CELLULAR-LOCALIZATION OF ENZYMATICALLY ACTIVE THROMBIN IN INTACT HUMAN TISSUES BY HIRUDIN BINDING

Cellular sites of coagulation activation within complex, intact tissue s have been studied by immunohistochemical techniques. Hirudin, a spec ific and high affinity inhibitor of the active site of thrombin, toget her with antibody to hirudin were applied to sections of AMeX-fixed sp ecimens of normal lung, kidney, placenta, freshly incised skin and unp erturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue ; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid sy novial, and placental macrophages that express an intact extrinsic coa gulation pathway. Staining was also observed restricted to the endothe lium of capillaries in freshly incised skin but not in either unpertur bed skin or in aged incisions. Staining of tumor cell bodies was obser ved in small cell carcinoma of the lung, renal cell carcinoma, and mal ignant melanoma tissues that we found previously to show tumor cell-as sociated procoagulant activity, This staining occurred commonly on cel ls within the tumor mass that were distant from stromal fibrinogen/fib rin, By contrast, tumor-associated macrophage but not tumor cell stain ing was seen in adenocarcinoma and squamous cell carcinoma of the lung , and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitte d failed to show staining. These results are in accord with previous f indings and suggest that such techniques may be useful for studying th e cellular sites of thrombin generation in intact tissues. We postulat e that administration of potent and specific thrombin antagonists, suc h as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local t hrombin generation on malignant progression can be determined.