myosin)

Microtubule tubulin dissolves at 4oC, when exposed to colchicine or vinca alkaloids, leading to platelet shape irregularities.

External coat

Microfilaments

Alpha granule

Open canalicular system

Dense tubular system

Microtubules

Golgi complex

Lysosome

Organelle zone Composed of all internal platelet

components, except microtubules, microfilaments (sol-gel zone) components and membrane system.

Main component of organelle zone are platelet granules, that are morphologically and biochemically heterogeneous, azurophilic granules (alpha-granules under electron microscope)

Alpha-granules are membrane bound, oval, round, electron dense, contain platelet factor 4 (antiheparin), congulation factor V, fibrinogen, beta-thromboglobulin (a thrombin-sensitive protein), fibronection, factor VIII- related antigen, and a mitogenic or growth factor.

Platelets in von Willebrand disease lack factor VIII related antigen

Electron dense granules, called delta granules, or dense bodies contain non metabolic pool ATP and ADP, Ca2+, mono-amines (serotonin, histamine).

Dense granules vary with species.

Lysosomal granules contain acid hydrolases; acid phosphatase, β-glucuromidase

Contraction of microtubules forces all internal organelles towards the centre squeezing or without squeezing their contents to the exterior via open canalicular system.

Platelet activation triggers secretion of various platelet constituents.

Membrane system Memberane system comprises the Open canalicular system Dense tubular systemOpen canalicular system provides a passage

for externalization of platelet secretory products and internalization of substances from plasma into the platelet.

Dense tubular system provides a site for sequestration of Ca2+ and localization of enzymes needed for prostaglandin synthesis

Release of Ca2+ from the dense tribular system triggers platelet aggregation

Platelet constituentsPlatelet location

Constituent Function

Exterior coat Fibrinogen Platelet aggregation

Membrane Arachidonic acid

Prostaglandin synthesis

Plaletet factor 3(phospholipid)

EnhancesCoagulation

cAMP Inhibits release reaction

Platelet constituentsMicrotubules Tubulin Cytoskeleton

Contractility

Microfilaments

Thrombosthenin

Shape change, clot reaction, release reaction

Alpha-granules

Beta-thromboglobulin

Impedes prostacylin production from endothelial cells

Catalase Enzymic process

Factor VIII-relatedantigen

Platelet adhesion to subendothelium

Fibrinogen Shape change, clot reaction, release reaction

Beta-thromboglobulin

Impedes prostacylin production from endothelial cells

Fibronectin Adherence to extracellular matrixPromote wound healing

Endothelial damageVasoconstriction

Plasmin

Anticoagulation

Clot dissolution

Thrombin (IIA) PGI2

Anti-aggregation

tpPF3

Platelet adherence

ADPCollagenvWF

Platelet release and aggregation

Fibrin depositionPlatelet plague

Clot retractionTS

Endothelial regeneration

Growth factor

ADP

Extrinsic activation

Intrinsic activation

STRUCTURAL ABNORMALITIES OF PLATELETS

Morphologic changes occur in platelets after

Contact with non physiologic surfaces.

Exposure to platelet aggregation agents.

The structural abnormalities involving:

Sphering Degranulation (ECF) Budding Indentations Pseudopods Vacuolation.

PLATELET METABOLISM The dry platelet has 50% proteins,

8.5% carbohydrates, the rest are lipids, and others chemicals. Platelets get energy from

Anaerobic glycolysis Hexose monophosphate pathway Oxidative phosphorylation in

mitochondria

Arachidonic acid metabolism Arachidonate metabolites function in

haemostasis and thrombosis involving platelet-vessel wall interactions and synthesis of prostaglandins and thromboxanes. Stimulated platelets liberate arachidomic acid from membrane phospholipids, by phospholipase and corphospholipase A.

Platelets stimulators Thrombin ADP Collagen Epinephrine Calcium ionophores.

FUNCTIONS OF PLATELETS

Platelets have been observed to play a role in the following;

Maintain haemostasis Maintain vascular integrity (with endothelial

cells) Blood coagulation, (provide platelet

phospholipid (platelet factor 3), carry coagulation factors on their surfaces.

Clot retraction (contractile protein system involving thrombosthenin).

Role in thrombosis and embolism Role in flammatory responses

(activation of chemotactic substances release of cationic proteins and vasoactive amines)

Phagocytosis of small particles and bacteria

Role in atherosclerosis Platelets are secretory cells,

producing proteins, procoagulant, anti-heparin, inflammatory and growth-promoting activities

QUALITATIVE AND QUANTITATIVE DISORDERS OF PLATELETS

Platelet disorders are characterized as qualitative or functional and qualitative or both. Qualitative platelet disorders include;

Hereditary, e.g Glanzmann’s thrombosthenia

Acquired Quantitative platelet disorders include;

Thrombocytopaenia is the most frequent, causes hemorrhagic diathesis

Thrombocytosis may be physiologic or reactive

Thrombosythemia a proliferative disorder of megakaryocytes in bone marrow, associated with severe thrombocytosis

Signs, diagnosis and common abnormalities

There are hereditary or acquired qualitative platelets disorders, and vary in severity.

They involve multiple functional platelet abnormalities, and caused by; extrinsic abnormalities

defects in morphological and biochemical components of platelets or megakaryocytes

Qualitative and quantitative platelet defects have similar clinical signs despite of differences in origin and may differ in pathogenesis. Signs of qualitative platelet disorders

(1) Increased tendency to bleed (2) Prolonged bleeding time, in a

situation of increased or normal platelet count.

(3) Set on early in life. (4) Familial occurrence

Diagnostic examination of qualitative platelet disorders

(1) Platelet count (2) Platelet distribution on blood films (3) Platelet morphology (4) Bleeding time (5) Prothrombin time (6) Partial prothrombin time. (7) In vitro platelet aggregation test

(adrenalin, collagen, ADP ristocetin).

(8) Platelet retention test in glass bead column

(9) In vivo platelet adhesion test

Common abnormalities in platelet function

Aggregation Retention in glass bead column Availability of platelet factors 3 (PF-3)

Cause of acquired qualitative platelet functional disorders

Acquired platelet disorders occur with or without hemorrhagic manifestations, occur in;

Renal disease, with uremia Liver disease Myeloproliferative disorders Lymphoproliferative disorders Macroglobulinemia Plasma cell myeloma

Immune mediated disorders (autoimmune thrombocytopenia, autoimmune hemolytic anemia, systemic lupus erythrematosus, circulating fibrin, circulating fibronogen split products – cirrhosis, disseminated intravascular coagulation)

Vitamin deficiency Congenital heart disease Acquired storage pool disease

(deficiency of platelet dense bodies) Drug therapy (non-steroid anti-

inflammatory drugs eg. asprin

Causes of hereditary qualitative platelet disorders

1. Glanzmann’s thrombasthenia Glanzmann’s thrombasthenia, a

hemorrhagic disorder due to autosomal recessive inheritance characterized by;

Greatly prolonged bleeding time in presence of normal platelet counts and coagulation factors

Spontaneous purpuric mucosal and cultaneous bleeding

Early onset in life

The defects include Lack of platelet aggregation with ADP,

collagen, thrombin, clot retraction PF-3 availability

Absence of membrane glycoprotein IIb, IIIa,

Lack of receptors for fibrinogen Short life of platelets (4 days, normal 7

days).

2. Hereditary thrombopathia Hereditary thrombopathia occurs in

dogs due to autosomal inheritance, characterized by markedly abnormal platelet function, depressed ADP and collagen aggregation and slightly prolonged bleeding time.

3. Von Willebrand’s disease A hereditary bleeding disorder Prolonged bleeding time, normal clot

retraction Quantitative and qualitative disorder of

von Willbrand’s factor associated with factor VIII – related antigen (VIII-Ag).

Decreased adherence of platelet to injured vessels and glass beeds.