Gabriel E. Weinreb

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

Factor Xa (FXa) binding to factor Va (FVa) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the “prothrombinase complex” that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FVa, FVa1 and FVa2. These two isoforms differ by a 3-kDa polysaccharide chain (at Asn2181 in human FVa1 (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999)Biochemistry 38, 11448–11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FXa, finding no significant difference. A soluble form of PS (C6PS) bound to FVa1 and FVa2 with comparable affinities (K d = 11–12 μm) and changes in FVa intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FVa2 enhanced its affinity for FXa in solution by nearly 3 orders of magnitude (K d eff = 40–2 nm over a C6PS range of 30–400 μm) but had no effect on the affinity of FVa1for FXa (K d = 1 μm). This results in a soluble complex between FXa and FVa2, whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparentk cat/K m ≅ 109 m −1 s−1). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.

Mechanical and Biochemical Modeling of Cortical Oscillations in Spreading Cells

Actomyosin-based cortical contractility is a common feature of eukaryotic cells and is involved in cell motility, cell division, and apoptosis. In nonmuscle cells, oscillations in contractility are induced by microtubule depolymerization during cell spreading. We developed an ordinary differential equation model to describe this behavior. The computational model includes 36 parameters. The values for all but two of the model parameters were taken from experimental measurements found in the literature. Using these values, we demonstrate that the model generates oscillatory behavior consistent with current experimental observations. The rhythmic behavior occurs because of the antagonistic effects of calcium-induced contractility and stretch-activated calcium channels. The model makes several experimentally testable predictions: 1), buffering intracellular calcium increases the period and decreases the amplitude of cortical oscillations; 2), increasing the number or activity of stretch activated channels leads to an increase in period and amplitude of cortical oscillations; 3), inhibiting Ca(2+) pump activity increases the period and amplitude of oscillations; and 4), a threshold exists for the calcium concentration below which oscillations cease.

Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXas active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.

RhoA Regulates Calcium-Independent Periodic Contractions of the Cell Cortex

When microtubules are depolymerized in spreading cells, they experience morphological oscillations characterized by a period of about a minute, indicating that normal interactions between the microfilament and microtubule systems have been significantly altered. This experimental system provides a test bed for the development of both fine- and coarse-grained models of complex motile processes, but such models need to be adequately informed by experiment. Using criteria based on Fourier transform analysis, we detect spontaneous oscillations in spreading cells. However, their amplitude and tendency to operate at a single frequency are greatly enhanced by microtubule depolymerization. Knockdown of RhoA and addition of various inhibitors of the downstream effector of RhoA, Rho kinase, block oscillatory behavior. Inhibiting calcium fluxes from endoplasmic reticulum stores and from the extracellular medium does not significantly affect the ability of cells to oscillate, indicating that calcium plays a subordinate regulatory role compared to Rho. We characterized the dynamic structure of the oscillating cell by light, fluorescence, and electron microscopy, showing how oscillating cells are dynamically polarized in terms of their overall morphology, f-actin and phosphorylated myosin light chain distribution, and nuclear position and shape. Not only will these studies guide future experiments, they will also provide a framework for the development of refined mathematical models of the oscillatory process.

In Silico Generation of Alternative Hypotheses Using Causal Mapping (CMAP)

Previously, we introduced causal mapping (CMAP) as an easy to use systems biology tool for studying the behavior of biological processes that occur at the cellular and molecular level. CMAP is a coarse-grained graphical modeling approach in which the system of interest is modeled as an interaction map between functional elements of the system, in a manner similar to portrayals of signaling pathways commonly used by molecular cell biologists. CMAP describes details of the interactions while maintaining the simplicity of other qualitative methods (e.g., Boolean networks).In this paper, we use the CMAP methodology as a tool for generating hypotheses about the mechanisms that regulate molecular and cellular systems. Furthermore, our approach allows competing hypotheses to be ranked according to a fitness index and suggests experimental tests to distinguish competing high fitness hypotheses. To motivate the CMAP as a hypotheses generating tool and demonstrate the methodology, we first apply this protocol to a simple test-case of a three-element signaling module. Our methods are next applied to the more complex phenomenon of cortical oscillations observed in spreading cells. This analysis produces two high fitness hypotheses for the mechanism that underlies this dynamic behavior and suggests experiments to distinguish the hypotheses. The method can be widely applied to other cellular systems to generate and compare alternative hypotheses based on experimentally observed data and using computer simulations.

Fuzzy Causal Mapping (F-Cmap) — A Proposal To Develop A New Systems Biology Tool

Biological systems are complex, consisting of many elements of different nature. As a whole, they are robust, and a general system description can be done in a semi-quantitative way when it comes to phenotype behaviors. We used these properties earlier1 to develop a new systems biology method, causal mapping (CMAP). In this paper, we pinpoint some problems with the earlier version of CMAP, and develop it further. CMAP used linguistic variables (LV) to describe the behaviour of biological systems, and here we use the procedure of fuzzyfications to improve CMAP. The numerical methods to calculate the ranges of LV are agreeable to reality in a very intuitive manner. The new version of CMAP reproduced the physical data on cortical oscillations2 in spreading cells with depolymerized microtubules. Further, predictions were made on the dependency of the myosin activity on the period of oscillations.The presented development lies on the way to a more general approach that should be able to address questions of biological robustness, modularity and hierarchy.

Visinets: A Web-Based Pathway Modeling and Dynamic Visualization Tool

In this report we describe a novel graphically oriented method for pathway modeling and a software package that allows for both modeling and visualization of biological networks in a user-friendly format. The Visinets mathematical approach is based on causal mapping (CMAP) that has been fully integrated with graphical interface. Such integration allows for fully graphical and interactive process of modeling, from building the network to simulation of the finished model. To test the performance of Visinets software we have applied it to: a) create executable EGFR-MAPK pathway model using an intuitive graphical way of modeling based on biological data, and b) translate existing ordinary differential equation (ODE) based insulin signaling model into CMAP formalism and compare the results. Our testing fully confirmed the potential of the CMAP method for broad application for pathway modeling and visualization and, additionally, showed significant advantage in computational efficiency. Furthermore, we showed that Visinets web-based graphical platform, along with standardized method of pathway analysis, may offer a novel and attractive alternative for dynamic simulation in real time for broader use in biomedical research. Since Visinets uses graphical elements with mathematical formulas hidden from the users, we believe that this tool may be particularly suited for those who are new to pathway modeling and without the in-depth modeling skills often required when using other software packages.