Reversible Exposure of Phosphatidylserine on Thrombin-Stimulated Platelets Detected by Binding of Lactadherin.

Platelets stimulated by thrombin support activity of the phosphatidylserine (PS)-dependent prothrombinase and factor Xase complexes. However, annexin V, a PS-detecting protein, does not bind to thrombin-stimulated platelets. Thus, the exposure of PS and its necessity for the prothrombinase and factor Xase complexes on platelets has been questioned. We hypothesized that platelets expose limited quantities of plasma membrane PS at levels that remain insufficient to support annexin V binding. We tested the hypothesis by investigating the capacity of platelets to support binding of lactadherin, a PS-binding milk protein. Binding of fluorescein-labeled lactadherin vs. fluorescence-labeled annexin V to synthetic membranes and to platelets was measured by flow cytometry. Lactadherin binding was proportional to PS content over the 0–4% range while annexin V required a threshold of approx. 2.5% PS. Platelets stimulated with 0.1–0.5 μM A23187 bound lactadherin but not annexin V. Binding of lactadherin was Ca++-independent, inhibited by PS-containing phospholipid vesicles and by antibodies to the C2 domain of lactadherin indicating that binding occurs via the phospholipid-binding rather than the integrin-binding motif of lactadherin. Platelets stimulated by thrombin or via the PAR-1 thrombin receptor also bound lactadherin but not annexin V. The KD was 84 ± 1 nM and there were 380 binding sites/platelet. Experiments in which platelets were labeled with fluorescent NBD-PS indicated that PS exposure occurred over 2 min and involved less than 15% of platelet PS. Exposed PS subsequently decreased, with more than 75% loss over 2 hr. Lactadherin inhibited greater than 98% of prothrombinase and factor Xase activity on platelets stimulated by A23187 or by thrombin. These results indicate that thrombin-stimulated platelets support the prothrombinase and factor Xase complexes through reversible exposure of a small fraction of membrane PS. Exposure of PS can be detected with Ca++-independent lactadherin binding in dilute platelet rich plasma, a method suitable for clinical studies.