H. Redlich, J. Vickers, P. Spangenberg, University of Applied Sciences of Jena, Faculty of Medical Engineering, W. L¨ osche, University Hospital Jena, Centre for Vascular Biology and Medicine Erfurt, W. L¨ osche, S. Heptinstall, P. Spangenberg, University Hospital Notting- ham, Cardiovascular Medicine, UK, B. Kehrel University Hospital M¨ unster, Department of Internal Medicine, Germany. Correspondence to: Professor Dr Peter Spangenberg, University of Applied Sciences of Jena, Faculty of Medical Engineering, Tatzendpro- menade 1b, P.O. Box 10 03 14, 07703 Jena, Germany. Formation of platelet–leukocyte conjugates in whole blood H. Redlich, J. Vickers, W. L¨ osche, S. Heptinstall, B. Kehrel, P. Spangenberg The purpose of this investigation was to obtain information on platelet–leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet–monocyte and platelet–polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes, conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L -formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb–IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet–monocyte conjugate formation, but did inhibit the platelet–PMNL interaction; this indicates that GpIIb–IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interfers with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet–leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet–leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation. Introduction Adhesion of platelets to white blood cells is usually studied by quantitating the heterotypic conjugates (rosettes) that form after mixing suspensions of isolated platelets and polymorphonuclear leukocytes (PMNL). 1 Quantitation is usually via microscopy. More recently attempts have been made to quantify such conjugates using flow cytometry, 2–4 a method that is applicable to conjugate formation in whole blood as well as after mixing individual cell types in vitro. Here we have used a method to quantitate platelet adhesion to PMNL, to monocytes and to lymphocytes using a flow cytometric approach involving the use of three antibodies, anti-CD14, anti-CD45 and anti-CD42a. These were used to identify the three types of leukocytes independently and to quantify the amount of platelet label (anti-CD42a) associated with each of the leukocyte populations. Simultaneously, exposure of the activation- dependent antigen CD62P (on platelets) and CD11b (on leukocytes) were measured. Blood samples from healthy volunteers were studied immediately after blood sampling and also after stirring the blood in an aggregometer. We also determined the 0953-7104/97/060419-07 $9.00 © 1997 Carfax Publishing Ltd Platelets (1997) 8, 419–425 Platelets Downloaded from informahealthcare.com by Univ of Nottingham - Periodicals Acq Group on 02/20/15 For personal use only.