Irwin A. Feuerstein

Serotonin transport and storage in rabbit blood platelets—The effects of reserpine and imipramine☆

Abstract Serotonin transport and storage in suspensions of washed rabbit platelets were investigated by following the exchange of platelet-bound [ 3 H]serotonin and [ 14 C]serotonin added to the suspending medium. Assuming a three-compartment system (suspending medium, platelet cytoplasm and platelet storage organelles), the transfer rates between the different compartments were calculated from the exchange data by statistical analysis. Reserpine reduced the storage organelle serotonin content by inhibiting the transfer of serotonin from the cytoplasm into the amine storage organelles. It also reduced the fraction of serotonin in the cytoplasm transferred per unit of time into the suspending medium. Imipramine (20 μM) inhibited the uptake of serotonin across the platelet plasma membrane into the cytoplasm and reduced the fraction of cytoplasmic serotonin transferred per unit of time into the suspending medium. At this concentration it had no effect on serotonin transport across the amine storage organelle membrane in either direction. The method used allows the serotonin transfer rates across the platelet plasma membrane to be distinguished from those across the amine storage organelle membrane in intact cells, and permits these transfer rates to be estimated simultaneously. The method may be used for determining the effects of drugs that interfere with transport and storage of biogenic amines and in defining the defects in diseases with abnormal transport or storage of biogenic amines.

Video microscopic and immunochemical evaluation of cells at surfaces.

Blood contact with nonbiological surfaces has been studied in various ways with both in vitro and in vivo protocols.‘,* Early experimentation frequently dealt with measurements of thrombus mass and time spans for occlusion of conduits to occur. These results circumscribed the magnitude of the problem and indicated that material properties and flow parameters were important features of the process. It was recognized that protein adsorption occurred before cell contact and that fibrin formation was both surface dependent and flow dependent.’.’ High flow rates resulted in low fibrin deposition. Workers then focused on the several components of blood contact independently and in controlled ways. Several large bodies of work emerged that fall into a number of well-defined categories: (1) adsorption of single plasma proteins to solid substrates; (2) adhesion of single cell types, mainly platelets; and (3) plasma interaction with solids where an indication of clotting time was sought. Continuing efforts with proteins have led to studies of protein mixtures, exchange of proteins between the surface-bound and solution states, and the dependence of adsorbed protein molecule type on surface ~ h e m i s t r y . ~ The importance of flow conditions as key variables to control came out of several studies that pointed out the role of transport phenomena (diffusion and convection) in blood contact.e The role of the red cell as a physical mover of platelets and proteins to surfaces was elucidated along with the idea of a dispersion coefficient (same units as a diffusion coefficient) to characterize such t ran~port .~” Thus, the meaningfulness of experiments dealing with adsorption of proteins and adhesion of cells done in the absence of flow and red cells could be questioned. The protein coating, the cells on the surface, and the moving fluid adjacent to the surface may be viewed as a system of interacting components. A number of time-dependent dynamic events may occur with this system. Of these, the adhesion and detachment of platelets is one such important event since detached platelets are likely to be altered from their normal, unstimulated state. Also, those cells remaining for longer times may alter or remove the protein between them and the solid surface and that protein adjacent to the cell. These topics form the subject of this paper.

Flow patterns in a platelet adhesion test cell-implications for adhesion measurement.

Flow patterns in a test cell used by Lyman et al. (1) to study adhesion of platelets to polymer surfaces were determined using a tracer particle technique. Flow was found to be characterized by low average shear rate of the order of 3 to 4 sec−1 and by vortex formation at the entrance to the cell. The vortices, however, would not have impinged on the surface regions where platelet adhesion was measured. The implications of these results for the platelet adhesion data obtained with this cell are that flow conditions were mild, rouleaux were present and red cell motions were minimal. Insofar as these motions can be critical for platelet transport and the provision of energy for surface reaction, the adhesion process was probably dominated by the platelet-surface forces. By contrast the data of Friedman et al. (2,16), obtained at higher shear rates could have been dominated by red cell motions leading to independence of adhesion on surface properties.

Adhesion and aggregation of thrombin prestimulated human platelets: evaluation of surface-bound fibrinogen and surface-bound albumin

The effect of prestimulation of human washed platelets with thrombin on their ability to adhere to and form aggregates on fibrinogen- and albumin-coated glass has been studied. Prestimulation of platelets does not alter platelet adhesion to surface bound albumin and fibrinogen. Our data for both protein coatings support a mechanism of adherence of single prestimulated platelets before formation of surface-bound aggregates from single platelets arriving from the flow. When whole blood contacts an artificial surface, thrombin will likely influence the platelet component of thrombus formation.

Computer tracking of white blood cells on protein-coated surfaces

The kinetics of human white blood cell adhesion and detachment is of interest in evaluating the effects of synthetic materials for blood damage. In an experimental setup, a suspension of fluorescently labeled white blood cells and unlabeled red blood cells is drawn through a protein-coated glass tube at a constant flow rate. Interaction of the white cells with the inside surface of the tube is monitored using a microscope coupled to an SIT television camera. A videotape recorder provides a permanent record on 1/2 in videotape to permit subsequent measurement of cell dynamics through the field of view. Instrumentation based on an image digitizer and a Zenith PC has been developed to automate the visual analysis of the videotapes. The new system tracks the cell motion on a frame-by-frame basis; once initiated, the entire process is controlled by the computer. Measurements include the number and nature of cell-to-surface collisions, residence times (the duration of the collisions), average distances traveled, and the size of clusters. Conditions have been uncovered where for two cases, the net cellular adhesion is the same while the pathways to this result are different. The design of the system is described together with experimental results.