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Platelet FunctionPlatelet Function

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

The functions of platelets in homeostasis include;The functions of platelets in homeostasis include;

Maintenance of vascular integrityMaintenance of vascular integrity

Initial arrest of bleeding by platelet plug Initial arrest of bleeding by platelet plug formationformation

Stabilization of hemostatic plug by contributing Stabilization of hemostatic plug by contributing to fibrin formationto fibrin formation..

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Adhesion

Key Mediators in Platelet Adhesion, Key Mediators in Platelet Adhesion, Activation and AggregationActivation and Aggregation

INJURY

• vWF• Thrombin• Collagen• Fibronectin

Shear Forces

vWFADP-receptor

THROMBUS

Activation Aggregation

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

• GPIIb/IIIa-mediated• Fibrinogen• vWF

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Aggregation of platelets into a

thrombus

Platelets

Endothelial cells

Platelets adhering to subendothelial space

Platelet thrombus

Normal platelets in flowing blood

Platelets adhering to damaged endothelium

and undergoing activation

Platelet Adhesion and ActivationPlatelet Adhesion and Activation

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: MartinDunitz; 2000: pp.15–35.

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Platelet Function (Adhesion)Platelet Function (Adhesion)

Following blood vessel injury, platelets adhere to Following blood vessel injury, platelets adhere to the exposed subendothelial connective tissues. the exposed subendothelial connective tissues.

Subendothelial bind to the larger multimers of Subendothelial bind to the larger multimers of vWF and through these react with Platelet vWF and through these react with Platelet membrane GPIb .membrane GPIb .

large number of adhesion proteins are involved large number of adhesion proteins are involved in platelet—vessel wall and platelet- platelet in platelet—vessel wall and platelet- platelet interaction.interaction.

Adhesion to collagen is facilitated by GPIa.Adhesion to collagen is facilitated by GPIa.

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Platelet FunctionPlatelet Function

Platelet adhesion induces a series of metabolic Platelet adhesion induces a series of metabolic reactions which initiate the platelet release reactions which initiate the platelet release reactions, shape change and aggregation. reactions, shape change and aggregation.

Following adhesion, platelets become more Following adhesion, platelets become more spherical and extrude long pseudopods which spherical and extrude long pseudopods which enhance interaction between adjacent platelets.enhance interaction between adjacent platelets.

von Willebrand factor (VWF) is involved in von Willebrand factor (VWF) is involved in platelet adhesion to the vessel wall and to other platelet adhesion to the vessel wall and to other platelets (aggregation).platelets (aggregation).

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Platelet FunctionPlatelet Function

vWF is encoded by a gene on chromosome 12 vWF is encoded by a gene on chromosome 12 and is synthesized by endothelial cells and and is synthesized by endothelial cells and Megakaryocytes.Megakaryocytes.

Release of vWF from endothelial cells occurs Release of vWF from endothelial cells occurs under the influence of several hormones.under the influence of several hormones.

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Adapted from: Kuwahara M et al. Arterioscler Thromb Vasc Biol 2002; 22: 329–34.

Platelet AggregationPlatelet Aggregation

FIRM, BUT REVERSIBLEADHESION

IRREVERSIBLEADHESION

Scanning electron micrograph of discoid, dormant platelets

Activated, aggregating platelets illustrating fibrin strands

Flowingdisc-shaped

platelet

Rollingball-shaped

platelet

Hemisphere-shapedplatelet

Spreadingplatelet

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Platelet Function (Release Reaction)Platelet Function (Release Reaction)

Collagen exposure or thrombin action results in Collagen exposure or thrombin action results in the secretion of platelet granule contents which the secretion of platelet granule contents which include ADP, serotonin, fibrinogen, lysosomal include ADP, serotonin, fibrinogen, lysosomal enzymes, enzymes, ββ-thromboglobulin.-thromboglobulin.

Collagen and thrombin activate platelet Collagen and thrombin activate platelet prostaglandin synthesis.prostaglandin synthesis.

Released ADP and thromboxane A2 cause Released ADP and thromboxane A2 cause additional platelets to aggregate at the site of additional platelets to aggregate at the site of vascular injury.vascular injury.

ADP causes platelets to swell and encourages the ADP causes platelets to swell and encourages the platelet membranes of adjacent platelets to platelet membranes of adjacent platelets to adhere to each other.adhere to each other.

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Platelet Function (Platelet aggregation)Platelet Function (Platelet aggregation)

As they do so further release reactions occur As they do so further release reactions occur liberating more ADP and thromboxane A2 liberating more ADP and thromboxane A2 causing secondary platelet aggregation. causing secondary platelet aggregation.

This positive feedback process results in the This positive feedback process results in the formation of a platelet mass large enough to plug formation of a platelet mass large enough to plug the area of endothelial injury.the area of endothelial injury.

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Platelet Function TestPlatelet Function Test

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

standardized skin wound to stop bleeding.standardized skin wound to stop bleeding.

Platelet aggregationPlatelet aggregation

Platelets function in primary hemeostasis by Platelets function in primary hemeostasis by forming an initial platelet plug at the site of forming an initial platelet plug at the site of vascular injury. The phenomenon occurs partly vascular injury. The phenomenon occurs partly through the ability of platelets to adhere to one through the ability of platelets to adhere to one another, a process known as platelet aggregation.another, a process known as platelet aggregation.

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Platelet Function TestPlatelet Function Test

Substances that can induce platelet aggregation Substances that can induce platelet aggregation include;include;

Collagen, ADP, epinephrine, thrombin, serotonin, Collagen, ADP, epinephrine, thrombin, serotonin, arachidonic acid, restocetin, snake venoms.arachidonic acid, restocetin, snake venoms.

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

Platelet aggregation is studied by means a Platelet aggregation is studied by means a platelet aggregometer.platelet aggregometer.

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Platelet Function TestPlatelet Function Test

A photo-optical instrument connected to a chart A photo-optical instrument connected to a chart recorder.recorder.

Platelet rich plasma (PRP) which is turbid in Platelet rich plasma (PRP) which is turbid in appearance, is placed in a cuvette warmed to appearance, is placed in a cuvette warmed to 37°C in the heat block of the instrument, and 37°C in the heat block of the instrument, and stirred by a small magnetic bar.stirred by a small magnetic bar.

Light transmittance through the PRP is recorded.Light transmittance through the PRP is recorded.

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Platelet Function TestPlatelet Function Test

The addition of aggregating agent cause the The addition of aggregating agent cause the formation of larger platelet aggregates with a formation of larger platelet aggregates with a corresponding increase in light transmittance, corresponding increase in light transmittance, owing to a clearing in the PRP.owing to a clearing in the PRP.

The change in the light transmittance is The change in the light transmittance is converted to electric signal and recorded as a converted to electric signal and recorded as a tracing by the chart recorder.tracing by the chart recorder.

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Platelet Function TestPlatelet Function Test

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

1- 1- A clean venipuncture is crucial. Hemolyzed A clean venipuncture is crucial. Hemolyzed samples should not be studied because RBCs samples should not be studied because RBCs contained ADP.contained ADP.

2- Plasma from fasting patients is preferred for 2- Plasma from fasting patients is preferred for testing. Lipemic samples may obscure change in testing. Lipemic samples may obscure change in optical density owing to platelet aggregation.optical density owing to platelet aggregation.

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Platelet Function TestPlatelet Function Test

3- Sodium citrate is the anticoagulant used in 3- Sodium citrate is the anticoagulant used in aggregation studies. In aggregation studies. In vitrovitro aggregation is aggregation is dependent on the presence of calcium ions.dependent on the presence of calcium ions.

4- Fibrinogen must be present in the test sample 4- Fibrinogen must be present in the test sample for aggregation to occur.for aggregation to occur.

5- Aggregation studies should be performed at 5- Aggregation studies should be performed at 37°C at a pH of 6.5 to 8.5. To help maintain 37°C at a pH of 6.5 to 8.5. To help maintain platelet values, all samples, once collected, platelet values, all samples, once collected, should be capped to prevent Coshould be capped to prevent Co22 loss. loss.

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Platelet Function TestPlatelet Function Test

6- 6- Test samples, should be maintained at room temp Test samples, should be maintained at room temp during processing. Cooling inhibits the platelet during processing. Cooling inhibits the platelet aggregation response.aggregation response.

7- Stirring is necessary to bring the platelets in close 7- Stirring is necessary to bring the platelets in close contact with one another to allow aggregation to contact with one another to allow aggregation to occur.occur.

8- It is essential that the patient refrain from taking 8- It is essential that the patient refrain from taking any anti-inflammatory drugs, one week before the any anti-inflammatory drugs, one week before the test. The drugs inhibit the platelet’s release reaction.test. The drugs inhibit the platelet’s release reaction.

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