Structural Alterations in Hereditary Dysfibrinogens Pp. 239-247
Molecular Views and Measurements of Hemostatic Processes
Using Atomic Force Microscopy Pp. 249-274
Dissecting Functional Interactions in Coagulation Protein
Complexes by Use of NMR Spectroscopy Pp.
275-285
Structure, Function, and Activation of Coagulation Factor
VII Pp. 287-299
Structure and Function of the Von Willebrand Factor A1
Domain Pp. 301-312
New Insights into Binding Interfaces of Coagulation
Factors V and VIII and their Homologues – Lessons from High Resolution Crystal
Structures Pp. 313-339
Structural Bioinformatics: Methods, Concepts and
Applications to Blood Coagulation Proteins Pp.
341-364
Back to top] Structural
Alterations in Hereditary Dysfibrinogens
Dysfibrinogens can be grossly
divided in two groups: (1) defective thrombin-catalyzed conversion of
fibrinogen molecules to fibrin monomers, and (2) defective fibrin
polymerization due to structural alterations in polymerization sites, that
include "A" and "a" sites, end-to-end D:D abutment
surfaces, and lateral association sites involving the carboxyl terminal region
of the fibrin a-chain. Recently, a
number of mutations in the fibrinogen genes have been identified, and many of
these encode changes that occur in regions of fibrinogen that have been
elucidated by high-resolution structural studies. Here we focus on the
structure-function relationships of fibrinogen that can be inferred from
studies involving these abnormal molecules.
Back to top] Molecular Views and Measurements
of Hemostatic Processes Using Atomic Force Microscopy
Hemostasis and thrombosis are
highly complex and coordinated interfacial responses to vascular injury. In
recent years, atomic force microscopy (AFM) has proven to be a very useful
approach for studying hemostatic processes under near physiologic conditions.
In this report, we review recent progress in the use of AFM for studying
hemostatic processes, including molecular level visualization of plasma
proteins, protein aggregation and multimer assembly, and structural and
morphological details of vascular cells under aqueous conditions. AFM offers
opportunities for visualizing surface-dependent molecular and cellular
interactions in three dimensions on a nanoscale and for sensitive, picoNewton
level, measurements of intermolecular forces. AFM has been used to obtain
molecular and sub-molecular, resolution of many biological molecules and
assemblies, including coagulation proteins and cell surfaces. Surface-dependent
molecular processes including protein adsorption, conformational changes, and
subsequent interactions with cellular components have been described. This
review outlines the basic principles and utility of AFM for imaging and force
measurements, and offers objective perspectives on both the advantages and disadvantages.
We focus primarily on molecular level events related to hemostasis and
thrombosis, particularly coagulation proteins, and blood platelets, but also
explore the use of AFM in force measurements and surface property mapping.
Back to top] Dissecting Functional
Interactions in Coagulation Protein Complexes by Use of NMR Spectroscopy
The blood coagulation cascade can be considered as a system of well-orchestrated protein activation reactions involving and leading to the formation of large macromolecular assemblies. NMR investigations performed during the last six years have focused on the structural, motional and binding properties of some protein domains and interfaces critical for the formation of these protein complexes, outlining sophisticated intermolecular adaptations. The studied protein domains are either single molecules or covalently-linked heterodimers of the epidermal growth factor (EGF) homology domains, calcium-binding EGF domains and g-carboxyglutamic(Gla)-containing domains responsible for calcium-dependent binding to cell membranes. The characterized binding interfaces have included those between thrombin and fibrinogen, between thrombin and thrombomodulin, between factor VIIIa and the cell membrane, between tissue factor and factor VIIa, and most recently between factor Va and prothrombin. The obtained results indicate that the regulation of blood coagulation by protein and low molecular weight cofactors may involve a significant degree of protein folding transitions with changes in molecular and conformational motions coupled to enzymatic activities. This new level of complexity of the molecular processes controlling coagulation may lead to novel strategies for the development of more effective therapeutic anticoagulants.
Back to top] Structure, Function, and
Activation of Coagulation Factor VII
Factor VII is the coagulation protease responsible for starting a cascade of proteolytic events that lead to thrombin generation, fibrin deposition, and platelet activation. As such, FVII has attracted wide interest as a target for clinical anti-coagulant applications. Commensurate with the critical importance of maintaining balance between thrombosis and hemostasis and its place at the beginning of the coagulation process, FVII is subject to a variety of biological and biochemical control mechanisms, among them allosteric influences exerted by cofactors, substrates, and inhibitors. Sites on FVIIa where allosteric influences are exerted and manifested have been identified and characterized in considerable detail. In recent years, a three-dimensional context for the interpretation of these results has become available from structural studies. New X-ray structures have augmented specific aspects of our understanding, in particular the X-ray structure of a fragment of the FVII zymogen. This review summarizes general allosteric behaviors of FVIIa and recapitulates structural findings since 1996, with particular emphasis on the recently determined zymogen structure.
Back to top] Structure and Function of the Von
Willebrand Factor A1 Domain
The role of von Willebrand factor (VWF) in blocking hemorrhage is centered on its ability to act as a bridging adhesive molecule between platelets and components of the extracellular matrix or other platelets. In the course of chronic vascular diseases, moreover, the same properties of VWF may become the cause of pathological thrombus formation leading to arterial occlusion. There is convincing evidence that VWF functions involving interactions with platelets ultimately depend on binding to the membrane glycoprotein (GP) Iba receptor mediated by the A1 domain. In this review, we present the current knowledge on the structural features of the VWF A1 domain that support its functions.
Back to top] New Insights into Binding
Interfaces of Coagulation Factors V and VIII and their Homologues – Lessons
from High Resolution Crystal Structures
The large, multifunctional proteins Factors V and VIII are cofactors in the coagulation cascade and possess a similar domain structure, A1–A2–B–A3–C1–C2. The C domains are related to the discoidin protein family, while the A domains are homologous to the copper-binding protein ceruloplasmin. After proteolytic activation, Factors V and VIII behave as peripheral membrane proteins, binding to negatively charged membranes containing phosphatidylserine, primarily via specific sites on their C2 domains. This type of membrane surface is exposed at sites of tissue damage, where platelets have become activated. The cofactors then accelerate sequential proteolytic activations that occur at critical control points in the blood coagulation cascade via complex formation with specific serine proteinases. Here we compare recent structural and functional studies of the C2 domains of Factors V and VIII, and discuss their respective roles. The membrane-binding motifs consist of several exposed hydrophobic side chains surrounded by a ring of basic residues, and the C2 domains appear poised to insert their hydrophobic “feet” into the membrane interior as basic residues interact favorably with phosphatidylserine head groups. In line with their physiological roles, the membrane-binding surfaces of the C2 domains display a good deal of mobility. We then extend our analysis to other members of the discoidin protein family, which perform diverse physiological functions involving signaling pathways at cell surfaces. Finally, structural similarities between discoidin proteins and the topologically distinct but functionally related membrane-binding “classic C2 domains”, including signal-transduction proteins such as Protein Kinase C and phospholipases, are noted.
Back to top] Structural Bioinformatics:
Methods, Concepts and Applications to Blood Coagulation Proteins
Structural and theoretical analyses of proteins are central to the understanding of complex molecular mechanisms and are fundamental to the drug discovery process. Computational techniques yield useful insights into an ever-wider range of biomolecular systems. Protein three-dimensional structures and molecular functions can be predicted in some circumstances, while experimental structures can be analyzed in depth via such computational approaches. Non-covalent binding of biomolecules can be understood by considering structural, thermodynamic and kinetic issues, and theoretical simulations of such events can be attempted. The central role of electrostatic interactions with regard to protein function, structure and stability has been investigated and some electrostatic properties can be modeled theoretically. Computer methods thus help to prioritize, design, analyze and rationalize biochemical experiments. Cardiovascular diseases and associated blood coagulation disorders are leading causes of death worldwide. Blood coagulation involves more than 30 proteins that interact specifically with various degrees of affinity. Many of these molecules can also bind transiently to phospholipid surfaces. Numerous point mutations in the genes of coagulation proteins and regulators have been identified. Understanding the coagulation cascade, its regulation and the impact of mutations is required for the development of new therapies and diagnostic tools. In this review, we describe concepts and methods pertaining to the field of structural bioinformatics. We provide examples of applications of these approaches to blood coagulation proteins and show that such studies can give insights about molecular mechanisms contributing to cardiovascular disease susceptibility.