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The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium.

We have asked whether treatment of normal cultured cells with proteases, other hydrolytic enzymes, or serum can convert them into transient phenocopies of transformed cells with respect to the very high rate of hexose transport characteristic of transformed cells. Treatment of density-inhibited cultures of normal chick embryo fibroblasts with trypsin, plasmin, neuraminidase, or hyaluronidase stimulated their rate of 2-deoxyglucose uptake to a level only marginally higher than that seen in normal exponentially growing cultures, and only 35-45% of that seen in transformed cultures. Addition of the hydrolytic enzymes to growing cell cultures had little effect on 2-deoxyglucose uptake. Serum, however, could stimulate 2-deoxyglucose uptake all the way up to the transformed level. Even though the hydrolases and serum differed in their ability to stimulate 2-deoxyglucose uptake, both reagents were capable of stimulating cell division equally well. Evidence is presented suggesting that the hexose transport rate is controlled by serum factors, and that proteolysis can affect the response of the cells of these factors.

The influence of various components of plasma hemostatic system on the assimilation of glucose by isolated rat hemidiaphragm in the presence of insulin was investigated. The addition of streptokinase and human plasminogen to the incubation medium decreased the rate of glucose assimilation. This effect did not occur when trasylol, an inhibitor of plasmin, was added to the assay system. The glucose assimilation by hemidiaphragms incubated in buffer with glucose and insulin was not influenced by thrombin, fibrinopeptides, fibrin degradation products, plasminogen, streptokinase or trasylol added separately to the assay system. The mechanism of the influence of plasmin on insulin action was partly elucidated in further experiments. Thus, less 125I-insulin was bound by isolated fat cells preincubated with plasmin than by control cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.

The conversion of plasminogen proactivator to plasminogen activator by Hageman factor fragments results in the generation of chemotactic activity for human neutrophils. This chemotactic activity can be distinguished from that generated by Hageman factor activation of prekallikrein and is demonstrable in plasma that is genetically deficient in prekallikrein (Fletcher factor deficiency). Both the plasminogen-activating activity and chemotactic activity produced by the interaction of Hageman factor fragments and plasminogen proactivator to yield plasminogen activator were inhibited by diisopropyl fluorophosphate (DFP) indicating an essential role for the enzymatic site in both these activities. The finding that the plasminogen proactivator tolerated a dose of DFP, which completely inactivated the plasminogen activator, reveals that the active site is protected in the precursor protein.

Chemotaxis of rabbit mononuclear cells was studied by the micropore-filter technique. Mononuclear cells obtained from mineral oil-induced peritoneal exudates respond chemotactically to rabbit serum treated with immune complexes or with streptokinase and plasminogen, to soluble factors produced by bacteria, and to lysates obtained from rabbit neutrophils. The first chemotactic factor requires heat-labile factors in serum for its generation, but, once formed, the chemotactic factor is relatively heat stable. This factor has been compared with the complement-associated factor in serum, C'(5, 6, 7)a, that is chemotactic for neutrophils. The ability to generate the mononuclear cell chemotactic factor in serum that has been treated with potassium thiocyanate suggests that complement is not required. The position of the chemotactic factor in preparative electrophoresis and density-gradient ultracentrifugation indicates that on the basis of physical-chemical criteria, this factor is not C'(5, 6, 7)a. The mononuclear cell chemotactic factor present in lysates of neutrophils sediments slowly in the ultracentrifuge and may be related, at least in part, to cationic peptides of lysosomal granules. A study in the time course of the chemotactic response of mononuclear cells reveals that the response begins to level off after 4 or 5 hr. This is in sharp contrast to the time course for the chemotactic response of neutrophils, in which the reaction is complete within 1.5 hr. Rabbit mononuclear cells obtained from a starch-induced peritoneal exudate respond to serum treated with plasminogen and streptokinase and to the soluble factor produced by bacteria, but no chemotactic response is elicited to serum treated with immune complexes. This indicates a functional difference between two populations of mononuclear cells. Rabbit alveolar macrophages respond poorly to all agents tested, although a weak chemotactic response to bacterial factors was found. The requirement of a serine esterase in the mononuclear cell for the cell to respond chemotactically was defined by the use of organophosphorus inhibitors. Pretreatment of mononuclear cells with several series of phosphonates renders them unresponsive in the chemotactic system. The effect is similar for mononuclear cells responding chemotactically to activated serum and to the bacterial chemotactic factor. Several points of contrast with inhibition profiles obtained in the chemotactic system of the neutrophil suggest that, while the mononuclear cell requires a serine esterase for chemotactic responsiveness, this enzyme is different from the one previously defined in the neutrophil.

Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by beta-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the alpha-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded alpha-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.

Dilute whole blood clots were prepared by addition of thrombin to blood diluted 1:10 in phosphate buffer. The pH of this buffer was 7.4 and the ionic strength was 0.084. Though the ionic strength was low, there was no hemolysis of red corpuscles due to the contribution to the osmotic gradient by plasma salts and proteins. In the standard assay the clot was formed by addition of thrombin at 4 degrees C then incubated at 37 degrees C. Retraction and lysis of these clots were inhibited by removal of platelets and by increasing concentrations of purified thrombin. Retraction and lysis were also inhibited by inactivation of any one of the following factors: gammaM globulin, complement components C4 and 3, and (in the case of lysis) plasminogen. Evidence that some of the above serum factors were adsorbed to the platelet membrane was obtained by aggregation of washed platelets by antisera to these factors (i.e. fibrinogen, gammaM, and C4 or C3). These platelets were not aggregated by antisera to other serum proteins (by albumin, transferrin, gammaG globulin). These and other studies suggested that platelets, thrombin, fibrinogen, gammaM globulin (cold agglutinin), complement components, and plasminogen influenced and facilitated retraction and lysis of clots. These studies also suggested that platelets and some of these factors were physically associated. Because of this physical association, and because of the fact that clot retraction is associated with aggregation and retraction of platelets, we extended the above observations to include a study of the effect of these same serum factors on serum-induced aggregation and retraction of washed platelets. (Other terms which have been in use in the past to describe serum-induced platelet aggregation and retraction have included those such as platelet "fusion" and "viscous metamorphosis," neither of which fully described the phenomena.)Platelet aggregation and retraction induced by serum was markedly accelerated by addition of increasing concentrations of thrombin and (or) cold agglutinin. Hirudin and antisera to gammaM globulin inhibited seruminduced aggregation and retraction of platelets. Reconstitution of inactivated serum with purified C4, 3, and 5 and thrombin restored its capacity to induce aggregation and retraction of platelets.Therefore, we postulated that platelet aggregation and retraction were necessary for clot retraction and that platelet aggregation and clot retraction facilitated clot lysis. More specifically we postulated that thrombin, in addition to catalyzing clot formation, also modified the platelet membrane such that gammaM globulin (cold agglutinin) and complement components can act on the platelet membrane leading to (a) aggregation and retraction of the platelets, (b) retraction of the clot, and (c) to the activation of plasminogen either on the surface of the platelet by C8i and (or) by release of platelet activators of plasminogen.