Platelet Function Platelet must be adequate in number

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. drmsaiem

Key Mediators in Platelet Adhesion, Activation and Aggregation INJURY v. WF ADP-receptor THROMBUS Shear Forces Adhesion • • v. WF Thrombin Collagen Fibronectin Activation • • • Membrane changes Granule secretion GPIIb/IIIa expression Multiple agonists Feedback loops drmsaiem Aggregation • GPIIb/IIIa-mediated • Fibrinogen • v. WF

Platelet Adhesion and Activation Normal platelets in flowing blood Platelets adhering to damaged endothelium and undergoing activation Aggregation of platelets into a thrombus Platelets adhering to subendothelial space 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. 15– 35.

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

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). drmsaiem

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

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: 329– 34. drmsaiem

Platelet Function (Release Reaction) Collagen exposure or thrombin action results in the secretion of platelet granule contents which include ADP, serotonin, fibrinogen, lysosomal enzymes, βthromboglobulin. Collagen and thrombin activate platelet prostaglandin synthesis. Released ADP and thromboxane A 2 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. drmsaiem

Platelet Function (Platelet aggregation) As they do so further release reactions occur liberating more ADP and thromboxane A 2 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. drmsaiem

Platelet Function Test Bleeding Time v Principle: Bleeding time is defined as the time taken for a standardized skin wound to stop bleeding. v Platelet aggregation v 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. drmsaiem

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. drmsaiem

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 37°C in the heat block of the instrument, and stirred by a small magnetic bar. Light transmittance through the PRP is recorded. drmsaiem

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. drmsaiem

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. drmsaiem

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 37°C at a p. H of 6. 5 to 8. 5. To help maintain platelet values, all samples, once collected, should be capped to prevent Co 2 loss. drmsaiem

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 platelet’s 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. drmsaiem