Direct Observation of Membrane Tethers Formed during Neutrophil Attachment to Platelets or P-selectin under Physiological Flow David W. Schmidtke and Scott L. Diamond Institute for Medicine and Engineering, Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Abstract. Adhesion and subsequent aggregation be- tween neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the mi- crovilli of neutrophils. High speed, high resolution videomicroscopy of ﬂowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 6 4% of the interactions. After capture by spread platelets, neutro- phil membrane tethers (length of 5.9 6 4.1 mm, n 5 63) were pulled at an average rate of 6–40 mm/s as the wall shear rate was increased from 100–250 s21. The average tether lifetime decreased signiﬁcantly (P , 0.001) from 630 to 133 ms as the shear rate was increased from 100 s21 (Fbond 5 86 pN) to 250 s21 (Fbond 5 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics un- der stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at Introduction Adhesion and aggregation of platelets and neutrophils to each other and to the blood vessel wall are critical events in inflammation and thrombosis, and are relevant to pa- tients with stroke, acute myocardial infarction, extracor- poreal circulation, and coronary angioplasty (Rinder et al., 1992; Kostantopoulos et al., 1995; Mickelson et al., 1996; Neumann et al., 1997; Hagberg et al., 1998). The initial bonding event between neutrophils and platelets is medi- ated by the P-selectin present on the surface of activated platelets (z12,000 molecules/cell) with P-selectin glycopro- tein ligand 1 (PSGL-1;1 z104 molecules/cell) located on the tips of neutrophil microvilli (Moore et al., 1995; Bruehl et Address correspondence to Scott L. Diamond, Institute for Medicine and Engineering, Department of Chemical Engineering, University of Penn- sylvania, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadel- phia, PA 19104. Tel.: (215) 573-5702. Fax: (215) 573-2093. E-mail: sld@ seas.upenn.edu 1Abbreviations used in this paper: DIC, differential interference micros- copy; PSGL-1, P-selectin glycoprotein ligand 1. 150 s21, thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 6 8.8 mm long), whereas tether breakage (average lifetime of 3.79 6 3.32 s) caused an acute jump in the rolling veloc- ity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (.40 mm) were sometimes pulled, which de- tached in a ﬂow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the ﬁrst visualization of the often hy- pothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inﬂammation and thrombosis. Key words: thrombosis • P-selectin • PSGL-1 • hemodynamics • tether al., 1997). Under hemodynamic conditions, the rolling and arrest of neutrophils on spread platelets or activated endo- thelium involves the transition from P-selectin–mediated tethering to more stable b2-integrin bonding (Lawrence and Springer, 1991; Buttrum et al., 1993; Diacovo et al., 1996; Yeo et al., 1994; Weber and Springer, 1997). The mechanics and kinetics of the P-selectin/PSGL-1 bond that regulate the adhesive dynamics have been stud- ied using several different experimental techniques. Paral- lel-plate flow assays have employed flowing neutrophils over monolayers of adherent platelets (Hamburger and McEver, 1990; Yeo et al., 1994; Lalor and Nash, 1995; Dia- covo et al., 1996), sparse islands of platelets (Bahara and Nash, 1998), and P-selectin–coated surfaces (Lawrence and Springer, 1991; Alon et al., 1995). The P-selectin/ PSGL-1 bond displays a modest increase in dissociation when force loaded, with an unstressed off rate ko0ff 5 1–2.4 s21 and a stressed off rate of 3.5–6.3 s21 at a wall shear stress of 1 dyne/cm2 (bond loading of 110 pN; Alon et al., ª The Rockefeller University Press, 0021-9525/2000/05/719/11 $5.00 The Journal of Cell Biology, Volume 149, Number 3, May 1, 2000 719–729 http://www.jcb.org 719