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The triple-helical cyanogen-bromide-derived fragment CB3 [IV] of collagen IV, located 100 nm from the N-terminus of the molecule, contains the binding sites for the integrins alp1 and a2pl. To investigate the interaction of these integrins and collagen IV, we performed solid-phase and inhibition assays using as receptor isolated alp1 and a2pl. The ligands used were the binding-site-bearing trimeric peptide CB3 [IV] and its shorter tryptic fragments Fl-F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for alp1 and a2pl were in different but adjacent areas of CB3 [IV]. Triple-helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Kd), for the affinity of integrins for collagen IV, were determined in the 1-nM range in the presence of Mn2+ and Mg". In the absence of Mn2+, the Kd values indicated a 30-60-fold decrease in the affinities, which for a2pl was further reduced by adding Ca". In the presence of Ca2+ and Mg2+ the affinity of collagen IV for alp1 was four-times higher than for a2pl.

Abstract. The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH: terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent.Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NHe and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, otl/31 and oe2/$1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors o¢1/~1 and o~2B1.

Background and Purpose-Transcranial color-coded duplex sonography (TCCS) of intracranial veins and sinuses in adults is a new, emerging application of ultrasonographic imaging. This study reports a standardized examination protocol for venous TCCS and provides reference data for clinical application. Methods-In 130 healthy volunteers (mean age, 45.9Ϯ16.9 years; range, 14 to 77 years) the intracranial venous system was examined using frequency-based transtemporal TCCS. Identification rate, blood flow velocity , resistance index, and systolic/diastolic ratio were recorded for each examined venous vessel. Results-Intracranial veins and sinuses show a low pulsatile forward flow with maximal systolic blood flow velocity up to 20 cm/s. Significant side differences of blood flow velocity in the paired venous structures could not be detected. Venous flow velocities decreased with age, whereas resistance indices and systolic/diastolic ratios increased. Women showed higher flow velocities than men. Mean identification rates for all age groups ranged from 70% to 90% for the deep middle cerebral vein, the basal cerebral vein, and the great cerebral vein of Galen. The straight sinus, the transverse sinus, and the rostral part of the superior sagittal sinus could be detected in 55% to 70% of cases. Detection rates were dependent on age and decreased as age increased. Conclusions-Venous TCCS can reliably image a significant part of the cerebral venous system. This method can provide information on venous hemodynamics in normal subjects and pathological cases.