It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.

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By using biotin-labeled proteoglycan core protein and an avidin-enzyme system, hyaluronic acid (HA) was visualized in the lungs of rats at different times (4, 10, and 20 days) after bleomycin injury. Four days after an intratracheal injection of bleomycin, HA was accumulated in the edematous alveolar septa of the focal areas with lung tissue injury. An interstitial cellular infiltrate of mainly lymphocytes was present. In normal rat lung, HA was not seen in the alveolar tissue but confined to peribronchial and perivascular spaces. Ten and twenty days after bleomycin administration, increasing numbers of macrophages were apparent in the alveolar space. Proliferating fibroblasts and deposition of collagen in the alveolar tissue were observed while the diffuse HA accumulation was becoming less prominent in the alveolar interstitial tissue. HA was more distinctly located in the surroundings of proliferating fibroblasts. A few scattered alveolar macrophages showed a positive staining for HA. An increased water content of the lung was most apparent 4 days after bleomycin administration. The accumulation of HA, a glycosaminoglycan with unique qualities to immobilize water, in the alveolar interstitium suggests a role for HA in the alveolar interstitial edema. The appearance of HA in alveolar macrophages might indicate that macrophage phagocytosis contributes to the elimination of HA from inflamed lung tissue.

Human lymph was collected from patients with leaking lymph vessels after thoracic surgery. Ovine lymph was obtained from the mesenteric, lumbar, popliteal and prescapular lymph ducts by cannulation. The concentration of hyaluronate varied considerably (between 0.2 and 50 mg/l) and the highest concentrations were found in mesenteric lymph. The Mr of the polysaccharide showed a great polydispersity and variation between individuals and in different regions of the lymphatic system. High-Mr hyaluronate (greater than 10(6) was present in lymph both from man and sheep. Hyaluronate was also isolated by affinity chromatography in 70-80% yield from human serum and plasma obtained from healthy individuals and patients with rheumatoid arthritis and primary biliary cirrhosis. The weight (Mw)- and number (Mn)-average relative molecular masses were roughly the same in the three groups [(1.4-2.7) X 10(5) and (2.1-5.7) X 10(4) respectively]. The low Mr of hyaluronate in blood compared with that in lymph is explained by a preferential uptake of the large molecules by the liver endothelial cells.

By using biotin-labeled proteoglycan core protein, hyaluronan (hyaluronic acid; HA) was visualized in rat heart grafts at different times (2, 4, and 6 d) after transplantation. In normal, nontransplanted hearts HA was present in the adventitia of arteries and veins and in the myocardial interstitial tissue. An increased accumulation of HA was evident in the edematous interstitial tissue, infiltrated with lymphocytes, on day 4 after allogeneic transplantation, and was even more pronounced by day 6. No apparent increase in HA was seen in syngeneic grafts. Biochemical assay of HA in heart tissue demonstrated that the myocardial content of HA had increased 60% by day 2 after transplantation in allogeneic as well as syngeneic grafts, indicating that surgical trauma may induce some HA accumulation in heart grafts. The extractable amounts of HA declined during the following days in the syngeneic grafts, but increased progressively during the development of rejection in the allogeneic grafts, and increased on average three times by day 6. The relative water content also increased progressively during rejection of allogeneic grafts and correlated with the HA accumulation. The interstitial accumulation of HA, a glycosaminoglycan with unique water-binding qualities, is presumably implicated in the development of interstitial edema during rejection of heart grafts. (J. Clin. Invest. 1990. 85:668-673.) hyaluronic acid * heart graft * allograft -rejection * edema