Platelet Aggregation

Individuals with inherited platelet disorders experience lifelong abnormal bleeding, usually at mucocutaneous sites or elsewhere after trauma or invasive procedures. Inherited platelet disorders should be suspected in patients with lifelong thrombocytopenia; bleeding not explained by a coagulation factor deficiency; presentation with an associated condition; and affected family members. Inherited platelet disorders can be caused by defects in surface membrane receptors, signaling pathways, granule formation and secretion, cytoskeletal remodeling and expression of procoagulant activity. 

Some of the most widely recognized inherited platelet disorders are listed in the following table.

Platelet Disorder Inheritance Biochemistry Clinical Findings
Von Willebrands Type 2B Autosomal Dominant Increased affinity of vWF for platelet GP1b Mild to severe bleeding
Bernard Soulier Autosomal recessive

Gp1b alpha (CD42b) and GP IX (CD42a) deficiency

Adhesion defect

Mild to moderate bleeding
Glanzmann’s Thrombasthenia Autosomal recessive

GpIIb (CD41) and GpIIIa (CD61) deficiency, Decreased fibrinogen binding

Aggregation defect

Moderate to severe bleeding

Normal platelet count, long bleeding time

Storage Pool Disease Autosomal recessive Dense granule defect Mild to severe bleeding
Hermansky Pudlak Autosomal recessive

Dense granule

Secretion defect

Albinism, poor vision, mild bleeding
Wiskott-Aldrich X-linked

WASp deficiency


Eczema, infections
Chediak Higashi Autosomal recessive

Dense granule

Storage pool

Albinism, infections, hemorrhage
Gray Platelet syndrome Autosomal recessive


Alpha granule deficiency

Thombocytopenia, myelofibrosis


Antiplatelet medications are prescribed to prevent thrombosis in patients with coronary artery disease, stroke, transient ischemic attacks and peripheral arterial disease. Aspirin is immediately absorbed after ingestion and irreversibly inhibits platelets within 15 to 30 minutes by binding to cyclooxygenase 1 (COX1). In patients with normal platelet lifespan, platelet inhibition lasts for approximately 3 days after the last dose.

Other nonsteroidal anti-inflammatory drugs (NSAIDS) such as ibuprofen and naproxen, also inhibit COX1 in platelets but not as strongly as aspirin and only while drug is circulating. Ibuprofen has as relatively short half-life of 2 hours. Platelet inhibition is usually undetectable by 24 hours after discontinuation. Naproxen has a half-life of 15 hours and platelets revert to normal function within 2 days after discontinuation.

Dipyridamole (Persantin) inhibits 2,3 phosphodiesterase enzyme in platelets and inhibits ADP induced platelet aggregation by increasing cAMP concentration. By itself, dipyridamole is considered to be a weak antiplatelet medication that seldom causes bleeding. Aggrenox is a more effective antiplatelet medication that is often prescribed for patients with a history of stroke. It is a combination of 25 mg aspirin and 200 mg of dipyridamole. Platelet transfusion is usually not necessary to control bleeding. The effect of dipyridamole can only be assessed by whole blood platelet aggregation.

Drugs inhibiting the platelet ADP P2Y12 receptor include clopidogrel, prasugrel, and ticagrelor. They are in widespread use, often in conjunction with low-dose aspirin. The inhibitory effects of these drugs on platelets are stronger than those seen with aspirin or NSAIDS.

Clopidogrel (Plavix) irreversibly inhibits platelet function by preventing binding of ADP to the platelet P2Y12 receptor. Clopidogrel is a prodrug which is metabolized to its active form by the cytochrome P450 enzyme pathway. A loading dose of clopidogrel (300-600 mg) inhibits platelet function within 2 to 4 hours, while a daily dose of 75 mg becomes effective within 24 hours and has maximum effect within 4 to 7 days. Circulating half-life is 7 to 8 days.

Like clopidogrel, prasugrel (Efient) also irreversibly inhibits binding of ADP to the platelet P2Y12 receptor. It is a prodrug which is metabolized to its active form by the cytochrome P450 enzyme pathway . Onset of action and circulating half-life are similar to clopidogrel.

Ticagrelor binds reversibly to the P2Y12 receptor and its active metabolite has a circulating half-life of approximately 9 hours. Platelet function returns to normal approximately 3 days after discontinuation. Ticagrelor inhibits platelet function more strongly than clopidogrel.

Patients undergoing percutaneous coronary intervention may be treated with platelet glycoprotein IIb/IIIa (GpIIb/IIIa) inhibitors such as Reopro, Integrilin and Aggrastat to prevent thrombosis. The platelet binding characteristics of these medications are summarized in the following table.

  Abciximab Eptifibatide Tirofiban
Brand Name ReoPro Integrilin Aggrastat
Structure Fab Ig Peptide Peptide
Plasma half life 20-30 minutes 2.5 hours 1.5 hours
Receptor half life 24-48 hours 2-4 hours 2-6 hours


Selective serotonin reuptake inhibitors, such as fluoxetine, sertraline, paroxetine, and clomipramine, decrease the uptake of serotonin by platelets. Since platelets are unable to synthesize serotonin, these medications lower the intracellular serotonin concentration, inhibit platelet aggregation and increase the risk of abnormal bleeding.

All of these drugs have an immediate onset of action. Although abciximab is rapidly cleared from plasma, platelet aggregation may be inhibited for 36 hours after discontinuation of the infusion. Platelet aggregation returns to normal within 2 to 6 hours after discontinuation of eptifibatide or tirofiban.

Activation of platelets causes them to change shape, secrete their intracellular granules, and aggregate with each other. Platelets normally contain two major types of granules – alpha and dense granules. Alpha granules contain fibrinogen, factor V, and von Willebrand factor while the dense granules contain platelet factor 4, ADP, ATP, calcium and serotonin. 

Light transmission platelet aggregation testing is useful in the evaluation of suspected hereditary and acquired disorders of platelet function. Whole blood is centrifuged at low centrifugal force to obtain platelet-rich plasma (PRP). PRP is pipetted into a cuvette that is positioned between a light source and a photocell.  Light transmission aggregometry measures the transmission of light through a sample of platelet-rich plasma (PRP) in response to a panel of platelet agonists. PRP, which is turbid, is stirred in a test cuvette maintained at 37° C. Light transmittance through PRP is measured relative to a reference cuvette containing platelet poor plasma. Light transmission is set at 100% in the reference cuvette and 0% in the platelet rich plasma. When a platelet agonist is added, platelets form increasingly larger aggregates and PRP begins to clear, allowing more light to pass through. This increase in light transmittance is directly proportional to the amount of platelet aggregation. Light transmission is recorded as a function of time after addition of each agonist. 

Modern platelet aggregometers are also capable of evaluating platelet secretion by measuring release of ATP from the dense granules of aggregating platelets. ATP released by platelet dense granules binds with luciferin-luciferase reagent and generates luminescent light that is detected by a photomultiplier. Measurement of platelet secretion allows the laboratory diagnosis of secretion defects with greater sensitivity than platelet aggregation testing alone.

Typically, the following platelet agonists are used for light transmission aggregometry and ATP secretion: ADP, collagen, arachidonic acid, epinephrine, thrombin and ristocetin.

Thrombin is useful for determining the maximum amount of ATP secretable from the dense granules. Secretion is independent of thromboxane synthesis. Decreased ATP release in response to thrombin is seen in secretion defects and storage pool disease.

Collagen is useful for checking the platelet’s general ability to aggregate. A lag phase of up to a minute is typically seen. Aggregation and secretion in response to collagen are partially dependent on thromboxane synthesis. Aspirin, and similar drugs, inhibit the production of thromboxane A2 which, in turn, inhibits aggregation and ATP secretion in response to collagen. Secretion to collagen of less than half the secretion to thrombin may be indicative of impaired thromboxane synthesis.

Arachidonic Acid is converted to thromboxane A in the presence of cyclooxygenase. Aspirin inhibits the cyclooxygenase pathway, causing a significant reduction in aggregation with this agonist. Other defects in thromboxane synthesis will give a similar pattern to cyclooxygenase inhibition.

ADP exposes the fibrinogen binding site on the membrane glycoprotein GPIIb/IIIa complex.

Epinephrine does not induce a change in platelet shape. Epinephrine produces a biphasic curve with the second wave dependent on thromboxane A2 synthesis. Epinephrine is the least consistent agonist. An abnormality in epinephrine response alone usually does not have any clinical significance.

Ristocetin is an antibiotic used for the detection of von Willebrand disease and Bernard Soulier syndrome. Lower concentrations of ADP can detect the hyper-responsiveness associated with Type 2B or Platelet-Type vWD. A qualitative defect may be seen with subjects taking aspirin and a saw-tooth or aggregation-disaggregation pattern may be with Glanzmann’s Thrombasthenia.

Reference ranges for light transmission aggregometry and ATP secretion using platelet rich plasma are summarized in the following table.

Agonist Aggregation Ref Range ATP Release Ref Range

ADP (20 uM)

45-94% 0.19-1.45 nmoles

Epinephrine (10 uM)

48-93% >0.52 nmoles

Collagen (0.19 mg/mL

52-88% 0.44-1.68 nmoles
Arachidonic (500 ug/mL) 42-79% 0.04-1.10 nmoles

Ristocetin High (1.5 mg/mL)

30-100% NA

Ristocetin Low (0.5 mg/mL)

0-5% NA


There are no known clinical reasons for measuring ATP Release with ristocetin.

The following table summarizes the responses to platelet agonists most commonly seen in hereditary platelet disorders and drug induced platelet dysfunction (including aspirin, NSAID’s, clopidogrel, antibiotics, and various cardiovascular and psychotropic drugs), uremia, and myeloproliferative disorders.

Platelet aggregation and ATP secretion are clinically significant in the detection and diagnosis of acquired or congenital qualitative platelet defects.  The platelet’s ability or inability to respond to particular aggregating reagents is the basis for differentiating platelet dysfunctions as shown in the table below.

Disorder ADP Epinephrine Arachidonic Collagen Thrombin Ristocetin
Glanzmann Absent Absent Absent Absent Decreased Disaggregation
BS or vWD Normal Normal Normal Normal N or D Absent
VWD 2b Normal Normal Normal Normal Normal Increased
Storage Pool D or A Decreased D or A N or D   Normal
Aspirin N or D N or D Absent Decreased   Normal
P2Y12 D or A Normal Normal Normal   Normal
Aspirin + P2Y12 Decreased N or D Absent Decreased   Normal
GPIIb/IIIa D or A D or A D or A D or A   Normal
Fish Oil Decreased   Absent Normal    
Uremia N or D N or D Decreased N or D   Normal
MPN Normal D or delayed Normal Normal   Normal

BS = Bernard Soulier syndrome; vWD = von Willebrand Disease; MPN = myeloproliferative neoplasm; N = normal; D = decreased, A=absent

Aggregation abnormalities with ADP or epinephrine alone are nondiagnostic and may be a false positive result.

Interpretation of ATP secretion results is summarized in the following table.

Disorder ADP Epinephrine Arachidonic Collagen Thrombin
Glanzmann Absent Absent Decreased Decreased Decreased
Bernard Soulier Normal Normal Normal Normal Normal
VWD 2b Normal Normal Normal Normal Normal
Storage Pool D or A D or A D or A D or A D or A
Aspirin D or A Absent Absent Decreased Normal


Decreased response to ristocetin and normal aggregation with the other agonists is seen in von Willebrand disease and Bernard Soulier Syndrome.  For diagnosis of von Willebrand disease, platelet response to both concentrations of ristocetin must be evaluated together with von Willebrand disease screening tests such as VW factor activity, VW factor antigen, and factor VIII assay. Glanzmann's thrombasthenia is characterized by absent aggregation with ADP, collagen and arachidonic acid and normal response to ristocetin. Inherited disorders of platelet secretion usually demonstrate a decreased secondary aggregation wave with ADP, decreased response to collagen and variable response to arachidonic acid; however, the pattern is not always typical.

Aspirin and other non-steroidal anti-inflammation drugs produce slightly decreased aggregation with ADP, collagen and arachidonic acid.  Decreased aggregation in response to ADP and collagen and absent aggregation response to arachidonic acid, is compatible with a combined aspirin/ clopidogrel effect.

Antibiotics and other drugs produce variably decreased platelet aggregation. The abnormal platelet function commonly seen in patients with uremia, dysproteinemia and liver disease is also associated with variably decreased platelet aggregation. Epinephrine induced aggregation and secretion may be absent in thrombocytosis secondary to chronic myeloproliferative disorders, in addition to the presence of a variety of other aggregation abnormalities.

Indications for ordering platelet aggregation and secretion testing include a bleeding diathesis secondary to suspected platelet dysfunction, especially if the PFA-100 platelet function screening test is abnormal and drug-induced platelet dysfunction is ruled out. If the patient is taking aspirin, NSAID’s or other platelet-inhibitory drugs, the drug should be discontinued for at least 2 weeks prior to aggregation testing.   

Seven 4mL sodium citrate (3.2%, light blue-top) tubes of blood are required for full aggregation and secretion testing. Platelet aggregation testing alone (without secretion) may be ordered and requires four 4mL tubes of blood. Specimens must be kept at room temperature. Testing should be performed within 4 hours after venipuncture.

Red Blood Cells in PRP can inhibit the ability of the Aggregometer to detect changes in light intensity.  This may cause the appearance of a decrease in platelet aggregation. Lipids in PRP can interfere with light transmission readings & prevent recording of aggregation. If the platelet count is <200,000 an abnormal result may be due to a low platelet count rather than a true platelet dysfunction

Reference value is normal aggregation with ADP, arachidonic acid, collagen, and ristocetin.  A clinical pathologist interprets results. 

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