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Biomedical subjects

D Hoppensteadt

Publications and source records attributed to D Hoppensteadt.

At least 19 recordsLinked to original sources
PubMed1996

Preclinical studies on a low molecular weight heparin.

Some major developments in the area of antithrombotic therapy have occurred during the past decade. Of these, the concept of fractionation of heparin has resulted in the development of several products from this agent. The introduction of low molecular weight heparins (LMWHs) has added a new chapter to the prophylactic and therapeutic management of thromboembolic disorders. These agents are now globally accepted as drugs of choice for post-surgical prophylaxis of deep vein thrombosis (DVT). Currently, the LMWHs are being developed for various therapeutic and cardiovascular indications. Reviparin is an optimized LMWH prepared by controlled nitrous acid digestion of porcine mucosal heparin. This drug has been developed using validated procedures and exhibits a relatively narrow molecular weight distribution in contrast to most other commercially available LMWHs. The specific activity in anticoagulant assays is approximately 32 U/mg whereas the specific activity in terms of anti-Xa units is 120 anti-Xa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects initially occur at dosages that are antithrombotic, this agent has been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. This agent has also been found to release tissue factor pathway inhibitor (TFPI) after both intravenous and subcutaneous administration. Repeated administration of reviparin produces progressively stronger antithrombotic effects. The current studies are designed to provide additional data on its molecular profile using new calibration methods and additional results on the pharmacological studies in a dose-dependent manner. In particular, the release of TFPI following i.v. and s.c. 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 increase in the antithrombotic or haemorrhagic effects of this agent. Comparative antithrombotic and pharmacological studies are also reported to compare the pharmacological profiles of reviparin, nadroparin and enoxaparin.

Animals↗
PubMed1996

Laboratory assays and duplex scanning outcomes after symptomatic deep vein thrombosis: preliminary results.

PURPOSE: The purpose of this article was to assess a number of hematologic and fibrinolytic assays at the time of diagnosis of deep vein thrombosis (DVT) and at several intervals over a period of 6 months afterward and to correlate these results with the results of serial duplex scanning. METHODS: Thirty-five patients (average age 61, range 18 to 82) with acute symptomatic DVT confirmed by duplex scanning were included. On diagnosis, blood was drawn, and plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI), D-dimer (DD), and tissue factor pathway inhibitor (TFPI) were determined. Duplex scanning and all laboratory assays were repeated 1 week, 1 month, 3 months, and 6 months thereafter. RESULTS: The rate of DVT complete resolution 6 months after diagnosis was 57%. Whereas plasma levels of PAI were similar throughout the 6-month follow-up period, t-PA increased significantly 1 week after diagnosis and decreased thereafter. Both DD and TFPI levels decreased significantly after diagnosis compared with presentation values. Comparing these assay levels between patients with complete resolution versus partial or no resolution, PAI levels were significantly higher during the first week in patients with poor outcome. Plasma levels of t-PA were higher in cases with good outcome, and DD levels were higher in patients with poor outcome. TFPI levels were similar in both outcome groups. CONCLUSIONS: Patients with complete DVT resolution on duplex scanning at 6 months had significantly lower levels of PAI on presentation and after 1 week than did those with incomplete lysis. Although differences were not significant, t-PA levels were higher and DD lower in patients with good outcome. Our results suggest that certain plasma fibrinolytic assays might correlate with the outcome of DVT, as assessed by duplex ultrasonography.

Adolescent↗
PubMed1996

Pharmacological profiling of recombinant tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor found within the vascular system that is known to be released upon heparin administration. Tissue factor pathway inhibitor regulates the extrinsic pathway by inhibiting both factors VIIa and Xa. The role of TFPI in the prevention of thrombosis is currently being elucidated. These studies describe the pharmacological profile of recombinant variants of this inhibitor that have recently been made available for study. Recombinant TFPI (rTFPI) exhibits potent anticoagulant (PT) and antiprotease actions (anti-Xa). While carboxy truncated forms of the inhibitor were not observed to prolong the PT, potent anti-Xa effects were still noted. In a rabbit model of stasis-thrombosis, full-length rTFPI dose-dependently inhibited thrombus formation, producing a complete inhibition of thrombosis at a dose of 500 micrograms/kg. At doses that were antithrombotically effective, minor increases in blood loss were observed in a rabbit ear bleeding model. The effect of TFPI on the inhibition of platelet activation was assessed using a flow cytometric assay. Three hundred nmol/L of rTFPI were observed to block the activation of platelets by 4.5 pmol/L recombinant tissue factor. In addition to participating in the maintenance of hemostasis, rTFPI may prove to be an effective therapeutic modality for preventing thrombus formation.

Amino Acid Sequence↗
PubMed1996

Plasma levels of the molecular markers of coagulation and fibrinolysis in patients with peripheral arterial disease.

In 103 patients with peripheral arterial disease (PAD) of the lower limbs, coagulation and fibrinolytic parameters were evaluated to identify hemostatic abnormalities characteristic of this patient population. PAD was defined as clinically stable Leriche stage 2 (based on clinical history, peripheral pulses, ankle-arm index, and treadmill test) for at least 3 months, walking distance > 100 m, and no other major illnesses, rest pain, or trophic lesions. Defibrotide, a polydeoxyribonucleotide derivative with vascular effects, was administered to the patients as part of a multicenter trial. The PAD patients exhibited a prothrombotic state as evidenced by high D-dimer in all but 24% of the patients (average 797 +/- 802 vs. 163 +/- 54 ng/mL normal population; p < 0.001) and high thrombin-antithrombin III complex (TAT) levels (10.2 +/- 8.9 vs. 2.5 + 1.5 ng/mL; p < 0.001) with low to normal levels of protein C (86 +/- 25 vs. 102 +/- 18%; p < 0.01) and plasminogen activator inhibitor-1 (PAI-1) antigen (5.9 +/- 4.5 vs. 1.3 + 0.7 ng/mL; p < 0.001) were elevated in 79% of the patients. These results suggest that there is ongoing thrombosis in the majority of PAD patients. Differences from normal controls were observed for t-PA, PAI-1, protein C, and protein S; however, it is not certain that the thrombosis in patients with PAD is due to these factors.

Adult↗
PubMed1996

Are the available low-molecular-weight heparin preparations the same?

Low molecular weight heparins (LMWHs) are now universally accepted as drugs of choice for post-surgical prophylaxis of DVT. Currently these agents are also being developed for therapeutic and cardiovascular indications. Because of manufacturing differences, each of the LMWHs exhibit distinct pharmacologic and biochemical profiles. The specific activity of these agents in the anticoagulant assays ranges from 35 to 45 anti-IIa U/mg whereas the specific activity in terms of anti-Xa units is designated as 80 to 120 anti-Xa U/mg. These LMWHs are capable of producing product specific dose and time dependent antithrombotic effects in animal models of thrombosis. While the ex vivo effects are initially present at dosages that are antithrombotic, these agents have been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. In the experimental animal models and in various clinical trials, these agents have also been found to release tissue factor pathway inhibitor (TFPI) after both intravenous (i.v.) and subcutaneous (s.c.) administration. Repeated administration of LMWHs produces progressively stronger antithrombotic effects; however, the hemorrhagic responses vary and are largely dependent on products. The release of TFPI following i.v. and s.c. administration in a primate model also demonstrated the product individuality and the relevance of this inhibitor to the actions of LMWHs. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT also exhibited product based augmentation of the antithrombotic or hemorrhagic effects. Antithrombotic and hemorrhagic studies are reported that compare the pharmacologic profile of some of the available LMWHs. Product individuality in terms of relative potency in different assays and the failure of standardization protocols to provide any guidelines for product substitution and prediction of the clinical effects is also addressed.

Animals↗
PubMed1995

Molecular profiling and weight determination of heparins and depolymerized heparins.

The recently proposed calibrant LHN-1 (lot F537; henceforth designated F537), for the molecular weight (MW) determination by high-performance size-exclusion chromatography of heparins, is shown here to have a range too narrow to allow for the accurate MW determination of all low molecular weight heparins (LMWHs). We have recently demonstrated, by this same methodology, that a chemically degraded benzyl ester of unfractionated heparin, heparin mass calibrator (HMC), is a better calibrant. Weight-average MW, number-average MW, peak MW, and dispersity values were calculated with F537, HMC, and by a reference narrow-range-calibration method for various LMWHs and unfractionated heparins. Values for these parameters determined with HMC were not significantly different from those determined by the reference method until the MW of the substance exceeded 15.0 kDa. In contrast, the MW profile obtained with F537 was appreciably different from that obtained by the reference method for samples with MWs > 7.5 kDa. The range exhibited by HMC should allow this calibrant to be used for both LMWHs and unfractionated heparins.

Heparin↗
PubMed1995

Impact of biotechnology in the diagnostic and therapeutic management of cardiovascular disorders.

Although biotechnology has been useful in the development of new diagnostic methods and drugs for the management of cardiovascular disorders, there are several issues which raise certain questions on the global use of biotechnology based drugs and diagnostic methods (Piascik, 1991; Fareed, 1993a; Fareed, 1994a). The cost is rather prohibitive in the development of this type of technology. Most diagnostic methods and drugs developed utilizing biotechnology based methods are relatively expensive. The second important consideration is the equivalence of the newer biotechnology derived drugs to the natural products. Many of the biotechnology derived drugs are obtained in prokaryotic systems (E. coli). Post-transcriptional modifications such as glycosylation are often important in determining the function of various proteins. On the other hand, biotechnology based diagnostic methods exhibit somewhat different specificity in comparison to conventional methods. Thus, it is rather important to assess the developments in this area in a careful manner. Furthermore, validation of the clinical, diagnostic and therapeutic efficacy of biotechnology derived diagnostic devices and drugs is a prerequisite for their use in cardiovascular medicine.

Amino Acid Sequence↗
PubMed1995

Modifications of plasma levels of tissue factor pathway inhibitor and endothelin-1 induced by a reverse Trendelenburg position: influence of elastic compression--preliminary results.

PURPOSE: The purpose of this study was to assess the effects of the passive 45-degree reverse Trendelenburg position and graduated compression stockings (GCS) on plasma tissue factor pathway inhibitor (TFPI) and endothelin-1 levels in a group of volunteers. METHODS: Ten healthy subjects lay on an examining table for 30 minutes while baseline measurements were made. The table was then tilted to a 45-degree upright position for 60 minutes, and measurements were repeated. On a different day, subjects were tilted again, but on this occasion they wore thigh-length GCS. Blood was drawn before and 60 minutes after tilting during both sessions, and plasma TFPI and endothelin-1 were obtained. Cross-sectional areas of the calf medial gastrocnemius vein, before and after tilting, were measured by a duplex scanner. RESULTS: Upright tilting induced a significant dilation of the medial gastrocnemius veins that was partially corrected by the use of elastic stockings. Similarly, endothelin-1 levels significantly increased after tilting. The use of GCS did not modify these differences. On the other hand, although TFPI levels were not affected by tilting without stockings, they were significantly elevated after tilting when GCS were used. CONCLUSIONS: Upright passive tilting induces significant dilation of the deep calf veins and is associated with a significant increase in plasma levels of endothelin-1, whereas TFPI levels remain unchanged. The use of elastic stockings reduces the degree of calf distention but does not prevent an increase in endothelin-1. TFPI levels are significantly increased after tilting when GCS are used. This might represent a previously unknown mechanism of action of elastic stockings with interesting potential for deep vein thrombosis prophylaxis. More studies are warranted in a larger series to confirm these results.

Adult↗
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

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 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

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

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↗