Search PubMed⌕ Search

Biomedical subjects

J I Weitz

Publications and source records attributed to J I Weitz.

At least 55 records · Page 3Linked to original sources
PubMed1999

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II.

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.

Anticoagulants↗
PubMed1999

A histomorphometric evaluation of heparin-induced bone loss after discontinuation of heparin treatment in rats.

Although it is well established that long-term heparin therapy causes osteoporosis, it is unknown whether heparin-induced bone loss is reversible when heparin treatment is stopped. To address this question, we randomized rats to once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g) or saline for 28 days and then followed the rats for an additional 28 days off treatment. Based on histomorphometric analysis of the distal third of the right femur proximal to the epiphyseal growth plate, 1.0 U/g heparin caused a 30% loss in cancellous bone volume over the first 28 days. This was accompanied by a 137% increase in osteoclast surface and a 60% decrease in both osteoblast and osteoid surface. One month after cessation of heparin treatment, no recovery in these parameters was observed. Similarly, serum levels of alkaline phosphatase, a biochemical marker of bone formation, which continued to decrease over the course of heparin treatment, showed no signs of recovery in the subsequent 28 days off treatment. To explore the mechanism responsible for the prolonged effect of heparin on bone, we repeated the experiment giving 125I-labeled heparin in place of unlabeled heparin. 125I-labeled heparin was found to accumulate in bone during the course of its administration, and be retained in bone for at least 56 days after stopping heparin treatment. These findings suggest that heparin-induced osteoporosis is not rapidly reversible because heparin is sequestered in bone for an extended period.

Animals↗
PubMed1999

Vasoflux, a new anticoagulant with a novel mechanism of action.

BACKGROUND: Heparin and direct thrombin inhibitors, such as hirudin, have limitations in the treatment of acute coronary syndromes. Heparin does not inactivate fibrin-bound thrombin, whereas hirudin fails to block thrombin generation. In contrast, Vasoflux is a novel anticoagulant that inactivates fibrin-bound thrombin and attenuates factor Xa generation. METHODS AND RESULTS: Vasoflux is prepared by depolymerization of heparin, restricting molecular size to between 3000 and 8000 Da, and reducing antithrombin affinity by periodate oxidation. Vasoflux catalyzes fibrin-bound thrombin inactivation by heparin cofactor II (HCII) and inhibits factor IXa activation of factor X independently of antithrombin and HCII. Compared with other anticoagulants in a thrombogenic extracorporeal circuit, Vasoflux maintains filter patency at concentrations that produce an activated clotting time (ACT) of 220 seconds. In contrast, to maintain filter patency, heparin, low-molecular-weight heparin (LMWH), and hirudin require concentrations that produced an ACT of 720, 415, and >1500 seconds, respectively, whereas dermatan sulfate was ineffective at concentrations that produced an ACT of 360 seconds. CONCLUSIONS: Vasoflux is more effective than heparin and LMWH because it inactivates fibrin-bound thrombin and is superior to hirudin and dermatan sulfate because it also blocks factor Xa generation.

Anticoagulants↗
PubMed1999

Prevention of activation of blood coagulation during acute coronary ischemic syndromes: beyond aspirin and heparin.

Many of the acute coronary ischemic syndromes are triggered by spontaneous or mechanical disruption of atherosclerotic plaques with resultant activation of platelets and coagulation. Given the central role of platelets and thrombin in arterial thrombosis, current strategies for its prevention and treatment focus on both inhibition of platelet aggregation and control of thrombin generation and activity. Although aspirin and unfractionated heparin are the cornerstones of current treatment strategies, both have limitations. This review will describe these limitations and discuss new antithrombotic agents developed for use in acute coronary ischemic syndromes and as adjuncts for percutaneous coronary revascularization procedures.

Anticoagulants↗
PubMed1999

Identification of a unique glomerular factor X activator in murine lupus nephritis.

The role of glomerular procoagulant activity (PCA) was studied in mice (MRL/lpr, NZBxWF,, and BXSB) that are known to develop lupus nephritis. In young mice (6 to 8 wk) without renal disease, there was no increase in spontaneous glomerular PCA. In contrast, older (5 to 8 mo) autoimmune mice had significant augmentation in glomerular PCA, coinciding with the histologic appearance of severe glomerulonephritis and renal fibrin deposition. The PCA was characterized as a serine protease that directly activated factor X. This factor X activator is not tissue factor because (1) expression of PCA was not dependent on factor VII; (2) a monoclonal antibody against the factor X activator inhibited glomerular PCA, but not tissue factor; (3) the molecular weight (66 kD) of the activator was different from that of tissue factor; and (4) concanavalin A inhibited tissue factor but not glomerular PCA. Immunohistochemical studies localized the factor X activator to the glomerular mesangium and capillary wall of 4- to 6-mo-old diseased MRL/lpr mice. Immunogold-labeled antibody bound to the dense deposits, macrophages, and endothelial cells of diseased glomeruli. These studies define the role of a unique glomerular factor X activator in murine lupus nephritis.

Animals↗
PubMed1999

Thrombosis and anticoagulation.

Most of the major advances in thrombosis research have occurred in the last 50 years, reflecting progress in biomedical sciences and clinical trials methodology. Improved understanding of the mechanisms of thrombogenesis has led to the discovery of a plethora of new antithrombotic agents that target many of the key steps in blood coagulation and platelet activation. Although most of these compounds are still under development, low-molecular-weight heparins (LMWH), glycoprotein (GP) IIb/IIIa receptor antagonists, and inhibitors of the adenosine diphosphate (ADP) receptor on platelets have already established their niche in the clinic. The vessel wall has emerged as a major player, both in protecting against and in promoting thrombosis, and as we approach the new millennium, compounds are being developed that have the potential to prevent and treat thrombosis by modulating vessel wall function.

Anticoagulants↗
PubMed1998

Sensitivity and specificity of a rapid whole-blood assay for D-dimer in the diagnosis of pulmonary embolism.

BACKGROUND: Patients with suspected pulmonary embolism often have nondiagnostic lung scans and may present in circumstances where lung scanning is unavailable. Levels of D-dimer, a fibrin-specific product, are increased in patients with acute thrombosis; this may simplify the diagnosis of pulmonary embolism. OBJECTIVE: To determine the sensitivity and specificity of a whole-blood D-dimer assay in patients with suspected pulmonary embolism and in subgroups of patients with low pretest probability of pulmonary embolism or nondiagnostic lung scans. DESIGN: Prospective cohort. SETTING: Four tertiary care hospitals. PATIENTS: 1177 consecutive patients with suspected pulmonary embolism. MEASUREMENTS: All patients underwent an assessment of pretest probability by use of a standardized clinical model, a D-dimer assay, ventilation-perfusion lung scanning, and bilateral compression ultrasonography. Patients in whom pulmonary embolism was not initially diagnosed were followed for 3 months. Accordingly, patients were categorized as positive or negative for pulmonary embolism. RESULTS: Of the 1177 patients, 197 (17%) were classified as positive for pulmonary embolism. Overall, the D-dimer assay showed a sensitivity of 84.8% and a specificity of 68.4%. In 703 patients (3.4%) with a low pretest probability of pulmonary embolism, the likelihood ratio associated with a negative D-dimer test result was 0.27, resulting in a posterior probability of 1.0% (95% CI, 0.3% to 2.2%). In 698 patients with nondiagnostic lung scans (previous probability, 7.4%), the likelihood ratio associated with a negative D-dimer test result was 0.36, resulting in a posterior probability of 2.8% (CI, 1.4% to 4.8%). CONCLUSIONS: A normal D-dimer test result is useful in excluding pulmonary embolism in patients with a low pretest probability of pulmonary embolism or a nondiagnostic lung scan.

Algorithms↗
PubMed1998

Homocysteine-dependent alterations in mitochondrial gene expression, function and structure. Homocysteine and H2O2 act synergistically to enhance mitochondrial damage.

Mitochondrial abnormalities have been identified in hepatocytes of patients with hyperhomocysteinemia and in endothelial cells from the aortas of rats with diet-induced hyperhomocysteinemia. However, the mechanism by which homocysteine affects mitochondria is unknown. In this report, homocysteine-induced expression of the mitochondrial electron transport chain gene, cytochrome c oxidase III/ATPase 6,8 (CO3/ATPase 6,8), was identified in a human megakaryocytic cell line DAMI using mRNA differential display. Steady-state mRNA levels of CO3/ATPase 6,8, as well as other mitochondrial transcripts, were increased in DAMI cells by homocysteine in a concentration- and time-dependent manner. Despite an increase in mitochondrial RNA levels and changes in mitochondrial ultrastructure, no effect on either cell growth or mitochondrial respiration rates was observed in DAMI cells exposed to homocysteine at concentrations up to 1 mM. In contrast, 1 mM homocysteine in the presence of Cu2+, which is known to generate H2O2, significantly decreased mitochondrial RNA levels, caused gross morphological changes in mitochondrial ultrastructure, and inhibited both cell growth and mitochondrial respiration rates. However, precursors of cellular glutathione and preexposure to heat shock blocked the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The observations that (i) homocysteine and H2O2, but not H2O2 alone, caused a decrease in mitochondrial RNA levels, (ii) intracellular levels of H2O2 were significantly increased in the presence of homocysteine and Cu2+, and (iii) catalase, but not free radical scavengers, prevented a decrease in mitochondrial RNA levels, provide evidence that homocysteine and H2O2 act synergistically to cause mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione and heat shock proteins play a role in protecting mitochondria against the adverse effects elicited by homocysteine and H2O2.

Animals↗
PubMed1998

Direct thrombin inhibitors for treatment of arterial thrombosis: potential differences between bivalirudin and hirudin.

Given the central role of thrombin in arterial thrombogenesis, most treatment strategies for acute coronary syndromes are aimed at inhibiting its generation or blocking its activity. Although heparin has been widely used, it has limitations in the setting of arterial thrombosis. These limitations reflect the inability of heparin to inactivate thrombin bound to fibrin, a major stimulus for thrombus growth. In addition, the anticoagulant response to heparin varies from patient to patient, and heparin is neutralized by platelet Factor IV, large quantities of which are released from platelets activated at sites of plaque rupture. Consequently, heparin requires careful laboratory monitoring to ensure an adequate anticoagulant effect. Direct thrombin inhibitors, such as hirudin and bivalirudin, overcome the limitations of heparin. These agents inhibit fibrin-bound thrombin, as well as fluid-phase thrombin, and produce a predictable anticoagulant response. Bivalirudin has both safety and potential efficacy advantages over hirudin. Bivalirudin appears to have a wider therapeutic window than hirudin, possibly because bivalirudin only transiently inhibits the active site of thrombin. The better safety profile of bivalirudin permits administration of higher doses, which may give it an efficacy advantage. Hirudin prevents thrombin from activating protein C, thereby suppressing this natural anticoagulant pathway. In contrast, bivalirudin may promote protein C activation by transiently inhibiting thrombin until it can be bound by thrombomodulin. Differences between bivalirudin and hirudin, as well as other direct thrombin inhibitors, highlight the pitfalls of considering all direct thrombin inhibitors to have equivalent risk-benefit profiles.

Acute Disease↗
PubMed1998

Long-term oral anticoagulant therapy in patients with unstable angina or suspected non-Q-wave myocardial infarction: organization to assess strategies for ischemic syndromes (OASIS) pilot study results.

BACKGROUND: Patients with acute ischemic syndromes (AIS) suffer high rates of recurrent ischemic events despite aspirin treatment. Long-term therapy with oral anticoagulants in addition to aspirin may reduce this risk. We studied the effects of long-term warfarin at 2 intensities in patients with AIS without ST elevation in 2 consecutive randomized controlled studies. METHODS AND RESULTS: In phase 1, after the cessation of 3 days of intravenous antithrombotic therapy, 309 patients were randomized to receive fixed low-dose (3 mg/d) warfarin for 6 months that produced a mean international normalized ratio (INR) of 1.5+/-0.6 or to standard therapy. Eighty-seven percent of patients received aspirin in both groups. The rates of cardiovascular (CV) death, new myocardial infarction (MI), and refractory angina at 6 months were 6.5% in the warfarin group and 3.9% in the standard therapy group (relative risk [RR], 1. 66; 95% CI, 0.62 to 4.44; P=0.31). The rates of death, new MI, and stroke were 6.5% in the warfarin group and 2.6% in the standard therapy group (RR, 2.48; 95% CI, 0.80 to 7.75; P=0.10). The overall rate of rehospitalization for unstable angina was 21% and did not differ significantly between the groups. Four patients in the warfarin group (2.6%) and none in the control group experienced a major bleed (RR, 2.48; 95% CI, 0.80 to 7.75), and there was a significant excess of minor bleeds in the warfarin group (14.2% versus 2.6%; RR, 5.46; 95% CI, 1.93 to 15.5; P=0.001). In phase 2, the protocol was modified, and 197 patients were randomized <48 hours from the onset of symptoms to receive warfarin at an adjusted dose that produced a mean INR of 2.3+/-0.6 or standard therapy for 3 months. Eighty-five percent received aspirin in both groups. The rates of CV death, new MI, and refractory angina at 3 months were 5. 1% in the warfarin group and 12.1% in the standard group (RR, 0.42; 95% CI, 0.15 to 1.15; P=0.08). The rates of all death, new MI, and stroke were 5.1% in the warfarin group and 13.1% in the standard therapy group (RR, 0.39; 95% CI, 0.14 to 1.05; P=0.05). Significantly fewer patients were rehospitalized for unstable angina in the warfarin group than in the control group (7.1% and 17.2%, respectively; RR, 0.42; 95% CI, 0.18 to 0.96; P=0.03). Two patients in the warfarin group and 1 in the control group experienced a major bleed, and there was a significant excess of minor bleeds in the warfarin group (28.6% versus 12.1%; RR, 2.36; 95% CI, 1.37 to 4.36; P=0.004). CONCLUSIONS: Long-term treatment with moderate-intensity warfarin (INR, 2.0 to 2.5) plus aspirin but not low-intensity warfarin (INR, 1.5) plus aspirin appears to reduce the rate of recurrent ischemic events in patients with AIS without ST elevation.

Administration, Oral↗
PubMed1998

Characterization of the interactions of plasminogen and tissue and vampire bat plasminogen activators with fibrinogen, fibrin, and the complex of D-dimer noncovalently linked to fragment E.

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin with Kd values of 13 and 0.13 microM, respectively. t-PA binds fibrin through two classes of sites with Kd values of 0.05 and 2.6 microM, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with epsilon-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a Kd of 0.15 microM. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with a Kd of 5.4 microM. Lys-Pg binds to (DD)E and Fg with Kd values of 0.03 and 0.23 microM, respectively. t-PA binds to (DD)E and Fg with Kd values of 0.02 and 0.76 microM, respectively; interactions were eliminated with epsilon-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.

Aminocaproic Acid↗
PubMed1998

Characterization of the stress-inducing effects of homocysteine.

The mechanism by which homocysteine causes endothelial cell (EC) injury and/or dysfunction is not fully understood. To examine the stress-inducing effects of homocysteine on ECs, mRNA differential display and cDNA microarrays were used to evaluate changes in gene expression in cultured human umbilical-vein endothelial cells (HUVEC) exposed to homocysteine. Here we show that homocysteine increases the expression of GRP78 and GADD153, stress-response genes induced by agents or conditions that adversely affect the function of the endoplasmic reticulum (ER). Induction of GRP78 was specific for homocysteine because other thiol-containing amino acids, heat shock or H2O2 did not appreciably increase GRP78 mRNA levels. Homocysteine failed to elicit an oxidative stress response in HUVEC because it had no effect on the expression of heat shock proteins (HSPs) including HSP70, nor did it activate heat shock transcription factor 1. Furthermore homocysteine blocked the H2O2-induced expression of HSP70. In support of our findings in vitro, steady-state mRNA levels of GRP78, but not HSP70, were elevated in the livers of cystathionine beta-synthase-deficient mice with hyperhomocysteinaemia. These studies indicate that the activation of stress response genes by homocysteine involves reductive stress leading to altered ER function and is in contrast with that of most other EC perturbants. The observation that homocysteine also decreases the expression of the antioxidant enzymes glutathione peroxidase and natural killer-enhancing factor B suggests that homocysteine could potentially enhance the cytotoxic effect of agents or conditions known to cause oxidative stress.

Animals↗
PubMed1998

Localization of the thrombin-binding domain on prothrombin fragment 2.

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.

Amino Acid Sequence↗
PubMed1998

Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors.

BACKGROUND: Thrombolytic therapy induces a procoagulant state characterized by elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is uncertain. METHODS AND RESULTS: Washed plasma clots were incubated in citrated plasma in the presence or absence of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is progressive FPA generation even after clots are removed from t-PA-containing plasma, and (b) clot lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using thrombin-agarose affinity chromatography, (DD)E and fragment E but not D-dimer were identified as the thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free thrombin. In contrast, D-Phe-Pro-ArgCH2Cl, hirudin and hirugen inhibit free thrombin and thrombin bound to fibrin degradation products equally well. CONCLUSIONS: Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA-induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy.

Analysis of Variance↗
PubMed1998

Hirudin causes more bleeding than heparin in a rabbit ear bleeding model.

This study was undertaken to determine the appropriateness of the current practice of using the activated partial thromboplastin time (APTT) to select hirudin doses. A rabbit bleeding ear model was used to compare the effects of various doses of heparin and hirudin on the relationship between the APTT and bleeding. In addition, the effects of these agents on the thrombin clotting time (TCT) and factor Xa clotting time also were examined. Both heparin and hirudin produced a concentration-dependent increase in bleeding. When bleeding was plotted as a function of APTT ratio, even a small increase in APTT ratio within the therapeutic range of 1.5 to 2.5 resulted in a marked increase in bleeding with hirudin but not with heparin. The TCT was more responsive than the APTT or factor Xa clotting time to increases in hirudin-induced bleeding. In this model, hirudin produces more bleeding than heparin when the agents are used in doses that increase the APTT ratio to the same extent. These studies highlight the pitfalls of extrapolating from experience with heparin when choosing a test to monitor new antithrombotics. Our findings also suggest that the TCT may be more responsive than the APTT for monitoring hirudin therapy.

Animals↗