ABSTRACT: Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD61 and side-scatter gating. The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure. Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10³; neutrophils, 1.75 x 10³; monocytes, 2.45 x 10³; platelets, 0.14 x 10³. These levels represent <= 0.1% of the values obtained after maximal stimulation of PS exposure with calcium ionophore, confirming that virtually all PS is intracellular in normal circulating leukocytes and platelets. Pretreatment of whole-blood samples with ammonium chloride to lyse erythrocytes caused a 9- to 300-fold increase in annexin V binding to leukocytes, indicating that analysis of unlysed whole-blood samples is essential to avoid artifactual increases in annexin V binding to leukocytes. Comparison of annexin V with two other markers of platelet activation, CD62P and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC1 antibody, indicated that platelets from normal donors showed the least amount of activation with the annexin V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confirm that these cell types normally circulate with extremely low levels of exposed PS.    © 1999 Academic Press

Keywords: Phosphatidylserine, annexin V, flow cytometry, leukocytes, platelets.

Reprint requests to: Jonathan F. Tait, M.D.,Ph.D., University of Washington, Laboratory Medicine, Room NW-120, Box 357110, Seattle, WA 98195-7110, fax: (206) 598-6189, e-mail: tait@u.washington.edu.

ABSTRACT: C4b-binding protein (C4BP) regulates the complement system and the anticoagulant activity of protein S. Protein S can bind to C4BP, resulting in a decreased cofactor activity of protein S for anticoagulant activated protein C. C4BP contains several identical alpha-chains and a single beta-chain. Each chain contains Short Consensus Repeats (SCRs). By making chimeras of beta-chain SCRs fused to tissue-type plasminogen activator (tPA chimeras), we found that beta-chain SCR-2 contributed to the interaction of beta-chain SCR-1 with protein S (van de Poel RHL, Meijers JCM, Bouma BN. J Biol Chem 274:15144-15150, 1999). Chimeras containing C4BP alpha-chains with SCR-1, SCR-1+2 or SCR-1+2+3 replaced by their beta-chain counterpart had affinities for protein S similar to C4BP (Hrdig Y, Dahlbck B. J Biol Chem 271:20861-20867, 1996). This was not in agreement with the finding that beta-chain SCR-2 contributed to the interaction and could be explained by the possibility that alpha-chain SCR-2 in the alpha-chai n chimeras contributed comparable with beta-chain SCR-2 in the tPA chimeras. To investigate this we constructed a tPA chimera containing beta-chain SCR-1 and alpha-chain SCR-2 (beta1alpha2). Binding studies showed that beta1alpha2 had a lower affinity compared with SCR-1+2, indicating that alpha-chain SCR-2 did not contribute to the interaction. The difference with the alpha-chain chimeras may be explained by the fact that the alpha-chain chimeras were linked by their C-terminal cysteines, resulting in multiple binding sites in a single molecule. Thereby, the effect of a lower affinity of each alpha-chain chimera may have been masked. The studies performed here help to clarify the apparent inconsistencies in two previous reports about the contribution of the SCR-2 domain in C4BP to protein S binding. In conclusion, beta-chain SCR-2 specifically contributes to the interaction of SCR-1 with protein S.    © 1999 Academic Press

Keywords: Protein S, C4b-binding protein, protein C, blood coagulation, complement.

Reprint requests to: Bonno N. Bouma. Thrombosis and Haemostasis Laboratory, G03.647, Department of Haematology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, THE NETHERLANDS, fax: +31-30-2511893, e-mail: b.n.bouma@lab.azu.nl.