Jeff E. Harris

Nucleotide Sequences of the Three Genes Coding for Human Fibrinogen

Fibrinogen is synthesized in the liver by hepatic parenchymal cells and is secreted into the circulation (1). Hepatic synthesis of fibrinogen is constitutive but the rate can be modulated by a number of physiological and nonphysiological factors. The three chains of fibrinogen are encoded by distinct species of mRNA that are derived from the expression of three single copy genes (2, 3). Present evidence indicates that the three genes of human fibrinogen are linked and are located in a region that extends approximately 45 kb on chromosome 4q23-q32 (4). The genes are arranged in the order of γ-Aα-Bβ. The γ and Aα genes are transcribed in the same direction while the Bβ gene is transcribed in the opposite direction.

Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade

We have isolated and sequenced several cDNAs derived from the sea squirt Ciona intestinalis that encode vitamin K-dependent proteins. Four of these encode γ-carboxyglutamic acid (Gla) domain-containing proteins, which we have named Ci-Gla1 through Ci-Gla4. Two additional cDNAs encode the apparent orthologs of γ-glutamyl carboxylase and vitamin K epoxide reductase. Ci-Gla1 undergoes γ-glutamyl carboxylation when expressed in CHO cells and is homologous to Gla-RTK, a putative receptor tyrosine kinase previously identified in a related ascidian. The remaining three Gla domain proteins are similar to proteins that participate in fundamental developmental processes, complement regulation, and blood coagulation. These proteins are generally expressed at low levels throughout development and exhibit either relatively constant expression (Ci-Gla1, γ-glutamyl carboxylase, and vitamin K epoxide reductase) or spatiotemporal regulation (Ci-Gla2, -3, and -4). These results demonstrate the evolutionary emergence of the vitamin K-dependent Gla domain before the divergence of vertebrates and urochordates and suggest novel functions for Gla domain proteins distinct from their roles in vertebrate hemostasis. In addition, these findings highlight the usefulness of C. intestinalis as a model organism for investigating vitamin K-dependent physiological phenomena, which may be conserved among the chordate subphyla.

Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yes-associated protein

Proline-rich Gla protein 2 (PRGP2) is one of four known vertebrate transmembrane γ-carboxyglutamic acid (Gla) proteins. Members of this protein family are broadly expressed in fetal and adult human tissues and share a common architecture consisting of a predicted propeptide and Gla domain, a single-pass transmembrane segment, and tandem Pro/Leu-Pro-Xaa-Tyr (PY) motifs near their C termini. Using a methodology developed for the regulated expression of enzymatically biotinylated proteins in mammalian cells, we demonstrate that PRGP2 undergoes γ-glutamyl carboxylation in a manner that is both dependent upon the presence of a proteolytically cleavable propeptide and sensitive to warfarin, a vitamin K antagonist that is widely used as an antithrombotic agent. When expressed at physiologically relevant levels, the majority of PRGP2 is present in the γ-glutamyl carboxylated, propeptide-cleaved (mature) form. We additionally demonstrate, by Western blotting and flow cytometry, that mature PRGP2 is predominantly located on the cell surface with the Gla domain exposed extracellularly. In a yeast two-hybrid screen that used the C-terminal cytoplasmic region of PRGP2 as bait, we identified the WW domain-containing transcriptional coactivator Yes-associated protein (YAP) as a binding partner for PRGP2. In GST pull-down experiments, both PRGP2 PY motifs and both YAP WW domains were essential for complex formation, as were residues proximal to the core sequence of the first PY motif. These findings suggest that PRGP2 may be involved in a signal transduction pathway, the impairment of which may be an unintended consequence of warfarin therapy.

High density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

The ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D′D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1–GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1–GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.