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E F Plow

Publications and source records attributed to E F Plow.

At least 55 records · Page 3Linked to original sources
PubMed1997

Development of a structural model for the cytoplasmic domain of an integrin.

The cytoplasmic tails of integrin heterodimers play central roles in controlling the activation states of integrins and in transmitting intracellular signals. Despite their short length, no structure of any integrin cytoplasmic domain has been determined. Therefore, molecular models for the cytoplasmic domain of alpha(IIb)beta3, the major platelet integrin, were generated, including models for the individual cytoplasmic tails, the binary alphaIIb-calcium complex, and the ternary alphaIIb-beta3-calcium complex. Structural analysis of circular dichroism spectra were compiled with data obtained from short homologous sequences within crystallized proteins, and with secondary structural predictions to develop starting models for each subunit. These models were subjected to a series of energy minimization and molecular dynamic simulations to generate final models. AlphaIIb was predicted to be ordered at its N-terminus and its C-terminus could accommodate a cation in a multicoordinated complex. The structure of beta3 was dominated by a beta-turn at its NPXY motif (beta3 744-747). In docking of alphaIIb to different sites within beta3, the conformation of the beta3 juxta-transmembrane (beta3 716-721) was greatly altered. This region was confirmed to be a conformational 'hot-spot' by circular dichroism. The conformational flexibility of this juxta-transmembrane region, which is highly conserved amongst integrins, is ideally located to regulate signaling.

Amino Acid Sequence↗
PubMed1997

Ligand recognition by beta 3 integrins.

Integrins are alpha/beta heterodimeric adhesion receptors, alpha IIb beta 3 (GPIIb-IIIa) and alpha v beta 3 (the vitronectin receptor) share the same beta subunit, beta 3, but have distinct alpha subunits. These sister integrins not only recognize many of the same ligands but also have certain unique ligands. Based upon current information in the literature, we propose that four classes of beta 3 ligands can be distinguished. Since the beta 3 integrins have multiple functions in vivo and are targets for therapy, this classification system may be useful in the design and characterization of therapeutic agents.

Antigens, CD↗
PubMed1996

A discrete site modulates activation of I domains. Application to integrin alphaMbeta2.

A central characteristic of integrin adhesion receptors is their capacity to become activated, thereby enhancing their affinity for ligands. Here, we report the identification of a discrete site within the I domain of integrin alphaMbeta2, which modulates the adhesive activity of this receptor. Based upon the crystal structure, this region is composed of two short and spatially proximal loops, E162QLKKSKTL and Q190NNPNPRS. Mutations in these loops yield receptors which support spontaneous cell adhesion to fibrinogen, whereas mutation of an adjacent region and wild-type receptors require activation to adhere to this substrate. An activating monoclonal antibody enhanced the adhesive activity of one but not the other loop mutants, suggesting that the activation states of these two mutant receptors were not identical. Given that similar I domains exist in several other integrin alpha subunits and non-integrin proteins, and possibly in all integrin beta subunits, these two loop segments may represent a universal target for controlling integrin activation and the function of other I domain-containing proteins. In support of this hypothesis, several naturally occurring mutations that activate von Willebrand factor map to the same loops of its I(A) domain.

Amino Acid Sequence↗
PubMed1996

Regulation of plasminogen receptor expression on monocytoid cells by beta1-integrin-dependent cellular adherence to extracellular matrix proteins.

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

Carboxypeptidase B↗
PubMed1996

Proteolysis regulates exposure of the IIICS-1 adhesive sequence in plasma fibronectin.

The alternatively spliced type III connecting segment (IIICS) of fibronectin (Fn) contains an amino acid sequence, CS-1, which is recognized by the integrin receptor, alpha 4 beta 1. Plasma Fn inhibits alpha 4 beta 1-dependent binding of lymphocytes and monocytes to CS-1 containing Fn derivatives poorly, suggesting limited exposure of the CS-1 sequence in Fn. To test the availability of CS-1 in plasma Fn, an antibody was raised to the synthetic peptide CS-1. The CS-1 sequence was found to be minimally exposed in plasma Fn; and immobilization of Fn, a model of matrix deposition, caused only a modest increase in its exposure. Digestion of Fn with selected proteases, however, induced substantial expression of the CS-1 sequence. The acid protease cathepsin D generated fragments of 31-33.5 kDa from the COOH-terminal heparin-binding domain of Fn which possessed high immunoreactivity with anti-CS-1. Digestion of Fn with cathepsin B also resulted in the exposure of CS-1 sequence in a 140 kDa fragment. Although the digestion of Fn with neutral proteases (neutrophil elastase, cathepsin G, chymotrypsin, trypsin) generated fragments from the COOH-terminal heparin-binding domain of similar molecular weight as with cathepsin D, the exposure of CS-1 did not occur. Exposure of the CS-1 region by the cathepsins was supported by cell adhesion experiments; digestion of Fn with cathepsins D and B transformed inert plasma Fn to an effective inhibitor of adhesion of lymphoblastoid B and T cells (Ramos, Jurkat, Molt-4) to an immobilized CS-1 conjugate. These results suggest that exposure of the CS-1 sequence in plasma Fn by proteolysis with cathepsins D and B, enzymes implicated in several pathological processes, may serve a regulatory function in cell adhesion. The adhesive function of the CS-1 region in intact Fn appears to be suppressed by the native conformation of the molecule.

Alternative Splicing↗
PubMed1996

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen.

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue-type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

Animals↗
PubMed1996

Overlapping, but not identical, sites are involved in the recognition of C3bi, neutrophil inhibitory factor, and adhesive ligands by the alphaMbeta2 integrin.

The alphaMbeta2 (CD11b/CD18, Mac-1) integrin receptor binds numerous ligands, including neutrophil inhibitory factor (NIF), C3bi, and certain immobilized protein substrates, represented by denatured ovalbumin. These ligands share no obvious structural similarities, yet their interactions with receptor are inhibited by NIF and involve the I domain, a stretch of approximately 200 amino acids in the alphaM subunit. Recombinant wild-type and mutant forms of alphaMbeta2 have been used to compare the recognition requirements of these ligands. The various constructs were expressed efficiently on the surface of human embryonic kidney 293 cells and formed alpha.beta heterodimeric complexes. The wild-type transfectants bound the three ligands in a similar fashion to naturally occurring alphaMbeta2. NIF inhibited these interactions, and deletion of the D248PLGY from within the I domain abolished binding of all three ligands, suggesting an overlapping recognition specificity. A single point mutation of Ser138 to Ala in the beta2 subunit abolished C3bi binding and cell adhesion but did not affect NIF binding. A switch of the R281QELNTI sequence in helix 6 of the alphaM I domain to the corresponding sequence in the I domain of the alphaL (QETLHKF) subunit completely abrogated adhesion while not affecting C3bi and NIF binding. The two mutant receptors also did not support activation-dependent adhesion to fibrinogen. Thus, the contact sites for NIF, C3bi, and adhesive proteins, represented by denatured ovalbumin and fibrinogen, in alphaMbeta2 are overlapping but not identical.

Base Sequence↗
PubMed1996

The ligand recognition specificity of beta3 integrins.

AlphaIIbbeta3 (platelet membrane glycoprotein IIb-IIIa) and alphavbeta3 are members of the beta3 subfamily of integrin adhesion receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD), originally described by Scarborough et al. (Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol. Chem. 268, 1066-1073) has been employed as a high affinity ligand for alphaIIbbeta3 to examine the specificity of the beta3 integrins. cHarGD interacted with high affinity with purified alphaIIbbeta3 (Kd = 10 nM) or with platelets (Kd = 120 nM). While cHarGD was specific for alphaIIbbeta3 in the presence of Ca2+, it bound to both beta3 integrins in the presence of Mn2+. Barbourin, a snake venom disintegrin containing a reactive KGD sequence, remained alphaIIbbeta3-specific, even in the presence of Mn2+. cHarGD became cross-linked to a site in beta3 of alphaIIb beta3, which is distinct from that of RGD peptides. These results allow identification of at least four classes of beta3 ligands: Class I, represented by RGD peptides and vitronectin, react similarly with alphaIIbbeta3 and alphavbeta3; Class II, represented by cHarGD, gamma-chain peptides and fibrinogen, react with both receptors in the presence of Mn2+ but only with alphaIIbbeta3 in the presence of Ca2+; Class III, represented by barbourin, are alphaIIbbeta3-specific under all cation conditions; Class IV, represented by osteopontin, bind primarily to alphavbeta3.

Amino Acid Sequence↗
PubMed1996

Inducible carboxypeptidase activity. A role in clot lysis in vivo.

BACKGROUND: An inducible carboxypeptidase activity in human plasma delays tissue-type plasminogen activator (TPA)-induced clot lysis in vitro. We investigated whether carboxypeptidase activity is induced in vivo during thrombosis and thrombolytic therapy in a canine model of myocardial infarction. METHODS AND RESULTS: By use of synthetic substrate assays, dog plasma was shown to contain an inducible carboxypeptidase activity that is efficiently inhibited by potato carboxypeptidase inhibitor. This inhibitor accelerates TPA-mediated clot lysis in vitro by an average of 27% (n = 5, P = .046). Analysis of the inducible carboxypeptidase activity in plasma samples of dogs with electrically induced thrombosis of the circumflex coronary artery treated with TPA revealed that (1) inducible carboxypeptidase activity is increased during thrombosis (8.7 +/- 2.0 U/L, P < .013) and thrombolytic therapy (9.9 +/- 1.8 U/L, P < .024) compared with baseline (3.2 +/- 2.0 U/L); (2) thrombosis is a prerequisite of carboxypeptidase induction during and after TPA infusion, since carboxypeptidase levels were lower in dogs without a coronary thrombus; and (3) a significant positive correlation (r = .6, P < .0069) of carboxypeptidase activity with time to restoration of blood flow was observed. CONCLUSIONS: These data indicate that carboxypeptidase activity is induced in vivo and may influence thrombolysis.

Animals↗
PubMed1996

The cytoplasmic domain of alphaIIb beta3. A ternary complex of the integrin alpha and beta subunits and a divalent cation.

Peptides corresponding to the cytoplasmic tails of the alphaIIb (alphaIIb (985-1008)) and beta3 (beta3 (713-762)) subunits of the integrin receptor alphaIIb beta3 (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. alphaIIb (985-1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy. The binding of Tb3+ to alphaIIb (985-1008) was of high affinity (Kd = 8.8 +/- 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxy] terminus (alphaIIb (999-1008), PLEEDDEEGE). The affinity of this site for divalent cations was in the micromolar range, suggesting that this site would be constitutively occupied in the intracellular environment. Incubation of alphaIIb (999-1008) with beta3 (713-762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for alphaIIb (999-1008) in beta3 was mapped to beta3 (721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (alphaIIb (985-1008).beta3 (721-740)) and a ternary (Tb3+.alphaIIIb (999-1008).beta3 (721-740)) complex were detected by mass spectroscopy, but the distribution and intensity of the mass/charge peaks were distinct. These difference may reflect the involvement of distinct cation coordination sites and the formation of salt bridges in stabilizing the ternary complex. These data demonstrate the formation of a novel entity composed of the cytoplasmic tails of alphaIIb and beta3 and a cation which may constitute a functional intracellular domain.

Amino Acid Sequence↗
PubMed1996

Molecular determinants of acute inflammatory responses to biomaterials.

The frequent inflammatory responses to implanted medical devices are puzzling in view of the inert and nontoxic nature of most biomaterials. Because implant surfaces spontaneously adsorb host proteins, this proteinaceous film is probably important in the subsequent attraction of phagocytes. In fact, earlier we found that acute inflammatory responses to experimental polyethylene terephthalate implants in mice require the precedent adsorption of one particular host protein, fibrinogen. The present investigations were aimed at defining the molecular determinants of fibrinogen-mediated acute inflammatory responses to implanted biomaterials. We find: (a) plasmin degradation of purified fibrinogen into defined domains reveals that the proinflammatory activity resides within the D fragment, which contains neither the fibrin cross-linking sites nor RGD sequences; (b) the major (and, perhaps, exclusive) proinflammatory sequence appears to be fibrinogen gamma 190-202, previously shown to interact with CD11b/CD18 (Mac-1). The chemically synthesized peptide, cross-linked to albumin (which itself does not promote inflammatory responses), mimics the proinflammatory effect of adsorbed native fibrinogen; and (c) this sequence probably promotes inflammatory responses through interactions with Mac-1 because phagocyte accumulation on experimental implants is almost completely abrogated by administration of recombinant neutrophil inhibitory factor (which blocks CD11b-fibrin(ogen) interaction). We conclude that improved knowledge of such surface-protein-phagocyte interactions may permit the future development of more biocompatible implantable materials.

Acute Disease↗
PubMed1996

Selective inhibition of factor Xa is more efficient than factor VIIa-tissue factor complex blockade at facilitating coronary thrombolysis in the canine model.

OBJECTIVES: We determined the effect of adjunctive inhibition of the extrinsic coagulation pathway by factor VIIa-tissue factor complex inhibitors, DEGR VIIa and tissue factor pathway inhibitor (TFPI), and the selective factor Xa inhibitor, tick anticoagulant peptide (TAP), after thrombolytic therapy with tissue-type plasminogen activator (t-PA) in a canine model of electrically induced coronary thrombosis. BACKGROUND: Ongoing thrombin generation is considered an important component of the heightened thrombin activity associated with thrombolytic therapy and may be responsible for reperfusion failure and reocclusion. METHODS: Forty-two dogs with electrically induced coronary thrombus undergoing thrombolysis with t-PA (1 mg/kg over 20 min) were randomly assigned to one of the following adjunctive regimens: TAP (30 micrograms/kg body weight per min for 90 min, n = 10); TFPI (100 to 150 micrograms/kg per min for 90 min, n = 10); DEGR VIIa (1- to 2-mg/kg bolus, n = 10) and saline control (n = 12). The dogs were observed for 120 min after thrombolysis for reocclusion. RESULTS: All three active study agents accelerated the time to reperfusion by an average of 12 min (all p < 0.05). Duration of reflow was greatest with TAP (117 +/- 8 min, p < 0.05 compared with saline control), whereas DEGR VIIa and TFPI did not prolong the duration of reflow. Reocclusion rates were similar among control, DEGR VIIa and TFPI groups (70%, 78% and 67%, respectively). Tick anticoagulant peptide reduced the occurrence of reocclusion (0%, p < 0.05 compared with saline control). CONCLUSIONS: In this experimental model, during systematic blockade of various extrinsic coagulation pathway proteins, we demonstrated that whereas acceleration of thrombolysis occurs with factor VIIa-tissue factor complex inhibition, optimal enhancement of thrombolysis was achieved through specific factor Xa blockade.

Animals↗
PubMed1996

Role of platelets in restenosis after percutaneous coronary revascularization.

The role of platelets in the process of restenosis after percutaneous coronary intervention is not fully understood. After vascular injury there is extensive platelet activation, adhesion, aggregation and secretion. Through the liberation of growth factors, such as platelet-derived growth factor, and surface expression of cell adhesion molecules, such as the glycoprotein IIb/IIIa integrin, platelets appear to be a pivotal mediator of the vascular injury response. Experimental models have demonstrated that profound, prolonged thrombocytopenia, or blockade of the IIb/IIIa receptor, may reduce neointimal hyperplasia after arterial balloon injury. However, multiple clinical trials testing conventional or new platelet agents have not yielded any salutary effects. The recent finding that abciximab, a monoclonal antibody fragment directed against IIb/IIIa, reduced clinical restenosis after coronary angioplasty by 26% in patients raises questions about the mechanism of benefit. The alpha v beta 3 vitronectin receptor is responsible for binding endothelial cells to platelets, and it also has a key role in modulating smooth muscle cell migration. It is possible that the antibody fragment exerts its effect on restenosis by means of alpha v beta 3, because abciximab fully cross-reacts to this integrin owing to the shared beta 3 subunit. To date, the other platelet glycoprotein IIb/IIIa inhibitors, including Integrelin, Tirofiban, Lamifiban and Xemilofiban, are specific in binding to this particular integrin. Considerable further study is necessary to unravel the effects of platelets on the restenosis process.

Angioplasty, Balloon, Coronary↗
PubMed1996

Selective inhibition of factor Xa during thrombolytic therapy markedly improves coronary artery patency in a canine model of coronary thrombosis.

The success of current thrombolytic strategies is undermined by ongoing thrombin activity, but it is uncertain whether prevention of thrombin generation or direct thrombin antagonism is effective in achieving more optimal thrombolysis. To address this question, 24 dogs with electrically induced coronary thrombus undergoing thrombolysis with tissue-type plasminogen activator (1 mg/kg) over 20 min, were given one of the following adjunctive regimens in a random fashion. Twelve dogs received saline, and served as the control group; a direct thrombin antagonist, hirudin, was given at a dose of 20 micrograms/kg/min for 90 min to six dogs, and a selective factor Xa inhibitor, tick anticoagulant peptide (TAP), was administered to six dogs at a dose of 30 micrograms/kg/min for 90 min. The time to reperfusion was similar in the saline and hirudin groups (34 +/- 4 vs 37 +/- 7 min; P = NS) but shorter in the TAP group (21 +/- 4 min; P < 0.05). Coronary blood flow was restored to 100% of its baseline value for 7 +/- 2 min in control dogs, and for 20 +/- 6 min in the hirudin group (P < 0.05). In the TAP group, coronary blood flow was restored to 100% of its baseline value for more than 120 min in all dogs (P < 0.01 vs others treatments). Reocclusion occurred in 89% and 50% of dogs receiving saline and hirudin, respectively (P = NS), but in none of the TAP-treated dogs (P < 0.01). Plasma fibrinopeptide A (FpA) and thrombin-antithrombin III complex (TAT) levels were determined in all dogs as indicators of thrombin activation. In the saline group, FpA and TAT during reperfusion were 19 +/- 2 ng/ml and 104 +/- 24 ng/ml respectively (P < 0.02 vs baseline) indicating high thrombin activity. In contrast, during reperfusion in hirudin-treated dogs FpA and TAT remained similar to baseline (10 +/- 3 ng/ml and 53 +/- 4 ng/ml respectively; both P < 0.05 vs saline). Reperfusion in TAP-treated dogs did not alter FpA and TAT in plasma, which remained similar to baseline (9 +/- 1 ng/ml and 39 +/- 5 ng/ml respectively; both P < 0.05 vs saline). Scanning electron microscopy of coronary arteries showed residual thrombi with intense platelet and fibrin deposition adherent to the deendothelialized surface of the vessels following saline and hirudin therapy. In contrast, TAP-treated arteries were characterized by the absence of fibrin and minimal platelet deposition. In conclusion, these hemodynamic, biochemical and morphologic data suggest that adjunctive treatment with a higher tier blockade of the coagulation cascade is superior to direct thrombin inhibition in maintaining coronary artery patency following thrombolysis in the experimental canine electrolytic model. These findings highlight the potential adverse effects of unchecked thrombin generation in the setting of thrombolytic therapy.

Animals↗
PubMed1996

A quantitative immunoassay for the lysine-binding function of lipoprotein(a). Application to recombinant apo(a) and lipoprotein(a) in plasma.

Apo(a), the unique apoprotein of lipoprotein(a) (Lp[a]), can express lysine-binding sites(s) (LBS). However, the LBS activity of Lp(a) is variable, and this heterogeneity may influence its pathogenetic properties. An LBS-Lp(a) immunoassay has been developed to quantitatively assess the LBS function of Lp(a). Lp(a) within a sample is captured with an immobilized monoclonal antibody specific for apo(a), and the captured Lp(a) is reacted with an antibody specific for functional LBS. The binding of this LBS-specific antibody is then quantified by using an alkaline phosphatase-conjugated disclosing antibody. The critical LBS-specific antibody was raised to kringle 4 of plasminogen. When applied to plasma samples, the LBS activity of Lp(a) ranged from 0% to 100% of an isolated reference Lp(a); the signal corresponded to the percent retention of Lp(a) on a lysine-Sepharose but did not correlate well with total Lp(a) levels in plasma. Mutation of residues in the putative LBS in the carboxy-terminal kringle 4 repeat (K4-37) in an eight-kringle apo(a) construct resulted in marked but not complete loss of activity in the LBS-Lp(a) immunoassay. These data suggest that this kringle is the major but not the sole source of LBS activity in apo(a). The LBS-Lp(a) immunoassay should prove to be a useful tool in establishing the role of the LBS in the pathogenicity of Lp(a).

Amino Acid Sequence↗
PubMed1996

Characterization of cellular binding sites and interactive regions within reactants required for enhancement of plasminogen activation by tPA on the surface of leukocytic cells.

Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and THP-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and with peripheral blood monocytes and neutrophils. The interactions of plasminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturating amounts of U937 monocytoid cells (1.25 x 10(5)/ml) the rate of plasmin generation was 0.39 nM.s-1 versus 0.07 and 0.09 nM.s-1 without cells or without tPA, respectively. The catalytic efficiency of Glu- or Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, respectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 microM. These values are below the plasminogen concentration in plasma (1-2 microM). Moreover, we provide new data indicating that 1) only a specific subset of plasminogen binding sites, i.e. molecules exposing carboxyl terminal lysines on the cell surface, promotes plasminogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfaces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.

Cell Count↗
PubMed1995

Effects of disruption of the plasminogen gene on thrombosis, growth, and health in mice.

BACKGROUND: Circumstantial evidence suggests that the plasminogen/plasmin system plays a role in many biological processes, including hemostasis, cell migration, and development. METHODS AND RESULTS: The in vivo function of the plasminogen/plasmin system was studied by generation of plasminogen-deficient (Plg-/-) mice. Inactivation of the murine plasminogen-gene (Plg) was achieved by replacing, via homologous recombination in embryonic stem cells, genomic sequences encoding the exons containing the catalytic site amino acids His605 and Asp648 with a neomycin phosphotransferase expression cassette. Germline transmission of the mutated allele, as determined by Southern blot hybridization and polymerase chain reaction, was obtained via blastocyst injection. Mendelian inheritance of the inactivated plasminogen allele was observed, and homozygous-deficient mice (Plg-/-) displayed normal viability but retarded growth up to at least 12 weeks of age. At 8 weeks of age, body weight was 21.8 +/- 1.2 g (n = 10) for wild-type (Plg+/+) mice, 21.0 +/- 1.1 g (n = 16) for heterozygous-deficient (Plg+/-) mice, and 17.4 +/- 1.3 g (n = 12) for Plg-/- mice; P < .05 versus Plg+/+ or Plg+/-. None of 36 Plg+/+ or 65 Plg+/- mice but 7 of 37 Plg-/- mice (19%) developed rectal prolapse at 7.4 +/- 0.6 weeks of age (P = .03 versus Plg+/+ and P = .003 versus Plg+/-); 4 of 37 Plg-/- mice (11%) became runted and apathic at 5.3 +/- 0.3 weeks of age (P = .041 versus Plg+/-); and 6 of 37 Plg-/- mice (16%) died prematurely at 8.8 +/- 1.7 weeks of age (P = .057 versus Plg+/+ and P = .029 versus Plg+/-). Although male and female Plg-/- mice were able to sire offspring, the fertility of Plg-/- female mice was reduced, possibly owing to their impaired health. Levels of plasminogen-related antigen in plasma, measured by ELISA, were 84 +/- 8 micrograms/mL (n = 4) in Plg+/+, 35 +/- 2 micrograms/mL (n = 3) in Plg+/-, and 0.076 +/- 0.032 microgram/mL (n = 6) in Plg-/- mice (P < .001 versus Plg+/- and Plg+/+). Plasmin activity generated by urokinase activation was unmeasurable in Plg-/- mice (< 5% of Plg+/+ mice). Plasminogen-specific immunoreactivity was observed in hepatocytes from Plg+/+ mice but not from Plg-/- mice (< 10% of Plg+/+ mice). Neither native nor variant plasminogen mRNA nor translation products could be identified by Northern or Western blot of liver extracts from Plg-/- mice. Spontaneous lysis within 24 hours of a 125I-fibrin-labeled pulmonary plasma clot was 85 +/- 5% (n = 5) in Plg+/+ mice, 62 +/- 7% (n = 3) in Plg+/- mice, and -2 +/- 1% (n = 3) in Plg-/- mice (P < .001 versus Plg+/- and Plg+/+). Delayed clot lysis within 72 hours was 33 +/- 1% (n = 3) in tPA-/- mice and 26 +/- 2% (n = 3) in Plg-/- mice (P = .054). Histological examination of several organs revealed fibrin deposition in the liver; lung; and in the stomach, associated with gastric ulcers, in 6- to 12-week-old Plg-/- mice but not in Plg+/+ or Plg+/- littermates. CONCLUSIONS: Plasminogen-deficient mice survive embryonic development but develop spontaneous fibrin deposition due to impaired thrombolysis and suffer retarded growth and reduced fertility and survival. The Plg-/- phenotype is reminiscent of the combined tPA-/-:uPA-/- phenotype, which suggests that there is no significant additional pathway for physiological plasminogen activation in mice.

Animals↗