The long-term goal of this project is to define the role that blood coagulation Factor XIII (FXIII) plays in stabilizing the hemostatic plug. The focus of this grant will be on the interaction between FXIII with fibrin and platelets in the hemostatic plug.
The specific aim are: (1) to localize and sequence the fibrin binding and platelet binding site(s) on the a-chain of FXIII; (2) to identify the Factor XIIIa binding site(is on fibrin and the platelet receptor for Factor XIIIA; (3) to study the physiologic consequences of inhibiting FXIII binding to fibrin and platelets in vitro. A method to purify the fibrin binding domain from a partial trypsin digest of platelet FXIII a-chains has been developed. A 54 kDa peptide contains the active site and has been purified to homogeneity and sequenced. An unlabeled 30 kDa polypeptide from the C-terminus of the molecule will also be purified. The effect of polyclonal affinity purified antibodies and monoclonal antibodies to the 54 kDa and 30 kDa polypeptides will be utilized to investigate whether they: modify thrombin cleavage of plasma FXIII, inhibit FXIII binding to fibrin, prevent FXIII crosslinking of fibrin, or enhance clot lysis in vitro. The effects of affinity purified antibodies will be investigated to determine whether they reduce FXIIIA crosslinking at the platelet surface, inhibit crosslinking of platelets to adhesive glycoproteins or inhibit plasmin degradation of platelet proteins. In addition, FXIIIa substrates on thrombin-stimulated platelets will be identified using [3h]-putrescine incorporation. Platelet glycoproteins IIb-IIIa has been identified as a Factor XIIIa receptor. Further characterization of the platelet receptor for Factor XIIIa will be obtained using flow cytometric analysis and photoaffinity crosslinking studies. cDNA clones from the a-chain of Factor XIII will be used to investigate whether the cloned proteins retains fibrin binding sites and to localize the binding site. Results from these studies may have direct application to the development of diagnostic and therapeutic agents which could benefit several thrombotic disorders including myocardial infraction, stroke and pulmonary embolization.
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