George D. Wilner

Studies of the structure of canine fibrinogen.

Abstract In order to provide the basis for an investigation of fibrinogen metabolism through fibrinopeptide radioimmunoassay studies in an animal model, some structural features of canine fibrinogen have been elucidated. Canine fibrinogen and fibrin have been purified, their reduced, carboxymethylated chains separated, and their amino acid compositions and NH2-terminal sequences determined. Canine fibrinogen was found to be very similar to human fibrinogen both in amino acid composition of the individual chains and in their NH2-terminal amino acid sequences beyond the regions of fibrinopeptides A and B. The results confirm the structure of canine fibrinopeptides A and B by Blomback et al. (1).

SYNTHESIS AND RADIOIMMUNOASSAY OF CANINE FIBRINOPEPTIDE A

Abstract Canine fibrinopeptide A, the 16 residue NH 2 -terminal segment of the Aα chain of canine fibrinogen released by thrombin proteolysis and its NH 2 -tyrosyl analogue, were synthesized by stepwise solid-phase syntheses. Following cleavage, the synthetic peptides were purified by ion-exchange chromatography, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis and Edman degradation analysis. Rabbits immunized with carbodiimide-coupled synthetic canine fibrinopeptide A-bovine albumin conjugates produced antibodies which bound up to 70% of available counts using 125 I-N-tyrosyl canine fibrinopeptide A as tracer. Quantitative displacement of bound tracer could be effected by both synthetic canine fibrinopeptide A and clot supernates prepared from purified canine fibrinogen. Fifty percent displacement of radiolabelled tracer could be achieved with 1.1 picomole of peptide antigen. Antiserum to canine fibrinopeptide A was about 50-fold less sensitive on a molar basis to canine fibrinogen as compared to the free peptide in solution. This suggests that antigenic determinants on the canine fibrinopeptide are altered by attachment of the peptide to its parent molecule. The molar reactivity of human fibrinopeptide A was about 25% in comparison with canine fibrinopeptide A, indicating that both the human and canine peptides share some antigenic determinants. Using this assay, fibrinopeptide A immunoreactivity was measured in canine plasma following removal of fibrinogen by ethanol precipitation and dialysis. The mean fibrinopeptide A level 4 normal dogs (18 determinations) was found to be 1.5±0.98 pmols/ml anticoagulated plasma.

Change in Blood Banking Procedures Decreases Donor Exposure in a High Risk Neonatal Population † 1027

Change in Blood Banking Procedures Decreases Donor Exposure in a High Risk Neonatal Population † 1027

THROMBIN B-LOOP PEPTIDE (B-LP) HAS SPECIFIC RECEPTORS ON MACROPHAGES

α-Thrombin, a serine protease with potent procoagulant activity, has the capacity to stimulate the proliferation of fibroblasts, vascular endothelial cells and macrophages. However, for the latter cell type, esterolytically intact enzyme is not required and, in fact, the mitogenic potency of the molecule appears to reside in a limited unique region of thrombin termed the B-loop peptide (B-LP). In an attempt to determine the mechanism by which B-LP mediates mitogenesis, B-LP sensitive J774.1 macrophage cells were fixed in paraformaldehyde and incubated for 1 hr with 125I B-LP in the presence and absence of exogenous unlabeled ligand (10−10-10−7)(n=5). The data show that the J744.1 cells possess a specific, saturable B-LP receptor that exhibits a Kd of ∼1.12 nM; a value that coincides with the concentration of peptide necessary to optimally elicit a mitogenic response in these cells. Native α-thrombin, inactivated DIP-thrombin and unlabeled B-LP displace bound 125-labeled B-LP from J774 cells at equimolar concentrations suggesting that all the 3 peptides interact with a common receptor site on macrophages. Scatchard analysis indicates that ∼ 4000 such receptors are present per cell. We suggest that ligand activation of these receptors is responsible for triggering the biological response associated with thrombin-stimulated macrophages, and therefore, is likely pivotal in promoting the accumulation of these cells during the processes of inflammation and wound healing.