Tina M. Misenheimer

Endothelial cell mitogenesis induced by LPA: Inhibition by thrombospondin-1 and thrombospondin-2

We examined the effects of thrombospondin-1 (TSP1) and thrombospondin-2 (TSP2) on the uptake of tritiated thymidine by bovine aortic endothelial (BAE) cells in response to two growth factors, basic fibroblast growth factor (bFGF) and lysophosphatidic acid (LPA). bFGF and LPA stimulate cell proliferation through distinct receptors that have convergent signaling pathways. The doses of LPA that trigger proliferation of BAE cells, which have not been reported previously, were 1 to 30 micromol/L, as opposed to the 5 to 100 micromol/L concentrations required to stimulate proliferation of human foreskin fibroblasts. Baseline mitogenic activity and activity stimulated by either bFGF or LPA on BAE cells was inhibited by human TSP1 purified from platelets or a recombinant source with a similar dose response. These results demonstrate that the anti-proliferative effect of platelet TSP1 is not caused by contaminants from the stimulated platelet. Recombinant mouse TSP2 inhibited BAE cell proliferation in response to LPA in a dose range similar to that of TSP1. Inasmuch as TSP2 does not activate latent TGFbeta1 (Schultz-Cherry et al., J Biol Chem 1995;270: 7304), these results show that inhibition of angiogenesis by TSPs is not related to control of activation of TGFbeta. Together, these studies suggest that structural motifs common to TSP1 and TSP2 inhibit endothelial cell proliferation. Furthermore, TSPs inhibit cell proliferation stimulated by two growth factor receptors that act through distinct signaling pathways.

Structure of the calcium-rich signature domain of human thrombospondin-2

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-Å-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor–like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

Expression of recombinant matrix components using baculoviruses

Publisher Summary This chapter discusses the expression of recombinant matrix components using baculoviruses. The chapter develops an expression system that is complementary to the Timpl system, based on the pAcGP67A.coco (pCOCO) baculoviral transfer plasmid, pCOCO allows baculovirally mediated expression of a fusion protein with an N-terminal signal sequence, the matrix component of interest, a thrombin cleavage site, and a C-terminal polyhistidine tag. The fusion protein is produced in large amount late in the viral infectious cycle, is secreted efficiently, and can be purified by a general strategy, i.e., binding to a Ni 2+ -chelate resin. The polyhistidine tag then can be removed with thrombin. More than 30 different matrix molecules or modular segments of matrix molecules have been expressed utilizing pCOCO. Yields have ranged from 1 to 80 mg per liter of medium from infected cells. In all cases studied, the recombinant proteins have been shown to undergo the same post-translational modifications and to adopt the same structures as molecules purified from natural sources.

Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography.NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C isN-glycosylated, and all 10 cysteine residues ofC are in disulfides. By equilibrium ultracentrifugation,C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

Warfarin resistance in a chicago strain of rats

A warfarin-resistant strain of rats trapped in Chicago was studied to determine the mechanism of the warfarin resistance. The Chicago-resistant rats (CR) differ from a Welsh-resistant strain (WR) which has a vitamin K epoxide reductase that is insensitive to warfarin. The epoxide and dithiol-dependent quinone reductases of the CR rats were as sensitive to warfarin as the normal enzyme. Unlike the irreversible warfarin inhibition seen in normal rats, the warfarin inhibition of the epoxide reductase from the CR strain was partially reversible in vitro. In this respect, the CR rats appeared similar to a Scottish warfarin-resistant strain. The same steady-state level of warfarin (40 ng/mg protein) in liver microsomes could be achieved in normal and CR strain rats following a few days ingestion of a diet containing 50 ppm warfarin, but clearance of warfarin (1 mg/kg) from the liver microsomes was more rapid in the CR strain than in normal rats, and the recovery of epoxide reductase activity and prothrombin levels was more rapid. The mechanism of warfarin resistance in the CR strain differed from the warfarin resistance mechanisms of both the Scottish- and Welsh-resistant rat strains. The combination of an increased rate of warfarin clearance and the partially reversible inhibition of the epoxide reductase would be sufficient to allow the rats to survive a limited exposure to warfarin.

Biophysical characterization of the signature domains of thrombospondin-4 and thrombospondin-2.

The signature domain of thrombospondins consists of tandem epidermal growth factor-like modules, 13 calcium-binding repeats, and a lectin-like module. Although very similar, the signature domains of thrombospondin-1 and -2 differ in several potentially important ways from the domains of thrombospondin-3, -4, and -5. We have compared matching recombinant segments representing the signature domains of thrombospondin-2 and -4. In the presence of 2 mm CaCl2, the far UV circular dichroism spectra of thrombospondin-2 and -4 constructs contain a strong negative band at 202 nm, but only the thrombospondin-2 construct has a band at 216 nm. Chelation of calcium shifted the negative bands to lower magnitudes. Titrations of the spectra demonstrated lower cooperativity and affinity for binding of calcium to thrombospondin-4 compared with thrombospondin-2. Atomic absorption spectroscopy demonstrated that the thrombospondin-4 constructs bind seven less calcium than the thrombospondin-2 construct at 0.6 mm CaCl2. In 2 mm CaCl2, the near UV circular dichroism spectra of thrombospondin-2, but not thrombospondin-4, contain a positive band at 292 nm that disappears upon calcium chelation. Intrinsic fluorescence spectra for both proteins were also sensitive to calcium, but the changes were simpler and more marked for thrombospondin-2 than for thrombospondin-4. In differential scanning calorimetry, the thrombospondin-2 construct melted in two distinct transitions at 53.5 and 81.8 °C, whereas the first transition for thrombospondin-4 constructs was observed at 63.5 °C. Thus, the studies revealed significant differences between the signature domains of thrombospondin-2 and thrombospondin-4 in calcium binding, fine structure, and inter-modular interactions.

Modulation of Fibrinolysis by Thrombospondina

Thrombospondin is a large, trimeric glycoprotein secreted by activated platelets and growing cells. Thrombospondin copolymerizes with fibrin during blood coagulation and deposits in extracellular matrix. We found that thrombospondin is a slow (rate constant approximately 6.3 x 10(3) M-1 sec-1), tight-binding (Kd < 10(-9) M) inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to degrade fibrinogen, and decreased lysis zones in fibrin plate assays (Biochemistry 31: 265-269, 1992). Thrombospondin also slowly inhibits urokinase plasminogen activator. The lysis zone when urokinase is put on fibrin plates made from whole plasma is less if thrombospondin is present. The stoichiometry of inhibition is approximately one mole plasmin:one mole thrombospondin trimer, a somewhat surprising result considering the trimeric nature of thrombospondin. These results indicate that thrombospondin is an important regulator of fibrinolysis and degradation of extracellular matrix, particularly when these processes are initiated by urokinase and even when other inhibitors of fibrinolysis are present.

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism

Summary.u2002 Background:u2002 Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined.

Biochemical basis of warfarin and bromadiolone resistance in the house mouse, Mus musculus domesticus.

Danish mice (Mus musculus domesticus) genetically resistant to the anticoagulant action of two 4-hydroxycoumarins, warfarin and bromadiolone, were examined to determine their mechanism of resistance. The hepatic vitamin K epoxide reductase in the bromadiolone-resistant mice and in one phenotype of warfarin-resistant mice was highly insensitive to in vitro inhibition by warfarin and bromadiolone. The kinetic constants for the epoxide reductase from bromadiolone-resistant mice were also altered. The Vmax for this enzyme was decreased by 40%, and the Km for the reaction reductant, dithiothreitol, was 70% lower than that of normal mice. This phenotype of Danish resistant mice appears to have a resistance mechanism that is similar to that reported for a Welsh strain of warfarin-resistant rats. The other phenotype of Danish resistant mice had a hepatic epoxide reductase that was only slightly less sensitive to warfarin inhibition than normal. The mechanism of warfarin resistance in these mice is not apparent from the available data.

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.