Comparison of the Procoagulant Activity of Freeze-Dried and Fresh Platelets with Phospholipid in Three Phospholipid Dependent Assays.

Background and Objective: Platelet activation and coagulation together determine the haemostatic activity of plasma. When activated, platelets expose phosphatidylserine (PS) at their outer surface, on which the tenase and prothrombinase complexes assemble, forming FXa and thrombin, respectively. In in-vitro tests phospholipid vesicles are normally used as a platelet substitute. However it is not clear if PL vesicles behave in the same way as platelets. The objective of the study was to compare and contrast the procoagulant activity (PCA) of lyophilized platelets, a bovine brain phospholipid standard (NIBSC 91/542) against fresh platelets using three phospholipid (PL) dependent assays. Inhibition of this PCA by Annexin A5 (AA5) and an anti-PS antibody (3G4) was also studied.

Methods and Materials: Lyophilised fixed platelets were obtained from Bio/Data corporation. Fresh platelets were collected into ACD-A from healthy donors and then gel-filtered through Sepharose 2B. Fresh platelets were activated by A23187 (10μM) before use in the assay systems. Platelet counts over a range of 10–200 x 10⁹/L were used in the assays. FXa and prothrombinase generation were measured in purified chromogenic systems, and calibrated against FXa and IIa standards. The time course of thrombin generated in plasma activated by FIXa was monitored, and peak thrombin levels and the total amount of free thrombin measured over time (ETP). AA5 and 3G4 at concentrations of 3.3–33 μM and 50–400 μg/mL respectively were used to inhibit the assembly and function of macromolecular coagulant enzyme complexes.

Results: Fresh platelets were 2.3 and 2.8-fold more potent than lyophilized platelets and PL standard respectively, in supporting FXa generation. When prothrombinase activity was measured, fresh platelets were 3.4 and 38-fold more potent than the lyophilized platelets and PL standard, respectively. Lyophilised platelets were 11-fold more potent than the PL standard. When thrombin generation was investigated fresh platelets were 3.1 (peak thrombin) and 3.7-fold (ETP) more potent than lyophilized platelets and 2.2 (peak thrombin) and 7-fold (ETP) more active than the PL standard. Lyophilised platelets were 0.7 (peak thrombin) and 1.9-fold more active than the PL standard. The tenase and prothrombinase PCA supported by the PL standard and fresh platelets were more susceptible to AA5 (8.3μM) inhibition (>70%) than lyophilized platelets (45 – 59%) though this activity was totally inhibited at 33μM. In the same tests 3G4 (400μg/mL) completely inhibited the PL standard but was only able to inhibit fresh and lyophilized platelets by 40–80%. Thrombin generation supported by the PL standard was completely inhibited by AA5 (16.5μM). Lyophilised platelets were more susceptible to the action of AA5 than fresh platelets. However 3G4 (400μg/mL) was unable to completely inhibit thrombin generation activity supported by PL standard or platelet preparations. 3G4 for all preparations delayed the time taken for peak thrombin levels to be reached.

Conclusions: Lyophilized platelets showed similar activity patterns to the PL standard in supporting tenase and thrombin formation/generation, though they were more active than the PL standard. Fresh-platelets were significantly better able to support these complexes, probably because of the presence of specific receptors for FVa and FXa and the maintenance of membrane fluidity.