Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). In priliminary studies, the principal investigator: 1) developed a monoclonal antibody technique for differentiating plasmin-derived von Willebrand factor (vWF) fragments from the """"""""native"""""""" fragments normally present in plasma, 2) determined that the increase in vWF-fragments present in IIA vWD plasma is not due to plasmin, 3) found evidence that vWF cleavage fragments in disseminated intravascular coagulation (DIC) and deep vein thrombosts (DVT) are produced by plasmin and at least one other enzyme, and 4) obtained evidence by inhibition with a sulfhydryl reagent suggesting that the enzyme(s) responsible for """"""""native"""""""" fragments is a cysteine proteinase but probably not platelet calcium activated proteninase. In phase I, the enzyme(s) involved in IIA vWD will be characterized by: a) delineation of a plasma or blood cellular origin, b) compartmentalization of the enzyme(s) within cells (plasma membrane, lysosomes, cytosol), and c) inhibition by a panel of proteinase inhibitors with known mechanism of proteinase inactivation. The hypothesis that increased proteolysis of IIA vWF is due to increased susceptibility to cleavage will be tested. This will be accomplished by isolating IIA vWF from different families and evaluating cleavage by an enzyme shown to duplicate in vivo cleavage. A survey of hypo/hypercoaguable states (eg. DIC, venous thrombosis, myeloproliferative diseases (MPD)) will be performed to define the extent of vWF proteolysis and to determine the likely proteinase(s) responsible. In phase II, the responsible enzyme(s) will be isolated, if not identified in Phase I. The sites of vWF cleavage will be determined using amino and carboxy terminal sequencing of the cleavage fragments. Bridging peptides will be synthesized and monoclonal antibodies prepared to them to determine which cleavages are important to loss of large multimers and/or function. IIA vWF structural abnormalities responsible for the increased susceptibility to cleavage will be identified using isoelectric focusing of vWf peptides which will be identified with MoAb's reacting with known portions of the vWf molecule. A simple MoAb based test for identifying increased vWf proteolysis in disease states will be developed.
| Federici, A B; Berkowitz, S D; Lattuada, A et al. (1993) Degradation of von Willebrand factor in patients with acquired clinical conditions in which there is heightened proteolysis. Blood 81:720-5 |
| Berkowitz, S D; Nozaki, H; Titani, K et al. (1988) Evidence that calpains and elastase do not produce the von Willebrand factor fragments present in normal plasma and IIA von Willebrand disease. Blood 72:721-7 |
| Berkowitz, S D; Federici, A B (1988) Sialic acid prevents loss of large von Willebrand factor multimers by protecting against amino-terminal proteolytic cleavage. Blood 72:1790-6 |
