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Band 3 and Glycophorin C as Potential Mediators of Erythrocyte Aggregation During the Onset of Thermal Burn Injury
Author(s) -
Seidner Harrison S.,
Gunter Geoffrey,
WeberFishkin Samantha,
Frame Mary D.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.946.7
Subject(s) - band 3 , glycophorin , erythrocyte aggregation , chemistry , biophysics , extracellular , intracellular , hemorheology , hematocrit , biochemistry , medicine , membrane , erythrocyte membrane , biology
Our goal is to understand aggregation behavior of flowing erythrocytes in the zone of stasis near thermal burn injury. In the temperature range of 42°C to 45°C erythrocytes are known to exhibit hyperaggregation of which the mechanism is unknown. First, we sought to understand the contribution of membrane proteins to aggregation in a vertical flow model of the onset of stasis where temperature was changed from 37°C to a test temperature. Erythrocytes from healthy humans were isolated from other blood components and re‐suspended in PBS, 0.5 g/dL Dextran 500 kDa, or autologous plasma. Erythrocyte solutions were prepared at 20% hematocrit and incubated with rabbit polyclonal antibodies (Abcam) to band 3 and Glycophorin C (GYPC) with human reactivity to test the contribution of the extracellular domains band 3 and GYPC to aggregation. To test for non‐specific binding, anti‐α v β 3 (Chemicon International) was used. The involvement of the intracellular domain of band 3 with aggregation was tested using 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS), an intracellular‐binding band 3 inhibitor. Following monodispersion by higher Reynolds flow (Re = 0.254), flow was abruptly stopped and aggregates were allowed to form in a low shear environment where their subsequent center‐seeking behavior was observed. The sudden cessation of flow emulated the environment encountered by erythrocytes in the zone of stasis (Re < 0.01) immediately after the occlusion of microvessels in the zone of coagulation during the onset of thermal injury. To study the effects of hyperthermia on aggregation of erythrocytes in stasis the experimental procedure was performed for samples heated to 37 °C, 41 °C, 45 °C, and 49 °C. Temperature was controlled by a heated water chamber attached to the microchannel. Images were acquired for 3 minutes using a horizontally oriented Nikon microscope (20×) and intensified camera (Stanford Photonics, Inc.), passing through an acquisition board (EPIX, Inc.). Python‐based image processing software was used to quantify and analyze time‐dependent measures of aggregation rate and magnitude. Significant differences in aggregation magnitude and rate were observed between erythrocytes suspended in different solvents. Antibodies to band 3, and GYPC had minimal effects on aggregation parameters and temperature dependences were not observed. DIDS‐treated erythrocytes exhibited reduced aggregation that was temperature dependent. The lack of differences in aggregation in the presence of anti‐band 3, and anti‐GYPC indicate that the extracellular domains of these proteins are not mediators of aggregation but provide a strong basis for investigation of the intracellular domains of GYPC and band 3 as mediators of aggregation.