Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer.

Thrombin-catalyzed, cross-linked fibrin (XLF) formation is a characteristic histopathological finding in many human and experimental tumors and is thought to be of importance in the local host defense response. Although the pathogenesis of tumor-associated fibrin deposition is not entirely clear, several tumor procoagulants have been described as likely primary stimuli for the generation of thrombin (and XLF) in the tumor microenvironment (TME). In a previous study of a variety of human tumors we have shown that tissue factor (TF) is the major procoagulant. However, the relative contribution to fibrin deposition in the TME of tumor cell TF and host cell TF (eg, macrophage-derived) was not established. In addition, recent evidence has implicated TF in the regulation of the synthesis of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells. In the current study we used in situ techniques to determine the cellular localization of XLF, TF, VEGF, and an alternative tumor procoagulant, so-called cancer procoagulant (CP), a cysteine protease that activates clotting factor X. In lung cancer we have found XLF localized predominantly to the surface of tumor-associated macrophages, as well as to some endothelial cells and perivascular fibroblasts in the stromal area of the tumors co-distributed with TF at the interface of the tumor and host cells. Cancer pro-coagulant was localized to tumor cells in several cases but not in conjunction with the deposition of XLF. TF and VEGF were co-localized in both lung cancer and breast cancer cells by in situ hybridization and immunohistochemical staining. Furthermore, a strong relationship was found between the synthesis of TF and VEGF levels in human breast cancer cell lines (r2 = 0.84; P < 0.0001). Taken together, these data are consistent with a highly complex interaction between tumor cells, macrophages, and endothelial cells in the TME leading to fibrin formation and tumor angiogenesis.     

Characteristics of membrane and cytosol forms of the mammary tumor glycoprotein molecule mtgp in human breast carcinoma cell cultures and tumors

The presence, concentration and selected molecular characteristics of the human mammary carcinoma glycoprotein molecule set MTGP , a trace and apparently tumor-specific molecule, were examined in fifteen cell cultures established from mammary carcinomas, tissue from seven mammary carcinomas and control cultures. Both cytosol and membrane-associated forms of MTGP were analyzed, and each was phenotyped by reference to isoelectric point and buoyant density. All cells or tissues of mammary carcinoma origin contained membrane MTGP , whereas cytosol MTGP was undetectable in cell cultures from half of the mammary carcinomas. Neither membrane nor cytosol MTGP were detectable in cells other than mammary carcinomas. Cytosol MTGP could be assigned to three groups by reference to presence, isoelectric point and buoyant density. Membrane MTGP also exhibited heterogeneity between different tumors and could be assigned to three groups by isoelectric point and buoyant density. Each form of MTGP was homogeneous for a given single tumor or cell culture and retained its phenotypic features with passage and cloning. Four general types of MTGP are proposed, though there may be additional fine heterogeneity that cannot be further resolved at this time. These data provide an initial characterization of the membrane form of MTGP and an integrated characterization that is consistent with the concept that tumor-specific antigens may possess both constant regions by reference to antigens recognized by the antisera and variable structure by reference to physicochemical characteristics.     

Immune responses to the cleavage-associated neoantigens of fibrinogen in man. Identification and characterization of humoral antibodies specific for cleavage fragments.

Cleavage of human fibrinogen and fibrin by plasmin is associated with modification of native antigenic expression and the exposure of cleavage-associated neoantigenic sites on the derivative molecular fragments. In this study, the presence of humoral antibodies in man to cleavage-associated neoantigens has been demonstrated by primary antigen binding radioimmunochemical assays. Specific binding of radioiodinated human fibrinogen D fragment by serum immunoglobulins was demonstrated in 52 of 59 random normal human sera and was independent of immunoglobulin concentration. Binding was mediated by F (ab)2 fragments of IgG, and specificity for neoantigens was indicated by the capacity of the D fragment but not native fibrinogen to competitively inhibit the antibody. The population distribution of antibody to these cleavage-associated neoantigens indicated the presence of a major group of individuals (77%) with a mean antigen binding capacity of 11.8 pmol/ml serum. Two minor populations with : (a) low or undetectable binding capacities (less than 6.0 pmol/ml serum) and (b) exhibiting markedly elevated binding capacities (less 18.0 pmol/ml serum) were delineated. Independent of these features, sera could also be readily separated into two groups that differed with respect to relative antibody affinity. The antibodies in most sera exhibited marked heterogeneity of binding affinity, whereas a small group of sera contained antibodies exhibiting relative homogeneity of binding affinity. Specific antibody was rather equally distributed between the major immunoglobulin classes, and in no serum was the antibody restricted to a single immunoglobulin class. Antibodies capable of binding fibrinogen fragments X, Y, and D and fibrin D fragment were detected in most sera. The quantity of antibody differed for different fragments with X greater than Y congruent to D greater than fibrin D. The presence of antibody capable of binding any single fragment was statistically correlated with the presence of antibody capable of binding other cleavage fragments. No antibody to the E fragment was detected. Antibody to cleavage fragments was not demonstrable in sera containing fibrinogen or fibrin cleavage fragments. Demonstration of this humoral immune response to the products of the fibrinolytic systems provides a new interface between the coagulation and immune system.     

Clinical and immunologic features of transient cold agglutinin hemolytic anemia

Atypical pneumonitis, often induced by infection with Mycoplasma pneumoniae, is frequently associated with elevated cold agglutinin titers but only rarely with significant hemolytic anemia. An example and documentation of the clinical and immunologic course of such transient cold agglutinin hemolytic anemia is presented, and the immunochemical characteristics of cold agglutinins in this syndrome are assessed in regard to their biologic implications and diagnostic significance. Transient cold agglutinins, such as observed in this case, commonly appear to be of a restricted polyclonal character and as first demonstrated in this case may possess structural determinants usually considered unique for monoclonal cold agglutinins. Although the immunopathogenetic mechanisms and certain clinical manifestations of the various forms of cold agglutinin hemolytic anemia appear similar, the immunogenic basis of cold agglutinin production and the molecular structure of transient cold agglutinins are quite distinctive and provide reasonably reliable guidelines for the differential diagnosis of the cold agglutinin syndromes and for consideration of appropriate clinical management.