Catalytic oligodeoxynucleotides define a key regulatory role for early growth response factor-1 in the porcine model of coronary in-stent restenosis.

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.

Control of von Willebrand factor multimer size by thrombospondin-1.

Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury. Only the very large vWF multimers are effective in promoting platelet adhesion in flowing blood. A protein disulfide bond reductase in plasma reduces the average multimer size of vWF secreted by endothelial cells. This activity has been isolated from human endothelial cell conditioned medium and shown to be the trimeric glycoprotein, thrombospondin-1 (TSP-1). Incubation of purified TSP-1 with vWF resulted in formation of thiol-dependent complexes of TSP-1 and vWF, generation of new thiols in vWF, and reduction in the average multimer size of vWF. The ratio of the concentrations of TSP-1 and vWF in plasma reflected with average multimer size of vWF. The higher the plasma TSP-1/vWF molar ratio, the smaller the average vWF multimer size. In addition, administration of TSP-1 to mice resulted in reduction in the average multimer size of plasma vWF. Interaction of TSP-1 with vWF is mediated by TSP-1 type 1 properdin domains and the vWF A3 domain. These results indicate that TSP-1 regulates the multimeric size and therefore hemostatic activity of vWF.

Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid.

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.

Platelet-derived growth factor enhances granulopoiesis via bone marrow stromal cells.

Platelet-derived growth factor (PDGF), a growth factor for connective tissue cells, stimulates erythropoiesis and megakaryocytopoiesis in vitro but the effect of PDGF on granulocyte proliferation remains unknown. The effect of the recombinant human PDGF-BB isoform on granulopoiesis was investigated in this study. The results show that PDGF significantly stimulated murine colony-forming unit-granulocyte-monocyte (CFU-GM) proliferation in a dose-dependent manner (1 to 100 ng/mL) using murine bone marrow cells (n = 4). Maximum stimulation was obtained with 50 ng/mL of PDGF (P < .01). The effect of PDGF on murine CFU-GM proliferation was compared with that of interleukin (IL)-3, IL-6, granulocyte-monocyte colony-stimulating factor (GM-CSF), and acidic fibroblast growth factor (aFGF) at their optimal doses. The stimulating activity of PDGF was higher than that of aFGF but lower than that of IL-3, IL-6, or GM-CSF. There is no synergistic effect between PDGF and IL-3 or IL-6, but a significant enhancing effect was observed in IL-3 plus IL-6. PDGF also stimulated the growth of CFU-GM with CFU-megakaryocyte in the presence of bone marrow stromal cells. We also found that PDGF had similar a effect on human CFU-GM proliferation using bone marrow mononuclear cells (MNC). However, the increase in PDGF-stimulated CFU-GM proliferation was inhibited by anti-GM-CSF, anti-IL-3, and anti-IL-6 antibodies (n = 4), suggesting that endogenously produced GM-CSF, IL-3, and IL-6 may play a role in the PDGF-induced CFU-GM proliferation. Furthermore, PDGF (1 to 100 ng/mL) did not show any effect on CFU-GM proliferation when replacing bone marrow MNC with immunomagnetic selection-enriched CD34+ cells from human cord blood (n = 5; purity, 91% +/- 6.5%). This study indicates that PDGF may indirectly enhance CFU-GM proliferation by inducing the bone marrow stromal cells to produce GM-CSF, IL-3, or IL-6.

Early growth response factor-1 mediates insulin-inducible vascular endothelial cell proliferation and regrowth after injury.

Hyperinsulinemia in diabetes mellitus is a significant risk factor in the development of atherosclerosis and early restenosis after balloon angioplasty. These manifestations could be mediated by the ability of insulin to potentiate the cellular proliferative and reparative response of vascular cell types to local stimuli. Here we demonstrate that insulin stimulates DNA synthesis in aortic endothelial cells. Reverse transcription-polymerase chain reaction and Northern blotting revealed that insulin induces the expression and transcriptional activity of the immediate early gene and zinc finger transcription protein, early growth response factor-1 (Egr-1). Western immunoblot analysis revealed that insulin-inducible Egr-1 expression was inhibited using phosphorothioate-specific antisense oligonucleotides targeting Egr-1 mRNA. These agents blocked endothelial cell DNA synthesis stimulated by insulin in a dose-dependent manner and inhibited the capacity of insulin to potentiate the reparative response of endothelial cells to mechanical injury in vitro. These oligonucleotides also attenuated wound repair in smooth muscle cells. DNA synthesis induced by insulin was suppressed by inhibitors of two upstream activators of Egr-1, extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-phosphate (PI 3-K), whereas p38 kinase inhibitors had no effect. These present findings demonstrate that insulin-inducible DNA synthesis and repair after injury are processes critically dependent upon the activation of Egr-1. Additionally, they implicate this transcription factor as a potential target for the inhibition of restenosis in diabetics.

Anti-PF4-heparin immunoglobulin G is the major class of heparin-induced thrombocytopenia antibody: findings of an enzyme-linked immunofiltration assay using membrane-bound hPF4-heparin.

An enzyme-linked immunofiltration assay (ELIFA) was developed for detecting anti-human platelet factor 4 (hPF4)-heparin antibody in sera of patients with heparin-induced thrombocytopenia (HIT). The immunofiltration assay was developed to capture HIT antibody by hPF4-heparin complex adsorbed onto a positively charged nylon membrane, as an alternative to plastic bound hPF4. Of 75 sera with a positive serotonin-release assay (SRA), anti-PF4-heparin of the immunoglobulin (Ig)G class was detected in 72 (96%) sera. With SRA-negative sera from thrombocytopenic patients treated with heparin, anti-hPF4-heparin IgG and IgA were detected in 16% (n = 126) and 14% (n = 74) of sera respectively; 6% (n = 71) of SRA-negative sera contained both IgG and IgA anti-hPF4-heparin antibodies. The detection of anti-hPF4-heparin IgG in all HIT sera supports the assay of anti-PF4-heparin IgG as being a sensitive screening test for HIT. Alternatively, the absence of anti-hPF4-heparin IgA cannot be used as a test for excluding HIT, as it was detected in only 48% of SRA-positive HIT sera. However, it may be used to support the diagnosis of HIT, when HIT IgG is weak. This study emphasized the need to use different immobilizing media for the capture of anti-PF4-heparin antibody.

Prospective randomised open-label comparison of danaparoid with dextran 70 in the treatment of heparin-induced thrombocytopaenia with thrombosis: a clinical outcome study.

AIM: To compare clinical outcomes in a randomised comparison of treatment with danaparoid sodium (a heparinoid), or dextran 70, for heparin-induced thrombocytopaenia (HIT) plus thrombosis. METHODS: Forty-two patients with recent thrombosis and a clinical diagnosis of probable HIT who presented at ten Australian hospitals during a study period of six and one half years were randomly assigned to open-label treatment with intravenous danaparoid or dextran 70, each combined with oral warfarin. Thirty-four patients (83%) had a positive platelet aggregation or 14C-serotonin release test for HIT antibody. Twenty-five received danaparoid as a bolus injection of 2400 anti-Xa units followed by 400 units per hour for 2 h, 300 units per hour for 2 h, and then 200 units per hour for five days. Seventeen received 1000 mL dextran 70 on day one and then 500 mL on days 2-5. Patients were reviewed daily for clinical evidence of thrombus progression or resolution, fresh thrombosis or embolism, bleeding or other complications. The primary trial endpoint was the proportion of thromboembolic events with complete clinical resolution by the time of discharge from hospital. RESULTS: With danaparoid, there was complete clinical recovery from 56% of thromboembolic events compared to 14% after dextran 70 (Odds Ratio 10.53, 95% Confidence Interval 1.6-71.4; p = 0.02). Clinical recovery with danaparoid was complete or partial in 86% of thromboembolic events compared with 53% after dextran 70 (Odds Ratio 4.55, 95% Confidence Interval 1.2-16.7; p = 0.03). Overall clinical effectiveness of danaparoid was rated as high or moderate in 88% of patients compared with 47% for dextran 70 (p = 0.01). One patient given danaparoid died of thrombosis compared with three patients given dextran 70. The platelet count returned to normal after a mean of 6.7 days with danaparoid and 7.3 days with dextran 70. There was no major bleeding with either treatment. CONCLUSION: danaparoid plus warfarin treatment for HIT with thrombosis is effective, safe, and superior to dextran 70 plus warfarin.

Reduction of von Willebrand factor by endothelial cells.

The haemostatic activity of plasma von Willebrand factor (vWF) is a function of multimer size. Only the large vWF multimers are effective in promoting platelet adhesion to a site of vascular injury. We observed that the conditioned medium of cultured human umbilical vein, human microvascular and bovine aortic endothelial cells contained an activity which reduced the average multimer size of plasma or purified vWF. The average multimer size of vWF produced endogenously by human umbilical vein endothelial cells was similarly reduced following secretion. The reducing activity was ablated by pre-treatment with heat or the thiol blocking agents. iodoacetamide, N-ethylmaleimide or E-64, but not by a range of specific serine-, cysteine-, aspartic-, or metalloproteinase inhibitors. Reduction in vWF multimer size was associated with formation of new thiols in vWF and there was no evidence for additional proteolytic processing of vWF. The reducing activity was associated with a protein with an anionic pi that binds heparin and contains reactive thiol(s). These results suggested that the interchain disulfide bonds that link the vWF homodimers near the N-termini were being reduced by a vWF reductase secreted by endothelial cells. In support of this hypothesis, incubation of vWF with the protein reductants, protein disulfide isomerase and thioredoxin, resulted in formation of new thiols in vWF and reduction in the average multimer size of vWF. These findings may have consequences for control of vWF haemostatic activity.

A novel approach to the assessment of variations in the human platelet count.

This is the first report of a method to assess the significance of numerical changes in the platelet count based upon a result exceeding the normal intra-individual variation in platelet numbers. Serial platelet counts from 3,789 subjects were analysed to determine the intra-individual variation in platelet numbers. A platelet count difference of 98 x 10(9)/L in males was found to represent a change that would occur by chance in less than 1 in 1,000 platelet count determinations. Tables to determine the significance of platelet number variations, given N previous observations, are provided at two probability levels. The repeatability of the platelet count was calculated as 0.871 (males) and 0.849 (females) indicating that the heritability of platelet count is high and that the platelet count is predominantly genetically determined. A seasonal variation in platelet count was found with a 'winter' versus 'summer' difference of 5.10 X 10(9)/L (males) and 5.82 x 10(9)/L (females).

Intracoronary blood sampling using the multifunction probing catheter does not cause platelet activation.

We sought to test the hypothesis that intracoronary blood sampling in patients with angiographically demonstrated coronary artery disease could be performed using the multifunction probing catheter (Schneider, Bulach, Switzerland), without causing ex vivo platelet activation.

Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).