Procoagulant Function and Phosphatidylserine Distribution on Immortalized Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) causes diffuse intravascular coagulation that can worsen with chemotherapy. APL cells expose tissue factor (TF) that contributes to procoagulant activity but the relationship of procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Lactadherin, a milk protein, binds to membranes containing PS and the binding interaction is mediated by stereoselctive interaction with phosphatidyl-L-serine. As such, lactadherin can serve as a probe to detect low-level PS exposure on cell membranes when the quantity of PS remains below the threshold required for binding of annexin V. Lactadherin can also inhibit prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding Lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of NB4 cells was first measured in plasma with NB4 cells serving as thromboplastin. Activity of NB4 cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Plasma procoagulant activity of NB4 cells increased approx. 15-fold after cells were exposed to 1 μM etoposide for 48 hr and decreased by 80% after 48 hr treatment with 1 μM all trans retinoic acid (ATRA) or As2O3, demonstrating that the procoagulant phenotype of NB4 cells reflects the clinical pattern of diffuse intravascular coagulation in APL. Increased plasma procoagulant activity corresponded to increased intrinsic and extrinsic factor Xase activity as well as increased PS exposure on cells that were not yet frankly apoptotic. ATRA and As2O3 treatment decreased PS exposure and activity of intrinsic and extrinsic enzyme complexes. Lactadherin inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for ∼70% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by monoclonal Ab’s that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure. Following treatment with etoposide, staining increased. Many cells exhibited a faint, rim-pattern indicating low level, diffuse PS exposure. Factor VIII binding sites and TF distributed to discreet, but overlapping regions of the cells. Fluorescein-Glu-Gly-Arg-chloromethyl ketone derivatized factor IXa bound to the cells in a factor VIIIa-dependent manner in a pattern that reflected the factor VIII(a) binding pattern but not the lactadherin distribution pattern. These results indicate that PS exposure parallels procoagulant activity on NB4 cells and is required for at least 85% of intrinsic and extrinsic factor Xasee activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.