Heparin-associated thrombocytopenia: observations on the mechanism of platelet aggregation

We investigated the mechanism of heparin-mediated platelet aggregation in 11 patients with heparin-associated thrombocytopenia. Severe thrombocytopenia (16,000 to 66,000 platelets/microliters) developed in each patient during heparin therapy, and platelet aggregation occurred in vitro when heparin was added to mixtures of patient plasma and normal platelet-rich plasma. In 10 patients, heparin-initiated platelet aggregation was inhibited by preincubation of mixtures of normal platelet-rich plasma and heparin-associated thrombocytopenia plasma with monoclonal antiglycoprotein Ib antibodies 6D1 or LJ-Ib1. Both antibodies are directed against the von Willebrand factor binding site on glycoprotein Ib and inhibit only ristocetin-induced platelet agglutination. Purified immunoglobulin G (IgG) from patients with heparin-associated thrombocytopenia also supported heparin-induced aggregation, but equivalent amounts of antigen-binding fragments [F(ab')2] did not. We also found that F(ab')2 of LJ-Lb1 did not inhibit heparin-induced platelet aggregation but retained inhibitory activity against ristocetin-induced platelet agglutination. The monoclonal antibody 3G6, directed against the alpha-chain of glycoprotein Ib but not inhibitory of ristocetin-induced platelet agglutination, had no effect on heparin-induced platelet aggregation. Antibodies to von Willebrand factor that inhibit ristocetin-induced platelet agglutination did not inhibit heparin-mediated platelet aggregation, but antibodies to glycoprotein IIb-IIIa blocked aggregation. These data suggest that platelet aggregation in heparin-associated thrombocytopenia may be initiated by an interaction between patient IgG, heparin, and the platelet surface. Platelet activation appears to be mediated by a platelet surface crystallizable fragment (Fc) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and identification of platelet antibodies in clinical disorders.

Serologic assays to detect and identify platelet-reactive antibodies have progressed from less sensitive and specific Phase I tests based on platelet functional endpoints through more sensitive Phase II assays that detect platelet-associated immunoglobulins, to highly specific Phase III assays that detect antibodies bound to alloantigens located on isolated platelet surface glycoproteins. Phase II and III assays are useful in the evaluation of patients with suspected platelet alloimmune syndromes neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) as well as in platelet crossmatching. Flow cytometry, a Phase II assay, can be modified to detect drug-dependent platelet-reactive antibodies. 14C-serotonin release, a Phase I assay and the platelet factor 4 ELISA, a Phase III assay, are now used to diagnose patients with heparin-induced thrombocytopenia (HIT). A sufficiently sensitive and specific assay to diagnose idiopathic (autoimmune) thrombocytopenia (ITP) remains elusive.     

Human platelet alloantigens.

Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.     

Metabolite-specific (IgG) and drug-specific antibodies (IgG, IgM) in two cases of trimethoprim-sulfamethoxazole-induced immune thrombocytopenia

Two cases of trimethoprim-sulfamethoxazole (TMP-SMX)-induced immune thrombocytopenia are reported in which unusual drug-dependent platelet antibodies were demonstrated by immunofluorescence and enzyme-linked immunosorbent assay. Whereas two distinct sulfamethoxazole-dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N-4-acetyl-sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP-SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite-specific antibodies in drug-dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses.     

Fetoneonatal alloimmune thrombocytopenia (FNAIT): our experience.

OBJECTIVE: Fetoneonatal alloimmune thrombocytopenia (FNAIT) is a relatively rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. It occurs when fetal platelets are destroyed, after sensitization, by a transplacental passage of maternal antibodies directed against a fetal platelet alloantigen inherited from the father. This article reviews some pathophysiologic and clinical aspects of FNAIT. METHODS: We also present our experience with the management of 12 newborns affected with a symptomatic form of this disorder in order to verify what would be the best diagnostic and therapeutic protocols. RESULTS: Antibody identification in maternal serum showed 9 anti-HPA-1a (75% of cases), 2 anti-HPA-1b (17%) and 1 anti-HPA-1a+anti-Gp IV+anti-HLA class I (8%). CONCLUSION: Sixteen human platelet alloantigen (HPA) systems have been identified, six major (from HPA 1 to 5 and HPA 15) and ten rare or private, each composed of two allelic antigens (named "a" or "b", according to major or minor frequency in the population). All HPA systems, including private or low frequency, may play a role in determining FNAIT. Unfortunately FNAIT cannot be prevented, in fact no one of maternal parameters is predictive of thrombocytopenia or its magnitude.     

Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is a common immune-mediated drug reaction that can be complicated by life-threatening arterial thrombosis. The diagnosis can be confirmed by demonstrating heparin-dependent release of radiolabeled serotonin from washed normal platelets in the presence of patient serum. However, certain serum samples from these patients produce 14C-serotonin release from some but not other normal donor platelets. We investigated this problem of donor platelet variability by studying the reactivities of 10 serum samples from patients with heparin-induced thrombocytopenia with platelets from 10 normal donors (100 serum and platelet combinations). We observed a marked variability in reactivity for patient serum and platelets from normal donors; this initially appeared random. However, closer examination indicated that the reactivities varied hierarchically. Because heparin-induced thrombocytopenia is caused by binding of heparin-dependent IgG to platelet Fc receptors, we examined whether platelet Fc receptor number or function explained the variability in platelet reactivity. We observed that platelet Fc receptor function, as measured by platelet release associated with heat-aggregated IgG, was highly correlated with platelet reactivity to heparin-induced thrombocytopenia serum samples. No significant correlation, however, was found between Fc receptor number and platelet response. Reaction of murine monoclonal antibodies that activate human platelets by means of the platelet Fc receptors was not predictive of platelet reactivity to heparin-induced thrombocytopenia serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

血小板配合型输注在血液病患者中的应用

目的探讨经血小板交叉配合试验的配合单采血小板的临床输注效果。方法选择30例反复输注血小板的血液病患者,其中15例应用单克隆抗体固相血小板抗体试验(M A SPAT)进行血小板交叉配合试验,另15例患者采用血小板随机输注,比较其临床效果,并于输注前及输注后1 h和24 h检测血小板计数,以血小板校正计数增值(CC I)判定输注效果。结果交叉配合试验组与随机输注组有效率分别为85.7%,28.1%,差异十分显著(P<0.01)。交叉配合试验组其CC I显著高于随机输注组(P<0.01)。结论经M A SPAT进行血小板交叉配合试验,能显著提高血小板临床输注效果。     

Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management

Summary.  Drug-induced immune thrombocytopenia (DITP) can be triggered by a wide range of medications. Although many cases of DITP are mild, some are characterized by life-threatening bleeding symptoms. The pathogenesis of DITP is complex, in that at least six different mechanisms have been proposed by which drug-induced antibodies can promote platelet destruction. It is possible in many cases to identify antibodies that react with platelets in the presence of the sensitizing drug, but the required testing is technically demanding and not widely available. Therefore, a decision on whether to discontinue an implicated medication in a patient suspected of having DITP must be made on clinical grounds. An algorithm is available that can be helpful in assessing the likelihood that a particular drug caused thrombocytopenia, but the most important aspects of patient management are a high index of suspicion and a careful history of drug exposure in an individual who presents with acute, often severe thrombocytopenia of unknown etiology. How drugs induce platelet-reactive antibodies and how, once formed, the antibodies cause platelet destruction following exposure to the drug is poorly understood. Further studies to address these issues and characterize more completely the range of drugs and drug metabolites that can cause DITP are needed.     

Immune-mediated platelet transfusion refractoriness in a severely thrombocytopenic patient with myelodysplastic syndrome successfully treated with romiplostim

Immune-mediated platelet transfusion refractoriness due to anti-human leukocyte antigen (HLA) antibodies can occur in approximately 9% of patients with myelodysplastic syndromes (MDS) and can lead to an increased risk of clinically relevant bleeds and treatment delays. These patients are typically managed with frequent platelet transfusions; however, HLA-matched platelet transfusions are usually available only in large blood centers. For this reason, alloimunized thrombocytopenic MDS patients are notoriously difficult to manage. Here, we present a case of a MDS patient with an immune-mediated platelet transfusion refractoriness, severe thrombocytopenia and spontaneous subarachnoid hemorhage who we successfully treated with romiplostim, a thrombopoietin receptor agonist.     

Digitoxin intoxication with severe thrombocytopenia: reversal by digoxin-specific antibodies

As a result of overdosage a 77-year-old patient with heart disease developed digitoxin intoxication, associated with arrhythmias, extracardiac symptoms of intoxication and severe thrombocytopenia. Treatment with digoxin-specific antibody fragments relieved the signs and symptoms of intoxication within a few hours. The rise in platelet count from the pretreatment value of 26 000/mm3 to 47 000 within 12 h and to over 60 000/mm3 within 16 h of starting the antibody infusion may also be attributed to the treatment with antibodies. Such a rapid recovery from digitoxin-induced thrombocytopenia has not hitherto been described. Digoxin-specific antibodies, obtained by immunization of sheep with a digoxin-albumin conjugate, were used to treat intoxication with digitoxin, since cross-reaction had been demonstrated in vitro and in animal experiments. The present paper briefly discusses the mode of action and the general problems relating to the antibody therapy of digitalis poisoning.     

Heparin-induced thrombocytopenia

A summary of heparin-induced thrombocytopenia (HIT) is presented. HIT is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. The frequency of HIT ranges from 1 to 5% of patients receiving heparin with exact frequencies ranging between specific agents. Interestingly, this immune-mediated syndrome is ironically associated with thrombosis, not bleeding, with thrombin formation playing a major role. It is caused by heparin-dependent, platelet-activating antibodies that identifies a self-protein, PF4, bound to heparin that results in an antibody formation. The resulting platelet activation is associated with increased thrombin generation. Typically, the platelet count fall begins 5–10 days after starting heparin, although a rapid platelet count fall can occur in a patient who has antibodies from recent heparin use. Typical causes of HIT as well as the best diagnostic studies and treatment are discussed in this review. HIT was reviewed using a pubmed? search; google scholar? using key words: “Heparin-induced thrombocytopenia”; “heparin”, and “drug AND thrombocytopenia.”     

Gold-induced thrombocytopenia responsive to cyclophosphamide.

Thrombocytopenia and nephrotic syndrome developed in a 51-year-old patient receiving gold therapy for rheumatoid arthritis. Marrrow findings and platelet infusion studies were consistent with a pattern of increased platelet destruction, known to occur in gold-induced thrombocytopenia. Improvement in the platelet count after therapy with dimercaprol was transient, and although steroids and splenectomy were not effective, a response was achieved with cyclophosphamide. The use of immunosuppressive drugs can be considered in refractory cases of gold-induced thrombocytopenia in which a significant hemorrhagic risk is present.     

Immune thrombocytopenias: tests for platelet antibodies

In a study of 49 consecutive patients for whom a test for platelet antibodies had been ordered, tests were performed for both cell-bound and serum platelet antibodies. In cases of autoimmune thrombocytopenia (ITP), the test result for cell-bound antibody was more likely to be positive than was the test result for serum antibody. In three patients with ITP who were taking systemic corticosteroids, however, the result of the CBPAT was negative. A similar phenomenon occurred in a patient with SLE. The result of the SPAT correlated best with the presence of alloantibodies formed during pregnancy or after blood transfusion.     

The use of flow cytometry in the diagnosis of heparin-induced thrombocytopenia (HIT)

Heparin-induced thrombocytopenia (HIT) affects some of the patients exposed to heparin. It is mediated by antibodies that recognize neoepitopes on platelet factor 4 (PF4)/heparin complexes. A HIT diagnosis requires both clinical and laboratory evaluation and remains a challenge. Since many patients develop antibodies in response to heparin, but only a few of them generate anti-PF4/heparin antibodies capable of activating platelets which consequently cause clinical complications, the performance of serologic assays is not enough to diagnose HIT. Functional assays can identify pathogenic antibodies capable of platelet activation, but they are more demanding and their limited availability contributes to the problem of diagnosing HIT. Restricted laboratories usually collect sera of multiple patients to perform functional assays only once or twice a week; hence, a HIT diagnosis can take several days. The use of flow cytometry appears to be a promising alternative in the confirmation of pathogenic anti-PF4/heparin antibodies. Flow cytometric assays detect either activation markers on a healthy donor’s platelet surfaces or platelet derived microparticles formed after platelet incubation with a patient’s serum. Flow cytometers are readily available in many clinical laboratories, so this technology introduces the possibility of an earlier HIT diagnosis. The objective of this review was to collect findings on flow cytometric HIT confirmations to the present date, and to review the currently available flow cytometric assays used in the diagnosis of HIT.     

Are maternal antiplatelet antibodies a prothrombotic condition leading to miscarriage?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition characterized by thrombocytopenia in the newborn. If severe, the thrombocytopenia can lead to intracranial hemorrhage. FNAIT arises when maternal antibodies specific for platelet antigens, most commonly β3 integrin, cross the placenta and destroy fetal platelets. Surprisingly, few cases of FNAIT are associated with antibodies specific for the platelet antigen GPIbα, which is a common target in patients with immune thrombocytopenia. In this issue of the JCI, Li et al. have identified a potential reason for this - they find that in the majority of pregnant mice, anti-GPIbα antibodies enhance platelet activation and accelerate thrombus formation in the placenta and that this leads to miscarriage.     

Characterization of antibodies directed against platelet cryptantigens detected during the immunological study of 356 consecutive patients with presumed autoimmune thrombocytopenia (AITP)

SUMMARY. Cryptic antigens are detected by anti-bodies present in a wide spectrum of patients with or without thrombocytopenia, and even in healthy individuals. They are produced for unknown reasons and do not react with antigens of native platelets, but only with altered platelets. Cryptantigen antibodies may not only result in spuriously low platelet counts, but also in 'falsely' positive tests for platelet antibodies. We report our experience in the characterization of the different types of antibodies directed against cryptantigens of platelets: EDTA-dependent antibodies, PFA-dependent antibodies, EDTA-PFA-dependent antibodies and cold agglutinins. These antibodies were detected in the course of the serological study of 37 patients from a group of 356 (10%) whose blood was sent to our laboratory for platelet antibody testing. Pseudothrombocytopenia was diagnosed in 24 cases. Twenty-one of these showed EDTA-dependent or EDTA-PFA-dependent platelet agglutination and three were due to the presence of cold agglutinins. In 13 patients the thrombocytopenia was genuine. Eleven of these presented EDTA-dependent or EDTA-PFA-dependent antibodies in their serum and in the two remaining cases PFA-dependent antibodies were found. Cryptantigen antibodies were also detected in 9 out of 228 (4%) blood donors who were used as healthy controls in the platelet immunofluorescence test. In the light of the results obtained we put forward some guidelines to detect the presence of these antibodies and establish an accurate serological and clinical diagnosis of the autoimmune thrombocytopenias.     

Bivalirudin use in carotid endarterectomy in a patient with heparin-induced thrombocytopenia

OBJECTIVE: To describe the successful use of bivalirudin as the primary procedural anticoagulant in a patient with suspected heparin-induced thrombocytopenia (HIT) undergoing carotid endarterectomy (CEA). CASE SUMMARY: A 73-year-old white man presented for an elective CEA 3 weeks after emergent, on-pump coronary artery bypass grafting. Bivalirudin was used for procedural anticoagulation because of seropositivity for heparin-PF4 antibodies and a clinical history consistent with HIT. The dose was administered as a 0.75 mg/kg bolus and 1.75 mg/kg/h infusion as reported in percutaneous coronary intervention, based on review of the available bivalirudin literature. The dosage was adjusted for the patient's renal dysfunction. The outcome was successful, with the patient discharged home in 8 days without significant complications. DISCUSSION: During active HIT, when thrombocytopenia and heparin-PF4 antibodies are present, heparin therapy must be avoided. In patients with subacute HIT, when platelet counts have recovered but HIT antibodies are still present, it is also prudent to avoid heparin administration. In the case of a patient in whom anticoagulation is necessary but heparin use is contraindicated, a direct thrombin inhibitor, such as bivalirudin, may offer a viable alternative. Bivalirudin is not immunogenic and does not cross-react with the heparin-PF4 antibodies associated with HIT. To our knowledge, as of January 20, 2006, this is the first report of the use of bivalirudin for procedural anticoagulation during CEA in a patient with HIT antibodies and recent exposure to heparin. CONCLUSIONS: Further investigation is warranted to clarify the clinical benefits of bivalirudin for patients undergoing vascular surgery of the carotids, including potential advantages for vulnerable patient populations such as those with diagnosed or suspected HIT as well as those with renal dysfunction.     

Severe thrombocytopenia as a complication of acute Epstein-Barr virus infection

Severe thrombocytopenia is an extremely rare complication of acute Epstein-Barr virus (EBV) infection. EBV infection usually causes hematological abnormalities, mainly atypical lymphocytosis, which is a feature of infectious mononucleosis, and uncomplicated cases often present with mild decreases in platelet counts. Our otherwise healthy, 21-year-old male Caucasian patient had thrombocytopenia and bleeding diathesis with platelet counts of 8 x 10(9)/L without other signs and symptoms of infectious mononucleosis. We commenced treatment with intravenous methylprednisolone before the acute EBV infection was serologically confirmed. Platelet counts initially rose and then fell after we stopped administrating corticosteroids. Repeated administration of methylprednisolone was followed by full recovery of the platelet count and normalization of formerly elevated transaminases. EBV infection may happen in children, adolescents and adults and this differential diagnosis should be considered in every patient presenting with acute thrombocytopenia.     

Post-transfusion purpura in a patient with HPA-1a and GPIa/IIa antibodies

Post-transfusion purpura is a rare bleeding disorder characterized by severe and sudden thrombocytopenia within 3-12 days after blood transfusion. Typically, preformed antibodies directed against human platelet antigens, especially HPA-1a, are associated with the clinical symptoms. A 46-year-old female presenting to the hospital with acute progressive kidney insufficiency and anaemia received two units of packed red blood cells (RBC) within 2 days. On day 7, platelet count felt from 414 to 189 x 10(9) L(-1) and 1 day later dropped to 4 x 10(9) L(-1). Four platelet concentrates were applied without success. After serological confirmation of an HPA-1a antibody, the patient was treated with intravenous gamma immunoglobulin (ivIgG), and the platelet count increased to normal values on day 17. In addition to the persisting HPA-1a alloantibody, an antibody reactive with GPIa/IIa of HPA-5a- and HPA-5b-positive platelets was detected during the acute phase of thrombocytopenia. After complete remission, the patient was transfused with four units of packed RBC from HPA-1a-negative donors, and platelet counts remained normal.