The intron 6 G/T polymorphism of c-myb oncogene and the risk for coronary in-stent restenosis.

BACKGROUND: The principle mechanisms leading to the development of atherosclerosis are long-term accumulation of lipids and cell proliferation. We have recently shown that a single nucleotide polymorphism in the c-myb gene is associated with the development of coronary artery disease in humans and intracellular lipid accumulation. C-myb expression has been further shown to be up-regulated during cell proliferation. The development of in-stent restenosis is predominantly driven by smooth muscle cell proliferation. METHODS: To study a possible association of c-myb with neointima formation in humans we genotyped 485 consecutive patients undergoing coronary stenting for a G/T-single nucleotide polymorphism in intron 6 of the cmyb gene. Restenosis was assessed by quantitative coronary angiography and angiographic follow-up after 6 months. To study the effect of c-myb on smooth muscle cell proliferation primary human smooth muscle cells were infected with recombinant adenovirus expressing c-myb, a dominant negative myb-engrailed fusion protein or control virus. RESULTS AND CONCLUSION: Restenosis > 50% occurred in 27.6% of patients with at least one G-allele and in 20.8% of those without (p = 0.10). Even after adjustment for the independent risk factors diabetes mellitus, reference lumen diameter, smoking, dyslipidemia and number of diseased vessels, the observed difference in the distribution of the c-myb alleles did not reach statistical significance (p = 0.08). Adenoviral gene transfer of c-myb did not increase proliferation of cultured smooth muscle compared to control virus or untransfected cells, while the expression of the dominant negative mutant reduced proliferation of VSMC as previously shown. Our results indicate that expression of c-myb, while being important for cell cycle is not sufficient to induce smooth muscle cell proliferation. The G/T-nucleotide transversion polymorphism in intron 6 of the c-myb oncogene that has been associated with atherosclerosis and lipid accumulation is not a risk factor for human in-stent restenosis.

Prion protein stimulates tissue-type plasminogen activator-mediated plasmin generation via a lysine-binding site on kringle 2.

Recombinant human prion-protein (PrP23-231) stimulates plasminogen activation by tissue-type plasminogen activator (t-PA). The stimulatory activity is conserved in the N-terminal fragment (PrP23-110). It has further been shown by others that PrP(c) binds to kringle-domains of plasminogen. We compared the stimulatory activity of recombinant PrP23-231 and PrP23-110 on plasminogen activation catalyzed by t-PA, urokinase (u-PA), streptokinase and Desmodus salivary plasminogen activator (DSPAalpha1). As these plasminogen activators are distinct, with respect to their kringle domains we studied their binding to immobilized PrP23-110. Plasminogen activation was measured in a chromogenic assay in vitro and binding studies were carried out using surface plasmon resonance technology. We found that recombinant full-length prion protein, PrP23-231, and PrP23-110 specifically stimulate t-PA mediated plasminogen activation. Two hundred nanomoles per liter of PrP23-110 stimulated 1.8 nmol L(-1) t-PA 48-fold, 180 nmol L(-1) DSPA(alpha1) 2.5-fold, 1.8 nmol L(-1) u-PA 1.1-fold, and 1.8 nmol L(-1) streptokinase 1.8-fold. Our data show no specific binding for streptokinase. In contrast all plasminogen activators carrying a kringle domain bound to PrP23-110. We further studied the effect of lysine on binding to PrP23-110 and on plasminogen activation by DSPA(alpha1) or t-PA. Lysine decreased both the binding of t-PA to PrP23-110 and the stimulation of plasmin generation by t-PA. Both binding and plasminogen activation of DSPA(alpha1) were not influenced by the presence of lysine. All plasminogen activators tested bearing kringle domains bind to PrP23-110. Binding to PrP23-110 is not sufficient for stimulation of plasmin generation. Thus the lysine-binding site of kringle 2 that is unique to t-PA appears to mediate the specific stimulation of plasminogen activation by the cellular prion protein.

Both lysine-clusters of the NH2-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation.

We have recently shown that the NH(2)-terminal fragment (PrP23-110) of the human cellular prion protein (PrP(c) ) stimulates t-PA mediated plasminogen activation. PrP23-110 contains an N-terminal lysine cluster (LC1; K(23),K(24), K(27)) and a C-terminal one (LC2; K(101),K(104),K(106),K(110)). To study their biological function we have substituted all lysine residues of each cluster by alanine and generated the recombinant PrP proteins PrP23-110sLC1 and PrP23-110sLC2. The ability of the mutant proteins to stimulate plasminogen activation was assayed. We found that both lysine clusters are essential for t-PA mediated plasminogen activation. We further studied the binding of soluble PrP23-110 to immobilized t-PA or plasminogen using surface plasmon resonance. The recorded binding curves could not be modeled by classical 1:1 binding kinetics suggesting oligomerisation of PrP23-110. Further plasmon resonance studies show that indeed PrP23-110 binds to itself and that glycosaminoglycans modify this interaction. Binding of t-PA or plasminogen to PrP23-110 was no longer influenced by glycosaminoglycans when PrP23-110 was immobilized on the chip surface. Thus a possible role of heparin as a cofactor in the stimulation of plasminogen activation by t-PA could be the generation of a PrP23-110 form with both lysine clusters accessible for binding of t-PA and plasminogen.

Heparin-level-based anticoagulation management during cardiopulmonary bypass: a pilot investigation on the effects of a half-dose aprotinin protocol on postoperative blood loss and hemostatic activation and inflammatory response.

UNLABELLED: Cardiac surgery involving cardiopulmonary bypass (CPB) leads to activation of the hemostatic/inflammatory system. We compared the influence of a half-dose aprotinin regimen on postoperative blood loss and the activation of the hemostatic/inflammatory system during CPB, when used during a heparin-level-based heparin management for cardiac surgery. Two-hundred patients (n = 100 in each group) were enrolled in this randomized prospective study. In Group I only heparin was given according to the results of the Hepcon HMS Plus. In Group II aprotinin was added with a bolus of 1 x 10(6) kallikrein inhibiting units (KIU) for the patient immediately before initiation of CPB, 1 x 10(6) KIU in the priming solution of the CPB, and a continuous infusion of 250,000 KIU/h during CPB. Postoperative blood loss was determined after 12 h. Heparin and antithrombin activity were evaluated by an anti-Xa assay and measurement of antithrombin III activity. Hemostatic activation was evaluated by adenosine diphosphate-stimulated platelet aggregometry and by measurements of the generation/release of beta-thromboglobulin (beta-TG), soluble P-selectin (sPS), thrombin (TAT), prothrombin 1 and 2 fragments (PTF1+2), factor XIIa (FXIIa), plasmin (PAP), and D-dimers. Inflammatory response was evaluated by measuring complement factors 5b-9 (C5b-9), interleukin (IL)-6, and neutrophil elastase (NE). There were no differences in the pre-CPB values or duration of CPB between the two groups. There were no differences in the post-CPB values for platelet count, platelet aggregation, beta-TG, sPS, TAT, PTF1+2, C5b-9, NE, or IL-6. The additional use of aprotinin resulted in a significant decrease of PAP, D-dimers, and 12 h postoperative blood loss, whereas generation of the contact factor XIIa was increased. The administration of aprotinin significantly reduced postoperative blood loss after cardiac surgery and CPB. This most likely has to be attributed to the antifibrinolytic effects of aprotinin. No effects on thrombin generation, platelet activation, inflammatory response, or clinical outcome were noted. IMPLICATIONS: The use of half-dose aprotinin and heparin-level-based anticoagulation management during cardiopulmonary bypass leads to a significant reduction of postoperative blood loss after cardiac surgery. This effect can most likely be attributed to the antifibrinolytic effects of aprotinin, as we did not observe effects on other variables of activation of the hemostatic/inflammatory system.

Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH2-terminal fragment PrP23-110.

The cellular prion protein (PrP(c)), tissue-type plasminogen activator (t-PA) and plasminogen are expressed in synaptic membranes in vivo. In the central nervous system the fibrinolytic system is associated with excitotoxin-mediated neurotoxicity and Alzheimer's disease. Recently binding of the disease associated isoform of the prion protein (PrP(Sc)) to plasminogen and stimulation of t-PA activity have been reported. In this study the interaction of PrP(c) and plasminogen was investigated using chromogenic assays in vitro. We found that plasmin is able to cleave recombinant PrP(c) at lysine residue 110 generating an NH(2)-terminal truncated molecule that has previously been described as a major product of PrP(c) metabolism. We further characterized the proteolytic fragments with respect to their ability to stimulate plasminogen activation in vitro. Our results show that the NH(2)-terminal part of PrP(c) spanning amino acids 23-110 (PrP23-110) together with low molecular weight heparin stimulates t-PA mediated plasminogen activation in vitro. The apparent rate constant was increased 57 fold in the presence of 800 nM PrP23-110. Furthermore, we compared the stimulation of t-PA activity by PrP(c) and beta-amyloid peptide (1-42). While the activity of the beta-amyloid was independent of low molecular weight heparin, PrP23-110 was approximately 4- and 37 fold more active than beta-amyloid in the absence or presence of low molecular weight heparin. In summary, plasmin cleaves PrP(c) in vitro and the liberated NH(2)-terminal fragment accelerates plasminogen activation. Cleavage of PrP c has previously been reported. Thus cleavage of PrP(c) enhancing plasminogen activation at the cell surface could constitute a regulatory mechanism of pericellular proteolysis.

Both u-PA inhibition and vitronectin binding by plasminogen activator inhibitor 1 regulate HT1080 fibrosarcoma cell metastasis.

Overexpression of plasminogen activator inhibitor 1 (PAI-1) reduces tumor cell migration in vitro and metastasis in mice in vivo by mechanisms involving either inhibition of urokinase plasminogen activator (u-PA) activity or competition for an integrin binding site on vitronectin. To analyze the effects of PAI-1 on tumor cell migration in vitro and metastasis in vivo, recombinant adenoviral vectors expressing wild-type or mutant PAI-1 proteins were constructed. The mutant PAI-1 proteins were defective in either vitronectin binding (PAI-1(VN-)), plasminogen activator inhibition (PAI-1(INH-)) or both (PAI-1(VN-,INH-)). In vitro, migration of HT1080 human fibrosarcoma cells through a reconstituted extracellular matrix (ECM) was reduced 73% by overexpression of wild-type PAI-1 and 65% by PAI-1(VN-) compared with control virus-infected cells. Migration of cells infected by virus expressing either PAI-1(INH-) or PAI-1(VN-,INH-) was unaffected, indicating a requirement for plasminogen activator inhibitory activity. In vivo, however, only overexpression of wild-type PAI-1 reduced the burden of metastasis by 68% compared with the control group. This indicates that both u-PA inhibition and PAI-1 ECM interactions contribute to the mechanism of PAI-1-mediated regulation of cell migration.

Hemostatic activation and inflammatory response during cardiopulmonary bypass: impact of heparin management.

BACKGROUND: Cardiac surgery involving cardiopulmonary bypass (CPB) leads to fulminant activation of the hemostatic-inflammatory system. The authors hypothesized that heparin concentration-based anticoagulation management compared with activated clotting time-based heparin management during CPB leads to more effective attenuation of hemostatic activation and inflammatory response. In a randomized prospective study, the authors compared the influence of anticoagulation with a heparin concentration-based system (Hepcon HMS; Medtronic, Minneapolis, MN) to that of activated clotting time-based management on the activation of the hemostatic-inflammatory system during CPB. METHODS: Two hundred elective patients (100 in each group) undergoing standard cardiac surgery in normothermia were enrolled. No antifibrinolytic agents or aprotinin and no heparin-coated CPB systems were used. Samples were collected after administration of the heparin bolus before initiation of CPB and after conclusion of CPB before protamine infusion. RESULTS: There were no differences in the pre-CPB values between both groups. After CPB there were significantly higher concentrations ( < 0.05) for heparin and a significant reduction in thrombin generation (25.2 +/- 21.0 SD vs. 34.6 +/- 25.1), d-dimers (1.94 +/- 1.74 SD vs. 2.58 +/- 2.1 SD), and neutrophil elastase (715.5 +/- 412 SD vs. 856.8 +/- 428 SD), and a trend toward lower beta-thromboglobulin, C5b-9, and soluble P-selectin in the Hepcon HMS group. There were no differences in the post-CPB values for platelet count, adenosine diphosphate-stimulated platelet aggregation, antithrombin III, soluble fibrin, Factor XIIa, or postoperative blood loss. CONCLUSION: Compared with heparin management with the activated clotting time, heparin concentration-based anticoagulation management during CPB leads to a significant reduction of thrombin generation, fibrinolysis, and neutrophil activation, whereas there is no difference in the effect on platelet activation. The generation of fibrin even in the presence of high heparin concentrations most likely has to be attributed to the reduced antithrombin III concentrations or reduced inhibition of clot-bound thrombin. Therefore, in addition to maintenance of higher heparin concentrations, monitoring and substitution of antithrombin III should be considered to ensure more efficient antithrombin activity during CPB.

The plasma supplemented modified activated clotting time for monitoring of heparinization during cardiopulmonary bypass: a pilot investigation.

UNLABELLED: The standard celite or kaolin activated clotting time (ACT) correlates poorly with heparin levels during cardiopulmonary bypass (CPB). We compared a modified kaolin ACT, in which plasma was supplemented, to a standard undiluted kaolin ACT for monitoring heparin levels during CPB. Fifteen patients undergoing normothermic CPB were enrolled in this prospective study. Heparin management was performed according to the Hepcon HMS results (Medtronic, Minneapolis, MN). The ACTs were performed with the ACT II device (Medtronic). Hepcon HMS calculations, standard kaolin ACTs, and plasma supplemented modified ACTs (mACTs), prepared by diluting blood samples 1:1 with human plasma (Behring, Marburg, Germany), were measured every 30 min during CPB. The data obtained were correlated to the plasma chromogenic anti-Xa activity as a reference assay for heparin levels. A total of 64 samples were evaluated. The chromogenic anti-Xa activity ranged from 0.2 to 5.5 IU/mL. The Hepcon HMS calculations ranged from 2.7-8.2 IU/mL of heparin, the standard ACT ranged from 424 to >999 s, and the mACT ranged from 210 to 801 s. The correlation to the chromogenic anti-Xa method was r = 0.43 for the standard kaolin ACT and r = 0.69 for the plasma mACT. The plasma mACT provided an improved correlation to chromogenically measured levels of anti-Xa activity during CPB. The improved correlation most likely results from a correction of the effects of the impairment of the coagulation system caused by hemodilution and consumption of procoagulants on extracorporeal surfaces. IMPLICATIONS: During cardiopulmonary bypass, the plasma modified kaolin activated clotting time (ACT) provides a better correlation with heparin levels than the standard kaolin ACT.