Platelet Serotonin Release Research Articles

Fondaparinux treatment of acute heparin‐induced thrombocytopenia confirmed by the serotonin‐release assay: a 30‐month, 16‐patient case series

Fondaparinux is theoretically an attractive agent for the treatment of immune heparin-induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet-activating anti-platelet factor 4/heparin antibodies. Although reports of the use of fondaparinux for this indication have thus far been favorable, the diagnosis of HIT in most cases was not based on definitive laboratory confirmation of heparin-dependent, platelet-activating antibodies. To report thrombotic and major bleeding outcomes with fondaparinux in patients with a high likelihood of having acute HIT based on clinical features and a positive result in the confirmatory platelet serotonin-release assay (SRA), a sensitive and specific test for platelet-activating HIT antibodies. We reviewed consecutive eligible patients with SRA-positive HIT (mean peak serotonin release, 91% [normal, < 20%]; mean IgG-specific PF4/heparin enzyme immunoassay result, 2.53 optical density units [normal, < 0.45 units]) in one medical center over a 30-month period who received fondaparinux for anticoagulation during acute HIT (platelet count, < 150 × 10(9) L(-1)). Where available, plasma samples were used to measure thrombin-antithrombin (TAT) complex levels. Sixteen patients with SRA-positive HIT received fondaparinux: 14 surgical (11 after cardiac surgery; three after vascular surgery) and two medical (acute stroke). Fifty-six per cent of patients had HIT-associated thrombosis at the time of diagnosis. No patient developed new, recurrent or progressive thrombosis; one patient developed a major bleed (calf hematoma). One patient judged to have irreversible tissue necrosis before receiving fondaparinux therapy ultimately required limb amputation. TAT complex levels were reduced within 24 h of starting fondaparinux, and 13 of 13 patients were successfully switched to warfarin. Fondaparinux shows promise for the treatment of patients with SRA-positive acute HIT.

Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides

Abstract 2231Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT pentasaccharide-binding sites within their structures which is comparable to the pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the pentasaccharide in heparin compromised patients. Disclosures:No relevant conflicts of interest to declare.

Serotonin regulates the cardiopulmonary effects of pulmonary embolism through vagal C‐fiber activation

We have addressed the hypothesis that the tachypnea, tachycardia and pulmonary hypertension evoked by pulmonary embolism (PE) occur secondary to platelet activation/degranulation followed by serotonin-dependent pulmonary C-fiber activation. In anesthetized guinea pigs, intravenously administered equine collagen induced immediate, sustained (>20 minutes) and pronounced (~200%) increases in respiratory rate, transient falls in blood pressure and sustained increases in right ventricular pressure (n=10). These effects were mimicked by 5-HT and the P2Y receptor agonist and platelet stimulant ADP (n=5/treatment group), and prevented by prior platelet depletion (>90% reduction) with busulfan (n=5). The serotonin reuptake inhibitor fluoxetine (1mg/kg iv) rendered ADP challenges lethal (n=5). Vagotomy prevented the tachypnea and systemic hypotension evoked by PE (n=4). The 5-HT3 receptor antagonist ondansetron (1 mg/kg iv) nearly abolished the responses to 5-HT and prevented the lethal effects of ADP challenge following fluoxetine (n=5/treatment group). Ondansetron also markedly attenuated the tachypnea and hypotension accompanying PE (n=8). Based on these results we speculate that the cardiopulmonary reflexes evoked by PE depend upon platelet serotonin release and subsequent activation of pulmonary C-fibers via 5-HT3 receptors.

Isolation of Bioactive Compounds That Relate to the Anti‐Platelet Activity of Cymbopogon ambiguus

Infusions and decoctions of Cymbopogon ambiguus have been used traditionally in Australia for the treatment of headache, chest infections and muscle cramps. The aim of the present study was to screen and identify bioactive compounds from C. ambiguus that could explain this plant's anti-headache activity. A dichloromethane extract of C. ambiguus was identified as having activity in adenosine-diphosphate-induced human platelet aggregation and serotonin-release inhibition bioassays. Subsequent fractionation of this extract led to the isolation of four phenylpropenoids, eugenol, elemicin, eugenol methylether and trans-isoelemicin. While both eugenol and elemicin exhibited dose-dependent inhibition of ADP-induced human platelet serotonin release, only eugenol displayed potent inhibitory activity with an IC50 value of 46.6 μM, in comparison to aspirin, with an IC50 value of 46.1 μM. These findings provide evidence to support the therapeutic efficacy of C. ambiguus in the non-conventional treatment of headache and inflammatory conditions.

CDP7657, and Other Anti-CD40L Antibody Formats Lacking a Functional Fc Region, Do Not Cause Platelet Activation In Vitro

AbstractAbstract 5128 Objectives:Hu5c8, a whole IgG1 anti-CD40L antibody, is known to cause thrombotic side effects in patients with Systemic Lupus Erythematosus (SLE). Anti-CD40L-mediated activation of platelets in vitro was shown to be FcγRIIa-dependent1, suggesting that the adverse effects of hu5c8 could be due to this mechanism, and that anti-CD40L antibodies lacking a functional Fc region would not activate platelets. CDP7657 is a monovalent Fab' PEG anti-CD40L antibody being developed for the treatment of SLE. The aim of the present work was to study a range of anti-CD40L antibody formats in isolated platelet assays and to evaluate the role of the Fc region and of valency. Methods:Isolated human platelets from healthy donors were used in the platelet aggregometry and serotonin release assay systems. Antibodies were added either alone, or pre-complexed with soluble CD40L trimer (sCD40L) at optimal stoichiometry. Antibody IV.3 was used to block FcγRIIa. The antibodies and antibody fragments tested included hu5c8 in IgG1, aglycosyl IgG, Fab′, & (Fab′)2 formats, CDP7657, and the CDP7657 variable regions in di-Fab' PEG, tri-Fab' PEG and aglycosyl formats. Results:Hu5c8 in complex with sCD40L caused strong aggregation (in 2–3 minutes) and >85% serotonin release, both alone and in complex with sCD40L. This activation was completely abrogated by blocking FcγRIIa. In contrast, platelet activation did not occur with CDP7657 or other antibody formats, in any assay, regardless of the valency or presence of sCD40L. Conclusions:The data support the hypothesis that platelet activation by anti-CD40L antibodies is dependent on the presence of an active Fc moiety, via interactions with FcγRIIa, and indicate that CDP7657 has the potential to deliver the therapeutic effect of anti-CD40L blockade in SLE without thrombotic toxicity.

Formation of anti–platelet factor 4/heparin antibodies after cardiac surgery: Influence of perioperative platelet activation, the inflammatory response, and histocompatibility leukocyte antigen status

Anticoagulation therapy with heparin induces antibodies that recognize multimolecular complexes of platelet factor 4 bound to heparin (anti-platelet factor 4/heparin antibodies). Considering that cardiac surgery induces an intense platelet activation and proinflammatory response, we examined the relationship between formation of anti-platelet factor 4/heparin antibodies and plasma levels of platelet factor 4 and interleukin 6. We also examined the relationship between anti-platelet factor 4/heparin seroconversion and the histocompatibility leukocyte antigen system. In 71 patients undergoing cardiac surgery, anti-platelet factor 4/heparin antibody levels were evaluated by means of enzyme-linked immunosorbent assay preoperatively and 14 days postoperatively. Platelet serotonin release assays were performed to assess the platelet-activating potential of the antibodies. Plasma levels of platelet factor 4 and interleukin 6 were assayed at prespecified time points. Histocompatibility leukocyte antigen status was assessed preoperatively in all patients and was compared with that of 6156 healthy subjects. Thirty-seven (52%) patients had anti-platelet factor 4/heparin antibodies with an OD value of 0.45 or greater in 1 or more of the assays. Applying strict seroconversion criteria (>2-fold increase in Optical Density), only 16 (22.5%) patients had evidence of anti-platelet factor 4/heparin antibody seroconversion after the operation. Neither the presence of anti-platelet factor 4/heparin antibodies nor seroconversion influenced postoperative outcomes. The CW4 allele was significantly more frequent among seroconverted patients (46.9% vs 19.1%, P = .002). Platelet factor 4 levels did not influence seroconversion. Patients with anti-platelet factor 4/heparin levels of 0.45 OD units or greater 14 days after the operation had significantly higher interleukin 6 levels measured 1 hour after protamine administration. Patients with a greater amount of perioperative inflammation could be more likely to have anti-platelet factor 4/heparin antibodies 1 to 2 weeks later. We provide additional evidence that the histocompatibility leukocyte antigen CW4 confers genetic susceptibility in an acquired inflammatory disorder that includes the anti-platelet factor 4/heparin immune response.

IL-1 beta and IL-6 stimulate the production of platelet-activating factor (PAF) by cultured rabbit synovial cells.

The aim of this study was to determine whether synovial cells are capable of producing PAF in the presence of cytokines such as IL-1 beta and IL-6 and other stimuli. Synovial cells were obtained from joints of healthy rabbits. PAF production was assayed by measurement of serotonin release in rabbit platelets and the incorporation of 3H-acetate into PAF. Synovial cells produced PAF after 5 min of incubation with ionophore A23187, reaching the maximal amount at 15 min (4.3 +/- 0.7 x 10(-3) pmol of PAF/mg protein, P < 0.005, n = 4), and declining afterwards. The treatment of synoviocytes with IL-1 beta and IL-6 induced synthesis of PAF after 5 min of stimulation, reaching the greatest production at 15 min with IL-6 and 30 min with IL-1 beta (3.6 +/- 1.1 x 10(-3) and 3.3 +/- 1.2 pmol of PAF/mg protein, respectively, P < 0.05, n = 4). The incubation of the cells with PMSF, an inhibitor of acetylhydrolase, before the addition of the stimuli, increased the incorporation rate of 3H-acetate, suggesting a rapid degradation of PAF by synoviocytes. These results demonstrate that synovial cells can produce PAF after stimulation with agonists, such as ionophore, and cytokines. Thus, PAF, acting alone or with other mediators, could amplify the inflammatory joint reaction.

A Spontaneous Prothrombotic Disorder Resembling Heparin-induced Thrombocytopenia

Background Antibodies against the “self” protein, platelet factor 4 (PF4), bound to heparin—the cause of immune heparin-induced thrombocytopenia—are believed invariably to be triggered by preceding heparin therapy. We describe a novel syndrome, spontaneous heparin-induced thrombocytopenia, in which clinical and serologic features characteristic of this adverse drug reaction develop in patients despite the absence of preceding heparin therapy. Methods Three patients met the study criteria (clinical and serologic features of heparin-induced thrombocytopenia without preceding heparin exposure), of whom 2 patients were identified among 225 patients (0.89%, 95% confidence interval, 0.11%-3.17%) with serologically confirmed heparin-induced thrombocytopenia recognized during an 18-year period at 1 hospital. The platelet serotonin-release assay was used to detect heparin-dependent immunoglobulin G-induced platelet activation, and 2 enzyme immunoassays were used to detect antibodies against PF4/heparin. Results Two patients presented with thrombocytopenia and multiple arterial thrombosis, and 1 patient presented with anaphylactoid reactions after 2 subcutaneous injections of low-molecular-weight heparin. All 3 patients had high levels of platelet-activating anti-PF4/heparin antibodies of immunoglobulin G class at presentation despite the absence of previous heparin exposure. However, each patient did have a preceding infectious or inflammatory event; 1 patient had concomitant antiphospholipid antibodies. Conclusion Circumstances other than heparin use can trigger a spontaneous disorder that closely mimics heparin-induced thrombocytopenia, further supporting the autoimmune nature of this adverse drug reaction.

Increased mortality in hemodialysis patients having specific antibodies to the platelet factor 4-heparin complex

Heparin-induced thrombocytopenia is a serious complication of heparin therapy that can lead to thromboembolism, cardiovascular events, and death. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing antibodies to the platelet factor 4-heparin (PF4-H) complex. We sought to determine the association between PF4-H antibodies and mortality in a prospective cohort of 419 asymptomatic hemodialysis patients. Pre-dialysis blood samples were screened for nonspecific PF4-H antibodies, and all positive and indeterminate samples were subsequently tested using immunoglobulin (Ig)G-specific PF4-H and platelet serotonin-release assays. During a median follow-up of 2.5 years there were 129 all-cause deaths. After controlling for potential confounding variables, the relative risk of death was significantly increased for patients with IgG-specific PF4-H antibodies and further elevated with an indeterminate serotonin-release assay. Our study suggests that IgG-specific PF4-H antibody formation is associated with increased mortality in hemodialysis patients.

What is the potential for overdiagnosis of heparin‐induced thrombocytopenia?

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4//heparin (PF4/H) complexes. According to the "iceberg model," only a subset of anti-PF4/heparin antibodies of IgG class evincing strong platelet-activating properties cause clinical HIT. Since many centers rely predominantly on an anti-PF4/polyanion enzyme-immunoassay (EIA) to diagnose HIT, we estimated the potential for overdiagnosis when only this single test is available. We examined a database of 100 patients in whom the probability of HIT had been estimated using a clinical scoring system (4Ts), and where patients underwent systematic testing for HIT antibodies using three assays: the platelet serotonin release assay (SRA), an "in-house" EIA that detects IgG anti-PF4/heparin antibodies (EIA-IgG), and a commercial EIA that detects anti-PF4/polyanion antibodies of all three immunoglobulin classes (EIA-GTI). Whereas 16 of 100 patients fulfilled a "classic" definition of HIT (intermediate/high probability plus strong platelet-activating anti-PF4/heparin IgG antibodies), an additional 16 patients fulfilled a "liberal" definition in which any investigated patient (irrespective of the pretest probability) who had a positive EIA-GTI was considered to have HIT. The clinical features of these 16 additional patients--including generally weak antibodies and low risk for thrombosis--suggest underlying non-HIT explanations for thrombocytopenia. Patients with a positive SRA generally corresponded to those with intermediate or high pretest probability of HIT who also had strong EIA-GTI reactivity (>1.20 OD units). We conclude there is the potential to overdiagnose HIT by approximately 100% if any positive EIA is considered to "confirm" the diagnosis of HIT irrespective of the clinical scenario.

Defibrotide Does Not Cross-React with HIT Antibodies. Implications in the Management of HIT.

Defibrotide is a mammalian DNA derived antithrombotic and anti-ischemic agent that does not produce systemic anticoagulation. Heparin-induced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1 – 100 μg/ml) and unfractionated heparin (1 – 100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 4 samples (2%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with platelet factor 4 (PF4), and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Studies carried out on the effect of purified IgG from HIT patients revealed that defibrotide blunts platelet activation and microparticle formation as measured by flow cytometry. These studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Moreover, unlike heparin, defibrotide does not promote platelet activation, rather it is capable of suppressing the hypercoagulable state associated with HIT. Defibrotide is orally bioavailable and can be used for extended anticoagulant management of HIT patients.

An Algorithm for “Indeterminate” Test Results in the Platelet Serotonin Release Assay for Heparin-Induced Thrombocytopenia (HIT).

HIT is a prothrombotic complication of heparin caused by antibodies that recognize complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions. These antibodies produce thrombocytopenia by activating platelets via their Fc receptors. A “functional” (platelet activation) assay that utilizes washed platelets, known as the 14C-serotonin release assay (SRA), has the highest reported sensitivity-specificity tradeoff for detecting clinically-significant antibodies that recognize PF4/heparin complexes (“HIT antibodies”). Platelet activation (% serotonin release) induced by patient (or control) serum is assessed under several different reaction conditions, including absence of heparin, therapeutic concentrations of unfractionated heparin (UFH, 0.1 to 0.3 U/mL) and low-molecular-weight heparin (LMWH, 0.2 U/mL), supratherapeutic concentrations of UFH (100 U/mL), and with 0.1 U/mL UFH in the presence of a platelet Fc receptor-blocking monoclonal antibody. Various technical aspects of the assay optimize test sensitivity and specificity (e.g., using heat-inactivated patient serum; washing the platelets with apyrase; resuspending the platelets in albumin-free Tyrode's buffer; using platelets from healthy volunteers known to react well to IgG platelet agonists, etc.). The classic HIT platelet activation profile is strong platelet activation (>50% serotonin release) in the presence of therapeutic UFH or LMWH that is inhibited by supratherapeutic heparin and the platelet Fc receptor-blocking monoclonal antibody. One drawback to the assay is that some patient sera activate platelets via the Fc receptors in a heparin-independent fashion, i.e., the platelets are activated at all heparin concentrations. This is known as an “indeterminate” reaction profile, since the presence of in vitro immune complexes (generated by the heat-inactivation process) or in vivo immune complexes or other platelet-activating factors could “mask” the presence of a true HIT antibody. We developed an algorithm for dealing with such indeterminate reaction profiles. First, we repeat the SRA using another aliquot of patient serum that is newly heat-inactivated, and also use different platelet donors to perform the assay. Often, this results in an interpretable test result. However, if the repeat SRA also gives an indeterminate result, we then use an in-house anti-PF4/heparin ELISA (that detects only IgG class antibodies) to determine whether HIT antibodies could be present. From 2091 patient serum samples tested for HIT antibodies using the SRA, we identified 199 (9.5%) samples that gave an initial indeterminate result. Using our algorithm, 81 samples subsequently gave clearly negative results, and 35 samples gave clearly positive results. However, 83 samples (representing 41.7% of the retested samples, or 4.0% of the total samples) gave a repeat indeterminate test result. When this last group of samples was tested using the anti-PF4/heparin-IgG ELISA, 53 of the samples tested negative (OD<0.45) and 30 tested positive (OD >0.45). With this algorithmic approach, about 96% of patients can be classified as negative or positive using the SRA. However, 4% of patients require the use of a complementary assay- the anti-PF4/heparin-IgG ELISA, to evaluate for the presence of HIT antibodies. Further studies are required to determine the causes of persistent indeterminate results in the SRA, which may lead to new approaches to further optimize this assay.

Antibodies to the Platelet Factor 4-Heparin Complex and Mortality in Chronic Hemodialysis Patients.

Background: Heparin-induced thrombocytopenia is a serious complication of heparin therapy that can lead to thromboembolism, cardiovascular events or death. Patients with this disorder develop antibodies to the platelet factor 4-heparin (PF4-H) complex. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing PF4-H antibodies. The clinical impact of asymptomatic PF4-H antibodies in patients on chronic hemodialysis is not known.Objective: To determine the association between asymptomatic PF4-H antibodies and mortality in a cohort of chronic hemodialysis patients repeatedly exposed to heparin.Methods: Pre-dialysis blood samples were drawn from 419 asymptomatic patients. All patients received unfractionated heparin (Baxter) while on dialysis. All samples were screened for PF4-H antibodies using an ELISA assay (GTI PF4 Enhanced, GTI Diagnostics). All positive and indeterminate samples were then tested using an IgG-specific PF4-H ELISA assay and a platelet serotonin-release assay. Participants were then followed up prospectively for thromboembolic events, cardiovascular events, or death.Results: During a median follow-up of 2.5 years there were 129 deaths. After controlling for important potential confounding variables, the relative risk of death was 2.92 (95% CI: 1.18-7.25; P= 0.02) in patients with IgG-specific PF4-H antibodies and 4.08 (95% CI: 1.26–13.2; P= 0.02) in patients with IgG-specific antibodies and an indeterminate serotonin-release assay. Conclusions: PF4-H antibody formation is associated with increased all-cause mortality in patients on chronic hemodialysis. Further investigation is needed to determine if anticoagulation with alternative agents would improve survival in this population.

The prevalence of antibodies to the platelet factor 4 -heparin complex and association with access thrombosis in patients on chronic hemodialysis

IntroductionHeparin-induced thrombocytopenia is a serious complication that can lead to thrombocytopenia, venous and arterial thrombosis. Patients with this disorder develop antibodies to the platelet factor 4-heparin (PF4-H) complex. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing PF4-H antibodies.We sought to determine the prevalence of PF4-H antibodies in a large cohort of patients on chronic hemodialysis and to evaluate the relationship between PF4-H antibodies and hemodialysis vascular access thrombosis in a case-control study. Material and methodsPre-dialysis blood samples were drawn on 419 patients; 107 cases with access thrombosis and 312 controls that never had access thrombosis. All samples were screened for PF4-H antibodies using an ELISA assay (GTI PF4 Enhanced, GTI Diagnostics). All positive and indeterminate samples were then tested using an IgG-specific PF4-H ELISA assay and a platelet serotonin-release assay. ResultsAntibodies to PF4-H were positive in 54 (12.9%) patients using the screening ELISA assay. Nine (2.1%) patients had IgG-specific PF4-H antibodies. None of the patient's had a positive platelet serotonin-release assay. No relationship between hemodialysis access thrombosis and PF4-H antibodies was noted using the screening ELISA assay (unadjusted odds ratio 0.63; 95% CI 0.30–1.30; P = 0.21), the IgG-specific ELISA assay (unadjusted odds ratio 0.83; 95% CI 0.17–4.06; P = 0.82) or indeterminate platelet serotonin-release assay results (unadjusted odds ratio 0.97;95% CI 0.10–9.44;P = 0.98). ConclusionsHemodialysis with repeated exposure to unfractionated heparin was associated with a moderately elevated prevalence of PF4-H antibodies. However, our results do not support a relationship between PF4-H antibodies and hemodialysis vascular access thrombosis.

Defibrotide does not cross‐react with HIT antibodies. Implications in the management of HIT

Defibrotide is a mammalian DNA derived antithrombotic and antiischemic agent that does not produce systemic anticoagulation. Heparininduced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies formed. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1–100 μg/ml) and unfractionated heparin (1–100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 2 samples (1%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with PF4, and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Taken together these studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Defibrotide would not promote the HIT pathology of platelet activation; it would suppress the hypercoagulable state associated with HIT; and it is orally bioavailability making it applicable for both acute and long-term treatment.

Towards a Conceptual Framework for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

HIT can be regarded as a “clinicopathologic” syndrome whereby the diagnosis is based upon a compatible clinical picture and the presence of platelet-activating, anti-platelet factor 4 (PF4)/heparin antibodies of IgG class. However, both components of this conceptual framework are problematic. First, there are many potential non-HIT explanations for thrombocytopenia and/or thrombosis in hospitalized patients receiving heparin (“clinical confounders”); and second, prospective studies show that numerous heparin-treated patients develop non-pathogenic anti-PF4/heparin antibodies of no clinical significance (“antibody confounders”). A third issue is that the most specific laboratory assay for pathogenic HIT antibodies (platelet serotonin release assay [SRA]) is performed in only a few reference centers, whereas less specific commercial anti-PF4/polyanion enzyme-immunoassays (EIAs) are widely available. Recently, a clinical scoring system (“4 T's”) that classifies patients into low-, intermediate- and high-risk groups (LR, IR, and HR, respectively) has been shown to have predictive value in estimating the likelihood of a patient having clinically-significant HIT antibodies. Applying these various concepts, we sought to estimate the potential for HIT “over-diagnosis” inferred from a comparison of “conservative” and “liberal” conceptual frameworks for HIT. The conservative definition required an IR or HR clinical score, as well as that strict laboratory criteria be met for defining pathogenic antibodies (positive SRA [>50% serotonin release] and positive EIG-IgG [>0.45 units]). In comparison, a liberal definition considered HIT in the setting of any positive commercial anti-PF4/polyanion-EIA result (>0.40 units) in any patient evaluated for possible HIT irrespective of clinical score. Our data base consisted of 100 consecutive patients evaluated for possible clinical HIT over a 16-month period in which the “4 T's” clinical scoring system was applied prospectively. Patients underwent systematic serologic assessment by SRA, EIA-IgG, and EIA-GTI. We found that 16 patients (HR=8, IR=8) met the clinical and serologic criteria for clinical HIT using the conservative framework. In contrast, using the liberal framework, there were 32 patients who would have been considered to have clinical HIT (HR=8, IR=12, LR=12). Thus, twice as many patients (32 vs 16) would have been “diagnosed” as HIT from the liberal perspective. The EIA-GTI absorbance values (in units) for the 16 patients identified using the conservative framework (median=2.39; IQR=2.09–2.70; range=1.46–2.90) were significantly greater (p<0.001 by Mann-Whitney u test, 2-sided) than in the additional 16 patients identified using the liberal framework (median=0.89; IQR=0.54–1.13; range=0.42–2.13). In summary, there is considerable potential for over-diagnosis of HIT (by approximately 100%) depending upon one's conceptual framework (and available assay) for defining HIT. However, in a center relying upon the EIA-GTI for diagnosis, the designation of a patient as having clinical HIT by integrating the clinical score (IR or HR status required) with a strong positive result in the EIA-GTI (e.g., >1.20 units in this data set) yields a population of patients that overlaps considerably with that identified using a more conservative approach utilizing a more specific diagnostic assay. Thus, by integrating information from the clinical score together with the magnitude of a positive test result, accurate diagnosis of HIT is achievable even when very different assays are employed.

IN FOCUS: Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor

Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects. The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors. Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4. The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.

SRA Does Not Detect Potentially Relevant HIT Antibodies.

Even before the nature of the heparin:platelet factor 4 (H:PF4) antigenic target was identified, it was known that pathogenic activity in sera/plasma from Heparin-Induced Thrombocytopenia (HIT) patients could be recovered in the immunoglobulin G (IgG) fraction. In in vitro diagnostic tests, blockade of platelet FcγIIa receptors completely prevents platelet activation by HIT sera. For these reasons, the IgG isotype is considered the predominant HIT pathogenic agent. H:PF4 ELISA techniques have made it possible to distinguish between antibodies (Abs) of the IgG, IgA and IgM isotypes, and have shown that IgA and IgM Abs are more prevalent than previously thought. In a group of 247 H:PF4 ELISA-positive specimens studied at Loyola University Medical Center, 65% contained IgG, 53% had IgA, and 56% had an IgM component. The biological activity and pathogenic relevance of non-IgG H:PF4 Abs is as yet unknown. These studies were undertaken to specifically test whether H:PF4 IgA or IgM isotypes, in the absence of IgG, could cause platelet activation in the Serotonin Release Assay (SRA), the reference standard activation assay for diagnosis of HIT. Four SRA positive specimens with multiple isotypes were fractionated using Protein G (Amersham Biosciences) chromatography to separate IgGs. Isotyping of the fractions demonstrated that the H:PF4 IgA and IgM were present in the flow through peak; IgGs were in fractions eluted from the Protein G by low pH. Only fractions that bound to Protein G, ie IgG isotype, caused the characteristic SRA positive response of platelet activation in the presence of low heparin (0.1 U/ml) and not in the presence of excess heparin (100 U/ml). Study of an SRA positive specimen that tested negative for the IgG isotype, showed that the SRA activity was in fractions that bound to Protein G even though the antibody was not detected in the ELISA. Another four SRA positive specimen with multiple isotypes were fractionated using immobilized jacalin chromatography (Pierce, Rockford IL) to separate IgA Abs. Isotyping demonstrated that IgG Abs did not bind to the column; IgA Abs were in fractions eluted from the jacalin column in the presence of melibiose. Only the flow through H:PF4 IgG, and not the eluted IgA Abs, tested positive in the SRA. However, three of the isolated H:PF4 IgAs elicited some degree of platelet serotonin release in the absence of heparin only, without the characteristic 2-point response to heparin. In conclusion, the platelet activation by SRA positive specimens with multiple isotypes is attributable to the IgG component only. Neither IgA nor IgM H:PF4 Abs are detected in the 2-point SRA. While the SRA does not detect IgA or IgM Abs, these isotypes may still have pathogenic activity. Preliminary studies with H:PF4 IgA, indicate that these Abs can bind to platelets and cause serotonin release in the absence of heparin. Until there is better understanding of in vivo biological activity of non-IgG HIT isotypes, it should be noted that the SRA, the reference standard activation assay for HIT, may not detect all physiologically relevant H:PF4 Abs.

Prevalance of Heparin-Dependent Platelet-Activating Antibodies in Preterm Newborns After Exposure to Unfractionated Heparin

Heparin is frequently used in preterm infants to prolong the patency of intravascular catheters. The aim of this study was to evaluate the prevalence of heparin-dependent platelet-activating antibodies in newborns. A cross-section of all preterm newborn infants expected to require heparin to maintain patency of a central venous access line were enrolled. A blood sample was obtained soon after birth before heparin exposure to exclude the possibility of placental transfer of maternal heparin-dependent platelet-activating antibodies. A second sample was obtained at termination of heparin use (mean duration of heparin exposure was 23 +/- 13 days; range, 6-67). Paired samples, at birth and after heparin use, were available for 42 infants with a mean gestational age of 27.8 +/- 2.2 weeks and birth weight of 1036 +/- 267 g. Thrombocytopenia developed in 57% (24/42) of the infants. None of these infants had clinical suspicion of thrombosis during the study period. The etiology of thrombocytopenia was confirmed sepsis in six, presumed sepsis in three, necrotizing enterocolitis in one, and unclear in 14 infants. Anti-heparin/platelet factor 4 antibodies measured using the standard assays for heparin-induced thrombocytopenia (two commercially available enzyme-linked immunosorbent assay tests and the functional platelet serotonin release assay) were negative on all infants. Although it could be related to the poor ability of these infants to mount an immunologic response, further research is necessary to fully understand this lack of response to heparin and to elucidate further the reasons for thrombocytopenia in very-low-birth-weight infants.