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Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXβ (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP + -xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts., (© 2018 Nesbitt et al.)

Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to approximately 2.5 times the normal shear stress, or approximately 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation (cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca(2+)](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structure-function relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.

It has been assumed that cleavage of the N-terminal propeptide domain of membrane type-1 matrix metalloproteinase (MT1-MMP) is required for enzyme function. We recently demonstrated that the propeptide domain of MT1-MMP is not cleaved and actually is required for function of the membrane-bound enzyme in transfected COS-1 cells (Cao, J., Drews, M., Lee, H. M., Conner, C., Bahou, W. F., and Zucker, S. (1998) J. Biol. Chem. 273, 34745-34752). In this report, we have inserted the cDNA encoding the signal and propeptide sequences of MT1-MMP (MT(1-109)) and the cDNA encoding propeptide-deleted mature MT1-MMP (MT delta pro) in expression vectors that were then transfected into matrix metalloproteinase-deficient COS-1 cells. Co-expression of both the mature sequence and the prosequence of MT1-MMP as independent polypeptides (in trans) in COS-1 cells resulted in reconstitution of MT1-MMP function in terms of facilitating (125)I-labeled tissue inhibitor of metalloproteinase 2 binding to transfected cells and subsequent activation of progelatinase A. Transfection of cells with either cDNA alone resulted in non-functional cells. These results are consistent with the propeptide sequence of MT1-MMP functioning as an intramolecular chaperone involved in protein folding and trafficking to the cell surface.

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.

Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.

We have evaluated the properties of alpha-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 37 degrees C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha-thrombin interaction site on glycoprotein (GP) Ib alpha. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib alpha antibody. Results were similar at room temperature (22-25 degrees C), whereas the initial characteristics of alpha-thrombin interaction with platelets were preserved for at least 20 min at 4 degrees C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib alpha, completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10(-8) M) and relatively high capacity, rather than "high" affinity (kd on the order of 10(-10) M) and low capacity as currently thought. The parameters of alpha-thrombin binding to intact GP Ib alpha on platelets at 4 degrees C corresponded closely to those measured with isolated GP Ib alpha fragments regardless of temperature. Blocking the alpha-thrombin-GP Ib alpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib alpha contributes to the thrombogenic potential of alpha-thrombin.

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

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.

The seven-transmembrane segment thrombin receptor (TR) represents the prototype of a putative family of proteolytically cleaved receptors that may include the proteinase activated receptor-2. A panel of somatic cell hybrids retaining distinct portions of human chromosome 5 were used to establish that the human TR gene is present as a single-copy locus within the region 5q11.2 -->q13.3, confirming our previous localization using fluorescent in situ hybridization analysis. To further characterize the TR gene, overlapping clones from a human genomic library were isolated. Genomic analysis confirmed that the TR gene is of limited complexity, spanning approximately 27 kilobases and containing two exons separated by a large approximately 22-kilobase intron. The larger second exon contains the majority of the coding sequence and the thrombin cleavage site, remarkably similar to the organization of the proteinase activated receptor-2 gene in which the putative cleavage site is also contained within the large second exon. Primer extension analysis using two 30-mer oligonucleotide primers known to be contained within the first exon identified the predominant transcription initiation site 351 base pairs upstream from the initiator methionine in both human umbilical vein endothelial and human erythroleukemia cells. Sequence analysis of the 5'-flanking region revealed the TR promoter to be TATA-less, although nucleic acid motifspotentially involved in transcriptional gene regulation were evident and include a GATA motif, octamer enhancer sequences, AP-2-like sites, and Sp1 sites. These data provide evidence for remarkable similarity at the gene level between both proteolytically cleaved receptors described to date.

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's 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 alpha-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 metaloproteinase 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.

There is accumulating evidence that endothelial cells express a non-isopeptide-selective endothelin (ET) receptor, ETB, which may be responsible for ET-1-induced transient vasorelaxation. The purpose of the present study was to seek direct evidence for ETB receptor expression in human umbilical vein endothelial cells (HUVEC) and to characterize its functional role in HUVEC and in Chinese hamster ovary cells stably transfected with ETB receptor cDNA (CHO-ETB). Reverse polymerase chain reaction using HUVEC total RNA and ETB receptor-specific oligonucleotide primers firmly demonstrated the presence of an endogenous transcript of the appropriate molecular size. Next, a biotinylated ligand specifically recognizing the ETB receptor, IRL-1620, was synthesized, and immunocytochemical mapping of binding sites was performed in CHO-ETB cells. Specific binding of biotinylated IRL-1620 was evident in CHO-ETB cells, confirming appropriate cell surface receptor expression. Continuous nitric oxide (NO) monitoring with NO-selective electrode revealed a dose-dependent ET-1 stimulation of NO production by HUVEC. Stable transfection of CHO-ETB cells with endothelial nitric oxide synthase (NOS), but not mock-transfection, imparted responsiveness to ET-1 similar to that for HUVEC and was characterized by the immediate release of NO. Protein tyrosine kinase-dependent and calcium-calmodulin-dependent pathways were involved in ET-1-induced activation of the constitutive NOS in CHO-ETB/NOS cells, but coupling of the receptor to the enzyme in HUVEC appeared to be predominantly protein tyrosine kinase-dependent. Although sufficient, calcium/calmodulin system was not an obligatory prerequisite for the ET-1-induced activation of NOS in HUVEC. In conclusion, using two cell systems, we demonstrated that the ETB receptor is functionally coupled to NOS and coordinates the generation of NO via a tyrosine kinase-dependent and a calcium/calmodulin-dependent pathway.

Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma. Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000). Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library. Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs. No significant sequence similarity was observed on analysis of current GenBank databases. The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody. Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein. This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin. Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function. Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney. On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons. Its tissue-specific expression may suggest a functional role in hematopoiesis.