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Biomedical subjects

J M Walenga

Publications and source records attributed to J M Walenga.

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

Laboratory tests for the diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is associated with high morbidity and mortality. Because the pathophysiology of this complex disorder has remained unclear, so has the development of supportive diagnostic laboratory assays. The currently available laboratory methods for HIT diagnosis include several platelet function assays: the platelet aggregation assay, platelet aggregation with simultaneous measurement of ATP release (lumi-aggregometry), the serotonin release assay, and flow cytometric assays. ELISA assays, which quantitate anti-heparin/platelet factor 4 antibody titers, have recently become available. Assay characteristics for these assays were studied using sera collected from clinically diagnosed HIT patients with and without thrombosis, normal individuals, various types of hospitalized patients without HIT, heparin or low molecular weight heparin-treated patients without HIT, and patients with platelet-immune disorders other than HIT. The results of our studies suggest that none of the assays can be considered a "gold standard" for the laboratory diagnosis of HIT as many false-negative and false-positive results were obtained. Furthermore, antibodies against the heparin/platelet factor 4 complex, as identified by the current ELISA tests, are not the sole cause of HIT since many patients lacking clinical symptoms associated with HIT exhibited high antibody titers following heparin treatment. An assay using flow cytometry, being developed for HIT testing, will be described. At this time, clinical impression remains important for the diagnosis of HIT.

Adult↗
PubMed1999

Comparison of two PF4/heparin ELISA assays for the laboratory diagnosis of heparin-induced thrombocytopenia.

Serum samples from 105 patients with suspected heparin-induced thrombocytopenia (HIT) were evaluated using the 14C-serotonin release assay (SRA), considered the "gold standard" for the diagnosis of HIT, and two enzyme-linked immunosorbent assays (ELISA) that measure anti-platelet factor (PF) 4/heparin antibodies to determine the performance characteristics of the newly available ELISA assays. Relative to the SRA, the sensitivity and specificity of the Asserachrom HPIA assay were 73% and 77%, respectively, in this population of patients. The sensitivity and specificity of the GTI-HAT assay were 60% and 93%, respectively. In serum negative by SRA, GTI-HAT and HPIA detected antibodies in 9% and 25%, respectively. Antibodies were detected by HPIA in 18% of the sera negative by both SRA and GTI-HAT. In a second study, samples evaluated from patients (n = 10) treated for established thrombosis with a low-molecular-weight heparin and who had no decrease in platelet counts, showed a weak antibody titer in 50% of the patients after 12 days of therapy by GTI-HAT, whereas the HPIA identified a strong antibody titer in 75% of the patients after 4 days. These data suggest that the currently available ELISA methods for the detection of anti-PF4/heparin antibodies offer a limited sensitivity and specificity in comparison to SRA. The ELISA assays on their own are thus of limited value for the laboratory diagnosis of HIT. SRA-positive sera do not always have positive antibody titers, and antibodies can be present in SRA-negative sera. Furthermore, these data show that ELISA methods differ in their relative sensitivities and specificities for the detection of anti-PF4/heparin antibodies.

Adult↗
PubMed1999

Coagulation laboratory testing in patients treated with argatroban.

During clinical trials with the thrombin inhibitor argatroban, appropriate methods for drug monitoring were identified. Treated patients presented interesting challenges for coagulation laboratory parameter testing in the presence of argatroban. These issues are reported here. Regarding the monitoring of argatroban, the aPTT and ACT were effectively used clinically for low (0-2.5 microg/mL) or high (1-15 microg/mL) doses of argatroban. However, system (reagent and instrument) differences were noted in the time-to-clot values. A clot-based assay using Ecarin as the activator (ECT, Ecarin clotting time) appeared to be useful for monitoring both low and high drug levels with less interference from other drugs or coagulation defects. Also identified were the chromogenic antithrombin assay that could directly quantify argatroban and an HPLC based assay that could specifically quantify argatroban and its metabolites. With regard to assay interference by argatroban, several important effects were observed. The presence of argatroban synergistically interfered with the INR for those patients treated with oral anticoagulants. However, a chromogenic based method was able to determine factor X levels as a monitor of the oral anticoagulation without effect from argatroban. A similar synergistic response on the aPTT with heparin and argatroban was observed. Patients receiving argatroban evaluated for potential coagulation abnormalities could not be tested with the routine functional (clot based) assays for fibrinogen, factor levels or protein C. Argatroban acted as an inhibitor in these assays, causing a dose-dependent false decrease of fibrinogen and factor levels, and a false increase of protein C. Using a chromogenic assay for protein C, values equal to those obtained by an immunologic assay were achieved. These issues will most likely hold true for all thrombin inhibitors.

Antithrombins↗
PubMed1999

Heparin-induced thrombocytopenia: the role of platelet activation and therapeutic implications.

In the past, heparin has been the sole anticoagulant for interventional cardiovascular procedures. Today, several alternate approaches to anticoagulate patients with heparin-induced thrombocytopenia (HIT) are under consideration. Antiplatelet drugs, such as the ADP receptor antagonists and inhibitors of glycoprotein (GP) IIb/IIIa, are currently in development. We investigated the effect of two anti-platelet agents on platelet activation induced by HIT serum (n = 5 HIT positive sera, n = 5 HIT negative sera and n = 4 donor platelets) and heparin, using the traditional platelet aggregation assay, a Lumi-aggregation assay to also determine platelet release, and flow cytometry. By all methods, the GP IIb/IIIa inhibitor-GPI 562 (Novartis; Nürnberg, Germany)-produced a concentration dependent (6.25 to 125 ng/mL) decrease in platelet activation, as shown by platelet aggregation, platelet microparticle formation, P-selectin expression, and ATP release. Similar results were obtained with the thienopyridine ADP receptor antagonist ticlopidine (Sanofi Recherche; Toulouse, France) in vitro at high concentrations of 5.0 to 50 microg/mL and ex vivo in a patient dosed at 250 mg/day. These studies show that GP IIb/IIIa and ADP receptor inhibitors can block platelet activation induced by HIT serum/heparin, providing evidence that the mechanism of HIT may be multifactorial involving not only the generation of the heparin-PF4 or other antibodies but also involving platelet-specific processes and, potentially, the generation of proaggregatory substances. The new antiplatelet agents may be useful in the clinical management of HIT patients.

Adenosine Triphosphate↗
PubMed1999

Clinical experience with combined treatment of thrombin inhibitors and GPIIb/IIIa inhibitors in patients with HIT.

Despite the use of potent anticoagulants such as r-hirudin and argatroban, the morbidity/mortality of heparin-induced thrombocytopenia (HIT) patients remains high. In the last several months, we have treated three HIT-positive patients with a combined therapy of thrombin inhibitor and GPIIb/IIIa inhibitor when treatment with thrombin inhibitor alone failed to alleviate acute thrombosis. Combination therapies included r-hirudin (Refludan) with tirofiban (Aggrastat) or argatroban (Novastan) with ReoPro. A reduced dose of the thrombin inhibitor with the standard dose of the antiplatelet drug was the dosing regimen used. In all cases, there was no overt bleeding that required intervention and all patients had improved or fully recovered. This first report of the use of GPIIb/IIIa inhibitors with thrombin inhibitors in HIT patients with active thrombosis suggests that this combined therapy may be more effective than thrombin inhibitor treatment alone. The data from these three cases warrant testing of this therapeutic regimen in larger studies to determine optimal dosing strategies.

Abciximab↗
PubMed1999

Clinical laboratory monitoring of a synthetic antithrombin agent, argatroban, using high performance liquid chromatography and functional methods.

BACKGROUND: Argatroban is a peptidomimetic inhibitor of thrombin which is in clinical trials for thrombotic complications. Clot-based assays measure the cumulative anticoagulant effect of argatroban and its metabolites(s). To monitor the absolute concentrations of argatroban, a specific HPLC method was developed. METHODS: Validation studies included normal volunteers administered with escalating doses of argatroban (ARG 102 Study), patients undergoing coronary interventional procedures (ARG 310), and patients receiving argatroban in conjuction with streptokinase for acute myocardial infarction (ARG 230). Plasma samples were extracted with acetonitrile and reconstituted in a mobile phase. UV detection was made at 333 nm. Calibratrion curves were prepared with known standards of argatroban in normal human plasma. RESULTS: The retention time for argaeroban was 6.0+/-0.5 min and the extraction efficiency was >98% (r2=0.99). In the ARG102 Study, argatroban levels were: 0.84+/-0.23 (day 1), 1.55+/-0.34 (day 2), 2.92+/-0.15 (day 3), and 3.04+/-0.49 (day 4). In the ARG310 trial, the mean argatroban levels were: 0.23+0.09 microg/ml (preinfusion), 5.77+/-0.92 microg/ml (postinfusion/intraprocedure), and 2.23+/-0.29 microg/ml (postprocedure). In the ARG 230 Study, the mean argatroban levels at 2-8 hrs were between 1.5-2.0 microg/ml. Upon completion of the infusion, a time-dependent clearance of argatroban was noted. CONCLUSIONS: Since heparinization, hemodilution and hypofibrinogenemia due to thrombolysis influence the clotting tests, absolute quantitation of argatroban by HPLC in these patients provides a more reliable means of monitoring this anticoagulant and helps in the dosage-optimization of this agent. The current HPLC method is of value in the monitoring of patients who are simultaneously administered with thrombolytic drugs.

Anticoagulants↗
PubMed1999

Vascular damage correlates between heparin-induced thrombocytopenia and the antiphospholipid syndrome.

Antibody-mediated disorders of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome have remarkably similar clinical presentations, both of which can progressively result in severe vascular and thrombotic disorders. We hypothesized that the mechanism of platelet activation as occurs in heparin-induced thrombocytopenia may also occur in antiphospholipid antibody syndrome particularly at the vascular wall, that endothelial injury may be similar in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome, and that these alterations may be caused by related antibodies. Antibody titers and vascular endothelial damage in patients with heparin-induced thrombocytopenia and antiphospholipid antibody syndrome were studied in plasma samples collected from normals (n = 17), heparin-induced thrombocytopenia patients (n = 15), antiphospholipid antibody syndrome patients (n = 30), and patients clinically diagnosed with antiphospholipid antibody syndrome and heparin-induced thrombocytopenia (n = 8). Diagnosis of heparin-induced thrombocytopenia was confirmed by 14C-serotonin release assay or positive antiheparin-platelet factor 4 antibody titer, and antiphospholipid antibody syndrome was confirmed by positive anti-beta 2-glycoprotein (GP) 1/cardiolipin (IgG or IgM) antibody titer. The antiheparin-platelet factor 4 antibody was not detected in any patient with antiphospholipid antibody syndrome. Patients with heparin-induced thrombocytopenia did not have elevated IgG anti-beta 2-GP1 titers, but three (20%) patients had low-positive IgM anti-beta 2-GP1 titers. The endothelial damage markers of soluble thrombomodulin, soluble P-selectin (p < 0.05 vs. normal), plasminogen activator inhibitor-1 and tissue factor were elevated in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome patients. The soluble E-selectin was elevated only in the patients with both heparin-induced thrombocytopenia and antiphospholipid antibody syndrome (p < 0.05 vs. normal). Levels of soluble L-selectin and von Willebrand factor were not different from normals. The pathogenesis of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome appears to be due to two distinct antibodies but associated with similar damage to the vascular endothelium in both diseases.

Antiphospholipid Syndrome↗
PubMed1999

Laboratory tests for heparin-induced thrombocytopenia: a multicenter study.

A multicenter clinical trial of the thrombin inhibitor argatroban (Novastan; Texas Biotechnology, Houston, TX; Smith-Kline Beecham Pharmaceuticals, Philadelphia, PA) was recently conducted in patients with heparin-induced thrombocytopenia (HIT) and HIT that had progressed to thrombosis (HITTS). In patients defined by the inclusion/exclusion criteria, the utility of three diagnostic HIT assays was investigated: the platelet aggregation assay, the serotonin release assay (SRA), and the enzyme-linked immunosorbent assay (ELISA) for the antibody to the heparin-platelet factor 4 (H-PF4) complex. Confirmation was made in 26%, 55%, and 64% of the patients, respectively (n = 199 patients; 512 to 606 samples; P < .001 platelet aggregation assay v SRA v ELISA). Patients who progressed to HITTS (n = 98) were more often confirmed than were HIT patients without associated thrombosis (n = 101) (P < .05). Confirmation by platelet aggregation assay and SRA results generally was associated with a higher antibody titer. However, a minimum critical titer could not be identified, because all patterns of positive and negative results by the platelet aggregation assay, SRA, and ELISA were observed, and clinically ill patients had a wide range of antibody titers. Over a 30-day period, the percentage of positive responses did not change. Although multiple testing over several days enhanced the chance of confirmation, this difference was not significant. Combined results of the three assays enhanced the positive response to 83% of the total population (P < .005). These data demonstrate that there is no direct correlation between the positive response of these assays, and that clinically positive HIT patients can be missed by all three assays. With these limitations, the combination of platelet aggregation assay, SRA, and ELISA testing with multiple samples offers the best chance of confirming a positive HIT patient. Caution is advised, however, in interpreting all assay results, as no assay is optimal.

Acute Disease↗
PubMed1999

Antithrombin agents as anticoagulants and antithrombotics: implications in drug development.

The development of direct thrombin inhibitors goes back nearly four decades. Organic synthetic benzamidine derivatives were initially developed as direct antithrombin agents. Later, the structural analysis of fibrinogen, leading to the identification of thrombin cleavage sites, resulted in the recognition of specific peptide sequences where thrombin cleaved fibrinogen. These observations led to the development of synthetic peptide derivatives as inhibitors of thrombin. The leech salivary extract contained natural hirudin, the structural elucidation of which led to the development of a recombinant equivalent protein (r-hirudin). Understanding the biochemical actions of thrombin and the structure of various inhibitors prompted the development of hirulogs, a class of hybrid molecules with two sites of action. Currently, several of these thrombin inhibitors are being developed for various indications in both intravenous and subcutaneous protocols. The increased interest in thrombin inhibitors is also prompted by reports of heparin-induced thrombocytopenia (HIT) with heparin and the need to anticoagulate patients with alternate drugs. These agents produce a direct anticoagulant response by targeting thrombin. In addition, the amplification of the coagulation cascade by thrombin activation of factors V and VIII, stabilization of fibrin by activated factor XHI, and platelet activation is also inhibited by these thrombin inhibitors. Some of the synthetic thrombin inhibitors are also capable of inhibiting other enzymes in the coagulation cascade. Thrombin inhibitors therefore exert a complex effect on the coagulation network and should be carefully evaluated in clinical trials. These drugs can be used for prophylactic and therapeutic and surgical indications. However, the different thrombin inhibitors have shown distinct pharmacologic differences. There is now an interest in developing oral antithrombin inhibitors. Such issues as antagonism, laboratory monitoring, drug interactions, and long-term safety remain unresolved. Current research is focused on addressing these issues.

Amino Acid Sequence↗
PubMed1998

Low-molecular-weight heparins: pharmacologic profile and product differentiation.

The interchangeability of low-molecular-weight heparins (LMWHs) has been the subject of discussion since these products were first introduced for the prophylaxis of deep vein thrombosis. Experimental evidence now exists to show that LMWHs differ from each other in a number of characteristics. Products have been differentiated on the basis of molecular weight and biologic properties, but only limited information derived from the clinical setting is available. Potency has been described on the basis of anti-Factor Xa activity, but at equivalent anti-Xa activities, the anti-Factor IIa activity of different products shows marked variations. At the relatively small doses used for the management of postsurgical deep vein thrombosis, the effect of these interproduct differences may be relatively minor, but as LMWHs are developed for therapeutic use at much higher doses, such differences may become clinically important. Variations in safety and efficacy reported in clinical trials of LMWHs may reflect the known differences in their molecular composition and pharmacologic properties.

Animals↗
PubMed1998

Antithrombin agents as anticoagulants and antithrombotics. Implications in drug development.

Synthetic and recombinant thrombin inhibitors have undergone several clinical evaluations for thrombotic and cardiovascular indications. While the initial trials were focused in coronary indications, more recently, these agents are also developed for the prophylaxis and therapeutic management of thromboembolic disorders. Hirudin, PEG-hirudin and argatroban are in advanced clinical development. Recombinant hirudin has been approved in Europe as a substitute anticoagulant for the management of HIT patients. Several additional clinical trials are currently carried out to demonstrate the usefulness of these agents in thrombotic and cardiovascular indications. Despite these developments such issues as dosage optimization, laboratory monitoring, neutralization and drug interactions require additional studies for the optimal development of these drugs.

Animals↗
PubMed1998

Heparin-induced thrombocytopenia, paradoxical thromboembolism, and other side effects of heparin therapy.

Although several new anticoagulant drugs are in development, heparin remains the drug of choice for most anticoagulation needs. The clinical effects of heparin are meritorious, but side effects do exist. Important untoward effects of heparin therapy including heparin-induced thrombocytopenia, heparin-associated osteoporosis, eosinophilia, skin reactions, allergic reactions other than thrombocytopenia and alopecia will be discussed in this article.

Animals↗
PubMed1998

Molecular markers of hemostatic activation and inflammation following major injury: effect of therapy with IFN-gamma.

Interferon-gamma (IFN-gamma) has shown promise in treatment of injured patients. However, reactive states marked by immunologic and inflammatory responses constitute a potential deleterious effect of IFN-gamma administration. IFN-gamma therapy has been associated with high levels of tumor necrosis factor-alpha (TNF-alpha), with potential enhancement of coagulopathy after injury. This study evaluated TNF-alpha production and markers of hemostatic activation in patients receiving IFN-gamma therapy. Seventy-three patients, part of a larger multicenter trial, with severe injuries were randomized to IFN-gamma (100 microg/day s.c. for 21 days) or placebo treatment. Enrollment criteria included injury severity score (ISS) > or = 25 or significant bacterial contamination with ISS > or = 20. TNF-alpha and other cytokine production was assessed at baseline and on days 3, 8, and 22 following injury. Markers of coagulation activation and fibrinolysis were also evaluated. Plasma TNF-alpha and interleukin-6 (IL-6) levels were higher in IFN-treated relative to placebo-treated patients before and after IFN administration. Markers of coagulation and fibrinolysis were elevated at all times studied following injury in both treatment and control groups but did not differ between patients receiving IFN and those receiving placebo. Activation of coagulation and fibrinolysis diminished in a time-related manner following injury. We conclude that (1) IFN-gamma therapy at the dose employed was not associated with a significant increase in TNF-alpha or other inflammatory cytokine production beyond that seen in patients receiving placebo, (2) coagulation and fibrinolytic markers were increased following injury but decreased significantly in surviving patients, and (3) no changes in coagulation and fibrinolytic parameters were noted in relation to IFN-gamma therapy. These findings support previous observations that trauma is associated with hemostatic activation and that treatment of patients at the dose of IFN-gamma studied is safe in the setting of injury.

Adult↗
PubMed1998

Trauma and thermal injury: comparison of hemostatic and cytokine changes in the acute phase of injury.

BACKGROUND: Initiated either by thermal injury or mechanical trauma, the systemic inflammatory response syndrome stimulates activation of coagulation and fibrinolysis, evolving into a subclinical disseminated intravascular coagulation. METHOD: Hemostatic parameters, interleukin-6, and endothelin plasma levels were compared in burn and trauma patients. Nineteen patients with major burn injury (> or = 40% total body surface area) were compared with 35 trauma patients with Injury Severity Scores > 25 on day 1 and days 5 to 8. RESULTS: Thrombin-antithrombin levels were significantly higher in trauma patients than in burn patients (p < 0.0001) on day 1, and endothelin was significantly higher on days 1 and 5 (p < 0.0001) in trauma patients than in burn patients. Interleukin-6 plasminogen activator inhibitor-1, and tissue plasminogen activator levels were elevated above normal limits on both days in both groups. CONCLUSION: There was a difference in the degree and level to which homeostasis was perturbed between the two groups. The mechanism of injury did not affect the initiation of subclinical disseminated intravascular coagulation and cytokine release, and the physiologic response remained the same.

Adult↗
PubMed1998

Synthetic and recombinant antithrombin drugs.

As the final enzyme in the activation of the coagulation system, the serine protease, thrombin, is believed to be an important target for the development of new anticoagulant/antithrombotic drugs. Direct thrombin inhibitors are either derived from natural sources, such as hirudin or are chemically synthesised, such as argatroban. The coupling of hirudin or parts of it with other entities leads to novel agents with different pharmacokinetic and pharmacodynamic characteristics, such as polyethylene glycol (PEG)-hirudin or the hirulogs. Due to the reversible or irreversible inactivation of the enzyme, thrombin inhibitors exert strong anticoagulant effects that can be measured in global clotting assays. Furthermore, these compounds inhibit thrombin-induced platelet reactions and influence other cellular, receptor-mediated actions of thrombin, e.g., on vascular cells. Directly acting thrombin inhibitors prevent blood clotting and are also capable of inhibiting clot-associated thrombin; however, they do not effectively block the further generation of the enzyme. Comprehensive experimental studies suggest that thrombin inhibitors may be effective drugs in a wide range of intravascular thrombus formation, also including the inhibition of vascular restenosis. Recent clinical trials revealed the effectiveness of direct thrombin inhibitors in various thrombotic and cardiovascular indications, but also a tendency to an increased risk of bleeding complications. At present, thrombin inhibitors are the most promising class of drugs for the initial therapy of patients with heparin-induced thrombocytopaenia (HIT) or the heparin-induced thrombocytopaenia and thrombosis syndrome (HITTS). They are also useful for the management of venous thrombosis and for acute ischaemic syndromes as well as for invasive procedures. However, with regard to the long-term outcome, a superiority of thrombin inhibitors over heparin has not yet been demonstrated. Several important issues, such as monitoring, pharmacological antagonism and drug interactions will also play an important role in the development of these new drugs. Further clinical trials are required to confirm the effectiveness of direct thrombin inhibitors in the prophylaxis and treatment of various thromboembolic and cardiovascular disorders.

Journal Article↗
PubMed1998

Effect of SR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of human endothelial cells.

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

Adenosine Diphosphate↗