Regulation of L-selectin-dependent hydrodynamic shear thresholding by leukocyte deformability and shear dependent bond number.

BACKGROUND: During inflammation leukocyte attachment to the blood vessel wall is augmented by capture of near-wall flowing leukocytes by previously adherent leukocytes. Adhesive interactions between flowing and adherent leukocytes are mediated by L-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1) co-expressed on the leukocyte surface and ultimately regulated by hydrodynamic shear thresholding. OBJECTIVE: We hypothesized that leukocyte deformability is a significant contributory factor in shear thresholding and secondary capture. METHODS: Cytochalasin D (CD) was used to increase neutrophil deformability and fixation was used to reduce deformability. Neutrophil rolling on PSGL-1 coated planar surfaces and collisions with PSGL-1 coated microbeads were analyzed using high-speed videomicroscopy (250 fps). RESULTS: Increased deformability led to an increase in neutrophil rolling flux on PSGL-1 surfaces while fixation led to a decrease in rolling flux. Abrupt drops in flow below the shear threshold resulted in extended release times from the substrate for CD-treated neutrophils, suggesting increased bond number. In a cell-microbead collision assay lower flow rates were correlated with briefer adhesion lifetimes and smaller adhesive contact patches. CONCLUSIONS: Leukocyte deformation may control selectin bond number at the flow rates associated with hydrodynamic shear thresholding. Model analysis supported a requirement for both L-selectin catch-slip bond properties and multiple bond formation for shear thresholding.

The Escherichia coli clamp loader can actively pry open the ß-sliding clamp.

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds ß first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.

A slow ATP-induced conformational change limits the rate of DNA binding but not the rate of beta clamp binding by the escherichia coli gamma complex clamp loader.

In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 10(7) m(-1) s(-1) for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was preincubated with ATP before adding beta or added directly to beta and ATP. In contrast, when gamma complex is preincubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Clamp loaders are multisubunit complexes that use the energy derived from ATP binding and hydrolysis to assemble ring-shaped sliding clamps onto DNA. Sliding clamps in turn tether DNA polymerases to the templates being copied to increase the processivity of DNA synthesis. Here, the rate of clamp release during the clamp loading reaction was measured directly for the first time using a FRET-based assay in which the E. coli gamma complex clamp loader (gamma3deltadelta'chipsi) was labeled with a fluorescent donor, and the beta-clamp was labeled with a nonfluorescent quencher. When a beta.gamma complex is added to DNA, there is a significant time lag before the clamp is released onto DNA. To establish what events take place during this time lag, the timing of clamp release was compared to the timing of DNA binding and ATP hydrolysis by measuring these reactions directly side-by-side in assays. DNA binding is relatively rapid and triggers the hydrolysis of ATP. Both events occur prior to clamp release. Interestingly, the temporal correlation data and simple modeling studies indicate that the clamp loader releases DNA prior to the clamp and that DNA release may be coupled to clamp closing. Clamp release is relatively slow and likely to be the rate-limiting step in the overall clamp loading reaction cycle.

L-selectin shear thresholding modulates leukocyte secondary capture.

Transient homotypic adhesions between flowing leukocytes and those previously adherent on the vessel wall has been proposed to amplify the accumulation of leukocytes at sites of inflammation. While adhesion of leukocytes to the vessel wall (primary capture) is mediated primarily by P-selectin on the endothelium and P-selectin Glycoprotein Ligand-1 (PSGL-1) on the leukocyte, the homotypic interactions leading to downstream leukocyte adhesion (secondary capture) are mediated primarily by reciprocal interactions between PSGL-1 and L-selectin on apposing leukocytes. One consequence of leukocyte secondary capture events are the formation of strings of adherent leukocytes as each recently captured leukocyte in turn captures another one flowing over its surface. Interestingly, PSGL-1-L-selectin interactions also mediate leukocyte hydrodynamic shear thresholding, whereby leukocyte rolling on purified L-selectin ligands such as PSGL-1 is maximized at a wall shear stress of approximately 1 dyne/cm(2) and minimized at both higher and lower flow rates. Using a novel quantitative method, we analyzed leukocyte string formation in vitro and found that hydrodynamic shear thresholding precluded secondary capture at low shear stresses yet amplified it at high shear stresses. Addition of the L-selectin mAb DREG-56 strongly inhibited leukocyte string formation, suggesting adhesion contributed significantly to hydrodynamic interactions in secondary capture processes. Taken together, the data suggest that secondary capture is modulated by the shear thresholding property of L-selectin. L-selectin mediated shear thresholding may therefore play a significant role in the regulation of leukocyte secondary capture in addition to recently described hydrodynamic recruitment mechanisms.

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow.

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 +/- 0.04 bonds/microm at 0.2 dyn/cm(2) to 0.014 +/- 0.003 bonds/microm at 1.0 dyn/cm(2). In contrast, L-selectin tether bond formation increased from 0.017 +/- 0.005 bonds/microm at 0.2 dyn/cm(2) to 0.031 +/- 0.005 bonds/microm at 1.0 dyn/cm(2). L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.

A model for clinical estimation of perioperative hemorrhage.

The purpose of this study was to assess the accuracy of estimated blood loss (EBL) as a reliable predictor of actual blood loss during orthopedic procedures. Between 1999 and 2002, 198 orthopedic cases were reviewed. A retrospective review compiled preoperative and postoperative demographic and laboratory data from the surgical patients. Estimated blood loss data was collected from the perioperative and anesthesia reports. Statistical analysis of EBL vs. change in hemoglobin yielded a correlation coefficient of 0.189 and a p value of 0.008. We used multiple linear regression to obtain a model to predict change in hemoglobin based on EBL and the intravenous fluids received. The model is as follows: predicted change in hemoglobin = 1.001 x estimated blood loss (in liters) + 0.441 x intravenous fluids received (in liters) + 2.334. The study population included 198 patients, 126 males and 72 females, who met our inclusion criteria. The mean age was 68.1 years (range: SD 12.5), including 126 males (64%) and 72 females (37%). The mean amount of perioperative intravenous fluids given was 1,732 mL (SD: 773). The mean surgical time was 64.8 minutes (SD: 23.1). The mean preoperative hematocrit and hemoglobin levels were 40.9 g/dL (SD: 4.3) and 13.9 g/dL (SD: 1.6), respectively. The mean postoperative hematocrit and hemoglobin levels were 32.0 g/dL (SD: 6.0) and 10.7 g/dL (SD: 1.6), respectively. The mean difference of preoperative hemoglobin vs. postoperative hemoglobin was 3.3 g/dL (SD 2.1). In this retrospective study, clinical estimation of blood loss was closely correlated with actual change in perioperative hemoglobin. Accurately predicting the postoperative hemoglobin level may prevent many unnecessary blood transfusions and related complications.