Wadie F. Bahou

Thrombin Induces the Activation of Progelatinase A in Vascular Endothelial Cells PHYSIOLOGIC REGULATION OF ANGIOGENESIS

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombins multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of α-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metalloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Permissive Role of Nitric Oxide in Endothelin-induced Migration of Endothelial Cells

Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.

Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain.

Membrane type matrix metalloproteinase 1 (MT-MMP1), a novel 63-kDa member of the matrix metalloproteinase family, is a membrane-anchored enzyme and an activator for gelatinase A. In addition to its C-terminal hydrophobic transmembrane domain, MT-MMP1 has an insertion of 11 amino acids between its propeptide and catalytic domain encrypted with a RRKR recognition motif for the paired basic amino acid cleaving enzyme, furin. In this report, we investigated whether the cleavage of the RRKR motif of MT-MMP1 by Golgi-associated furin is analogous to a similar enzyme activation mechanism observed with stromelysin-3. Mutant forms of MT-MMP1 were cotransfected into COS-1 cells with cDNAs for pro-gelatinase A and/or furin. Immunoprecipitation and immunoblotting using specific antibodies were employed to characterize cell proteins. Whereas furin readily cleaved soluble MT-MMP1 lacking the transmembrane domain (ΔMT-MMP1), a soluble stromelysin-1/ΔMT-MMP1 chimera without the RRKR basic motif was resistant to furin-induced cleavage. COS-1 cells cotransfected with wild type MT-MMP1 cDNA and furin cDNA demonstrated a 63-kDa protein (latent enzyme) on SDS-polyacrylamide gel electrophoresis rather than the anticipated lower molecular weight activated enzyme. Inhibition of furin activity with α1-protease inhibitorPittsburgh (a furin inhibitor) did not affect the pro-gelatinase A activation mechanism in COS-1 cells cotransfected with MT-MMP1 and pro-gelatinase A cDNAs. Furthermore, substitution of the RRKR motif of MT-MMP1 with alanine residues by site-directed mutagenesis resulted in the same 63-kDa protein without loss of pro-gelatinase A activation function. These data indicate that furin-induced activation of MT-MMP1 is not a prerequisite for pro-gelatinase A activation. The mechanism of activation of cell-bound MT-MMP1 remains to be elucidated.

Characterization of the Initial α-Thrombin Interaction with Glycoprotein Ibα in Relation to Platelet Activation

We have evaluated the properties of α-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37u2009°C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the α-thrombin interaction site on glycoprotein (GP) Ibα. By 2–5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ibα antibody. Results were similar at room temperature (22–25u2009°C), whereas the initial characteristics of α-thrombin interaction with platelets were preserved for at least 20 min at 4u2009°C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ibα, completely inhibited by LJ-Ib10, had “moderate” affinity (k d on the order of 10−8 m) and relatively high capacity, rather than “high” affinity (k d on the order of 10−10 m) and low capacity as currently thought. The parameters of α-thrombin binding to intact GP Ibα on platelets at 4u2009°C corresponded closely to those measured with isolated GP Ibα fragments regardless of temperature. Blocking the α-thrombin-GP Ibα interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ibα contributes to the thrombogenic potential of α-thrombin.

Attacked from within, blood thins.

Thrombin leads to blood clotting through activation of specialized G protein–coupled receptors. In mice, small peptides call pepducins inhibit thrombin receptors and prevent blood clotting (pages 1161–1165).

CHAPTER 9 – Thrombin Receptors

Thrombin is a multifunctional serine protease that is distinctly unique among coagulation proteins, possessing both procoagulant and anticoagulant properties.1 These properties bestow upon thrombin a critical role in the regulation of normal hemostasis and exaggerated thrombosis. The precursor protein prothrombin is synthesized exclusively in the liver as an inactive zymogen with a tightly regulated plasmatic concentration of 100 μg/mL and a circulating half-life of about 72 hours. 2 The conversion of prothrombin to its 38-kD active protease (α-thrombin) requires the assembly of a prothrombinase complex comprised of prothrombin, coagulation factor Xa, and the active cofactor Va on the surface of a cellular phospholipid membrane requiring calcium ions (see Chapter 19). 3,4 α-thrombin contains a typical active site catalytic triad composed of histidine 365, aspartic acid 419, and serine 527 that are in close proximity and responsible for the charge relay system evident in all serine proteases. 5 An anion binding exosite confers binding specificity to various thrombin substrates. 6 Although thrombin substrate specificity is similar to that of trypsin for small peptides, thrombins action on larger proteins is more highly selective.7-9 Virtually all thrombin substrates contain an arginine or lysine adjacent to the sessile bond, a representative cleavage site containing the consensus X-Pro-Arg-u0891X. 7 Known thrombin substrates include coagulation factor VIII, factor V, and factor XIII10-13; critical cleavages of fibrinogen at Arg 16 -Gly 17 within the Aα-chain and Arg 14 -Gly 15 within the Bβ-chain regulate fibrin polymerization and stabilization of the fibrin clot. 7