Infiltrating monocytes, neoplastic cells and hyaluronic acid (HA) are components of cancers which can potentially contribute to thromboembolic phenomena in malignancy. We propose to examine the ability of these components to modify initial rates of coagulation reactions. Human peripheral blood monocytes differentiated during co-culture with human carcinoma cells will be examined for differentiation-related changes in expression of functional coagulant proteins. coagulant proteins expressed, and mechanisms regulating this expression will be investigated using clotting, radiometric and chromogenic tests and by total mRNA and DNA transcription-rate measurements. The family of vitamin K-dependent gamma- carboxylated proteins synthesized by carcinoma cells will be purified and characterized from scaled up cultures. Methodologic approach will be: 1) Vitamin K-dependent 14CO2-gamma-carboxylation of endogenous neoplastic cell proteins. 2) SDS-PAGE of these proteins. 3) Fluorography of gels. 4) Electroelution of radio labelled brands. 5) Polyclonal antibody production. 6) Immunoaffinity chromatography. 7) High pressure liquid chromatography (HPLC). Oligossacharides (HA5 to HA20) and larger molecular weight HA fragments (.5x10Da) will be purified from specific-hyaluronidase digests using polyacrylamide gel chromatography. These preparations will be used to characterize the nature of the interaction between HA and coagulation proteases. Effect of carcinoma derived gamma carboxylated proteins and of HA on catalytic reaction rates of protease complexes assembled on the membranes of tumor-differentiated monocytes will be investigated at nonsteady-state and steady-state phases of the reaction. Kinetic parameters of coagulation protease assembly and substrate catalysis during nonsteady-state phase of the reaction will be examined by analyses of tracer dilution curves after addition of purified reaction components to perfused monocytes anchored on microcarrier beads. These studies are focused on mechanistic and regulatory aspects of coagulation in tumors which have not been considered in previous studies.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Wake Forest University Health Sciences
Schools of Medicine
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