The flow and deformation of macromolecules is ubiquitous in nature and industry, and an understanding of this phenomenon at both macroscopic and microscopic length scales is of fundamental and practical importance. Here, we present the formulation of a general mathematical framework, which could be used to extract, from scattering experiments, the molecular relaxation of deformed polymers. By combining and modestly extending several key conceptual ingredients in the literature, we show how the anisotropic single-chain structure factor can be decomposed by spherical harmonics and experimentally reconstructed from its cross sections on the scattering planes. The resulting wave-number-dependent expansion coefficients constitute a characteristic fingerprint of the macromolecular deformation, permitting detailed examinations of polymer dynamics at the microscopic level. We apply this approach to survey a longstanding problem in polymer physics regarding the molecular relaxation in entangled polymers after a large step deformation. The classical tube theory of Doi and Edwards predicts a fast chain retraction process immediately after the deformation, followed by a slow orientation relaxation through the reptation mechanism. This chain retraction hypothesis, which is the keystone of the tube theory for macromolecular flow and deformation, is critically examined by analyzing the fine features of the two-dimensional anisotropic spectra from small-angle neutron scattering by entangled polystyrenes. We show that the unique scattering patterns associated with the chain retraction mechanism are not experimentally observed. This result calls for a fundamental revision of the current theoretical picture for nonlinear rheological behavior of entangled polymeric liquids.

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In a previous study, it was shown that fibrin fragment E-2 selectively promotes the activation of plasminogen by pro-urokinase (pro-UK) [Liu, J., & Gurewich, V. (1991) J. Clin. Invest. 88, 2012-2017]. In this study, the kinetics of this promotion by fragment E-2 was studied. Alanine-158-rpro-UK (A-pro-UK), a recombinant plasmin-resistant mutant, was used in order to avoid interference by UK generation during the reaction. In some experiments, pro-UK was substituted in order to validate the mutant as a surrogate. In the presence of a range of concentrations (0-20 microM) of fragment E-2, a linear promotion of the catalytic efficiency of A-pro-UK against native Glu-plasminogen was seen which was 245.5-fold at the highest concentration of fragment E-2 and 450-fold at the highest ratio of E-2/plasminogen used. The promotion was largely a function of an increase in kcat, since fragment E-2 induced a less than 10-fold reduction in KM (8.50-1.40 microM). In contrast to this ligand, epsilon-aminocaproic acid (EACA) induced a biphasic promotion of the activation of Glu-plasminogen which was only 18-fold at maximum. Fragment E-2 did not promote the activation of Lys-plasminogen, but the catalytic efficiency of A-pro-UK was 19.7-fold greater against the open Lys-form than against the closed Glu- form of plasminogen. Fragment E-2 had no effect on the amidolytic activity of A-pro-UK or pro-UK, suggesting that the promotion of their activities was indirect and related to a fragment E-2-induced conformational change in Glu-plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmin generation by equimolar concentrations of tissue plasminogen activator (t-PA), pro-urokinase (pro-UK), and urokinase (UK), and a twofold higher concentration of a plasmin-resistant mutant rpro-UK (Ala-158-pro-UK) was measured on a microtiter plate reader. The promoting effects on this reaction of equimolar concentrations of fibrinogen, soluble fibrin (Desafib), CNBr fragment FCB-2 (an analogue of fragment D), or purified fragment E-2 were compared. Plasmin generation by t-PA was moderately promoted by fibrinogen, substantially promoted by Desafib and FCB-2, but not at all promoted by fragment E-2. By contrast, plasmin generation by pro-UK or by Ala-158-pro-UK was not promoted either by fibrinogen, Desafib, or FCB-2, but was significantly promoted by fragment E-2. Plasmin generation by UK was not significantly promoted by any of the fibrin(ogen) preparations. Treatment of fragment E-2 by carboxypeptidase-B (CPB), eliminated its promotion of pro-UK and Ala-158-pro-UK-induced plasmin generation. Pretreatment of FCB-2 with plasmin slightly potentiated its promotion of t-PA activity. This effect of plasmin pretreatment of FCB-2 was reversed by CPB treatment. Plasmin pretreatment of FCB-2 did not induce any promotion of activity in pro-UK or Ala-158-pro-UK.The findings show that the intrinsic activity of pro-UK and the activity of t-PA are promoted by different regions of the fibrin(ogen) molecule. The latter is stimulated primarily by a determinant in the fragment D region, which is available in intact fibrin. By contrast, plasminogen activation by the intrinsic activity of pro-UK was stimulated exclusively by fragment E-2, which is unavailable in intact fibrin. The findings are believed relevant to fibrinolysis and support the concept that t-PA and pro-UK are complementary, sequential, and synergistic in their actions. (J. Clin. Invest.

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