Hemostasis, wound healing and tissue repair are integral constituents of our host defense mechanisms. The hypothesis to be tested is that there are multiple physiological roles of the glycosaminoglycan-binding protein, heparin cofactor II (HC) associated with its functional domains. Understanding these physiological functions requires us to compare and contrast HC with two other prominent glycosaminoglycan-binding proteins, antithrombin III (the prototypic coagulation proteinase inhibitor) and fibronectin (the model extracellular matrix cell-binding protein).
The specific aims of the research described In this project are: (1) Extravascular heparin cofactor II antithrombin activity on dermatan sulfate proteoglycans. (a) HC-dermatan sulfate and HC-heparin interactions will be characterized to understand the mechanism of glycosaminoglycan-dependent rate enhancement of thrombin inhibition; (b) Substratum-bound dermatan sulfate proteoglycans (from extracellular matrix) will be prepared to model an extravascular surface with HC-mediated antithrombin properties; (c) The protein-glycosaminoglycan interactions will be examined with monoclonal antibodies, synthetic peptides, molecular modeling and dynamics, quantitative binding analyses and equilibrium electrophoresis. (2) Heparin cofactor II cell motility and adhesion activities for leukocytes (neutrophils and monocytes) and fibroblasts. (a) The interactions of HC bioactive fragments with leukocytes will be studied to understand the mechanism of HC-directed effects on cell motility. The possible existence of a signal transduction mechanism distinct from that of the formyl- Met-Leu-Phe-receptor will be investigated using chemotaxis/haptotaxis techniques, localization and properties of plasma membrane receptors, and biochemical probes/toxins/inhibitors of known second messenger pathways; (b) The molecular requirements for the adhesion and spreading of fibroblasts to substratum-bound HC bioactive peptides will be determined. A comparison to extracellular matrix proteins. formation of focal adhesions, requirement for de novo protein synthesis, and the existence of specific receptors (including integrins), will also be examined. Once the biochemical and cell biological properties of HC are determined, and the contribution of extracellular matrix surfaces considered, it will be possible to assess the physiologic importance of the several activities of HC. Our overall goal will be to use the study of HC to expand our knowledge of (i) mechanism of protein-glycosaminoglycan interactions, (ii) generation and function of bioactive peptides, and (iii) the interrelationships of coagulation and wound healing/tissue repair processes.
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