Spatial variation of the charge and sulfur oxidation state in a surface gradient affects plasma protein adsorption.

A gradient of negative surface charge based on the 1D spatial variation from surface sulfhydryl to mixed sulfhydryl-sulfonate moieties was prepared by the controlled UV oxidation of a 3-mercaptopropylsilane monolayer on fused silica. The adsorption of three human plasma proteins--albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fgn)--onto such a surface gradient was studied using spatially resolved total internal reflection fluorescence (TIRF) and autoradiography. Adsorption was measured from dilute solutions equivalent to 1/100 (TIRF, autoradiography), 1/500, and 1/1000 (autoradiography) of protein physiological concentrations in plasma. All three proteins adsorbed more to the nonoxidized sulfhydryl region than to the oxidized, mixed sulfhydryl-sulfonate region of the gradient. In the case of HSA, the adsorption contrast along the gradient was largest when the adsorption took place from more dilute protein solutions. Increasing the concentration to 1/100 of the protein plasma concentration eliminated the effect of the gradient on HSA adsorption and, to the lesser extent, on IgG adsorption. In the case of Fgn, the greatest adsorption contrast was observed at the highest concentration used. On the basis of adsorption kinetics, the estimated binding affinity of HSA for the sulfhydryl region was twice the affinity for the mixed sulfhydryl-sulfonate region of the gradient. For IgG and Fgn, the initial adsorption was transport-limited and the initial adsorption rates approached the computed flux of the protein to the surface.

Competitive Adsorption of Three Human Plasma Proteins onto Sulfhydryl-to-sulfonate Gradient Surfaces.

Competitive adsorption of three human plasma proteins: albumin (HSA), fibrinogen (Fgn), and immunoglobulin G (IgG) from their ternary solution mixtures onto a sulfhydryl-to-sulfonate gradient surface was investigated using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. The concentration of each protein in the ternary solution mixture was kept at an equivalent of 1/100 of its physiological concentration in blood plasma. The three proteins displayed different adsorption and desorption characteristics. Each protein adsorbed less to the sulfonate region than to the sulfhydryl region of the gradient. The adsorption-desorption kinetics revealed large differences in the adsorption and desorption rates of three proteins. By fitting the experimental data to a simple model of competitive protein adsorption, the affinity of each protein to the surface at the gradient center position was ranked as: Fgn > HSA ≫ IgG. Competitive exchange of adsorbed proteins was related to the magnitude of desorption rate constants. Such competitive adsorption of the three major human plasma proteins illustrates the complex dynamics of blood proteins - biomaterials interactions.

K+-induced ion-exchanges trigger trypsin activation in pancreas acinar zymogen granules.

Trypsin premature activation has been thought to be a key event in the initiation phase of acute pancreatitis. Here we test a hypothesis that a sustained increase of cytosolic Ca(2+) concentration ([Ca(2+)](C)) can trigger K(+) influx into pancreas acinar zymogen granules (ZGs) via a Ca(2+)-activated K(+) channel (K(Ca)), and this influx of K(+) then mobilizes bound-Ca(2+) by K(+)/Ca(2+) ion-exchange to increase free Ca(2+) concentration in the ZGs ([Ca(2+)](G)) and release bound-H(+) by K(+)/H(+) ion-exchange to decrease the pH in ZGs (pH(G)). Both the increase of [Ca(2+)](G) and the decrease of pH(G) will facilitate trypsinogen autoactivation and stabilize active trypsin inside ZGs that could lead to acute pancreatitis. The experimental results are consistent with our hypothesis, suggesting that K(+) induced ion-exchanges play a critical role in the initiation of trypsin premature activation in ZGs.

Ethanol augments elevated-[Ca2+]C induced trypsin activation in pancreatic acinar zymogen granules.

It has been long recognized that significant percentage of patients with acute pancreatitis often presents with a history of excessive alcohol consumption; however, the patho-physiological effect of ethanol on acute pancreatitis remains poorly understood. Abnormally elevated cytosolic Ca2+ ([Ca2+]C) has been found to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation. Here, we present the effects of ethanol to sensitize zymogen granules (ZGs) of pancreas acinar cells to elevated [Ca2+]C leading to zymogen premature activation that could result in acute pancreatitis. The pH fluctuations ([pH]G), Ca2+ concentration ([Ca2+]G), and premature trypsin activation inside the ZGs were monitored directly with specific fluorescence indicators. Our results showed that ethanol could act directly on ZGs and cause ZGs more receptive to elevated [Ca2+]C that could induce premature activation of zymogen (trypsin). This alcohol-induced effect is concentration dependent and strongly influenced by the surrounding [Ca2+]C. The K+ channels on ZGs membranes are required in the sensitization process. Our observations provide a mechanistic understanding of the role of ethanol in the initiation phase of alcoholic pancreatitis.