The project is focused on a hypothesis that the formation of a clot is driven by forces originating from attraction of complementary polymerization sites located in either amino- or carboxy-terminal domains of the fibrin molecule. It is proposed to elucidate which amino acid sequence, in each of the three polypeptide chains of fibrin, is required to express binding affinity for fibrin clots. Peptides containing sequences of the Alpha and Beta chain amino-termini, and the Bety and Gamma chain carboxy-termini will be obtained either by enzymatic cleavage of fibrinogen and fibrin with various proteinases or by chemical synthesis. Preliminary data show dependence of binding affinity to fibrin on intact conformation of fragment D1 and t-NDSK. It is postulated that the structure of a polymerization site may contain more than one polypeptide chain segment. To prove this point, fibrinogen and fibrin fragments will be crosslinked with bifunctional reagents, the products cleaved by proteinases, and multi-chain derivatives with affinity for fibrin isolated and characterized. It will be measured whether crosslinked multi-chain derivatives possess higher affinity for clots than simple peptides. Peptides distinguished by fibrin binding will be used to obtain monoclonal antibodies against polymerization sites. These antibodies will be used for isolation of specific polymerization sites and for mapping of the sites on fibrin fragments. Fibrin polymerization sites will be utilized to construct fibrinolytic hybrids. The hybrids will contain a molecular vehicle with proven affinity for fibrin, for example fragment DD or E1, linked either non-covalently or covalently with a firbinolytic agent, especially with tissue plasminogen activator and urokinase. Preliminary results show that the activator in hybrids with fragments DD and (DD)E is protected against blood inhibitors. The effect is expressed by amplified fibrinolysis of plasma clots. Experimental results will provide an evidence as to the usefulness of fibrinolytic hybrids as clot-targeted species. The use of plasma and whole blood clots will allow quantification of the lytic effect of various hybrids. The long-term objectives of the proposal will provide explanation of molecular mechanisms involved in the formation of blood clots, develop new anticoagulants based on polymerization site structure and explore novel approach to thrombolytic therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL036221-02
Application #
3350997
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-05-01
Project End
1991-04-30
Budget Start
1987-05-01
Budget End
1988-04-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Sato, H; Swadesh, J K (1993) Structural difference between polymerized and non-polymerized fragment X, obtained by plasmin digest of fibrinogen. Int J Biol Macromol 15:323-7
Cierniewski, C S; Budzynski, A Z (1993) Localization of the cross-linking site of GPRVVERHK in the gamma-chain of human fibrinogen. Eur J Biochem 218:321-5
Ugarova, T P; Budzynski, A Z; Shattil, S J et al. (1993) Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa. J Biol Chem 268:21080-7
Ugarova, T P; Budzynski, A Z (1992) Interaction between complementary polymerization sites in the structural D and E domains of human fibrin. J Biol Chem 267:13687-93
Cierniewski, C S; Budzynski, A Z (1992) Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies. Biochemistry 31:4248-53
Hasan, A A; Chang, W S; Budzynski, A Z (1992) Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator. Blood 79:2313-21
Pandya, B V; Gabriel, J L; O'Brien, J et al. (1991) Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence. Biochemistry 30:162-8
Budzynski, A Z (1991) Interaction of hementin with fibrinogen and fibrin. Blood Coagul Fibrinolysis 2:149-52
Swadesh, J K; Huang, I Y; Budzynski, A Z (1990) Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii. J Chromatogr 502:359-69
Siebenlist, K R; DiOrio, J P; Budzynski, A Z et al. (1990) The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin. J Biol Chem 265:18650-5

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