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J Fareed

Publications and source records attributed to J Fareed.

At least 163 records · Page 9Linked to original sources
PubMed1995

Studies on the thrombogenic effects of recombinant tissue factor. In vivo versus ex vivo findings.

An exposure of blood to tissue factor (TF) activates the coagulation system by the extrinsic pathway and may cause clot formation. Recombinant TF (r-TF) has been produced and subsequently reconstituted into phospholipid vesicles. The aim of these studies was to elucidate the in vitro procoagulant effects and the in vivo thrombogenicity of r-TF using a rabbit jugular vein stasis thrombosis model. The in vitro studies exhibited a clear concentration-dependent decrease in the clotting time when rabbit brain thromboplastin was replaced by r-TF in the prothrombin time assay. The in vivo studies revealed a dose-dependent thrombogenicity between 1.6 ng/kg and 50 ng/kg. Electron microscope scanning of the surface of representative clots revealed fibrin-rich structures of heterogeneous density. In comparison, thrombi obtained when FEIBA was utilized as the thrombogenic agent were more homogeneous. The injection of r-TF caused a slight transient drop in blood pressure with little or no effects on the pulse rate, complete blood count (CBC) profile, clotting and amidolytic assays when compared to sham control animals. In contrast, the whole blood clotting parameters (activated clotting time and thrombelastograph) were prolonged dose-dependently after r-TF injection. The antithrombotic activity of heparin was assessed in this model and compared to the antithrombotic activity when FEIBA is used as the thrombogenic agent. The apparent ED50 of heparin was found to be 4 times higher in the r-TF system. In control studies, no thrombogenic effects were observed by the phospholipid vesicles alone nor by r-TF not embedded in phospholipid vesicles. These data demonstrate that lipidated r-TF is a potent thrombogenic challenge that activates the hemostatic system by the extrinsic pathway.

Animals↗
PubMed1995

Comparative studies on the anticoagulant and protease generation inhibitory actions of newly developed site-directed thrombin inhibitory drugs. Efegatran, argatroban, hirulog, and hirudin.

Site-directed thrombin inhibitors are being currently assessed clinically for their antithrombotic efficacy. Although these agents are claimed to be specific and direct thrombin inhibitors, their mechanism of inhibition varies. The objective of these studies was to compare four such agents in in vitro systems and to assess their relative anticoagulant efficacy. The four agents utilized in these studies were argatroban, Efegatran, hirulog, and hirudin. While hirulog and hirudin are specific irreversible inhibitors of thrombin, argatroban and Efegatran are reversible. All four agents were found to have a concentration-dependent anticoagulant effect when supplemented into normal human plasma, as assessed in the global clotting tests (PT, APTT, and Heptest). The most potent anticoagulant on a molar basis was hirudin in all three tests. The other three agents had similar anticoagulant actions. All four agents were also capable of inhibiting the generation of thrombin and factor Xa as determined by an amidolytic method after intrinsic or extrinsic activation of fibrinogen-deficient human plasma. Except for hirulog, all agents inhibited the extrinsic generation of thrombin, with hirudin being the most potent agent. The intrinsic generation of thrombin was blocked by the reversible thrombin inhibitors (argatroban and Efegatran) but not by the irreversible inhibitors (hirulog and hirudin). While all agents were capable of inhibiting the intrinsic generation of factor Xa at very low concentrations, only Efegatran was capable of blocking the extrinsic generation of the same factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Development and validation of two enzyme-linked immunosorbent assay (ELISA) methods for recombinant hirudin.

Recombinant hirudin is currently being developed as a potential prophylactic and therapeutic antithrombotic drug in various clinical indications such as angina and deep venous thrombosis. In this report, we have discussed the production of specific polyclonal antibodies to recombinant hirudin (rH) and the development of two ELISA methods to measure rH concentrations in biological fluids: a sandwich and a competitive ELISA method. Intra- and inter-assay variations in the two methods are extremely low (3-7%). The competitive ELISA method is rapid, simple and highly reproducible. Saturation binding curves, selection of appropriate incubation times, recovery of different hirudin variants and reactivity in the presence of thrombin are discussed. The methods can be easily adapted to monitor hirudin concentrations in the clinical laboratory for diagnostic purposes as well as for performing pharmacokinetic studies.

Antibody Specificity↗
PubMed1995

Antithrombin III affinity dependence on the anticoagulant, antiprotease, and tissue factor pathway inhibitor actions of heparins.

To investigate AT-III affinity dependence on heparin's actions, heparin (UH) was fractionated on an AT-III-Sepharose column into a high (HAH) and a low affinity (LAH) fraction. Molecular profiling revealed a molecular weight of 11.8 kDa for (UH), 12.6 kDa for HAH, and 10.6 kDa for LAH. The USP anticoagulant potencies were found to be: UH = 160 U/mg, HAH = 198 U/mg, and LAH = 42 U/mg. The anticoagulant effects of each of these fractions were proportionate to the USP potencies. However, protease generation inhibitory activities did not follow the same order. All fractions were also tested for their interactions with tissue factor pathway inhibitor (TFPI). No significant differences were noted on the anti-Xa effects of TFPI with these fractions. Administration of each fraction to primates resulted in equivalent release of TFPI. In a model of jugular vein clamping induced venous occlusion, all agents produced a dose-dependent antithrombotic action. HAH produced a 60 to 70% stronger antithrombotic effect than LAH or UH. Simultaneous administration of TFPI markedly augmented the antithrombotic actions of both UFH and LAH. The effect of TFPI on the antithrombotic activity of HAH was weaker than that on LAH. These observations suggest that AT-III affinity is not the sole determinant of the antithrombotic actions of heparin. The endogenous release of TFPI may contribute to the antithrombotic actions of heparin and related glycosaminoglycans. Furthermore, TFPI is capable of AT-III independent amplification of the antithrombotic actions of both UH and LAH, suggesting a crucial role of this polyvalent inhibitor in the control of thrombogenesis.

Animals↗
PubMed1995

Pharmacologic profile of a low-molecular-weight heparin depolymerized by gamma-irradiation.

Low molecular weight heparins (LMWHs) are considered to be the agent of choice for the prophylaxis of DVT in medical and surgical patients. Conventionally, these agents have been produced by fractionation of or by chemical or enzymatic depolymerization of native heparin. The fractionated heparin retains many of its biological properties such as AT III affinity and sulfate content gamma-irradiation (60Co) has been used to depolymerize GAGs (De Ambrosi et al. In: biomedical and Biotechnological Advances in Industrial Polysaccharides, pp. 45-53). This procedures has now been used for the preparation of LMWH derivatives of varying molecular weight. The current studies examine the biochemical and pharmacologic profile of one such gamma-irradiated depolymerized heparin. In standard clotting and amidolytic antiprotease assays (PT, APTT, AXa, Alla), gamma-irradiated depolymerized heparin produced equal or stronger activity when compared to a LMWH produced by nitrous acid depolymerization and retained the ability to active AT III and HCHII. Initial results indicate that LMWHs produced by gamma-irradiation exhibit comparable antithrombotic actions to those produced by chemical depolymerization when measured in animal models of thrombosis. gamma-Irradiation may be a useful method for the production of LMWHs.

Animals↗
PubMed1995

Pharmacologic validation of the clinical effects of an optimized low-molecular-weight heparin-reviparin.

Reviparin is a low-molecular-weight heparin (LMWH) prepared by controlled nitrous acid digestion of porcine mucosal heparin. The trade name designation for this agent is Clivarin. This agent has been released in Germany and France for the prophylaxis of deep venous thrombosis (DVT) in surgical patients. This agent is developed utilizing optimized procedures and exhibits a uniform, narrow-molecular-weight distribution in comparison to the other commercially available LMWHs. The specific activity in the anticoagulant assays is approximated to be 32 U/mg whereas the specific activity in terms of anti-Xa units is designated 120 aXa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects are initially presented at antithrombotically active dosages, this agent has been found to produce antithrombotic effects without any detectable ex vivo actions. This agent is also known to release tissue factor pathway inhibitor (TFPI) after both intravenous (IV) and subcutaneous (SC) administration. Repeated administration of Reviparin produces progressively stronger antithrombotic effects. Similarly, the bleeding as measured by rabbit ear blood loss is also progressively increased. However, the ratio between the dosage producing these effects is quite large. The current studies are designed to provide additional data on the molecular profile using new calibration methods and additional results on the pharmacologic studies in a dose-dependent manner. In particular, the release of TFPI following IV and SC administration in a primate model is described. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT is also reported in terms of any augmentation of the antithrombotic or hemorrhagic effects of these agents.

Animals↗
PubMed1995

Evaluation of hemostatic and fibrinolytic alterations associated with daily administration of low-molecular-weight heparin for a 12-week period.

The PTCA procedure fails in 30-50% of patients due to late restinosis, meaning that this is a problem of 100,000-150,000 people per year in the USA alone. It has been found that heparin and low-molecular-weight heparin (LMWH) have an inhibitory effect on smooth muscle cell (SMC) proliferation and migration, the major causes of restinosis. However, little is known about the toxicity and side effects of these drugs when used for a long period as may be required for prophylaxis of PTCA restinosis. To investigate possible side effects, non-human primates received daily injections of 1 mg/kg s.c. LMWH (Mono-Embolex) over a 12-week period. The hemostatic system was monitored through measurement of ACT-celite, APTT, Heptest, TT(10U/mL), TFPI, Anti-IIa activity, Anti-Xa activity, ACA-Heparin, AT III, factor VIII R:Ag, fibrinogen, and thrombomodulin levels. Elisa tests for t-PA, PAI-1, u-PA, FgDP, TDP, and D-Di levels were used for measurements of fibrinolytic activity. Increased values of ACT, APTT, Heptest, TT, Anti-IIa, Anti-Xa, ACA-Heparin, and TFPI were observed four hours after LMWH injections. AT III, vWFAg, fibrinogen and thrombomodulin showed no change from the pre-study baseline. An accumulation effect was seen in the APTT and Heptest over the 12 weeks. After the first week the blood levels of Anti-IIa activity remained elevated at 20% inhibition rather than 0% 24 hrs after drug administration. This activity slowly decreased after discontinuation of drug. The Anti-Xa blood level activity remained elevated at 40% inhibition 24 hrs after drug administration 2 weeks into the study, and this activity was detectable even 2 weeks after cessation of drug administration. There was increasing activity of the fibrinolytic system with LMWH treatment. After two weeks t-PA increased two-fold to 6 ng/mL but returned to baseline at six weeks. There was a corresponding increase of the TDP but not a clear increase in D-Di and FgDP. The increase of u-PA was limited to the first days of LMWH treatment only. The PAI-1 activity increased gradually over the entire study period. No bleeding complications occurred throughout the study. The long-term administration of Mono-Embolex as projected for the use in the prophylaxis of restinosis following PTCA appears to be safe for patients.

Animals↗
PubMed1995

Effect of repeated Aprosulate and Enoxaparin administration on tissue factor pathway inhibitor antigen levels.

Tissue factor pathway inhibitor (TFPI) is a naturally occurring, Kunitz-type serine protease inhibitor whose anticoagulant activity is due to an inhibition of the extrinsic coagulation pathway. Heparin injection has previously been shown to increase the plasma levels of TFPI. In this study, plasma samples were obtained from a multiple dose phase I tolerance study with a synthetic analogue of heparin, namely Aprosulate (PALLAS). Volunteers were randomized into four treatment groups: (A) 35 mg Aprosulate b.i.d. s.c.; (B) 70 mg Aprosulate b.i.d. s.c.; (C) 70 mg Aprosulate o.d. + placebo o.d. s.c.; (D) 40 mg of a low molecular weight heparin, Enoxaparin o.d. + placebo o.d. s.c. All treatments were for 7 days, with blood samples taken periodically over this time period. TFPI antigen levels were determined using Imubind TFPI ELISA kits (American Diagnostica, Greenwich, CT). TFPI antigen levels were observed to rapidly increase to levels two- to three-fold over baseline in all groups. Aprosulate caused a slightly larger increase in TFPI antigen levels than Enoxaparin, though this may be related to the doses chosen for this study. These data indicate that plasma concentrations of TFPI are increased following Aprosulate administration. TFPI may be important in mediating the antithrombotic activity of Aprosulate.

Analysis of Variance↗
PubMed1995

Tissue factor antigen levels in various biological fluids.

Tissue factor (TF), a transmembrane surface protein, is known to initiate thrombogenesis through plasmatic and cellular activation processes. Besides complexing with factor VII, eventually leading to fibrin generation via the extrinsic pathway, TF can also activate factor IX, resulting in the intrinsic activation of coagulation. Other functions of TF are currently unknown, although various cells are believed to have TF receptors. Many of the post-surgical and post-interventional thrombotic events are due to the release of TF. Increased levels of TF are associated with several pathologic conditions such as cancer, sepsis and inflammation. Cellular necrosis also results in an increase of TF as the cells in the traumatized area lyse and release endogenous cell surface-bound TF. An ELISA method (American Diagnostica, Greenwich, CT) has been developed to assay TF antigen levels in various biological fluids. This ELISA employs a murine monoclonal antibody raised against native human TF for antigen capture. In this study, cerebrospinal fluid, peritoneal fluid, pleural effusion and urine from patients were assayed for their TF content using this ELISA method. Normal individual serum and plasma were also assayed as controls against which the levels of TF in the patients' body fluids could be compared. The amount of TF antigen in normal human plasma and serum was 165 +/- 139 pg/ml and 165 +/- 110 pg/ml, respectively. Concentrations of TF antigen in other fluids were: cerebrospinal fluid 868 +/- 721 pg/ml, peritoneal fluid 124 +/- 247 pg/ml, pleural effusion 385 +/- 569 pg/ml, synovial fluid 97 +/- 23 pg/ml, seminal plasma 11,485 +/- 875 pg/ml and urine 86 +/- 57 pg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The role of tissue factor pathway inhibitor in the mediation of the antithrombotic actions of heparin and low-molecular-weight heparin.

It is widely accepted that antithrombin III (ATIII) mediated anti-Xa and anti-IIa effects are the sole determinant of the antithrombotic actions of unfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs). However, there are several unexpected observations such as the greater than 100% bioavailability of subcutaneously administered LMWH as measured by a chromogenic based anti-Xa method. The authors have proposed that, besides ATIII mediated antiprotease actions, additional endogenous factors may be responsible for the observed therapeutic and prophylactic actions of heparins. With the identification of tissue factor pathway inhibitor (TFPI) some of the unexpected effects of heparins can now be clarified. To investigate the role of heparin-releasable TFPI on LMWHs the anti-Xa and TFPI antigen levels after prophylactic and therapeutic administration of UFH and LMWHs have been studied in defined clinical trials. Regardless of the dosage designation (mg/kg or units/kg) each LMWH followed a distinct TFPI release profile. Similarly, in the intravenous studies these LMWHs produced an instantaneous increase in the TFPI antigen level. The anti-Xa effects did not always follow the same pattern as the TFPI antigen levels. These data suggest that the anti-Xa potency of a given LMWH is not the sole determinant of the antithrombotic actions of heparin and LMWH. In addition to pharmacologic agents, the effect of sequential compression devices (SCD) on the release of TFPI was also studied. A two-fold increase in TFPI antigen levels was observed in normal volunteers undergoing long leg compression for 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Enoxaparin↗
PubMed1995

Measurement of functional and immunologic levels of tissue factor pathway inhibitor. Some methodologic considerations.

Tissue factor pathway inhibitor (TFPI) is a newly identified inhibitor of proteases generated during activation processes. Several functional methods based on a chromogenic substrate technique measuring residual tissue thromboplastin/FVIIa catalytic activity using excess factor X and a chromogenic substrate for FXa have been published. Recently, a sandwich ELISA method has become available. A modified functional method was compared with the ELISA based antigen method in several groups of normal individuals. The functional assay is sensitive to TFPI concentrations of 125-200 ng/ml based on a rTFPI standard. The immunologic method is sensitive from 0 to 400 ng/ml. A marked dichotomy was observed between the two methods. The ratio between TFPI antigen and functional levels varies widely in patient groups. This TFPI standard was supplemented with buffer and normal human plasma and functional TFPI was measured before and after heat treatment at 56 degrees C for 10 min. Heat treatment of plasma (buffer) after the addition of TFPI resulted in decreased functional TFPI compared with the addition of TFPI after the plasma (buffer) was heat treated. In contrast, the immunologic method does not require heat treatment. Addition of exogenous TFPI to plasmas obtained from normal and the above patient plasmas demonstrated that recovery of functional TFPI activity was plasma dependent. Plasma proteins markedly influenced functional and antigenic levels of TFPI. The TFPI standard added to human pooled plasma gave varying recoveries in the immunologic and functional assays. These results indicate that currently available methods may provide highly variable results on TFPI levels. Several matrix related effects should be taken into account for proper evaluation of TFPI.

Buffers↗
PubMed1995

Flow cytometric evaluation of platelet activation by ionic or nonionic contrast media and modulation by heparin and recombinant hirudin.

RATIONALE AND OBJECTIVES: The purpose of this study was to investigate the platelet activation properties of ionic and nonionic contrast media in native heparinized or hirudinized human blood using flow cytometric analysis techniques. MATERIALS AND METHODS: Freshly drawn human blood samples were incubated at 37 degrees C with ionic (Hexabrix and Angiovist) and non-ionic (Isovue and Omnipaque) contrast media for varying periods of time. Using fluorescent monoclonal antibodies to platelet surface receptors glycoprotein IIIa and with granule membrane protein-140, platelet activation was measured in whole blood using flow cytometry. Platelet factor 4 and thromboxane B2 levels also were measured. RESULTS: Dose- and time-dependent platelet activation was observed in nonionic, but not in ionic, contrast media (P < 0.05). Whole blood drawn into heparin or hirudin showed reduced platelet activation in the presence of contrast media compared with nonanticoagulated whole blood. Platelet factor 4 and thromboxane B2 levels showed similar results. Significant platelet activation was seen by flow cytometry in high (2000 mOsmol/kg), but not in lower osmolality (800 mOsmol/kg) controls for ionic and nonionic contrast media, respectively (P < 0.001). CONCLUSION: This study suggests that nonionic contrast media use is associated with significant levels of platelet activation, and that heparin or recombinant hirudin (where heparin is contraindicated) is preferable for use with nonionic contrast media, as these anticoagulants reduce contrast media induced platelet activation.

Contrast Media↗
PubMed1995

Successful treatment of heparin-associated thrombocytopenia and thrombosis using Hirulog.

Heparin-associated thrombocytopenia is a serious medical problem, especially when the patient requires continued anticoagulation. Hirulog is an immediate-acting intravenous anticoagulant that can be substituted for heparin. A new use of Hirulog in the treatment of life-threatening heparin-associated thrombocytopenia with thrombosis (HATT) is presented. Two patients suffering from the HATT syndrome were successfully treated with Hirulog to prevent further thrombosis. A third patient had developed heparin-associated thrombocytopenia after coronary artery bypass surgery in the past and was subsequently treated with Hirulog during a peripheral angioplasty procedure. Hirulog was an effective and predictable anticoagulant for these patients and was free from adverse effects.

Aged↗
PubMed1995

Molecular markers of hemostatic activation. Implications in the diagnosis of thrombosis, vascular, and cardiovascular disorders.

Until recently, the diagnosis of thromboembolic disorders remained difficult to establish before the occurrence of a pathologic event. Clearly, thrombosis is the result of a progressive alteration of the blood and vasculature. Various molecular markers of hemostatic alteration are found in increased or decreased amounts predisposing to thrombosis or in increased circulating amounts during the activation process.

Biomarkers↗
PubMed1995

Comparative pharmacology of site directed antithrombin agents. Implication in drug development.

Beside the direct inhibition of thrombin and its regulatory functions, many of the newer antithrombin agents produce several additional effects, unrelated to their anticoagulant actions. Synthetic peptide inhibitors are capable of producing fibrinolytic compromise by virtue of their actions on fibrinolytic enzymes such as t-PA, plasmin, urokinase and protein Ca. In addition, the low molecular weight arginine-containing peptides are also known to produce hemodynamic and hemostatic deficits. The designs of the ongoing clinical trials are largely empirical because of the non-availability of valid pharmacologic and toxicologic data on thrombin inhibitors. In contrast to heparin, none of the thrombin inhibitors produce endogenous release of tissue factor pathway inhibitor (TFPI) in the experimental and clinical settings. These observations suggest that beside the direct inhibition of thrombin, these agents also produce multiple additional effects that can significantly contribute to their pharmacologic and toxicologic profile.

Amino Acid Sequence↗
PubMed1994

Inhibitory effects of TFPI variants on thrombin and factor Xa generation in fibrinogen-deficient human plasma.

Using a fast kinetic centrifugal analyzer, the inhibitory effects of glycosylated and unglycosylated full-length and truncated forms of TFPI on protease generation were studied in fibrinogen-deficient human plasma after extrinsic (EA) or intrinsic (IA) activation of coagulation. When the assay system was supplemented with increasing amounts of the TFPI variants the generation of both thrombin and factor Xa was inhibited in a concentration-dependent manner. Clear differences in the effectiveness of the TFPI variants were found. After EA, the unglycosylated full-length TFPI was most effective followed by the glycosylated full-length form. The C-terminal truncated TFPI showed the lowest inhibitory activity in this system. However, its efficiency increased several fold when coagulation was activated via the intrinsic pathway. Comparing the IC50 values after IA, the truncated TFPI was more effective than the unglycosylated full-length form and nearly as effective as the glycosylated full-length TFPI. After both EA and IA the thrombin generation inhibition by TFPI variants was more pronounced than the inhibition of factor Xa generation. The results show that chemical modifications of the TFPI structure can result in changes of TFPI's inhibitory properties to activated clotting factors leading to differences in protease generation inhibition.

Afibrinogenemia↗
PubMed1994

Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro--modulatory action of glycosaminoglycans.

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC50 255 ng/ml) was more pronounced than that of factor Xa (IC50 684 ng/ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 micrograms/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3-40 micrograms/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40-50% was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.

Animals↗