Studies from the applicant's laboratory, as well as those from other investigators, have focused on a novel but poorly understood unique protein, platelet FXI, which is the focus of the current application. Recent experiments from the applicant's laboratory utilizing reverse transcriptase polymerase chain reaction (RT-PCR) and molecular cloning from a megakaryocyte (CHRF-288 cell) cDNA library suggest the possibilities that platelet FXI is either an alternative splicing product of the plasma FXI gene lacking exon V or the product of a separate gene expressed exclusively in megakaryocytes. Platelet FXI migrates by SDS- polyacrylamide gel electrophoresis (PAGE) with an apparent Mr of approximately 220,000, approximately 55,000 after reduction, compared with plasma FXI which has an Mr approximately 160,000 and a subunit Mr of approximately 80,000. Platelet FXI coagulant activity and antigen are present in well-washed platelet suspensions constituting approximately 0.5% of the FXI activity in normal plasma, from which it can be calculated that there are approximately 300 molecules of platelet FXI per platelet. The objectives of this proposal are to accomplish a structural and functional characterization of platelet FXI both at the genomic and protein levels, to determine the molecular basis for the presence of platelet FXI with the platelet-plasma membrane in patients with plasma FXI deficiency, to determine the mechanism of association of platelet FXI with the platelet-plasma membrane, and to ascertain the mechanisms of its activation and the expression of its enzymatic activity.
The specific aims of this proposal are as follows: 1) To accomplish a structural characterization of the platelet FXI gene, mRNA and protein. Sub-aim 1a) To determine the relationship between the mRNA for platelet FXI and the FXI gene in order to resolve the issue where it is an alternative splicing of the plasma FXI gene or the product of a separate gene. Sub-aim 1b) To determine the relationship between platelet FXI mRNA and protein by in vitro translation. Sub-aim 1c) To accomplish a structural characterization of platelet FXI by purification and biochemical characterization of the platelet FXI protein. 2) To determine the molecular basis and functional relevance and the presence of platelet FXI in the platelets of patients with plasma FXI deficiency. Sub-aim 2a) To test the hypothesis that patients with type II FXI deficiency (characterized by a stop codon in exon V) are able to express platelet FXI normally because of the absence of exon V in platelet FXI. Sub-aim 2b) To test the hypothesis that platelet FXI can substitute for plasma FXI in hemostasis. 3) To determine the mechanism of association of platelet FXI with the platelet plasma membrane by exploring the hypothesis that one platelet FXI (Mr approximately 55,000) forms a disulfide-linked complex (Mr approximately 220,000) with platelet membrane glycoprotein Ib (GPIb) (Mr approximately 165,000). 4) To determine the mechanism of activation of platelet FXI by thrombin, by FXIa, by FXIIa, or by other proteases and to determine the mechanism of expression or its enzymatic activity and its normal macromolecular substrate (i.e., FXI or FIX).
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