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Essentials Many patients produce antibodies but few lead to heparin-induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non-pathogenic antibodies. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti-PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. Objectives To identify PF4 amino acids essential for binding pathogenic HIT antibodies. Methods Alanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet-activating murine monoclonal anti-PF4/heparin antibody (KKO) and HIT patient sera (n = 9). Results and Conclusions We identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n = 45) and non-pathogenic antibodies (EIA-positive but not platelet activating, n = 28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic. Am. J. Hematol. 90:629–633, 2015. © 2015 Wiley Periodicals, Inc.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

Summary Background A significant challenge in the management of heparin-induced thrombocytopenia (HIT) patients is making a timely and accurate diagnosis. The readily available enzyme immunoassays (EIAs) have low specificities. In contrast, platelet activation assays have higher specificities, but they are technically demanding and not widely available. In addition, ~ 10% of samples referred for HIT testing are initially classified as indeterminate by the serotonin release assay (SRA), which further delays accurate diagnosis. HIT is characterized by platelet activation, which leads to FcγRIIa proteolysis. This raises the possibility that identification of the proteolytic fragment of FcγRIIa could serve as a surrogate marker for HIT. Objectives To determine the specificity of platelet FcγRIIa proteolysis induced by sera from patients with HIT, and to correlate the results with those of the SRA. Methods/Patients Sera from HIT patients and control patients with other thrombocytopenic/prothrombotic disorders were tested for their ability to proteolyse FcγRIIa. The results were correlated with anti-platelet factor 4 (PF4)/heparin antibodies (EIA), and heparin-dependent platelet activation (SRA). Results Only HIT patient samples (20/20) caused heparin-dependent FcγRIIa proteolysis, similar to what was shown by the SRA. None of the samples from the other patient groups or hospital controls caused FcγRIIa proteolysis. Among nine additional samples that tested indeterminate in the SRA, FcγRIIa proteolysis resolved five samples that had a positive anti-PF4/heparin EIA result; three had no FcγRIIa proteolysis, and two were shown to have heparin-dependent FcγRIIa proteolysis Conclusions This study suggests that heparin-dependent FcγRIIa proteolysis is at least as specific as the SRA for the diagnosis of HIT.

Des anticorps monoclonaux spécifiques au virus de la mosaïque du panais (TuMV) ont été produits et utilisés dans un bioessai en sandwich à double anticorps afin de détecter des virus dans des plantes infectées. Un anticorps particulier d'un clone hybridome ayant les caractéristiques recherchées de croissance, de spécificité et de production d'anticorps a été décrit. Cet anticorps a été montré par microscopie électronique immunocytochimique and par immunodétection en point comme réagissant avec une protéine d'enrobage d'un virion. Les conditions procurant une extraction efficace du virus à partir des feuilles ont été étudiées par l'utilisation de l'anticorps dans les étapes de capture et de détection du bioessai en sandwich. Avec un système de tampons d'extraction contenant plusieurs détergents, un essai très sensible a été produit qui détecte des virus de façon fiable dans les plantes infectées. Cet essai est maintenant utilisé de façon routinière pour l'immunodiagnostic des infections causées par le virus de la mosaïque du panais., Monoclonal antibodies specifie for turnip mosaic virus (TuMV) were produced and used in a double antibody sandwich enzyme immunoassay to detect virus in infected plants. One particular antibody from a hybridoma clone having desirable growth, specificity and antibody production properties was characterized in detail. This antibody was shown by immunocytochemical electron microscopy and immunoblotting to react with a virion coat protein. Conditions providing efficient extraction of virus from leaves were investigated by using the antibody in both capture and detection steps of a sandwich immunoassay. With an extraction buffer System containing multiple detergents, a highly sensitive assay was produced that reliably detected virus in infected plants. This assay is now in routine use for immunodiagnosis of turnip mosaic virus infections.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic.

The frequency of immune heparin-induced thrombocytopenia (HIT) varies among prospective studies. It is unknown whether this is caused by differences in the heparin preparations, the patient populations, or the types of serologic assay used to confirm the diagnosis. Seven hundred forty-four patients were studied from 3 different clinical treatment settings, as follows: unfractionated heparin (UFH) during or after cardiac surgery (n = 100), UFH after orthopedic surgery (n = 205), and low-molecular-weight heparin (LMWH) after orthopedic surgery (n = 439). Both an activation assay and an antigen assay were used to detect heparin-dependent IgG (HIT-IgG) antibodies. By activation assay, the frequency of HIT-IgG formation ranged from a low of 3.2% in orthopedic patients receiving LMWH to a high of 20% in cardiac patients receiving UFH; by antigen assay, the corresponding frequencies ranged from 7.5% to 50%. Both UFH use (P = .002) and cardiac surgery (P = .01) were more likely to be associated with HIT-IgG formation. However, among patients in whom HIT-IgG formed and who were administered UFH, the probability for HIT was higher among orthopedic patients than among cardiac patients (by activation assay: 52.6% compared with 5%; odds ratio, 21.1 [95% CI, 2.2-962.8]; P = .001; by antigen assay: 34.5% compared with 2.0%; odds ratio, 25.8 [95% CI, 3.2-1141]; P

Heparin-induced thrombocytopenia (HIT) with thrombosis is a serious complication of heparin use. HIT sera can generate platelet-derived microparticles, which are produced in a heparin-dependent manner and are hypothesized to be important initial pathological participants because they promote vascular occlusion. To date, microparticles have been studied using flow cytometric techniques. However, it is uncertain whether the small-sized material seen in flow cytometric studies represents true platelet microparticles shed from activated platelets or whether they are platelets that have contracted after releasing their internal components. This report describes a morphological investigation of platelet-derived microparticles in HIT using, among other techniques, confocal, scanning electron, and transmission electron microscopy. Following incubation with HIT sera, the existence of small membrane-bound vesicles in the milieu of activated platelets was demonstrated. A population of microparticles, expressing platelet-specific glycoproteins, was separated from platelets by centrifugation over a sucrose layer. These microparticles had identical flow cytometric profiles, size heterogeneity, and GPIband GPIIb/IIIa staining intensity as the microparticle population in unfractionated samples. When microparticles were generated in situ and fixed onto grids, they were demonstrated to be distinct membrane-bound vesicles that originated near the platelet body and terminal ends of pseudopods on activated platelets. These microparticles appeared to be generated by localized swelling, budding, and release. Collectively, these morphological studies document the existence of true microparticles in platelets activated by HIT sera. The microparticles may play an important role in the pathogenesis of HIT.

IL-5 measurement in the fluid phase of induced sputum is considered to be important in the assessment of asthma, but the validity of these measurements is uncertain.We investigated the validity of sputum IL-5 measurements through a series of spiking experiments and examined the effect of dithiothreitol (DTT) on these measurements.Induced sputum from 26 asthmatic subjects was spiked with IL-5 and processed, and the percentage of recovery was measured by means of immunoassay. In 6 of the 26 samples the effect of adding albumin to the processing fluids was studied. In 3 separate samples radiolabeled IL-5 was added, and the recovery measured by means of gamma counting and immunoassay were compared. In addition, the effect of DTT on the immunoassay was examined.The mean +/- SD recovery of spiked IL-5 was 26.1% +/- 14.6% measured by means of immunoassay; adding albumin increased the recovery to 47.7% +/- 8.0% (P.001). The mean recovery measured by means of gamma counting was 84.8% +/- 5.7% (P.001); adding albumin had no effect on recovery. DTT had no significant effect on IL-5 measurement.The validity of IL-5 measurement by means of current methods is poor. The discrepancy in recovery as measured by gamma counting compared with immunoassay suggests that there is a problem with the recognition of IL-5 epitopes by immunoassay in induced sputum. This cannot be attributed to DTT but may be due to other interfering substances present in sputum, such as sputum proteases, soluble receptors, or autoantibodies.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies (HIT-Abs) that bind to a complex of heparin and platelet factor 4. We investigated the epitope specificity of the HIT-Abs, and found that the HIT-Abs recognized solid-phase immobilized complexes with an optimum ratio of four to eight molecules of PF4 per molecule of heparin. To try to define the epitopes within the PF4 molecule, intact and reduced (linearized) PF4 was tested against 29 different sera from patients with HIT. In addition, eight different peptides that spanned the PF4 molecule were studied for their ability to bind to the HIT-Abs either alone or in the presence of heparin. With the exception of a subpopulation of patient samples (5/29, 17%), we found that reduced PF4 and the peptides were uniformly non-reactive with the HIT-Abs in the presence of heparin. Reduced PF4 and PF4 carboxy-terminal peptides with a minimum size of 19 amino acids were recognized by a minority (5/29) of HIT-Abs samples but only when heparin was present. The specificity of this subgroup of samples from patients with HIT was highly restricted and the loss of one amino acid (peptide reduced in length from 19 to 18 amino acids) rendered the peptides non-reactive. The clinical characteristics of these patients were similar to the other HIT patients. These studies demonstrate that the majority of HIT-Abs recognize a noncontiguous conformational epitope on the PF4 molecule that is produced when four to eight PF4 molecules are bound together by heparin.

The Gova/b alloantigens are expressed on a 175-kD protein (GP175) on human platelets. Anti-Gov alloantibodies have been implicated in posttransfusion purpura and alloimmune neonatal thrombocytopenia. In this report we characterize the immunochemistry of the alloantigens and identify the platelet protein that expresses the Gov epitopes. Approximately 50% of GP175 containing the Gov epitope was released from platelets treated with phosphatidylinositol-specific phospholipase C, indicating that at least some of this protein exists as a glycosylphosphatidylinositol (GPI)-linked isoform. Radioimmunoprecipitation and immunodepletion studies indicated that the Gova/b alloantigens are expressed on the GPI-anchored CDw109 protein. The Gova/b epitopes were expressed on an extracellular, 120-kD soluble fragment (p120) of CDw109 produced by calcium-dependent protease cleavage. Anti-Gov immunoprecipitates of chymotryptic digests of p120 contained 70- and 52-kD fragments of CDw109. Deglycosylation of native CDw109 had no effect on recognition by Gov alloantisera; however, the epitopes were destroyed after exposure to sodium dodecyl sulfate. Gova/b alloantigens were expressed on platelets and PHA-activated T-cells, cultured human umbilical vein endothelial cells, and by many different tumor cell lines, consistent with the tissue distribution of CDw109.

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.

Increased serum cholesterol is a risk factor for atherosclerosis and coronary artery disease (CAD) (1)(2). Reduction of serum cholesterol has, therefore, been targeted for the prevention and management of CAD with cholesterol screening and is the primary tool for initial risk assessment (3)(4). Cholesterol, however, can also be quantified in skin. Skin reflects the vascular changes associated with age and aortic atherosclerosis. A simple, noninvasive procedure for the estimation of skin cholesterol, the “three-drop” test, has been proposed as an alternative screening method (5). The test, which uses three different concentrations of a digitonin–copolymer–horseradish peroxidase (HRP) conjugate and visual scoring, is capable of discriminating among healthy individuals, those at risk of developing atherosclerosis, and those with overt disease. Cholesterol 1,2,3TM is a refinement of this procedure. We describe here some properties of this test and report on our initial evaluation in a prospective clinical trial. Cholesterol 1,2,3 uses quantitative interpretation and a single concentration of detector. Briefly, the detector and the positive control each are added to die-cut wells in a foam template affixed to the palm. After a 1-min incubation, the reagents are removed by blotting and the HRP substrate is added to these wells and to a third well, which serves as negative control. After an additional 2-min incubation, color development in the center test well is measured by reflectance with a hand-held instrument (MD22 Spectrophotometer; X-Rite, Inc.) interfaced with a computer, and the result is reported numerically as the hue. Assay validity is assessed by visual interpretation of control wells. The specificities of digitonin conjugates have been demonstrated previously with several model systems [crystalline cholesterol, cholesterol-rich tissue sections, and antibody-immobilized LDL-cholesterol (LDL-C)], and the amount of skin-bound conjugate has been shown to correlate with epidermal cholesterol content (5). We extended the specificity …

Human platelets are activated by IgG immune complexes and undergo platelet aggregation and the release of granular contents. Recently, the receptor for IgG on human platelets has been identified and characterised. In contrast, it is not known if rabbit platelets carry Fc receptors. In this report we describe investigations to detect the presence of an Fc receptor on rabbit platelets. The Fc receptor was investigated using functional assays (platelet aggregation and (14)C-serotonin release), a direct binding assay, and an affinity precipitation assay. All of these methods gave consistently positive results using human platelets and consistently negative results using rabbit platelets, indicating that rabbit platelets do not carry Fc receptors. This inconsistency in the interactions of immune complexes with rabbit and human platelets indicates that rabbits are not ideal models for studying a number of human diseases that could be caused by immune complexes, including heparin-induced thrombocytopenia and certain immunological disorders such as systemic lupus erythematosus.

Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).

Platelets respond to a large number of stimuli by undergoing complex biochemical and morphological changes. These changes are involved in physiological processes including adhesion, aggregation, and coagulation. Platelet activation produces membrane alterations that can be recognized by monoclonal antibodies. In this report we describe a novel activation-dependent protein recognized by a monoclonal antibody, JS-1. The platelet glycoprotein was designated p-155 according to its apparent reduced molecular weight, p-155 exists in the native state as varying sized, large multimers held together by disulfide bonds. p-155 is released upon platelet activation and binds to the activated platelet surface. Although p-155 and platelet glycoprotein Ia migrate similarly on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodepletion and isoelectric focusing distinguished p-155 from glycoprotein Ia. p-155 differed from von Willebrand factor and from thrombospondin in its reduced molecular weight. Additionally, immunoblotting of immunoprecipitated p-155 with antisera to von Willebrand factor and to thrombospondin confirmed the unique identity of p-155. Evidence for a soluble, nonintegral membrane-associated protein was obtained by Triton X-114 phase separation studies, membrane elution studies, and by the demonstration of the protein in the aqueous phase of platelet releasate. Both radioimmunoprecipitation and direct binding techniques demonstrated the activation-dependent nature of p-155. The protein could not be detected in other blood cells, endothelial cells, HEL cells, liver, or in plasma. The functional role of p-155 in platelets is not yet known.

In this report we describe a platelet alloantigen system that is carried on a novel platelet protein of 175 Kd. Antisera against the two alleles (Gova/Govb) were found in two patients who had received large numbers of platelet transfusions. The anti-Gov alloantibodies could not be detected using a whole platelet solid phase enzyme immunoassay, or by a platelet glycoprotein capture enzyme immunoassay using monoclonal antibodies against glycoproteins Ib/IX, Ia/IIa, and IIb/IIIa. Using radioimmunoprecipitation techniques, a protein was precipitated that migrated at 175 Kd (reduced). Under nonreduced conditions, a 150-Kd protein was detected with a minor component at 175Kd. The detection of the alloantigens was not activation-dependent. Using immunodepletion studies, we demonstrated that each alloantiserum recognized an epitope on a discrete population of the 175-Kd platelet protein. Family studies demonstrated that the alloantigens designated as Gova and Govb were inherited in an autosomal codominant fashion. The phenotypic frequencies were Gova/Gova, 26%; Gova/Govb, 55%; Govb/Govb, 19%; giving gene frequencies of 0.532 and 0.468 for Gova and Govb, respectively (n = 33).

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, has been used as a therapeutic anticoagulant for a number of indications, including replacement of heparin in patients with heparin-induced thrombocytopenia. Ancrod has similar fibrinolytic activity to thrombin, but ancrod specifically cleaves only the alpha chain of fibrinogen, producing the characteristic fibrinopeptides A, AP and AY. Because ancrod has been used in patients with heparin-induced thrombocytopenia, it is important to ensure that ancrod does not directly affect the platelets and potentially increase the hemostatic effect. The effect of ancrod on platelets has not been well established, and there is not agreement in published studies. Additionally, some of the studies are over 15 years old and pre-date sensitive assays such as glycoprotein analysis. For these reasons, we investigated the interaction of ancrod with human platelets using direct and indirect, functional and biochemical techniques. Incubation of platelets with ancrod alone did not induce platelet aggregation or the release of dense-granule contents. Pre-incubation of platelets with ancrod did not augment or inhibit the maximal aggregation achieved with thrombin, nor did it affect the amount of serotonin release from dense granules caused by activation by thrombin. Studies of ancrod-treated platelets using monoclonal antibody-mediated radioimmunoprecipitation demonstrated that high concentrations of ancrod did not cause measurable cleavage of either the glycoproteins Ib-IX or IIb-IIIa. Incubation of radiolabeled platelets with ancrod-treated plasma also had no effect on the platelet glycoproteins, indicating that ancrod does not indirectly affect the major surface receptors. Direct binding studies using radiolabeled ancrod did not demonstrate specific binding to the platelet surface. Together these studies indicate that ancrod does not directly affect nor bind to platelets in vitro. The hypo-coagulant state and subsequent platelet function defect resulting from the use of ancrod appears to be limited to the removal of fibrinogen from the circulation.

Background: Heparin-induced thrombocytopenia (HIT) is a common drug reaction that causes arterial or venous thrombosis as a result of heparin therapy. Platelet-activating antibodies, against complexes of platelet factor 4 (PF4) and heparin, cause intense platelet activation, ultimately leading to an increased risk of thrombosis, limb-loss and even death. Most patients exposed to heparin will produce non-pathogenic anti-PF4/heparin antibodies while only a small number will produce platelet-activating and HIT-causing antibodies (pathogenic HIT antibodies). Among HIT tests, the functional assays, such as the serotonin release assay (SRA), correlate best with the disease because they can specifically identify the pathogenic HIT antibodies whereas the enzyme immunoassays (EIAs) cannot. We have previously shown that anti-PF4/heparin antibody production precedes thrombocytopenia in HIT patients (Warkentin et al., Blood 2009 113: 4963-4969) possibly indicating the need for a threshold plasma level of pathogenic HIT antibody, among other factors, to cause the disease. The objective of this study was to investigate the presence of low levels of pathogenic HIT antibodies in samples from patients suspected of HIT who had detectable anti-PF4/heparin antibodies in the EIA (EIA-positive), but who did not have platelet-activating antibodies in the standard SRA (SRA-negative). Methods: We used an in-house IgG-specific EIA to detect the presence of anti-PF4/heparin antibodies (EIA-positive: OD405nm> 0.45) and the standard SRA to detect the presence of heparin-dependent platelet-activating antibodies (SRA-positive: release >20% with 0.1-0.3 IU/mL of unfractionated heparin). We developed an enhanced SRA (eSRA) by adding increasing concentrations of exogenous PF4 (0-100 μg/mL) to detect sub-threshold levels of platelet activating antibodies undetectable in the standard SRA (eSRA-positive: release >20%). Samples tested were referred for HIT testing by the McMaster Platelet Immunology Laboratory (Hamilton, Canada). Results: Sera from healthy individuals (n=10) and from suspected HIT patients with a negative anti-PF4/heparin EIA (n=15) did not demonstrate platelet activation in the eSRA at any dose of exogenous PF4 added. SRA-positive sera (n = 7), diluted sufficiently that they were non-reactive in the standard SRA, demonstrated PF4 dose-dependent platelet activation in the eSRA. This confirmed the increased sensitivity of the eSRA in detecting low-titre platelet-activating antibodies. Reactivity in the eSRA was inhibited by high heparin (100 U/mL) and by blocking the platelet FcgRIIa receptor with the monoclonal antibody IV.3. We then tested samples (n=24) referred for HIT testing that were positive in the anti-PF4/heparin EIA (optical densities OD405nm 0.7 to 2.4) but negative in the standard SRA. Heparin-dependent platelet activation (20-99% release) was demonstrated in 11 of 24 (46%) in the eSRA. This reactivity directly correlated with the amount of PF4 added to the platelets (optimal concentration of PF4 12.5 - 100 μg/mL) but not with the strength (OD405nm) of the anti-PF4/heparin EIA. In further investigations, we concentrated (4-fold) 7 of the 11 eSRA-positive samples in an attempt to increase the concentration of the antibodies. Of those 7 samples, 5 (71%) became positive in the standard SRA upon testing of the concentrated sample. Conclusions: These data indicate that low-titre platelet-activating antibodies may be found in some patients suspected of having HIT that test negative in the standard SRA irrespective of the strength (OD405nm) of the anti-PF4/heparin EIA. The immune response during heparin therapy can produce both families of pathogenic and non-pathogenic anti-PF4/heparin antibodies but it is the titre of the pathogenic antibody that may be necessary for platelet activation. Perhaps under permissive clinical conditions and with patient-specific factors, the titre of the pathogenic HIT antibodies may increase and lead to HIT. Disclosures Warkentin: Pfizer Canada: Honoraria; Instrumentation Laboratory: Honoraria; GlaxoSmithKline: Consultancy, Research Funding; W.L. Gore: Consultancy, Research Funding.

The measurement of interleukin (IL)-;5 in sputum is problematic, with interfering factors affecting immunoassay. The authors investigated whether sputum proteases could be acting as interfering factors by studying the effect of protease inhibitors (PI) on sputum IL-;5 measurement.Induced sputa from 20 subjects with asthma were divided into aliquots, processed with and without protease inhibitors (in low and high concentrations) and the levels of IL-;5 (spiked and endogenous) measured by enzyme immunoassay were compared.The concentration of sputum IL-;5 was significantly increased by PI, with median (interquartile range) levels processed with no, low and high PI concentrations being 0 (0), 41.8 (75.6) and 66.1 (124.4) pg·mL−1, respectively. There was also a significant increase in percentage recovery of spiked IL-;5. Although high concentrations of PI reduced cell viability, there was no effect on total or differential cell counts and low concentrations of PI had no effect on cell counts or viability.Levels of endogenous interleukin-;5 in sputum of asthmatic subjects can be significantly increased by the addition of protease inhibitors, and samples which would be regarded as negative for interleukin-;5 without protease inhibitors may instead have considerable amounts of interleukin-;5 detected.

A small fraction of patients who receive heparin develop heparin-induced thrombocytopenia (HIT) and, of these patients, a still smaller proportion develop associated thrombotic complications. Heparin-induced thrombocytopenia is caused by the formation of antibodies that bind to specific complexes of platelet factor 4 (PF4) and heparin. However, it remains uncertain why certain patients form these antibodies and develop HIT or why certain patients have thrombotic events. In this report we describe studies on individuals with and without HIT to determined if a potential PF4 polymorphism could explain differences in susceptibility to HIT. In the 10 control individuals and the 10 patients we studied, we did not find a difference in the PF4 sequences. Genetic difference in the PF4 antigenic target does not explain the occurrence of HIT in susceptible patients.

Platelets express alloantigens that are platelet specific (eg, the HPA antigens) and alloantigens that are shared with other blood cells (eg, the ABH antigens). The blood group A and B determinants are expressed on glycolipids and on some intrinsic platelet membrane glycoproteins. This report characterizes multiple platelet proteins reacting with blood group antibodies in serum samples from mothers of children born with neonatal alloimmune thrombocytopenia. ABH antigens on additional platelet proteins are identified, including the glycosyl phosphatidylinositol-anchored protein CD109. The proteins that carry ABH antigens were identified by using monoclonal antibodies to glycoproteins Ib, IIb/IIIa, Ia/IIa, CD31, and CD109 and immunoprecipitation/immunoblotting techniques with monoclonal antibodies to A and B antigens. The maternal serum samples and anti-A and anti-B monoclonal antibodies immunoprecipitated identical radiolabeled platelet proteins including proteins at 220 and 175 kd and proteins with mobilities corresponding to glycoproteins Ib, IIb/IIIa, IV, and V. Treatment of platelets with phosphatidylinositol-specific phospholipase C released into the supernatant a 175-kd protein that expressed the blood group determinants. This protein comigrated with the glycosyl phosphatidylinositol-anchored protein CD109. When platelet proteins were purified by immunoprecipitation with monoclonal antibodies and then tested by immunoblotting, anti-A reacted with the glycosyl phosphatidylinositol-anchored protein CD109 and to glycoproteins Ib, IIb, IIa, IIIa, and CD31 (PECAM). These results indicate that structures for modification by glycosyltransferases exist on platelet CD109, which also expresses the Gov alloantigen system. This study indicates that certain platelet proteins express both platelet-specific and blood group antigens that may contribute to platelet transfusion refractoriness and to neonatal alloimmune thrombocytopenia.

In previous studies, we reported that vascular wall cells such as endothelial cells metabolize linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) via the 15-lipoxygenase pathway. Endothelial cell 13-HODE levels vary inversely with endothelial cell reactivity to platelets, which, in turn, varies directly with the expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells. We and others have also found that tumour cell adhesivity is dependent, in part, upon the relative amounts of intracellular 13-HODE and the arachidonic acid monohydroxide(s), 12- and/or 15-hydroxyeicosatetraenoic acids (12-, 15-HETE). In addition, we and others have found that platelet adhesivity is dependent upon the intraplatelet level of its major lipoxygenase metabolite, 12-HETE. Finally, we have demonstrated that 13-HODE and VnR co-localize in nonadhesive endothelial cells but dissociate following endothelial cell injury, at which time, the VnR relocates on the endothelial cell apical surface. These data suggest to us that lipoxygenase-derived monohydroxides regulate the ability of various receptors to recognize their specific ligands. The latter data also suggest that these monohydroxides act directly by a physiochemical mechanism. The present study supports this possibility. Thus, we demonstrate that 13-HODE downregulates VnR binding with vitronectin (Vn) > fibronectin (Fn) > fibrinogen (Fgn), whereas 12- and 15-HETE upregulate specific VnR/ligand binding, using purified VnR/liposomes and purified ligands in an adhesion assay; and that 12- and 15-HETE upregulate GPIIb/IIIa:liposome binding of Fgn > Fn > Vn. We conclude that cell-specific monohydroxides influence cell-specific receptor-ligand binding directly through a physiochemical mechanism.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder caused by heparin-dependent IgG (HIT-IgG) that recognizes a complex of heparin and platelet factor 4 (PF4), leading to platelet activation via the platelet Fc gammaIIa receptors (Fc gammaRIIa). Not all patients who generate HIT-IgG in response to heparin develop HIT, however, possibly because of observed differences in the ability of platelets from healthy individuals to be activated by HIT sera. It is known that a polymorphism in the platelet Fc gammaRIIa plays an important role in determining platelet reactivity to murine platelet-activating monoclonal antibodies of the IgG1 subclass: homozygous arg131 ("high responder" or HR) platelets respond well, and homozygous his131 ("low responder" or LR) platelets respond poorly, respectively, to these murine monoclonal antibodies. We sought to determine whether the differing risk for HIT among patients who receive heparin, as well as the variable platelet reactivity to HIT sera, could be explained by preferential activation by HIT-IgG of platelets bearing a particular Fc gammaRIIa phenotype. We found that the LR Fc gammaRIIa gene frequency was significantly overrepresented among 84 HIT patients, compared with that of 264 control subjects (0.565 versus 0.471; p = 0.03). We studied the subclass distribution of HIT-IgG against its major antigen, heparin/PF4 complexes, and found that 55 of 61 (90%) HIT sera expressed IgG1 antibodies either alone (n = 47) or in combination with IgG2 (n = 5) or IgG3 (n = 3). We then compared the platelet-activating profile of HIT sera with murine platelet-activating monoclonal antibodies. As expected, the murine IgG1 monoclonal antibodies preferentially activated platelets from homozygous HR individuals. In contrast, however, the LR homozygous platelets exhibited the greatest reactivity to HIT sera that contained predominantly anti-heparin/PF4 antibodies of the IgG1 subclass. We conclude that the significant overrepresentation of the LR (his131) gene among patients with HIT may be explained by the preferential activation of LR Fc gammaRIIa platelets by HIT antibodies of the IgG1 subclass, which is the predominant immunoglobulin subclass generated in HIT.

Heparin-induced thrombocytopenia, defined by the presence of heparin-dependent IgG antibodies, typically occurs five or more days after the start of heparin therapy and can be complicated by thrombotic events. The frequency of heparin-induced thrombocytopenia and of heparin-dependent IgG antibodies, as well as the relative risk of each in patients given low-molecular-weight heparin, is unknown.We obtained daily platelet counts in 665 patients in a randomized, double-blind clinical trial comparing unfractionated heparin with low-molecular-weight heparin as prophylaxis after hip surgery. Heparin-induced thrombocytopenia was defined as a decrease in the platelet count below 150,000 per cubic millimeter that began five or more days after the start of heparin therapy, and a positive test for heparin-dependent IgG antibodies. We also tested a representative subgroup of 387 patients for heparin-dependent IgG antibodies regardless of their platelet counts.Heparin-induced thrombocytopenia occurred in 9 of 332 patients who received unfractionated heparin and in none of 333 patients who received low-molecular-weight heparin (2.7 percent vs. 0 percent; P = 0.0018). Eight of the 9 patients with heparin-induced thrombocytopenia also had one or more thrombotic events (venous in 7 and arterial in 1), as compared with 117 of 656 patients without heparin-induced thrombocytopenia (88.9 percent vs. 17.8 percent; odds ratio, 36.9; 95 percent confidence interval, 4.8 to 1638; P0.001). In the subgroup of 387 patients, the frequency of heparin-dependent IgG antibodies was higher among patients who received unfractionated heparin (7.8 percent, vs. 2.2 percent among patients who received low-molecular-weight heparin; P = 0.02).Heparin-induced thrombocytopenia, associated thrombotic events, and heparin-dependent IgG antibodies are more common in patients treated with unfractionated heparin than in those treated with low-molecular-weight heparin.

LATELETS play a pivotal role in hemostasis. They participate in the formation of the he­ mostatic plug at sites of spontaneous and traumatic damage to the vessel wall, and then release internal components that recruit other platelets to bind to the growing platelet plug. The platelet surface components also serve as a site for activation ofthe coagulation factors. Finally, platelets release sub­ stances that cause the vessel wall to repair itself. Consequently, very low platelet counts can result in bleeding, which often requires treatment by platelet transfusions. When damage to the vessel wall is recurring and chronic, which can occur in patients with hypertension, the vessel wall gradu­ ally thickens, limiting the flow of blood through the narrowed opening. Often, a layer of platelets cap the damaged vessel wall and, depending on the tissue perfused by this vessel, the embolization of platelet clumps can produce a heart attack or stroke. The recognition of the participation of platelets in hemostasis and thrombosis has led to intense activity investigating the various mem­ brane components of platelets that participate in normal and pathological platelet function. Much of the information gained about platelet structure/ function relationships has been directly attributable to the use of monoclonal antibodies to study plate­ lets. In this artiele we will review some ofthe more important monoclonal antibodies that have been used to study the platelet glycoproteins (GP).

In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.

We have characterized monospecific sheep antisera to human IgG, IgA and IgM using Ouchterlony double diffusion, immunoelectrophoresis, quantitative precipitation and nephelometric activity. The antisera were conjugated at high and low F/P ratios, absorbed and subsequently evaluated in both direct and indirect immunofluorescence. Antisera which exhibited excellent reactivity in double diffusion, IEP and quantitative precipitation but gave a poor nephelometric response (Ab dilution >1/25 gave no measurable response in the Technicon Fluoronephelometer) showed a marked decreased effectiveness in both direct and indirect fluorescence. Antisera which exhibited a strong nephelometric response were invariably excellent in double diffusion, IEP and quantitative precipitation and gave strong positive fluorescence at reasonable dilutions in both direct and indirect fluorescence. We suggest nεphelometry prior to conjugation is the method of choice in screening antisera usable in immunofluorescence.

Label from [1-14C]-, [2-14C]-, [3-14C]-, and [1,3-14C2]phenylalanine is incorporated non-randomly into the Lythraceae alkaloids, decodine and decinine, in Decodonverticillatus. The incorporation pattern, determined by chemical degradation of the alkaloids, demonstrates incorporation of two intact C6—C3 units.

Summary The majority of platelet alloantigens are located on platelet glycoproteins IIb/IIIa. This report describes a codominant allelic system carried on the glycoprotein la/IIa complex, which we originally designated as Zava/Zavb but which is identical to the Bra/Brb system. Furthermore Zava was found to be identical to Hca. The alloantigens could not be detected using a direct binding enzyme immunoassay (EIA) with intact platelets, but were readily detected using a glycoprotein capture EIA and by radioimmunoprecipitation techniques. The two index cases (designated as homozygous Zava and Zavb) had alloantibodies against the corresponding antigen and did not react with their own platelets. Using these alloantibodies and a monoclonal antibody that reacts with the platelet glycoprotein la/IIa complex (12F1). We demonstrated that all Ia/IIa molecules carry either Zava or Zavb and we found that Zava and Zavb are on discrete populations of Ia/IIa. Following immunodepletion using either anti-Zava or anti-Zavb, all detectable Ia/IIa complexes from the respective homozygous platelets were removed. Immunodepletion of heterozygous Zava/Zavb with either anti-Zava or anti-Zavb did not reduce the amount of Ia/IIa complexes precipitable using the alternate alloantiserum. Population studies (n= 50) indicated the phenotypic frequency of Zava/Zava is less than 1%; Zava/Zavb is 18% and Zavb/Zavb is 82%. Four different alloantisera that had either anti-Zava or anti-Zavb reactivity also carried reactivity against the Baka or Bakb antigens which may suggest an association in the immune response to these alleles.

The amount of IgG on the surface of washed platelets from healthy individuals varies according to the assay used. An immunoradiometric assay (IRMA) for IgG was developed, validated and used to measure platelet associated IgG. The platelet-associated IgG (PAIgG) on washed platelets was allowed to react with excess 125I-labelled anti-IgG. The unbound 125I-anti-IgG was then quantitated by the addition of sepharose beads to which IgG had been covalently bound. The 125I-anti-IgG/IgG-beads were separated from the platelet suspension by passage across a density gradient. The mean amount of IgG present on washed platelets from healthy individuals was 0.8 fg IgG/platelet, or approximately 4,000 molecules of IgG per cell. Inverse Scatchard analysis confirmed the validity of the assay calibration and identical results were obtained when five different anti-IgG antibodies were used. The specific binding of 125I-anti-IgG to platelet-bound IgG was complete by 30 min, but non-specific binding of radioactivity continued thereafter. This non-specific binding occurred not only with anti-IgG but with all antibodies tested and could give elevated estimates of PAIgG in direct binding assays.

The biosynthesis of decodine (7) and decinine (9), Lythraceae alkaloids containing a trans-fused quinolizidine nucleus, was studied in intact plants of Decodonverticillatus (L.) Ell. Nonrandom incorporation of radioactivity from 14C-labelled samples of lysine, cadaverine, and Δ1-piperideine was demonstrated by partial degradation. Pelletierine did not serve as a precursor of the alkaloids. It was also shown, by means of 3H,14C-labelled tracers, that the alkaloids are derived from L-lysine whereas pipecolic acid, which is present in the plant, is derived from D-lysine.

Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In- labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

The glycoprotein localization of the platelet binding site for the Fc IgG has been the subject of debate. We attempted to resolve this issue by relating the binding of radiolabeled IgG immune complexes composed of heat-aggregated IgG to platelets from healthy individuals; an individual with Bernard-Soulier syndrome lacking glycoproteins IIb and IX; and a patient with Glanzmann's thrombasthenia lacking glycoproteins IIb and IIIa. The binding of IgG complexes to platelets was determined by measuring the specific binding of radiolabeled heat-aggregated IgG to washed platelets in a plasma-free mileu. 125I aggregated IgG bound to normal platelets in a saturable and concentration-dependent fashion. Specific binding could be inhibited by a 50-fold excess of purified Fc, but not by F(ab')2. Identical binding curves were obtained by using platelets from a patient with Glanzmann's thrombasthenia and a patient with Bernard-Soulier syndrome, indicating that the platelet Fc receptor is not carried on glycoproteins Ib, IIb, IIIa, or IX. We then measured the binding of radiolabeled detergent-solubilized platelets to IgG fixed to a solid matrix. A 40-kD platelet fragment bound to the immobilized IgG following passage across a density gradient. Confirmation of the Fc specificity of the interaction was shown by inhibition of platelet glycoprotein binding by excess IgG or purified Fc but not F(ab')2. The electrophoretic mobility decreased slightly after reduction, which indicated the existence of at least one intrachain disulfide bond. Treatment with high salt solutions or urea did not solubilize the receptor, which indicated that it was an integral protein. Enzyme studies showed that the platelet Fc receptor was not digested by neuraminidase, but neuraminidase treatment altered mobility by about 3%. In addition, treatment of platelets with trypsin or pronase did not affect its function as measured by the binding of 125I-IgG aggregates to enzyme-treated platelets, but did prevent its detection when using radioimmunoprecipitation studies. The platelet Fc receptor is a 40-kD, integral protein without interchain disulfide bonds.

Defective Fc receptor-specific reticuloendothelial (RE) function has been reported in certain patients with a variety of immunologic and nonimmunologic diseases. The mechanism responsible for the impaired RE function is uncertain, but it could be caused by immune complexes that are present in many of these disorders. Alternatively, the impaired RE function could be a secondary effect of the high concentrations of monomeric IgG in the serum of these patients, since monomeric IgG can compete with complexed IgG for macrophage receptors in vitro. We studied the Fc-dependent RE function in 30 healthy control subjects and 27 patients using IgG-coated radiolabeled autologous red cells. There was a significant relationship between the concentration of IgG in the serum and the rate of clearance of antibody-sensitized cells (r = 0.51, P less than .01). Patients with hypergammaglobulinemia had the slowest Fc-dependent clearance, whereas those with hypogammaglobulinemia had the most rapid clearance. Immune complexes (Raji or polyethylene glycol) could not be shown to contribute to Fc-dependent RE clearance above the effect of the IgG in the serum. The unusually rapid clearance in a patient with hypogammaglobulinemia could be returned to normal by raising the concentration of IgG in the serum. This study supports the concept that serum (monomeric) IgG competes with immune complexed IgG for macrophage Fc receptors in vivo. The competition for Fc receptors determines the level of competence of Fc-dependent RE function. Based on the results of this study, one can predict that a number of disorders characterized by hypergammaglobulinemia also will have impaired Fc-dependent RE function.

Linear pharmacokinetic models were obtained for fentanyl in anaesthetized rabbits. A composite model was used to predict the bolus dose and infusion rates necessary to achieve rapidly a steady-state concentration of fentanyl in plasma. When this was tested in a further six rabbits, steady-state concentrations were greater than predicted. The most likely reasons for this are imperfect mixing between plasma and the central compartment, and substantial uptake of fentanyl by the lung. These sources of error were allowed for in modifications to the model. On further testing, a 4-h infusion set to achieve a plasma fentanyl concentration of 10 ng ml-1 yielded actual concentrations ranging from a mean low of 8.7 ng ml-1 to a mean high of 18.0 ng ml-1. The second model described more precisely the disposition of fentanyl, although a tendency to overestimate dosing requirement remained. This tendency is probably a consequence of continuing lung uptake of fentanyl. The demonstrated ability to obtain rapidly and maintain for 4h "steady" blood fentanyl concentrations suggests that the methods merits further investigation with other drugs both in animals and man.

A dipstick suitable for immunoassay procedures has been developed, and its utility demonstrated by the development of tests for the determination of immunoglobulin levels in newborn bovine blood, and for the detection of bovine antibodies against Brucella abortus. The dipstick is of simple design, consisting of three components: a polycarbonate membrane, an adhesive, and a support material. The polycarbonate membrane is a porous filter material upon which the immunoassay is undertaken. Biomolecules bind to the membrane with sufficient affinity to permit the development of useful immunoassay procedures; however, this membrane exhibits low non-specific binding of reagents. Collectively, these properties make polycarbonate a useful material for solid-phase immunoassay procedures.

In this report, we describe a sensitive immunoradiometric assay (IRMA) for quantitating IgG on the surface of red cells. Washed red cells were prepared to a purity of greater than 99.9 percent. Varying dilutions of these cells were incubated with a fixed concentration of 125I-anti-IgG. After equilibrium was achieved, the unbound 125I-anti-IgG was measured by the addition of IgG covalently linked to agarose beads. The red cells were lysed by detergent, and the 125I-anti-IgG bound to the IgG- beads was measured. The amount of IgG on the red cells was determined by relating the concentration of test red cells causing 50 percent inhibition of binding of the 125I-anti-IgG to the IgG-beads to 50 percent inhibition of binding caused by the IgG standard. Using this assay, the red cell-associated IgG (RCA-IgG) of 20 healthy male and female controls with normal hemoglobin concentrations was 7.23 +/− 6.11 fg IgG per 10(3) cells (mean +/− 2 SD). The mean RCA-IgG on washed cells from 34 different tests performed on 19 anemic patients with clinically diagnosed autoimmune hemolytic anemia was 176.1 +/− 375.6 fg IgG per 10(3) cells. There was no correlation between the levels of RCA- IgG and the hemoglobin levels or reticulocyte counts in these patients.

The phenomenal rise in the past decade of clinical chemistry methods has brought about increased demands for more rapid, precise and economic assays. In the assay of serum proteins for either diagnostic or medical care purposes, these demands have been met largely by the development of automated immunoassays employing modern instrumental hardware. Occupying a prominent position among the analytical methods are the related light scattering techniques of nephelometry and turbidimetry since they meet many of the requirements for fast, efficient assays. While these light scattering methods may not have the glamour of newer immunoassay methods, they still remain an essential workhorse for most large clinical laboratories.

Background: Alloantibodies to HPA-1a (PlA1) are the major cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura and have been implicated in refractoriness to random-donor platelet transfusions. However, most assays used to phenotype platelets are cumbersome or time-consuming for large numbers of samples. Study Design and Methods: A simple, competitive (inhibition) enzyme-linked immunosorbent assay for HPA-1a phenotyping of donor platelets was developed. A segment from the donor platelet unit transfer line was sealed to obtain a small aliquot of platelets. These platelets were washed once and added to a predetermined dilution of serum containing alloantibodies to HPA-1a. Residual anti-HPA-1a binding to the glycoprotein IIb/IIIa purified by lectin and high-performance liquid chromatography and coated on microtiter wells was detected with a conjugated antihuman IgG. A lack of inhibition equivalent to control (no platelets) was used to determine that the platelets were HPA-1b/b. Results: Of the 557 platelet units tested, 14 (2.5%) were found to be HPA-1a negative, and they were confirmed to be HPA-1b/b by DNA genotyping. Two of the 14 HPA-1b/b units were also HPA-3b/b (approx. 0.35% of the random population). Use of the microtiter format allows 100 to 200 samples to be processed per day. Conclusion: This simple and inexpensive assay is useful for identifying HPA-1b/b units for platelet-compatible transfusions or for platelet antibody investigations.

Thebaine (1) reacts reversibly with various nitroso-arenes to form cyclo-adducts by 1,4-addition of the nitrosogroup to the conjugated diene system of the alkaloid. Adducts (2) of thebaine with nitrosobenzene and with 4-chloro-, 4-methyl-, 3-methoxy-, and 4-nitro-nitrosobenzene have been prepared in good yield. The adduct with nitrosobenzene was hydrolysed by hydrochloric acid to give 14β-(N-hydroxyphenylamino)codeinone (3; Ar = Ph) in high yield. Catalytic hydrogenation of this derivative afforded 7,8-dihydro-14β-phenylaminocodeinone (4). 14β-(N-Hydroxyphenylamino)codeinone rearranged readily in alkaline solution to give the phenol, 5,O-dihydro-5β,14β-(N-phenylepoxyimino)codeinone (5).

Nitrosyl chloride and silver cyanide react at low temperatures to give nitrosyl cyanide (ONCN) which undergoes 1,4-cycloaddition reactions with conjugated dienes to form the corresponding 2-cyano-3,6-dihydro-2H-1,2-oxazines. Thus, the alkaloid, thebaine (1), reacted with a limited quantity of nitrosyl cyanide to form a single adduct (2; R = H, X = CN) convertible into 14-cyanamidocodeinone (3); with an excess of nitrosyl cyanide the related N-cyanomethyl derivative (2; R = X = CN) was formed. Similarly, N-cyano-oxazines have been prepared from buts-1,3-diene and its 2,3-dimethyl, 1-methoxycarbonyl-4-methyl, 1-cyano-4-methyl, and 1,4-bisethoxycarbonyl derivatives. Base-induced rearrangements reactions leading to a pyridone (7), a pyrrolinone (11), and a mixture of dihydrofurans (14) are described.

The piperidine ring of securinine is derived from a C5N unit, Δ1–piperideine, which originates from lysine in non-symmetrical fashion by loss of the carboxy carbon and the α-amino group.

Nitrosyl cyanide and 9,10-dimethylanthracene (DMA)(2) reacted at –25 °C to form the crystalline cycloadduct, 9,10-dihydro-9,10-(N-cyanoepoxyimino)-9,10-dimethylanthracene (1). The adduct (1) decomposed in the presence of the conjugated diene thebaine (3), to form DMA (2) and the adduct (4) of nitrosyl cyanide and thebaine. First-order kinetics, k= 6.9 × 10–5s–1, were observed for the release of DMA in benzene at 40 °C, consistent with slow dissociation of the adduct (1) followed by rapid capture of nitrosyl cyanide by thebaine. A similar first-order rate, k= 6.8 × 10–5s–1, was observed for the reaction of the adduct (1) and triphenylphosphine (2 mol equiv.) under the same conditions, the products being DMA, triphenylphosphine oxide, and triphenylphosphine N-cyanoimide (5). The reactions of nitrosyl cyanide, generated thermally from the adduct (1), were studied with a range of dienes. The conjugated dienes, N-cyanomethyl-N-northebaine (3; NCH2CN replacing NMe), trans,trans-1,4-diphenylbuta-1,3-diene (6; R = H), and ergosteryl acetate (11) all gave the expected cycloadducts (3,6-dihydro-2H-1,2-oxazines). Norbornadiene gave the tetracyclic adduct (10) arising from 1,4-conjugate addition of nitrosyl cyanide. The reactions of the adduct (1) with tetraphenylcyclopentadienone (14), 1,3-diphenylisobenzofuran (18), 2-methyl-1,3-diphenylisoindole (23), diazofluorene (24), and diphenyldiazomethane all took a more complex course leading in each case to the formation of an N-cyano-ketimine (alkylidene-cyanamide)

Radioactivity from [14C]glycolaldehyde enters pyridoxal phosphate specifically, and is confined to the two-carbon unit C(5)–C(5′). Glycolaldehyde enters as an intact two-carbon unit, the aldehyde carbon atom supplying C-5, and the carbinol carbon atom C-5′ of the vitamin. These observations are interpreted in terms of the early stages of the biosynthesis of vitamin B6.

Conjugated dienes react with silver cyanide and nitrosyl chloride to give N-cyano-3,6-dihydro-2H-1,2-oxazines which may arise from cycloaddition with the reactive intermediate, nitrosyl cyanide.

Thebaine reacts reversibly with nitroso-arenes to form Diels–Alder adducts readily convertible into previously inaccessible derivatives of 14-aminocodeinone.