Cellular Localization of Activated Factor X by Xa-Specific Probes

Summary

A probe, recombinant antistasin, that reacts specifically with the activated form of factor X (Xa) was used in immunohistochemical procedures to detect cellular sites of Xa generation within intact tissues. Factor Xa was detected on tumor cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma. Tumor-associated macrophages (but not tumor cells) expressed Xa in adenocarcinoma and squamous cell carcinoma of the lung, and Hodgkin’s disease. Factor Xa in these locations corresponded to evidence reported previously for an intact coagulation pathway and thrombin formation associated with these tumor cells and macrophages. By contrast, only rare connective tissue cells stained for Xa in breast and colon cancer, tumor types shown previously to lack an intratumoral coagulation pathway and thrombin generation, and in normal liver, lung, breast, kidney, and placental tissues. Hepatocytes did not stain. These results suggest that such probes may be useful for studying the activation state of cell-associated factor X in situ within intact tissues.

• References

• 1 Zacharski LR, Memoli VA, Costantini Y, Wojtukiewicz MZ, Ornstein DL. Clotting factors in tumor tissue: Implications for cancer therapy. Blood Coagulation Fibrinolysis 1990; 1: 71-78
• 2 Wojtukiewicz MZ, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Absence of components of coagulation and fibrinolysis pathways in situ in mesothelioma. Thromb Res 1989; 55: 279-284
• 3 Wojtukiewicz MZ, Zacharski LR, Memoli VA, Moritz TE, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Fibrin formation on vessel walls in hyperplastic and malignant prostate tissue. Cancer 1991; 67: 1377-1383
• 4 Wojtukiewicz MZ, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Abnormal regulation of coagulation/fibrinolysis in small cell carcinoma of the lung. Cancer 1990; 65: 481-485
• 5 Wojtukiewicz MZ, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Fibrinogen-fibrin transformation in situ in renal cell carcinoma. Anticancer Res 1990; 10: 579-582
• 6 Wojtukiewicz MZ, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Malignant melanoma: interaction with coagulation and fibrinolysis pathways in situ. Am J Clin Pathol 1990; 93: 516-521
• 7 Ornstein DL, Costantini V, Zacharski LR, Memoli VA, Hunt J, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Coexistent macrophage procoagulant and tumor cell plasminogen activator in primary squamous cell and adenocarcinoma of the lung. Blood 1989; 74 Suppl (Suppl. 01) 292a
• 8 Costantini Y, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Fibrinogen deposition and macrophageassociated fibrin formation in malignant and non-malignant lymphoid tissue. Blood 1989; 74 Suppl (Suppl. 01) 37a
• 9 Costantini V, Zacharski LR, Memoli VA, Rousseau SM, Kisiel W, Kudryk BJ. Occurrence of coagulation and fibrinolysis pathway components in situ in primary breast cancer tissue. Thromb Haemostas 1989 62. 39 (Abstr)
• 10 Wojtukiewicz MZ, Zacharcki LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Stump DC. Indirect activation of blood coagulation in colon cancer. Thomb Haemostas 1989; 62: 1062-1066
• 11 Tirszynski GR, Gasic TB, Gasic GJ. Isolation and characterrzation of antistasin, an inhibitor of metastasis and coagulation. J Biol Chem 1987; 262: 9718-9723
• 12 Nutt E, Gasic T, Rodkey J, Gasic GJ, Jacobs JW, Friedman PA, Simpson E. The amino acid sequence of antistasin, a potent inhibitor of factor Xa reveals a repeated internal structure. J Biol Chem 1988; 263: 10162-10167
• 13 Han JH, Law SW, Keller PM, Kniskern PJ, Silberklang M, Tung J-S, Gasic TB, Gasic GJ, Friedman PA, Ellis RW. CloqinB and expression of cDNA encoding antistasin, a leech-derived protein having anticoagulant and antimetastasic properties. Gene 1989; 75: 47-57
• 14 Nutt EM, Jain D, Lenny A, Schaffer L, Siegl P, Dunwiddie C. Purification and charactertzation of recombinant antistasin: a leechderived inhibitor of coagulation factor Xa. Arch Biochem Biophys (in press)
• 15 Dunwiddie C, Thornberry NA, Bull HG, Sardana M, Friedman PA, Jacobs JW, Simpson E. Antistasin, a leech-derived inhibitor of factor Xa. J Biol Chem 1989; 264: 16694-16699
• 16 Holz GD, Krivan HC, Gasic GJ, Ginsburg V. Antistasin, an inhibitor of coagulation and metastasis, binds to sulfatide (Gal (3–SO₄) β1–1Cer) and has a sequence hemology with other proteins that bind sulfated glycoconjugates. J Biol Chem 1989; 264: 12138-12140
• 17 Donati MB, Gambactori-Passerini C, Casali B. et al. Cancer procoagulant in human tumor cells: evidence from melanoma patients. Cancer Res 1986 46. 6471-6474
• 18 Sakai T, Kisiel W. Binding of human factors X and Xa to Hep G2 and J82 human tumor cell lines. Evidence that factor Xa binds to tumor cells independent of factor Va. J Biol Chem 1990 265. 9105-9113
• 19 Altieri DC, Edgington TS. Identification of effector cell protease receptor–1, a leukocyte-distributed receptor for the serine protease factor Xa. J Immunol 1990; 145: 246-253
• 20 Gajdusek C, Carbon S, Ross R, Nawroth R, Stern D. Activation of coagulation releases endothelial cell mitogens. J Cell Biol 1986; 103: 419-428