Welcome to the Ruggeri Laboratory

Research

Cardiovascular disease and stroke remain the major health concern in the adult population of developed countries, including the United States. Investigators in the Division of Experimental Thrombosis and Hemostasis are conducting basic research to address the main pathogenetic mechanisms responsible for arterial and venous thrombosis, laying the foundation for novel and more efficient therapeutic approaches. Efforts are also devoted to further the understanding of endothelial cell function in normal and pathological conditions, as well as to clarify the role of the immune system in determining the success of failure of organ transplantation. Thus, several themes relevant to vascular biology are being developed in the Division, using all the advanced tools of cell, molecular and structural biology.

Knowledge of the detailed three-dimensional structure of adhesive proteins mediating platelet thrombus formation is indispensable to explain in detail the mechanisms responsible for normal hemostasis and pathological thrombosis. The crystallographic work performed in the Division has been the result of a close collaboration initiated with Kottayil I. Varughese when he was still at the University of California, San Diego, before moving to The Scripps Research Institute. Now, a team of investigators is working on fundamental structural and biomechanical issues pertaining to the process of platelet adhesion. Two projects have been completed, setting the bases for elucidating the molecular mechanisms that support the initiation of platelet thrombus formation through the interaction between the adhesive protein, von Willebrand factor (vWF), and the platelet receptor, glycoprotein (GP) Ib .

GP Ib , a component of the GP Ib-IX-V receptor complex, and vWF have a unique role in platelet function. Their interaction is necessary to initiate platelet deposition at sites of vascular injury when blood flow velocity is elevated, such as in arterioles during hemostasis or in stenosed atherosclerotic coronary arteries. Fast on rate and high resistance to tensile stress are the key properties that allow the bond between GP Ib and vWF A1 domain to support the initial tethering of rapidly flowing platelets to thrombogenic surfaces, thus reducing drastically their velocity relative to the vessel wall. Ensuing activation then results in interaction of the integrin IIb 3 (GP IIb-IIIa complex) with the Arg-Gly-Asp sequence in the carboxyl terminal C1 domain of vWF, rendering adhesion irreversible and favoring the accrual of additional platelets.

As a step towards elucidating the molecular bases of vWF and GP Ib function, we have solved the crystal structure of the Fab fragment of NMC-4, a monoclonal antibody that binds to the A1 domain with high affinity blocking its interaction with GP Ib . Two Asp and three Tyr residues in the complementarity determining regions (CDR's) 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the GP Ib amino terminal domain known to be critically involved in vWF binding. These results, therefore, define an antibody structure that may reflect the spatial orientation of corresponding GP Ib residues involved in supporting contact with the vWF A1 domain.

The definition of the contact interface between vWF A1 domain and the amino terminal region of GP Ib is essential to elucidate the structural bases of an interaction that plays a key role in normal platelet function and may be a major pathogenetic factor in arterial thrombosis. We have crystallized the complex between recombinant vWF A1 domain and NMC-4 Fab, and solved its structure at 2.2 � resolution using a combination of molecular replacement and single isomorphous replacement techniques. The vWF A1 domain displays an / Rossmann fold with a six strand central sheet sandwiched between two sets of three helices, one on each side. NMC-4 interacts with the A1 domain bridging to one of these helices, 4, that is separated by a break occurring after the first turn into two segments, a and b, with different longitudinal axes. The short segment 4a is a 310 helix and comprises residues 627 to 631 of mature vWF. Residues 631-634 form a type I -turn and residues 633-643 form 4b. The entire segment 4a, the -turn and the N-terminal portion of 4b wedge into the antigen binding site of NMC-4 created primarily by the CDR loops L3, H2 and H3 assembled as a tripod. Work is still in progress on this structure to elucidate aspects that may be relevant to the vWF A1 domain interaction with platelet GP Ib . In particular, targeted mutagenesis studies are being used to confirm the functional and structural role of specific residues with a key role in A1 domain/NMC-4 complex formation.

Thrombi consisting of aggregated platelets support hemostasis, but may occlude atherosclerotic arteries causing ischemic damage to vital organs. We have developed a new approach, based on laser confocal microscopy, to measure thrombus formation in real time during blood flow in experimental chambers, obtaining three-dimensional volumetric information and elucidating the effects of blood flow on the mechanisms of platelet aggregation. When blood flows slowly, at the venous shear rate of 100 s-1, the integrin IIb 3, but not GP Ib , is essential for platelet attachment to one another, and fibrinogen is the most efficient bridging ligand. In contrast, when blood flows rapidly, at the arteriolar shear rate of 1,500 s-1, thrombus growth is totally dependent on vWF and its two receptors, GP Ib as well as IIb 3. At intermediate velocity, with wall shear rate of 300 s-1, thrombus volume is reduced by more than 50% if GP Ib function is blocked. These results indicate that synergistic adhesive mechanisms support platelet aggregation and determine the rate of thrombus growth acting as continuous variables dependent on blood flow conditions, thus identifying distinct targets for selective anti-thrombotic intervention. Ongoing work in this area may substantiate the concept that optimal anti-thrombotic therapy depends on the combination of different inhibitors of adhesive interactions each responsible for specific biomechanical attributes of a developing thrombus.

Our long term objectives are to gain an understanding of the molecular events controlling megakaryocytopoiesis and platelet production. As compared to other hematopoietic cells of the bone marrow, the generation of basic information on megakaryocytpoiesis and platelet production has been slow due to the lack of cell lines mimicking the normal megakaryocytic progenitor cell and an inability to manipulate experimentally platelet formation. Clearly, thrombopoietin is essential for the differentiation of the pluripotent stem cell to a polyploid megakaryocyte, but the late events of the process, specifically those controlling the release of platelets are still poorly understood. However, the expression of megakaryocytic or platelet-specific antigens represents one strictly unique aspect of megakaryocytopoiesis that can be examined. Such experiments define the molecular events and factors regulating the commitment of cells to the megakaryocytic lineage, and ultimately, the release of platelets into the bloodstream. With regard to this latter point, the glycoprotein (GP) Ib-IX-V complex is an essential multi-subunit platelet membrane receptor critical for hemostasis and implicated in normal platelet release and structure as evidenced by the congenital absence of the complex and the release of abnormal or "giant" platelets, a condition referred to as the Bernard-Soulier syndrome.

The genes encoding GP Ib and the smaller -subunit of GP Ib (GP Ib ) both contain promoter elements necessary for megakaryocytic gene expression. A GP Ib is composed of two subunits (GP Ib and GP Ib ) each synthesized from separate genes. The 206 amino acid precursor of GP Ib is synthesized from a 1.0 kilobase (kb) mRNA expressed by megakaryocytes and was originally characterized from cDNA clones of human erythroleukemic (HEL) cell mRNA, a cell line exhibiting megakaryocytic-like properties. The cell line CHRF-288-11 also exhibits megakaryocytic-like properties, but synthesizes two related GP Ib mRNA species of 3.5 and 1.0 kb. We performed cDNA cloning experiments to identify the origin of the 3.5 kb transcript and determine its relationship to the 1.0 kb GP Ib mRNA found in megakaryocytes, platelets and human erythroleukemia (HEL) cells. Our cloning experiments demonstrated that the longer transcript results from a non consensus polyadenylation recognition sequence, 5'AACAAT3', within a separate gene located 250 nucleotides 5' to the transcription start site of the platelet GP Ib gene. In the absence of normal polyadenylation the more 5' gene uses the polyadenylation site within its 3' neighbor, the platelet GP Ib gene. This newly identified 5' gene contains an open reading frame encoding 369 amino acids with a high degree of sequence similarity to an expanding family of GTP-binding proteins, referred to as septins.

Originally described as essential for yeast budding, the functional relevance of septins in higher eukaryotes has not been examined with one exception; a Drosophila protein designated PNUT, that is embryonically-lethal in the homozygous state and produces an over accumulation of embryonic polyploid cells. However, a unifying functional property of septin proteins is their involvement in cytokinesis. The spatial proximity of the newly-identified human septin gene and the human platelet GP Ib gene is also present within the homologous mouse locus with a remarkable conservation of gene sequence and structure. Future studies will examine the structural and functional role of human septins and their ability to influence the expression of the GP Ib-IX-V platelet receptor.

Hematogenous metastasis requires tumor cell attachment to the vessel wall during blood flow. In an in vitro perfusion system, tumor cell arrest involved tumor cell interaction with activated platelet via beta-3 integrins and crosslinking plasma proteins. Beta-3 integrin-ligand recognition involves the Arg-Gly-Asp (RGD) ligand motif. Therefore, we analyzed critical flanking sequences surrounding the RGD site and the conformational contstraints of the RGD domain that are crucial in determining the ligand specificity for tumor cell integrin alpha-v beta-3 and platelet integrin alpha-IIb beta-3. This information will be important for the development of inhibitors of tumor cell-platelet interaction. Another receptor of potential importance in tumor cell adhesion to vascular cells is ICAM-1. We found a clear correlation between ICAM-1 expression and tumor progression in melanoma patients indicating a role for this molecule in melanoma cell adhesion during the metastatic cascade. The clinical significance of integrin alpha-v beta-3 and ICAM-1 expression in melanoma lesions identifies these receptors as targets for future studies on melanoma cell arrest and extravasation. For this purpose we have developed an experimental system that allows us to analyze both, tumor cell attachment to the endothelium and transendothelial migration during blood flow. Critical parameters in this in vitro system are the stimulation of the endothelium and its presentation on a shear resistant 3-D cushion that allows to analyze transmigrated cells, as well as the identification and quantification of attached and penetrated cells through 3-D reconstruction of confocal images acquired during the perfusion. This technique will enable us to study the vascular cell types and their receptors and ligands that are involved in adhesive and invasive tumor cell interaction with the vessel wall and which impact upon the rate of hematogenous tumor metastasis.