Modulation of Platelet Function by Recombinant Thrombomodulin Hematologic Implications

Several recombinant thrombomodulin (r-TM) preparations have been developed for different clinical indications. While the in vitro effects of r-TM on blood coagulation parameters are extensively studied, the effect of this agent on platelet function tests such as the adhesion, aggregation, activation and secretion are not fully explored. The purpose of this study was to investigate the effect of a recombinant version of thrombomodulin (ART 123, Asahi, Pharmaceutical, Japan) on various platelet function tests. Platelet aggregation, platelet release and platelet activation by tissue factor (TF) utilizing flow cytometry studies were carried out. In the platelet aggregation studies, citrated whole blood was supplemented with graded amounts of r-TM, in a concentration range of 0–10 ug/ml in the blood of normal healthy volunteers (n=25). Platelet rich plasma (PRP) was prepared by controlled centrifugation (800g) for 15 minutes. Platelet count in the PRP was adjusted to 250,000/ul and aggregation studies were carried out using ADP (5 and 2.5 uM), and alpha thrombin (0.5 U/ml). Platelet activation studies were carried out using flow cytometric method utilizing citrated whole blood and recombinant TF and ADP as activators. In this procedure whole blood was supplemented with TM in a concentration range of 0–10 ug/ml and incubated. TF was then added and further incubated for an additional 2 minutes. Platelets were fixed and incubated with CD 61 and CD 62 antibodies and analyzed using the flow cytometer. Platelet release assays included the 14C serotonin release (SRA) from the washed platelets. In the platelet aggregation assay r-TM did not produce any significant inhibition of agonist induced aggregation with ADP and epinephrine, however, r-TM produced a strong concentration inhibition of thrombin induced aggregation with an IC 50 of 0.42 ug/ml. In the flow studies, r-TM produced an initial augmentation of the generation of microparticles at concentrations up to 0.31 ug/ml (ranges; 5–20%). However at concentrations > 0.31 ug/ml r-TM produced a concentration dependent inhibition of the microparticle formation with an IC 50 of 2.5 ug/ml. At concentrations of >5.0 ug/ml a complete inhibition of TF mediated microparticle formation was noted. Interestingly, r-TM did not produce any inhibition of the p-selectin expression at all concentrations studied. In the SRA, r-TM did not produce any release at concentrations up to 10 ug/ml. However, r-TM produced a strong inhibition of the alpha thrombin induced SRA release. These studies demonstrate that although in the agonist induced platelet aggregation studies r-TM does not produce any modulation of platelet aggregation responses with the exception of thrombin, in the flow cytometric studies it produces a biophasic response. In a concentration range of 0 - .31 ug/ml it produced a slight augmentation of the TF mediated platelet activation. However, at higher concentrations it produced an inhibition of the platelet microparticle formation. Interestingly, there was no effect of r-TM on p-selectin activation. These studies suggest that although r-TM does not produce any inhibition of the agonist induced aggregation of platelets, it can inhibit the TF mediated microparticle formation. Moreover, since r-TM at concentrations of up to 10 ug/ml does not produce any effect on p-selectin expression. It is unlikely to produce any primary hemostatic compromise in a therapeutic range of 2–6 ug/ml.