General Approach in Investigation of Haemostasis Platelets aggregation

Studying Platelets Disorders Numbers CBC PLT count PLT morphology Function Bleeding Time (BT) Platelet Aggregation Whole blood aggregation Platelet-rich plasma aggregation

Platelets contribute to Hemostasis in two main ways: Primary haemostatic plug : Adhesion Aggregation Secretion Secondary Haemostatic plug: Procoagulant activities are generated

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

Platelets Aggregation Platelet-Platelet interaction Mechanism components ATP Ionized calcium Fibrinogen PLT receptor GPIIb/IIIa Initial aggregation – REVERSIBLE Secondary aggregation – IRREVERSIBLE* = white clot, also know as platelet plug formed. * The transformation of irreversible aggregated platelets into a mass of degenerative platelet material without membranes is termed viscous metamorphosis. (by platelet lysosomes)

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Why Platelet aggregation testing Evaluation of suspected hereditary and acquired disorders of platelet function. Platelets normally contain three major types of granules . The alpha granules contain fibrinogen, PF4, factor V, von Willebrand factor…. The dense granules contain ADP or ATP, calcium and serotonin. lambda granules – similar to lysosomes and contain several hydrolytic enzymes. Hereditary platelet function disorders includes Rare defects of adhesion (Bernard Soulier syndrome) Rare defects of aggregation (Glanzmann thrombasthenia) More common defects of secretion (alpha or dense granule deficiency, aspirin-like defects, or other primary secretion defects). Acquired platelet function disorders are more common than the hereditary disorders and include drug-induced platelet dysfunction (including aspirin, NSAID’s, clopidogrel, antibiotics, various cardiovascular and psychotropic drugs), uremia, and myeloproliferative disorders. NSAIDs: Nonsteroidal anti-inflammatory drugs, like Ibubrufin, asprin, Naproxen are drugs with analgesic (also known as a painkiller) and antipyretic (fever-reducing) effects and which have, in higher doses, anti-inflammatory effects. Clopidogrel is an oral, thienopyridine class antiplatelet agent used to inhibit blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. Uremia is a term used to loosely describe the illness accompanying kidney failure .

Principle Using Aggregating agents to induce platelet aggregation or cause platelets to release endogenous ADP, or both. Platelet aggregation is studied by means of a platelet aggregometer, Used Principle: Photo-optical Method luminescence technology (Platelet Lumiaggregometry) Electrical Impedance Method

AGGREGATING AGENTS

Arachidonic acid: used to assess the viability of the thromboxane pathway. Thrombin: reacts with several membrane sites to induce full aggregation and secretion of organelle contents independent of the prostaglandin or ADP pathways. ADP: binds to a specific platelet membrane receptor and causes platelet activation and release of dense granule stored ADP. Shows biphasic aggregation. Epinephrine: binds to specific receptor and causes ADP secretion, but does not cause aggregation in storage pool disorder or release defects. Collagen: Shows no primary wave of aggregation and depends on intact membrane receptors, membrane phospholipase pathway integrity and normal cyclooxygenase and thromboxane pathway function. Ristocetin: requires vWF and intact surface membrane including a functional vWF receptor site (GPIb).

Electrical Impedance Method These types of analyzers may use citrated whole blood, as the test sample. As platelets aggregate, the coat an electrode, impeding the electrical current through the analyzer. http://www.platelet-research.org/3/pfa.htm

Luminescence technology (Platelet Lumi aggregometry) The lumi-aggregometer may be used to simultaneously measure platelet aggregation and secretion. The instrument records both aggregation and secretion of dense-granule ATP. The ATP is measured by its reaction with firefly luciferin to give chemiluminescence. The resulting light emission is detected, amplified, and recorded by the instrument. Performed by using whole blood or PRP. This modification of aggregation is particularly sensitive to ATP release, and is as sensitive measure of platelet activation. Firefly luciferin is the luciferin, or light-emitting compound, found in many firefly (Lampyridae) species, a yellow light emission from. As with all other luciferins, oxygen is required to elicit light; however, it has also been found that ATP and magnesium are required for light emission [A] + [B] → [AB◊] → [Products] + light

Photo-optical Aggregometer (PLT Aggregometry Using PRP) The Platelet-rich plasma, which is turbid in appearance, is placed in a cuvettes, warmed to 37°C in the heating block of the instrument, and stirred via a small magnetic bar. Baseline light transmittance through the platelet-rich plasma is recorded. The addition of an aggregating agent causes the formation of larger platelet aggregates with a corresponding increase in light transmittance, because of a clearing in the platelet-rich plasma. The change in light transmittance is converted to electronic signals and recorded as a tracing by the chart recorder. The optical density of platelet-rich plasma falls as platelets form aggregates. The amount, and to some extent the rate, of fall is largely dependent on platelet reactivity, provided that all other variables (e.g. platelet count, mixing speed, and temperature) are controlled. The optical density changes are monitored, wherein the optical density of platelet-rich plasma reflects the degree of platelet aggregation induced by one of a variety of agonists. The optical density is monitored using an aggregometer connected to a chart recorder so results may be recorded graphically.

Patient Sample – 3.2% citrated WB Approximately 20 ml of blood is needed for a full aggregation study. Test Sample – PRP ( Platelets count fall (200–600 × 109/L) Principle – photometry: optical density of PRP warmed to 37° C is determined before and after the addition of various aggregating agents Issues Sample quality is critical Fibrinogen levels are important Agonists must be prepared fresh daily Thrombocytopenia makes result interpretation difficult Complete patient history is essential Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is approximately 200 × 109/L, and platelets are maintained in suspension by a magnetic stir bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.) Figure 2 – Five possible phases of PLT aggregation: 1) baseline, 2) agonist addition and shape change, 3) primary wave, 4) secretion, and 5) secondary wave. http://www.platelet-research.org/3/aggregometry.htm

Sample Quality for PRP PLT Aggregometry Clean venipunture Hemolysis = increased plasma ADP = prematurely activated platelets Lipemia may obscure OD during PLT aggregation PRP contact with glass = prematurely activated PLTs Keep samples capped to prevent loss of CO2 – change sample pH ( PRP PH should be maintained within the range 7.7–8.0) Store samples @ room temp – prevent inhibition of PLT aggregation Perform testing within 3 hours of sample collection

Procedure: Switch on aggregometer to warm to 37°C. Select AGGR TEST Select the type of agonist (ADP, AA, RISTO, THR, COLL, ADR) Place 0.27 ml PRP in a test cup and put in the incubation channels. Put beads into the cups, wait 1 minute to warm. Transfer the cups to testing channels. Press PPP, it will appear the status <PPP> Add 0.03 ml of agonist to bottom of cuvettes and press start key then monitor optical density changes for three minutes. Repeat this procedure for each agonist Curve Display. * ADP should placed in ICE.

PRP Aggregometry Agonist & Patterns ADP (at appropriate concentration) Biphasic curve: 1o and 2o waves (requires intact prostaglandin pathway) Note: if ADP is added at too low or too high concentration, it will not get biphasic response Epinephrine Biphasic curve; requires intact prostaglandin pathway Collagen Lag phase followed by 2o wave only

Thrombin Arachidonic acid Serotonin Ristocetin A biphasic however, often only a single broad wave Binds to vWF/GPIb/IX complex and results in agglutination Evaluates adhesion reaction Thrombin Biphasic curve. Irreversible aggregation only (does not require cyclooxygenase) Arachidonic acid 2o wave only; assesses cyclooxygenase pathway Serotonin A primary wave of aggregation with a maximum of 10% to 30% transmittance followed by disaggregation. Ristocetin is an antibiotic, It causes platelet agglutination and blood coagulation and is used to assay those functions in vitro, e.g., to diagnose von Willebrand disease (vWD) or the Bernard-Soulier syndrome. Platelet agglutination caused by ristocetin can occur only in the presence of large multimers of von Willebrand factor, so if ristocetin is added to blood lacking the factor , it will not coagulate.

Interpretation Platelet aggregation occurs as a two-step process, known as primary and secondary waves of aggregation. The primary wave of aggregation is observed when platelets adhere to one another in the presence of an external agent (agonist) such as ADP, epinephrine, or ristocetin. Secondary aggregation is characterized as the aggregation that occurs after the platelets have been stimulated to secrete the substances contained in their organelles. It should be noted that some agonists will stimulate primary aggregation and some will stimulate secondary aggregation. Others will stimulate both primary and secondary aggregation, yielding a "biphasic" aggregation curve.

In addition, different concentrations of the same agonist can produce varying patterns of primary and secondary aggregation. For example, Low concentrations of ADP induce biphasic aggregation (i.e., both a primary and a secondary wave of aggregation); Very low concentrations of ADP (l.5 ug/ml. final concentration) induce a primary wave followed by disaggregation; And high concentrations of ADP (10 ug/ml, final concentration) induce a single, broad wave of aggregation“ A biphasic aggregation response to ADP will not be seen in patients with platelet release disorders. Patients with Glanzrnann's thrombasthenia show incomplete aggregation with ADP regardless of the final concentration.

In patients with severe von Willebrand disease, aggregation to ristocetin is characteristically absent. Decreased to normal aggregation to ristocetin can be seen in patients with mild von Willebrand disease. Correction of the abnormal ristocetin aggregation curves can be seen by the addition of normal, platelet-poor plasma to the patient's platelet-rich plasma. Abnormal ristocetin-induced platelet aggregation may also occur in patients with Bernard-Soulier syndrome, Platelet storage pool defects Idiopathic thrombocytopenia purpura (ITP).

Glanzmann thrombasthenia Normal PLT count, but abnormal clot retraction Absence of secondary aggregation to ADP, epinephrine, collagen, (thrombin) Normal response to ristocetin

Bernard-Soulier syndrome Platelet aggregation test Failure to aggregate in the presence of ristocetin Aggregation by other agonists (ADP, collagen, epinephrine): normal Response to low-dose thrombin: may be delayed Platelet storage granule defects Dense (δ) granule defects ~ storage pool deficiency α granule defects ~ gray platelet syndrome Heterogeneous group of disorders Mild to moderate bleeding diathesis Abnormalities in platelet aggregation bleeding diathesis (bleeding tendency) : is an unusual susceptibility to bleeding (hemorrhage) mostly due to hypocoagulability, in turn caused by a coagulopathy

Precautions Prior To Studying Platelet Aggregation Aspirin-containing compounds should be excluded for at least 10 days prior to testing, as Aspirin interferes with the release reaction. Ingestion of other drugs known to influence platelet function should also be avoided for at least the time required for their elimination from the circulation. These include certain antihistamines, antibiotics, and anti-depressants. A check should be made of any drugs being prescribed before performing platelet function testing. Chylomicrons can interfere with the measurement of platelet aggregation, studies should not be carried out shortly after a fatty meal. Many other “normal” dietary constituents, including alcohol, onions, garlic, peppers, and ginger, may also inhibit platelet aggregation. Chilling activates platelets, and so the blood is processed at 20°C – 25°C.

Comment In evaluating patients with suspected platelet disorders, the aggregating agents most commonly used are ADP in varying concentrations, collagen, epinephrine, and ristocetin. Aspirin, aspirin compounds, and anti-inflammatory drugs inhibit the secondary wave of aggregation by inhibiting the release reaction of the platelet. The intensity of platelet aggregation may be estimated by recording the change in absorbance as a percentage of the difference in absorbance between platelet-rich and platelet- poor plasma. This has limited usefulness because absorbance is dependent on the size and density of platelet clumping and the number of platelets that aggregate.

Drugs and PLT Function Aspirin: Acetylsalicyclic acid  Irreversibly inhibits Cyclooxygenase Clopidogrel : Plavix Irreversibly inhibits P2Y12 Dipyridamole:  inhibits Thromboxane synthase Abciximab ReoPro  inhibits GP IIb/IIIa