Platelet Aggregation

One milliliter (or 0.5 mL) of platelet-rich plasma (PRP) (obtained by centrifuging anticoagulated blood for 15 min at 135g and 37°C) is placed in a cuvet containing a metal stir bar (20). In an aggregometer, the stir bar is rotated magnetically at 1100 rpm, and light transmission through the PRP is recorded by a photometer. On the addition ofmost aggregating agents, the platelets change shape from discs to a more rounded form with pseudo-pods, resulting in a transient, small decrease in light transmission that is followed by a large increase as the platelets aggregate; the rate and extent of the increase in light transmission are measured. Platelet aggregation is highly variable within patient populations, is highly dependent on the skill and experience of the investigators performing the measurements, and may be affected by the preparation and handling of blood samples (21). It is affected by very low platelet counts (<100 ^ 109 L-1) and the concentration of agonist. High concentrations of all these agonists are associated with the formation of TXA2, the secretion of granule contents, and the appearance of P-selectin on the platelet surface.

ADP is a weak agonist compared with collagen or thrombin. In citrated PRP, low concentrations of ADP cause only a primary, incomplete, reversible phase of aggregation, but at concentrations above 1-3 pM, primary aggregation ofhuman platelets does not reverse and is followed by a secondary, irreversible phase (Fig. 2). This biphasic aggregation depends on TXA2 formation. At high concentrations of ADP, the two phases fuse, result-

Fig. 2. ADP-induced aggregation in citrated PRP, showing primary phase of aggregation induced by low concentration of ADP (left), primary aggregation followed by secondary wave induced by slightly higher concentration of ADP (middle), and fusion of two phases of aggregation at high concentration of ADP (right). The arrows indicate the points of addition of ADP.

Fig. 2. ADP-induced aggregation in citrated PRP, showing primary phase of aggregation induced by low concentration of ADP (left), primary aggregation followed by secondary wave induced by slightly higher concentration of ADP (middle), and fusion of two phases of aggregation at high concentration of ADP (right). The arrows indicate the points of addition of ADP.

ing in a smooth aggregation curve resembling that seen on stimulation with collagen or thrombin. The drugs ticlopidine and clopidogrel act through this receptor to cause a similar selective inhibition of responses to ADP. Drugs that inhibit TXA2 formation, such as aspirin, prevent the secondary phase of ADP-induced aggregation.

The weak agonist epinephrine (5-10 ^M) aggregates platelets in citrated PRP without an initial change in platelet shape, but epinephrine is the least consistent agonist. If a subject has taken aspirin or other drugs that inhibit TXA2 formation, the platelets will not aggregate in response to any concentration of epinephrine. Aspirin and other drugs that inhibit TXA2 formation can block aggregation in response to low concentrations of collagen, although platelets change shape.

Thrombin receptor-activating peptide (TRAP) mimics the strong aggregating effect of thrombin on platelets. The response is only slightly reduced when TXA2 formation is blocked. Secretion defects, particularly the lack of the potentiating effects of ADP from the dense granules, result in abnormal patterns of aggregation characterized by a normal primary phase but absent secondary phase, and impaired aggregation induced by low concentrations of collagen or TRAP.

Whole-Blood Platelet Aggregation

Platelet aggregation is measured in diluted, anticoagulated whole blood by impedance aggregometry. Two electrodes are immersed in the sample, and when a current is passed through it, platelets adhere to the electrodes. On the addition of an aggregating agent with stirring, platelets aggregate around the platelets on the electrodes, and the rate and extent ofthe increase in impedance are recorded. The advantages ofthis test are that the preparation of PRP is avoided, and platelet function in lipemic blood can be evaluated. Again, the test is not suitable at very low platelet counts (22).

Fig. 3. Schematic of RPFA device.

Ultegra Rapid Platelet Function Assay

The Ultegra Rapid Platelet Function Assay (RPFA) provides a simple, rapid, accurate measure of platelet function that is used at the bedside to monitor patients receiving treatment with Gpllb/IIIa antagonists (23,24). Immediately after blood has been taken into citrate, 0.16-mL samples are drawn into two sample channels in a disposable cartridge (Fig. 3). The blood is mixed with the platelet agonist (iso-S) FLLRN and fibrinogen-coated polystyrene beads for 70 s by movement of a microprocessor-driven steel ball, and light transmission through the sample is measured. Agglutination occurs between the activated platelets and the fibrinogen-coated beads such that they fall out of suspension, leading to an increase in light transmission. The rate and extent of agglutination are used to calculate the platelet aggregation unit, which decreases in the presence of GpIIb/ IIIa antagonists, because agglutination occurs in direct proportion to the number of unblocked GpIIb/IIIa receptors on the activated platelets. It has been used successfully to monitor the inhibitory effects of the GpIIb/IIIa antagonists (25,26).

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