Platelet Function Platelet must be adequate in number and function to participate optimally in homeostasis.

The functions of platelets in homeostasis include; Maintenance of vascular integrity Initial arrest of bleeding by platelet plug formation Stabilization of hemostatic plug by contributing to fibrin formation.

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Key Mediators in Platelet Adhesion, Activation and Aggregation

INJURY
vWF ADP-receptor

THROMBUS
Shear Forces

Adhesion
vWF Thrombin Collagen Fibronectin

Activation
Membrane changes Granule secretion GPIIb/IIIa expression Multiple agonists Feedback loops

Aggregation
GPIIb/IIIa-mediated Fibrinogen vWF

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Platelet Adhesion and Activation

Platelets adhering to damaged endothelium and undergoing activation Aggregation of platelets into a thrombus

Normal platelets in flowing blood

Platelet thrombus Platelets adhering to subendothelial space

Platelets

Endothelial cells Subendothelial space

Adapted from: Ferguson JJ. The Physiology of Normal Platelet Function. In: Ferguson JJ, Chronos N, Harrington RA (Eds). Antiplatelet Therapy in Clinical Practice. London: Martin drmsaiem Dunitz; 2000: pp.1535.

Platelet Function (Adhesion) Following blood vessel injury, platelets adhere to the exposed subendothelial connective tissues. Subendothelial bind to the larger multimers of vWF and through these react with Platelet membrane GPIb . large number of adhesion proteins are involved in plateletvessel wall and platelet- platelet interaction. Adhesion to collagen is facilitated by GPIa.

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Platelet Function Platelet adhesion induces a series of metabolic reactions which initiate the platelet release reactions, shape change and aggregation. Following adhesion, platelets become more spherical and extrude long pseudopods which enhance interaction between adjacent platelets. von Willebrand factor (VWF) is involved in platelet adhesion to the vessel wall and to other platelets (aggregation).

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Platelet Function vWF is encoded by a gene on chromosome 12 and is synthesized by endothelial cells and Megakaryocytes. Release of vWF from endothelial cells occurs under the influence of several hormones.

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Platelet Aggregation

Flowing disc-shaped platelet

Rolling ball-shaped platelet

Hemisphere-shaped platelet

Spreading platelet

FIRM, BUT REVERSIBLE ADHESION

IRREVERSIBLE ADHESION

Scanning electron micrograph of discoid, dormant platelets

Activated, aggregating platelets illustrating fibrin strands

Adapted from: Kuwahara M et al. Arterioscler Thromb Vasc Biol 2002; 22: 32934.

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Platelet Function (Release Reaction) Collagen exposure or thrombin action results in the secretion of platelet granule contents which include ADP, serotonin, fibrinogen, lysosomal enzymes, thromboglobulin. Collagen and thrombin activate platelet prostaglandin synthesis. Released ADP and thromboxane A2 cause additional platelets to aggregate at the site of vascular injury. ADP causes platelets to swell and encourages the platelet membranes of adjacent platelets to adhere to each other.

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Platelet Function (Platelet aggregation) As they do so further release reactions occur liberating more ADP and thromboxane A2 causing secondary platelet aggregation. This positive feedback process results in the formation of a platelet mass large enough to plug the area of endothelial injury.

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Platelet Function Test

Bleeding Time
Principle: Bleeding time is defined as the time taken for a

standardized skin wound to stop bleeding.

Platelet aggregation Platelets function in primary hemeostasis by forming an

initial platelet plug at the site of vascular injury. The phenomenon occurs partly through the ability of platelets to adhere to one another, a process known as platelet aggregation.
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Platelet Function Test

Substances that can induce platelet aggregation include; Collagen, ADP, epinephrine, thrombin, serotonin, arachidonic acid, restocetin, snake venoms.

Platelet aggregation is an essential part of the investigation of any patient with a suspected platelet dysfunction. Platelet aggregation is studied by means a platelet aggregometer.

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Platelet Function Test

A photo-optical instrument connected to a chart recorder. Platelet rich plasma (PRP) which is turbid in appearance, is placed in a cuvette warmed to 37C in the heat block of the instrument, and stirred by a small magnetic bar. Light transmittance through the PRP is recorded.

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Platelet Function Test

The addition of aggregating agent cause the formation of larger platelet aggregates with a corresponding increase in light transmittance, owing to a clearing in the PRP. The change in the light transmittance is converted to electric signal and recorded as a tracing by the chart recorder.

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Platelet Function Test

There are some basic requirements for platelet aggregation as in vitro means of evaluating platelet functions.

1- A clean venipuncture is crucial. Hemolyzed samples

should not be studied because RBCs contained ADP. 2- Plasma from fasting patients is preferred for testing. Lipemic samples may obscure change in optical density owing to platelet aggregation.

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Platelet Function Test

3- Sodium citrate is the anticoagulant used in aggregation studies. In vitro aggregation is dependent on the presence of calcium ions. 4- Fibrinogen must be present in the test sample for aggregation to occur. 5- Aggregation studies should be performed at 37C at a pH of 6.5 to 8.5. To help maintain platelet values, all samples, once collected, should be capped to prevent Co2 loss.
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Platelet Function Test

6- Test

samples, should be maintained at room temp during processing. Cooling inhibits the platelet aggregation response. 7- Stirring is necessary to bring the platelets in close contact with one another to allow aggregation to occur. 8- It is essential that the patient refrain from taking any anti-inflammatory drugs, one week before the test. The drugs inhibit the platelets release reaction.

9- Aggregating reagents should be prepared fresh daily and brought to room temp before use. They must have known potency and be added in small volume.
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