The new heparins.
Explore the source record for details and available documents.
Clinical Trials as Topic↗
Biomedical subjects
J I Weitz
Publications and source records attributed to J I Weitz.
The effects of standard and low molecular weight heparin on bone nodule formation in vitro.
Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.
Animals↗
Evidence for allosteric linkage between exosites 1 and 2 of thrombin.
Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH2Cl (FPR)-thrombin. Hirudin-(54-65) and sF2, a synthetic peptide corresponding to residues 63-116 of prothrombin fragment 2, were used as ligands for exosites 1 and 2 of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPR-thrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF2 and hirudin-(54-65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF2 is titrated with hirudin-(54-65), complete displacement of fluorescein-sF2 is observed. Likewise, when thrombin-bound fluorescein-hirudin-(54-65) is titrated with sF2, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.
Allosteric Regulation↗
Low-molecular-weight heparins.
Explore the source record for details and available documents.
Anticoagulants↗
Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide.
Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide were undertaken to evaluate a gamma ray emitting ODN ligand for thrombus imaging in vivo . Synthesis of the ODN was based on modified automatedbeta-cyanoethyl phosphoramidite chemistry with an organotin nucleoside (dU*) coupled to a thrombin binding aptamer sequence to give d(U*GGTTGGTGTGGTTGG). The synthesis accommodated dU*, which is destannylated by iodine or acids. Fourteen standard synthesis cycles were followed by one 'stannyl synthesis cycle', distinguished by Fmoc protection, omission of capping, oxidation by an organic peroxide and cleavage by ammonium hydroxide. The organotin nucleoside phosphoramidite {5'-[fluorenylmethoxycarbonyl]-5-(E)-[2-tri-n -butylstannylvinyl]-2'-deoxyuridine-3'-(2-cyanoethyl N,N-diisopropyl phosphoramidite)} was prepared from 5-iodo-2'-deoxyuridine. A customized mild rapid workup included deprotection with methylamine, and reverse phase HPLC with CH3CN/triethylammonium bicarbonate. Pure stannyl ODN was highly retained by reverse phase HPLC. Radioiodination of stannyl ODN (100 microg) provided 123I-labeling yields up to 97%. Five alternative oxidants were effective. High specific activity [123I]- ODN (15 000 Ci/mmol) was recovered, separated from unlabeled isomers. Excellent reverse phase HPLC resolution of ODN isomers (alternatively I, Cl, H or Br in vinyl deoxyuridine) was essential. The affinity of the iodovinyl aptamer analog (Kd = 36 nM) for human alpha-thrombin was similar to the native aptamer (Kd = 45 nM).
Gamma Rays↗
The use of D-dimer testing and impedance plethysmographic examination in patients with clinical indications of deep vein thrombosis.
OBJECTIVE: To prospectively test the hypothesis that a diagnosis of deep vein thrombosis can be excluded in outpatients who present with clinical indications of deep vein thrombosis and whose results of D-dimer testing and impedance plethysmographic examination on the day of presentation are normal. DESIGN: Prospective cohort study. SETTING: Four university-affiliated hospitals. METHODS: Three hundred ninety-eight consecutive patients with clinical indications of deep vein thrombosis were included in the final analysis. All patients underwent an assessment of pretest probability, bedside D-dimer testing, and impedance plethysmographic examination. In most patients, if the results of D-dimer testing and impedance plethysmographic examination were negative for deep vein thrombosis, anticoagulants were withheld and patients were followed up for 3 months. If the results of one or both tests were abnormal, an examination using venous compression ultrasonography or phlebography was performed. RESULTS: In the majority of patients (69%), the results of D-dimer testing and impedance plethysmographic examination were normal. This combination had a negative predictive value of 98.5% (95% confidence interval, 96.3-99.6) for deep vein thrombosis. CONCLUSION: The results of the D-dimer assay and impedance plethysmographic examination on the day of presentation can be used to treat the majority of outpatients who present with clinical indications of deep vein thrombosis without further testing.
Adult↗
A histomorphometric comparison of the effects of heparin and low-molecular-weight heparin on cancellous bone in rats.
Long-term heparin treatment causes osteoporosis through, an as yet, undefined mechanism. To investigate this phenomenon and to determine the relative benefits of low-molecular-weight heparin (LMWH) use, we treated rats with once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g), the LMWH, Tinzaparin (1.0 U/g or 0.5 U/g), or placebo (saline) for a period of 32 days. The effects on bone were then compared both histomorphometrically and biochemically by measuring urinary type I collagen cross-linked pyridinoline (PYD) and serum alkaline phosphatase, markers of bone resorption and formation, respectively. Histomorphometric analysis of the distal third of the right femur, in the region proximal to the epiphyseal growth plate, demonstrated that both heparin and LMWH decrease cancellous bone volume in a dose-dependent fashion, but that heparin causes significantly more cancellous bone loss than does LMWH. Although both heparin and LMWH decrease osteoblast and osteoid surface to a similar extent, only heparin increases osteoclast surface. In support of these histomorphometric findings, biochemical markers of bone turnover demonstrated that both heparin and LMWH treatment produce a dose-dependent decrease in serum alkaline phosphatase, consistent with reduced bone formation, whereas only heparin causes a transient increase in urinary PYD, consistent with an increase in bone resorption. Based on these observations, we conclude that heparin decreases cancellous bone volume both by decreasing the rate of bone formation and increasing the rate of bone resorption. In contrast, LMWH, causes less osteopenia than heparin because it only decreases the rate of bone formation.
Alkaline Phosphatase↗
Effect of nonspecific binding to plasma proteins on the antithrombin activities of unfractionated heparin, low-molecular-weight heparin, and dermatan sulfate.
BACKGROUND: Nonspecific binding to plasma proteins decreases the anti-factor Xa (anti-Xa) activity of unfractionated heparin (UFH) but not that of low-molecular-weight heparin (LMWH). However, plasma proteins could influence the anti-thrombin (anti-IIa) activity of LMWH. To explore this possibility, we compared the effects of plasma proteins on the anti-IIa activities of UFH and LMWH. We also examined their effects on the anti-IIa activity of dermatan sulfate (DS) because, like UFH, DS binds to plasma proteins. METHODS AND RESULTS: There was almost complete recovery of anti-IIa activity when UFH, LMWH, or DS was added to plasma from each of 20 healthy volunteers. The addition of a chemically modified heparin with low affinity for antithrombin III to plasma containing UFH increased the anti-IIa activity in a concentration-dependent fashion by displacing UFH from plasma proteins. In contrast, addition of low-affinity heparin had no effect on the anti-IIa activity of LMWH. LMWH does not bind to plasma proteins because the bulk of the LMWH chains are < 6000 D, and only heparin fractions > 6000 D bind nonspecifically to plasma proteins. As further evidence that plasma proteins do not influence the anti-IIa activity of LMWH, the rate of thrombin inhibition in plasma in the presence of LMWH is virtually identical to that in buffer containing physiological amounts of the major antithrombins. In contrast, with UFH or DS, the rate of thrombin inhibition is twofold slower in plasma than in buffer. CONCLUSIONS: Nonspecific binding of UFH to plasma proteins most likely contributes to the variable anti-IIa response to UFH in patients with thromboembolic disease. Although DS also binds to plasma proteins, the clinical significance of this finding is unclear. In contrast, because LMWH does not bind to plasma proteins, the anti-IIa activity of LMWH should be just as predictable as its anti-Xa activity.
Anticoagulants↗
Molecular basis for the resistance of fibrin-bound thrombin to inactivation by heparin/serpin complexes.
Explore the source record for details and available documents.
Animals↗
The variable anticoagulant response to unfractionated heparin in vivo reflects binding to plasma proteins rather than clearance.
The anticoagulant response to fixed doses of unfractionated heparin is variable in patients with acute illness, and some patients with venous thromboembolism require high doses of heparin to achieve a therapeutic anticoagulant response. To investigate the mechanism responsible for the variable anticoagulant response to heparin in acute illness, heparin clearance and nonspecific protein binding were compared in control and endotoxin-treated rabbits. The plasma half-life (t 1/2) of radiolabeled heparin increased in a dose-dependent fashion. At all doses of heparin studied, the t 1/2 of radiolabeled heparin was unaffected by experimental endotoxemia when compared with control animals. In contrast, the amount of heparin recovered was lower in the plasma of endotoxemic animals because of increased binding to plasma proteins. A chemically modified heparin with low affinity for antithrombin III was added ex vivo or in vivo to displace heparin bound nonspecifically to plasma proteins. The proportion of heparin bound to plasma proteins was significantly greater in the plasma of endotoxemic animals than in controls. These findings indicate that acute inflammation alters heparin recovery but does not affect heparin clearance. The variability of the anticoagulant response to heparin seen in patients with thromboembolism may, in part, be due to this effect of the underlying disease process.
Animals↗
Diagnosis and treatment of chronic arterial insufficiency of the lower extremities: a critical review.
Explore the source record for details and available documents.
Arteries↗
Histomorphometric analysis of the effects of standard heparin on trabecular bone in vivo.
Long-term heparin treatment causes osteoporosis through an as yet undefined mechanism. To investigate this phenomenon, we treated rats with once daily subcutaneous injections of heparin (in doses ranging from 0.25 to 1.0 U/g) or saline for 8 to 32 days and monitored the effects on bone both histomorphometrically and by serial measurements of urinary type 1 collagen cross linked-pyridinoline (PYD) and serum alkaline phosphatase, markers of bone resorption and formation, respectively. Histomorphometric analysis of the distal third of the right femur in the region proximal to the epiphyseal growth plate showed that heparin induces both a time- and dose-dependent decreased in trabecular bone volume, with the majority of trabecular bone loss occurring within the first 8 days of treatment. Thus, heparin doses of 1.0 U/g/d resulted in a 32% loss of trabecular bone. Heparin-treated rats also showed a 37% decrease in osteoblast surface as well as a 75% decrease in osteoid surface. In contrast, heparin treatment had the opposite effect on osteoclast surface, which was 43% higher in heparin-treated rats, as compared with that in control rats. Biochemical markers of bone turnover showed that heparin treatment produced a dose-dependent decrease in serum alkaline phosphatase and a transient increase in urinary PYD, thus confirming the histomorphometric data. Based on these observations, we conclude that heparin decreases trabecular bone volume both by decreasing the rate of bone formation and increasing the rate of bone resorption.
Alkaline Phosphatase↗
Lysis of surface-localized fibrin clots by adsorbed plasminogen in the presence of tissue plasminogen activator.
The ability of plasminogen adsorbed from buffer onto sulphonated silica glass or lysine-derivatized silica glass to lyse fibrin I clots has been investigated. Clots were formed around the test surface by adding reptilase to fibrinogen solutions in which the surfaces were immersed. Tissue plasminogen activator (t-PA) was then added and the extent of clot lysis was determined by measuring the levels of the specific plasmin cleavage product of fibrinogen, B beta 1-42 peptide. The data indicate that in the presence of t-PA, B beta 1-42 generation per mole of bound plasminogen on the lysinized material is approximately two-fold higher than on the sulphonated material. It is concluded that a preformed clot may be lysed by adsorbed plasminogen in the presence of t-PA, and that clot lysis is significantly enhanced when the plasminogen is adsorbed via its lysine binding sites.
Adsorption↗
Elevated fibrinopeptide A and B levels during thrombolytic therapy: real or artefactual?
There is good evidence that thrombolytic therapy induces a procoagulant state that retards the lytic process and triggers reocclusion. Thus, both in experimental animal systems and in humans, potent inhibitors of platelets and antithrombin III-independent thrombin inhibitors have been shown to be better than heparin at accelerating thrombolysis and preventing reocclusion. These in vivo observations have lent credence to the concept that elevated FPA levels that are suppressed by heparin reflect systemic activation of coagulation during the thrombolytic process. However, this concept may not be correct. Thus, when heparin is given in conjunction with plasminogen activators only modest increases in FPA levels are found, and our data suggest that this is due, at least in part, to the activity of enzymes other than thrombin. In the absence of concomitant heparin, thrombolytic therapy causes a much greater increase in the FPA values. These high FPA values are rapidly reduced by heparin, even though heparin has limited antithrombotic activity in the setting of pharmacologic thrombolysis. Based on these considerations, we believe that plasma FPA and desarginine FPB levels should not be used as specific markers of thrombin activity during the course of thrombolytic therapy and suppression of elevated FPA values by heparin should not be accepted as evidence that heparin is effective in this clinical setting.
Amino Acid Sequence↗
Evaluation of a soluble fibrin assay in patients with suspected pulmonary embolism.
In order to determine the clinical utility of an enzyme immunoassay (EIA) for soluble fibrin in patients with suspected pulmonary embolism (PE), 195 unselected patients with suspected PE underwent blood sampling for measurement of plasma levels of soluble fibrin, and objective testing for PE. A soluble fibrin result of < or = 0.75 micrograms/ml showed a sensitivity of 100% for PE and a specificity of 12.8%, whereas a soluble fibrin result of < or = 1.35 micrograms/ml showed a sensitivity of 90.3% and a specificity of 49.4% for PE. The soluble fibrin assay has potential clinical utility in excluding PE.
Adolescent↗
An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice.
Pinworm infection, a common problem in laboratory rodent colonies, is difficult to control because anthelmintics like ivermectin eliminate adult worms but have no effect on ova, which can survive ex vivo for prolonged periods. On the premise that repeated treatments with ivermectin would keep rodents parasite-free until all ova matured into ivermectin-susceptible worms in vivo or died in vivo or ex vivo, 80 rats and 25 mice heavily infected with pinworms (Syphacia obvelata and S. muris) were randomized to receive two to five courses of ivermectin 3 days apart or no treatment. During each treatment, ivermectin was given for 4 days in the drinking water; based on water consumption, the mean ivermectin dose was 2.9 and 4.0 mg/kg of body weight per day in rats and mice respectively. Ova production was monitored by weekly cellophane tape tests; 29 to 32 weeks after treatment ended, all rodents were euthanized, and their evacuated large intestinal contents were examined for adult pinworms and ova. Despite intermittently negative cellophane tape test results in untreated rodents (10 rats and 5 mice), all were infected with parasites at the end of the follow-up period. These findings underscore the limitations of the tape test for diagnosis of pinworm infection. After two courses of ivermectin, 1 of 10 rats and four of five mice were infected, whereas after three courses only 1 of 40 rats and one of five mice had parasites. In contrast, none of the 20 rats or 10 mice given either four or five courses of ivermectin had parasites at 30 to 32 weeks of follow-up evaluation. This simple and well-tolerated ivermectin regimen may help to treat and control pinworm infection in laboratory rodent colonies.
Administration, Oral↗
Biological rationale for the therapeutic role of specific antithrombins.
Explore the source record for details and available documents.
Animals↗
More effective suppression of hemostatic system activation in patients undergoing cardiac surgery by heparin dosing based on heparin blood concentrations rather than ACT.
This study was designed to determine whether the maintenance of higher than usual patient-specific heparin concentrations during cardiopulmonary bypass (CPB) was associated with more effective suppression of hemostasis system activation. Thirty-one patients scheduled for repeat cardiac surgery or combined procedures (i.e., coronary revascularization + valve repair/replacement) were consented and enrolled in this study. All patients received porcine heparin and protamine and were randomly assigned to monitoring of anticoagulation by either celite ACT alone (Control, n = 16) or by kaolin ACT combined with on-site measurements of whole blood heparin concentration (Intervention, n = 15). Blood specimens collected before administration of heparin, before weaning from CPB and after administration of protamine were analyzed with a battery of coagulation assays. Patients in the intervention cohort received appreciably greater heparin doses than control patients, resulting in higher anti-Xa heparin levels at the end of CPB. Fibrinopeptide A and D-dimer levels were higher in the control group before discontinuation of CPB. Percent decrease during CPB were greater in the control group for factors V and VIII, fibrinogen and antithrombin III. Percent decrease in complement 3 was greater in the control group after protamine and bleeding times measured in the Intensive Care Unit were significantly more prolonged in this group. Maintenance of higher patient-specific heparin concentrations during CPB more effectively suppresses excessive hemostatic system activation than do standard heparin doses chosen based on measurement of ACT. These findings may explain, at least in part, the significant reduction in perioperative blood loss and blood product use when higher heparin concentrations are maintained.
Aged↗