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Biomedical subjects

D C Sane

Publications and source records attributed to D C Sane.

At least 37 records · Page 2Linked to original sources
PubMed2000

Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2).

Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. As the first step to test this hypothesis, we demonstrate that MGP is a binding protein for 125I-BMP-2. Optimal binding is dependent on metals which suggests that the metal binding Gla region in MGP is involved. MGP is shown to undergo a Ca++ induced conformational change despite the presence of the gamma-carboxylase binding site being part of the mature protein sequence. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. Antibodies were used in an attempt to identify MGP in bovine serum. Conformational specific MGP antibodies were shown to also recognize the Gla region in prothrombin and factor X but did not identify MGP in serum. This finding is supported by electrophoresis data which demonstrate the absence of MGP among Ba-citrate absorbed vitamin K-dependent serum proteins. We conclude that MGP does not exist in normal bovine serum.

Animals↗
PubMed1999

Vitaxin, a humanized monoclonal antibody to the vitronectin receptor (alphavbeta3), reduces neointimal hyperplasia and total vessel area after balloon injury in hypercholesterolemic rabbits.

The vitronectin receptor (alphavbeta3) mediates several biological processes that are critical to the formation of a neointima after coronary interventions. Blockade of alphavbeta3 could reduce neointima formation by inhibiting smooth muscle cell migration, decreasing transforming growth factor-beta1 expression, enhancing apoptosis, or reducing neovasculature. The effects of short-term administration of Vitaxin, a humanized monoclonal antibody to alphavbeta3, on the responses to balloon injury were tested in hyperlipidemic rabbits. Balloon angioplasty was performed on the iliac arteries of male New Zealand White rabbits that were fed an atherogenic diet for 1 week before injury and until euthanization at 4 weeks. Rabbits were given either saline (control) or 1 of 2 dosing regimens of Vitaxin (high dose, 5.0 mg/kg, and low dose, 0.5 mg/kg), which were administered intra-arterially before injury and intramuscularly on days 2 and 3. High-dose and low-dose Vitaxin were equally effective in decreasing neointima formation even in the presence of hypercholesterolemia, a stimulus to alphavbeta3 expression. Vitaxin reduced transforming growth factor-beta1 and enhanced apoptosis in injured arteries. Despite these positive effects, Vitaxin administration was associated with a reduction in artery size, indicating a negative effect on remodeling. Vitaxin has a potential role in preventing intimal hyperplasia, especially if the negative effects on remodeling can be overcome, by dose adjustment or other strategies.

Angioplasty, Balloon↗
PubMed1999

Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth muscle cell proliferation and migration In vitro.

Angiostatin is an inhibitor of angiogenesis that is known to reduce endothelial cell proliferation and consequently prevent the progression of tumor metastases. However, the modest effect of angiostatin on endothelial cell proliferation raises the possibility that angiostatin might exert its effects on other cells. To determine the cellular distribution of angiostatin binding in tissues with neovasculature (atherosclerotic coronary arteries), we developed a fusion protein consisting of placental alkaline phosphatase and the first 3 kringles of plasminogen. Angiostatin binding colocalized with smooth muscle cells and could be inhibited by a 50-fold molar excess of plasminogen and 10 mmol/L epsilon-amino-n-caproic acid. The fusion protein also bound to smooth muscle cells in culture. Angiostatin inhibited hepatocyte growth factor-induced proliferation and migration of smooth muscle cells, suggesting that they are a target for the antiangiogenic effect of angiostatin.

Alkaline Phosphatase↗
PubMed1999

Matrix Gla protein synthesis and gamma-carboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells--a cell system which resembles the system in bone cells.

Matrix GLA protein (MGP) is an inhibitor of calcification in the arterial wall and its activity is dependent upon vitamin K-dependent gamma-carboxylation. This modification is carried out by a warfarin sensitive enzyme system that converts specific Glu residues to gamma-carboxyglutamic acid (GLA) residues. Recent studies have demonstrated that the gamma-carboxylation system in the arterial wall, in contrast to that in the liver, is unable to use vitamin K as an antidote to warfarin. By use of immunohistochemistry we demonstrate that MGP is expressed in the arterial wall and immunocytochemistry localized the MGP precursors to the endoplasmic reticulum in vascular smooth muscle cells. Resting smooth vascular muscle cells in the aortic wall and proliferating cells from explants of the aorta have all the enzymes needed for gamma-carboxylation of MGP. However, when compared to the liver system, expression of the enzymes of the gamma-carboxylation system in vascular smooth muscle cells is different. Of particular interest is the finding that the specific activity of the warfarin sensitive enzyme vitamin K epoxide reductase is 3-fold higher in vascular smooth muscle cells than in liver. DT-diaphorase, which catalyses the antidotal pathway for vitamin K reduction in liver, is 100-fold less active in resting vascular smooth muscle cells than in liver. Data obtained from an in vitro gamma-carboxylation system suggest that the antidotal pathway catalyzed by DT-diaphorase in the vessel wall is unable to provide the carboxylase with enough reduced vitamin K to trigger gamma-carboxylation of MGP. This finding provides an explanation to the inability of vitamin K to work as an antidote to warfarin intoxication of the arterial wall. Therefore the vitamin K dependent gamma-carboxylation system in the arterial wall share a common feature with the system in bone cells by being unable to utilize vitamin K as an antidote.

Animals↗
PubMed1998

Occurrence and clinical significance of thrombocytopenia in a population undergoing high-risk percutaneous coronary revascularization. Evaluation of c7E3 for the Prevention of Ischemic Complications (EPIC) Study Group.

OBJECTIVES: This study sought to determine the frequency of thrombocytopenia and its relation with clinical outcomes in high risk patients undergoing percutaneous coronary revascularization who received either the platelet glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (ReoPro, c7E3 Fab) or conventional therapy. BACKGROUND: The development of thrombocytopenia on exposure to GPIIb/IIIa antagonists threatens the utility and economic viability of this drug class for patients with vascular disease. METHODS: We analyzed data from the Evaluation of c7E3 for the Prevention of Ischemic Complications trial (EPIC), a 2,099-patient, randomized trial of placebo, abciximab bolus or abciximab bolus plus a 12-h infusion during high-risk coronary revascularization. RESULTS: Thrombocytopenia (nadir platelet count <100 x 10(9)/ liter) developed in 81 patients (3.9%) during their hospital stay, with 19 (0.9%) developing severe (<50 x 10(9)/liter) thrombocytopenia. Both thrombocytopenia and severe thrombocytopenia were more frequent in the bolus-plus-infusion arm (5.2% and 1.6%, respectively) than in the bolus-only and placebo arms combined (p = 0.020 and p = 0.025, respectively). Acute profound thrombocytopenia developed in two patients in the bolus-plus-infusion arm. Patients with thrombocytopenia experienced more unfavorable clinical outcomes than those who did not develop thrombocytopenia, regardless of treatment assignment, but those with thrombocytopenia who received abciximab had fewer worse outcomes at 30 days. Multivariable logistic modeling revealed a lower baseline platelet count, older age and lighter weight to be important predictors of thrombocytopenia. In a logistic regression model, bolus-plus-infusion treatment was a significant predictor of thrombocytopenia (p = 0.016) and remained so after adjustment for procedures and baseline risk factors (p = 0.0077). CONCLUSIONS: Thrombocytopenia was associated with adverse clinical outcomes and excessive bleeding, but patients receiving abciximab fared better than those receiving placebo.

Abciximab↗
PubMed1995

Localization of a vitronectin binding region of plasminogen activator inhibitor-1.

The PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70, 1-105, 1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

Amino Acid Sequence↗
PubMed1994

Clinical importance of thrombocytopenia occurring in the hospital phase after administration of thrombolytic therapy for acute myocardial infarction. The Thrombolysis and Angioplasty in Myocardial Infarction Study Group.

OBJECTIVES: The purpose of this study was to examine the incidence and clinical implications of thrombocytopenia that occurs in hospital after administration of thrombolytic therapy for acute myocardial infarction. BACKGROUND: The use of thrombolytic therapy in patients with acute myocardial infarction has improved mortaltiy rates, but hemorrhage remains a major complication. Because thrombocytopenia may be associated with hemorrhage after thrombolytic therapy, we examined the incidence and clinical implications of thrombocytopenia after administration of thrombolytic therapy for acute myocardial infarction. METHODS: The patient population comprised 1,001 patients enrolled in Phases 2, 3 and 5 of the Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) trial and the urokinase trial. Patients received recombinant tissue-type plasminogen activator, urokinase or combination therapy in various dosing schemes. All patients received heparin, aspirin and a calcium-channel blocking agent. Thrombocytopenia occurring anytime after thrombolytic therapy was defined as a nadir platelet count either < 100,000/microliters or < 1/2 baseline. Blood loss was quantified by a bleeding index. Multiple logistic regression was used to evaluate the independent contribution of thrombocytopenia in a model predicting in-hospital mortality. RESULTS: Thrombocytopenia occurred in 16.4% of patients, with no difference among the thrombolytic regimens. Patients with thrombocytopenia had a lower median acute ejection fraction and a higher likelihood of three-vessel coronary artery disease than patients without thrombocytopenia. Patients with thrombocytopenia had more hemorrhage, a higher in-hospital mortality rate and a more complicated hospital course than patients without thrombocytopenia, even after consideration of other important variables, including age, acute ejection fraction, number of diseased vessels, bypass surgery and use of intraaortic balloon counterpulsation. CONCLUSIONS: Thrombocytopenia after thrombolytic therapy is a common event and is associated with excess hemorrhage and mortality. Platelet counts should be monitored daily after administration of thrombolytic therapy because the appearance of thrombocytopenia identifies a subset of patients at increased risk for hemorrhage and death.

Aged↗
PubMed1993

Expression of a recombinant baculovirus for vitronectin in insect cells: purification, characterization of post-translational modifications and functional studies of the recombinant protein.

Vitronectin (VN) is a cell attachment glycoprotein present in plasma and the extracellular matrix that also has multiple regulatory roles in the complement, coagulation, and fibrinolytic systems. VN stabilizes plasminogen activator inhibitor-1 (PAI-1) with potential binding sites reported at both the somatomedin B and heparin binding domains. In order to perform detailed structure-function analyses of VN, we have produced and expressed a recombinant baculovirus containing the cDNA of human VN. Recombinant VN (rVN) was secreted as a single chain 70-kDa protein into the serum-free culture medium of infected Spodoptera frugiperda cells. Maximum rVN levels of 33.9 mg/liter were obtained 48 hours post-infection. Purification of rVN from serum-free medium was achieved with single-step anion-exchange chromatography. Several native post-translational modifications were demonstrated in rVN including the presence of N-linked oligosaccharides, tyrosine sulfation, and serine phosphorylation. Recombinant VN retained cell and PAI-1 binding capacity. Therefore, rVN expressed for the first time in insect cells retains many post-translational modifications and functional activities and will be useful for additional structure-function analyses.

Animals↗
PubMed1993

The cell attachment and spreading activity of vitronectin is dependent on the Arg-Gly-Asp sequence. Analysis by construction of RGD and domain deletion mutants.

The cell attachment activity of vitronectin has been ascribed to an Arg-Gly-Asp (RGD) sequence near the amino terminus. To verify the importance of the RGD sequence for cell binding, we created RAD and RGE vitronectin mutants and also deleted either the somatomedin B (delta S-rVN) or heparin (delta H-rVN) binding domains. These mutants were expressed as fusion proteins, purified using Ni+2 affinity chromatography, and assayed for cell attachment. EAhy.926 cells bound equally well to wild-type, delta S-rVN, and to delta H-rVN, but binding to RAD-rVN and RGE-rVN was inhibited by more than 90%. We therefore conclude that the RGD sequence of vitronectin is the most important cell recognition site and that neither the somatomedin B nor heparin domains contribute significantly to the cell adhesive activity of vitronectin.

Amino Acid Sequence↗
PubMed1993

Human platelets possess tyrosylprotein sulfotransferase (TPST) activity.

Tyrosine sulfation is a widespread modification of secreted proteins including several coagulation proteins synthesized in the liver. Since factor V, a tyrosine-sulfated protein, is also synthesized in megakaryocytes, we determined whether platelets posses tyrosylprotein sulfotransferase (TPST) activity. Using the synthetic substrate EAY, substantial TPST activity (0.405 +/- 0.049 pmol EAY-SO4 formed min-1 mg-1) was detected in platelet homogenates. This activity could not be accounted for by contaminating leukocytes, erythrocytes or plasma. The Km of platelet TPST for EAY was 3.7 microM and Vmax 0.09 pmol/min. For the cofactor 3' phosphoadenosine 5' phosphosulfate (PAPS) the Km was 1.7 microM and Vmax 0.11 pmol/min. The PAPS analogue 3',5'-adenosine diphosphate inhibited platelet TPST with an IC50 of 15.4 microM. These findings suggest that tyrosine sulfation of factor V will occur in megakaryocytes. Platelets may be a useful source for further study of TPST.

Animals↗
PubMed1992

Clinical risks of thrombolytic therapy.

Understanding the clinical risks of intravenous thrombolytic therapy is critical to appropriate patient selection. The major risks can be classified into 5 major categories: intracranial hemorrhage, systemic hemorrhage, immunologic complications, hypotension, and myocardial rupture. Although theoretical concern exists about thromboembolic complications, they rarely occur. Although cardiac rhythm disturbances are somewhat more likely to occur at the time of reperfusion, the clinical significance of "reperfusion arrhythmias" is minimal. Intracranial hemorrhage, the most devastating complication, occurs in 0.2-1% of patients treated with thrombolytic therapy. Factors associated with incremental risk are now being identified from large clinical trials. Systemic hemorrhage is uncommon in patients without major vascular punctures and seldom leads to serious adverse outcomes. Immunologic complications--including anaphylaxis, which is rare, and immune complex disease, which is more common--occur only with streptokinase or agents with a streptokinase moiety, including anistreplase (anisoylated plasminogen--streptokinase activator complex, APSAC). Hypotension, which can be managed easily in most patients, is also observed much more frequently with streptokinase and anistreplase. Myocardial rupture is increasingly being recognized as a possible complication of late thrombolysis. A proper perspective on clinical risk can only be gained in the context of potential benefit of therapy. In many cases individual patients considered to be at highest risk for complications also stand to gain the most from treatment. Many of the questions raised by currently available data about bleeding risk are being addressed in the ongoing Global Utilization of t-PA and Streptokinase (GUSTO) Trial. A paradigm for considering this decision making problem is presented.

Anistreplase↗
PubMed1992

Exploration of rapid bedside monitoring of coagulation and fibrinolysis parameters during thrombolytic therapy.

Monitoring coagulation parameters during thrombolytic therapy could be useful for prediction and treatment of haemorrhagic episodes. Technology based on dry reagent chemistry has been developed that allows rapid (less than 10 min) assays on small samples of whole blood. The assay principle is based on the restriction of motion of paramagnetic particles during fibrin polymerization, and subsequent liberation of particle motion during fibrinolysis. This technology was used to monitor prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen levels and fibrinolysis profiles during thrombolytic therapy with tissue plasminogen activator for acute myocardial infarction. The PT and aPTT obtained with the COAG-1 correlated well with conventional assays (r = 0.93 and 0.92 for PT and aPTT, respectively; p = 0.0001). Fibrinogen estimates, obtained by COAG-2 also correlated well with modified Clauss assays (r = 0.86, p = 0.0001). The rapid determination of the aPTT may improve management of adjunctive anticoagulant therapy following thrombolysis. The fibrinolysis profile may be useful during thrombolytic therapy to verify that a lytic state has been achieved, to monitor the lytic state throughout therapy, and to verify that the lytic state normalizes once therapy has been completed.

Blood Coagulation↗
PubMed1991

Limited proteolysis of vitronectin by plasmin destroys heparin binding activity.

Vitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN. Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

Blood Proteins↗
PubMed1991

Correlation between baseline plasminogen activator inhibitor levels and clinical outcome during therapy with tissue plasminogen activator for acute myocardial infarction.

Baseline plasminogen activator inhibitor (PAI) levels were examined for their influence on the responses to thrombolysis with recombinant tissue plasminogen activator (rt-PA) administered for acute myocardial infarction during the Thrombolysis and Myocardial Infarction (TAMI)-I study. Baseline PAI activity was 19 +/- 21 IU/ml (normal less than 5 IU/ml) and baseline PAI-1 antigen 54 +/- 53 ng/ml (normal 27 +/- 16 ng/ml), confirming previous findings of elevated PAI levels during acute myocardial infarction. Among clinical outcomes, lower PAI-1 antigen levels correlated weakly with greater patency at the 90 min angiogram. Thus, high baseline plasma PAI-1 levels may be detrimental to reperfusion with t-PA. There was no correlation with other major in-hospital clinical outcomes including reocclusion at the 7-10 day angiogram, survival to discharge, or bleeding. During the follow up period of 2.0 +/- 0.4 years, no relationship between baseline PAI levels and post-discharge reinfarction was observed.

Angioplasty, Balloon, Coronary↗
PubMed1991

Vitronectin in the substratum of endothelial cells is cross-linked and phosphorylated.

Vitronectin (VN), previously shown to be a substrate for purified transglutaminases, was demonstrated in this study to be cross-linked when incubated with HUVEC and EAhy926 cells. The cross-linking was calcium-dependent and required that VN be plated at the substratum of the cells. These cells also phosphorylated VN, but in contrast to a previous study demonstrating a cAMP-dependent protein kinase in platelets, the phosphorylation of VN by was decreased with the addition of 1mM cAMP. The cross-linking of VN by endothelial cells demonstrates that the adhesion of these cells to VN is a dynamic process in which the substratum may be enzymatically altered. Furthermore, the modifications of VN by cross-linking and phosphorylation could modulate the functions of VN and influence events such as endothelial cell proliferation and angiogenesis.

Adenosine Triphosphate↗