Low-affinity platelet factor 4 1H NMR derived aggregate equilibria indicate a physiologic preference for monomers over dimers and tetramers.
Low-affinity platelet factor 4 (LA-PF4), unlike another related, sequentially homologous (about 50%) platelet-specific protein, platelet factor 4 (PF4), is an active mitogenic and chemotactic agent. PF4 exhibits a high binding affinity for heparin, while LA-PF4 does not. Both PF4 and LA-PF4 can exist in dimer and tetramer aggregate states. Equilibrium constants for PF4 aggregation have recently been estimated from fractional populations derived from proton nuclear magnetic resonance (NMR) integrals assigned to resonances in monomer, dimer, and tetramer states [Mayo & Chen (1989) Biochemistry 28, 9469]. On a 500-MHz NMR time scale, relatively slow exchange among LA-PF4 aggregate species has also allowed Tyr 15 ring proton resonances to be assigned for monomer, dimer, and tetramer states in LA-PF4. As a function of pH and ionic strength, equilibrium association constants for LA-PF4 dimer (KD) and tetramer (KT) formation have been estimated from Tyr 15 ring proton resonance integrals. At low ionic strength, KD reaches a minimum value of 12 M-1 at pH 3 where KT is at its maximum value of 1.6 x 10(5) M-1. At pH 4.1, KD and KT have the same value, 1.1 x 10(3) M-1, which is the minimum value for KT. KD plateaus off to its maximum value of 2.2 x 10(4) M-1 by pH 5.5. These values are significantly lower than those for PF4. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions probably among Glu/Asp and Lys/Arg side chains form the predominant force in the monomer-monomer binding process, i.e., KD, while like-charge repulsion due to proximal, intersubunit Glu/Asp residues decreases KT as the pH is raised. At pH 7 and low ionic strength, the dimer state is highly favored over the tetramer state. Elevating the solvent ionic strength at pH 7 destabilizes the dimer state. Under these more physiologic conditions, i.e., pH 7 and 0.1-0.2 M NaCl, LA-PF4 monomers are highly favored over dimers and tetramers. For PF4 under similar solvent conditions, tetramers predominate. Differences in biological activities between these homologous platelet-specific proteins may be the result, at least in part, of differing aggregation properties. The biologically active state for PF4 is tetramer, while for LA-PF4 it is monomer. Quaternary structure may, therefore, account for strong heparin binding in PF4, most likely by presenting a more favorable structural matrix for effective glycosaminoglycan interactions.
Mayo
K H
KH
Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122.
D000595:Amino Acid Sequence; D000818:Animals; D001779:Blood Coagulation Factors / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006493:Heparin / Q000378:metabolism; D006494:Heparin Antagonists / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides; D010974:Platelet Aggregation; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D001779:Blood Coagulation Factors; D006494:Heparin Antagonists; D010455:Peptides; C009862:connective tissue-activating peptide; C018329:low affinity platelet factor 4; D006859:Hydrogen; D006493:Heparin