We have determined the structure of plasma fibronectin by electron microscopy of Shadowed specimens . The 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand . The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. The large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends . This model is supported by images of fibronectinfibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand .Fibronectin is a high molecular weight glycoprotein that is found in a soluble form in blood and other extracellular tissue fluids, and in an insoluble form in connective tissues and attached to cell surfaces . Fibronectin is thought to mediate the attachment of cells to the other components of the extracellular matrix, in particular to collagen and, where it occurs, fibrinogen or fibrin (for reviews see 14,16,22,26). Both plasma fibronectin and the cell surface or extracellular matrix forms are dimers, comprising two subunits of 220,000 mol wt, covalently linked by a single disulfide bond near their carboxyl termini . The two polypeptide chains are identical by most criteria but are frequently separated as a closely spaced doublet in gel electrophoresis ; the basis for this separation is not known (11) . Cell surface fibronectin differs from plasma fibronectin in carbohydrate content and solubility and exists as oligomers of the basic dimeric molecule, but the two forms are very similar in amino acid composition and are immunologically indistinguishable (14) .Plasma fibronectin has a sedimentation coefficient of 8 to 13S, depending on the pH and ionic strength (1) . This is too small for a globular protein of 440,000 mol wt, and suggests that the molecule has an elongated shape (1) . Studies with proteases have shown that fibronectin can be cleaved into a number of large fragments or domains, and it has been found that the different binding functions are retained by the separated domains . By analogy with the well established trinodular THE JOURNAL OF CELL BIOLOGY " VOLUME 91 DECEMBER 1981 673-678 © The Rockefeller University Press -0021-9525/81/12/0673/06 $1 .00 structure of fibrinogen, it has been suggested (1, 16) that fibronectin may have a nodular structure, consisting of large globular domains connected by flexible linking segments that can be readily attacked by proteases .Techniques of electron microscopy are now well established for determining the structure of large proteins . Molecular structures de...

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Extravascular coagulation is a prominent feature of such important pathological processes as cellular immunity and neoplasia and has been thought to result from procoagulants associated with the inflammatory or tumor cells peculiar to these entities. It was found that increased microvascular permeability alone is sufficient to induce equivalent extravascular coagulation in several normal tissues. The results indicate that saturating levels of procoagulant are present even in normal tissues and that microvascular permeability is a rate-limiting step in extravascular coagulation.

Factor XIII is a blood protransglutaminase that is distributed in plasma and platelets. The extracellular and intracellular zymogenic forms differ in that the plasma zymogen contains A and B subunits, while the platelet zymogen has A subunits only. Both zymogens form the same enzyme. Erythrocytes, in contrast, contain a tissue transglutaminase that is distinct from Factor XIII. In this study other bone marrow-derived cells were examined for transglutaminase activity. Criteria that were used to differentiate Factor XIII proteins from erythrocyte transglutaminase included: (a) immunochemical and immunohistochemical identification with monospecific polyclonal and monoclonal antibodies to Factor XIII proteins, (b) requirement for thrombin cleavage to express activity, (c) pattern of fibrin cross-linking catalyzed by the enzyme, and (d) different electrophoretic mobilities in nondenaturing gel systems. By these criteria human peripheral blood monocytes, peritoneal macrophages, and monocytes maintained in culture contain an intracellular protransglutaminase that is the same as platelet Factor XIII. The monocyte-macrophage protein is thrombin-sensitive, and under appropriate conditions there is no enzyme expression without activation of the zymogen. Both the monocyte-macrophage zymogen and enzyme have the same electrophoretic mobilities as platelet Factor XIII zymogen and enzyme. Antibody to A protein reacts with the monocyte-macrophage protein. B protein is not associated with this intracellular zymogen. By immunoperoxidase staining monocyte-macrophage protein seems to be localized in the cytoplasm, similar to the known cytoplasmic distribution of platelet and megakaryocyte Factor XIII. These procedures were also used to study populations of human granulocytes and lymphocytes, and protransglutaminase activity was not observed in these cells.

The effects of Ca2+ ion on the structure of thrombin-derived and reptilase-derived fibrin gels formed at various ionic strengths were studied turbidimetrically. For both enzymes clotting times were shorter, final gel turbidities were higher and fibre mass/length ratios were increased as the ionic strength was lowered. The addition of 5 mM-Ca2+ augmented each of these effects for any given ionic strength. In the thrombin system, Ca2+ increased the final gel turbidity from 0.04 to 0.26 A632.8 at ionic strength 0.15. Under identical conditions in the reptilase system, the final gel turbidity increased from 0.03 A632.8 in the absence of Ca2+ to 0.345 A632.8 in the presence of 5 mM-Ca2+. In the thrombin system, fibre mass/length ratios increased from 0.4 X 10(12) to 6.9 X 10(12) Da/cm in the absence of Ca2+, and from 4.4 X 10(12) to 7.9 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.11 respectively. In the reptilase system, the mass/length ratios increased from 0.9 X 10(12) to 5.8 X 10(12) Da/cm in the absence of Ca2+, and from 4.8 X 10(12) to 8.7 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.10 respectively. At ionic strengths below 0.10, the presence of 5 mM-Ca2+ caused precipitation and macroscopic aggregation of fibrinogen upon the addition of either enzyme. In the presence of 5 mM-Ca2+, the fibres composing thrombin-induced and reptilase-induced gels were virtually identical.