Kinetics and Force Dependence of P-selectin/PSGL-1 Interactions Measured by AFM

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory and thrombotic processes. The rolling under hydrodynamic shear forces is a first step in directing leukocytes out of the blood stream into sites of inflammation and is mediated by the selectins, a family of extended, modular, and calcium-dependent lectin receptors. The interactions between P-, E-or L-selectins and their count.

应用光阱技术测量P-selectin/PSGL-1键解离的断裂力

选择素(selectin)与配体相互作用在诸如炎症反应、肿瘤转移等生物学过程中具有重要作用;作用力影响受体-配体键解离.本文发展了基于光阱技术的新实验方法,用于考察P-选择素(P-selectin)与P-选择素糖蛋白配体-1(P-selectin Glycoprotein Ligand 1, PSGL-1)相互作用的解离过程.采用黏滞力法对光阱刚度系数进行标定,并通过分子在玻璃小球表面的功能化表征,研究力作用下P-selectin/PSGL-1键的解离,得到了在较低加载率(<25 pN/s)下键解离的断裂力分布,发现键的最可几断裂力随加载率而增加.实验结果在较低加载率下补充和验证了已有的结论.

Quantifying the Effects of Contact Duration, Loading Rate, and Approach Velocity on P-Selectin-PSGL-1 Interactions Using AFM

Kinetics and its regulation by extrinsic physical factors govern selectin-ligand interactions that mediate tethering and rolling of circulating cells on the vessel wall under hemodynamic forces. While the force regulation of off-rate for dissociation of selectin-ligand bonds has been extensively studied, much less is known about how transport impacts the on-rate for association of these bonds and their stability. We used atomic force microscopy (AFM) to quantify how the contact duration, loading rate, and approach velocity affected kinetic rates and strength of bonds of P-selectin interacting with P-selectin glycoprotein ligand I (PSGL-1). We found a saturable relationship between the contact time and the rupture force, a biphasic relationship between the adhesion probability and the retraction velocity, a piece-wise linear relationship between the rupture force and the logarithm of the loading rate, and a threshold relationship between the approach velocity and the rupture force. These results provide new insights into how physical factors regulate receptor-ligand interactions.

Forced extension of P-selectin construct using steered molecular dynamics

P-selectin, a 70-nm-long cellular adhesive molecule, possesses elastic and extensible properties when neutrophils roll over the activated endotheliam of blood vessel in inflammatory reaction. Transient formation and dissociation of P-selectin/ligand bond on applied force of blood flow induces the extension of P-selectin and relevant ligands. Steered molecular dynamics simulations were performed to stretch a single P-selectin construct consisting of a lectin (Lec) domain and an epithelial growth factor (EGF)-like domain, where P-selectin construct was forced to extend in water with pulling velocities of 0.005-0.05 nm/ps and with constant forces of 1000-2500 pN respectively. Resulting force-extension profiles exhibited a dual-peak pattern on various velocities, while both plateaus and shoulders appeared in the extension-time profiles on various forces. The force or extension profiles along stretching pathways were correlated to the conformational changes, suggesting that the structural collapses of P-selectin Lec/EGF domains were mainly attributed to the burst of hydrogen bonds within the major beta sheet of EGF domain and the disruptions of two hydrophobic cores of Lee domain. This work furthers the understanding of forced dissociation of P-selectin/ligand bond.

Low Spring Constant Regulates P-Selectin-Psgl-1 Bond Rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r(f) (>= 10(2) pN/s), defined as the product of spring constant k and retract velocity v, how the low r(f) (< 10(2) pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r(f) <= 20 pN/s with low k (similar to 10(-3)-10(-2) pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r(f) increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r(f). The most probable force, f

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.

Soluble P-selectin levels during normal pregnancy: a longitudinal study.

Objective: To investigate soluble P-selectin (sP-selectin) levels and platelet parameters in normal pregnant women compared with non-pregnant control subjects. Design: A longitudinal case-control study. Setting: Obstetric outpatient clinic in the Jubilee Maternity Hospital, Belfast. Population: One hundred and twenty normal pregnant women and 41 non-pregnant age matched control subjects. Main outcome measures Plasma sP-selectin as a measure of platelet activation in normal pregnancy. Methods: The plasma concentration of sP-selectin in pregnant women sampled at 12, 20 and 35 weeks of gestation, and, in a subgroup at 3 days post-partum, and non-pregnant controls sampled in parallel, was determined using a commercial quantitative sandwich immunoassay kit. Platelet parameters on each blood sample were also recorded using a SYSMEX SE 9500 analyser. Main outcome measures: Plasma sP-selectin as a measure of platelet activation in normal pregnancy. Results: Soluble P-selectin was significantly higher in pregnant women than in non-pregnant control subjects at 20 and 35 weeks of gestation, (p