Generation of platelet-derived microparticles and procoagulant activity by heparin-induced thrombocytopenia IgG/serum and other IgG platelet agonists: a comparison with standard platelet agonists

Heparin-induced thrombocytopenia (HIT) is a relatively common, immunoglobulin-mediated adverse drug reaction associated with in vivo thrombin generation and both venous and arterial thrombosis. Serum and purified IgG from patients with HIT induce normal platelets to generate procoagulant platelet-derived microparticles, but the magnitude of this response in comparison with other IgG and standard platelet agonists is unknown. We describe a comparison of IgG platelet agonists, including HIT-IgG/serum, heat-aggregated IgG, and platelet-activating murine monoclonal antibodies, with standard 'strong' and 'weak' platelet agonists, and have determined their relative ability to generate platelet procoagulant activity. Using washed normal platelets as targets, we observed that HIT sera as well as other IgG agonists produced similar or even greater numbers of microparticles and procoagulant activity than the standard strong platelet agonists (thrombin, collagen, and thrombin receptor agonist peptide). The only exception was the non-physiological platelet agonist, calcium ionophore, which consistently produced a platelet procoagulant response even greater than the IgG agonists. We conclude that the IgG class of platelet agonists (including pathogenic HIT antibodies) is an effective trigger of the platelet procoagulant response comparable at least to strong physiological platelet agonists. These results help to explain the association between HIT, in vivo thrombin generation, and thrombosis.     

Generation of platelet-derived microparticles and procoagulant activity by heparin-induced thrombocytopenia IgG/serum and other IgG platelet agonists: a comparison with standard platelet agonists

Heparin-induced thrombocytopenia (HIT) is a relatively common, immunoglobulin-mediated adverse drug reaction associated with in vivo thrombin generation and both venous and arterial thrombosis. Serum and purified IgG from patients with HIT induce normal platelets to generate procoagulant platelet-derived microparticles, but the magnitude of this response in comparison with other IgG and standard platelet agonists is unknown. We describe a comparison of IgG platelet agonists, including HIT-IgG/serum, heat-aggregated IgG, and platelet-activating murine monoclonal antibodies, with standard 'strong' and 'weak' platelet agonists, and have determined their relative ability to generate platelet procoagulant activity. Using washed normal platelets as targets, we observed that HIT sera as well as other IgG agonists produced similar or even greater numbers of microparticles and procoagulant activity than the standard strong platelet agonists (thrombin, collagen, and thrombin receptor agonist peptide). The only exception was the non-physiological platelet agonist, calcium ionophore, which consistently produced a platelet procoagulant response even greater than the IgG agonists. We conclude that the IgG class of platelet agonists (including pathogenic HIT antibodies) is an effective trigger of the platelet procoagulant response comparable at least to strong physiological platelet agonists. These results help to explain the association between HIT, in vivo thrombin generation, and thrombosis.     

Morphological analysis of microparticle generation in heparin-induced thrombocytopenia

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(alpha) 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.     

Correlation of a quinidine-induced platelet-specific antibody with development of thrombocytopenia

The rapidity by which drug-dependent antiplatelet antibodies can develop is not known, since patients are only studied during or after the episode of thrombocytopenia. This report describes the development of quinidine-induced immune thrombocytopenia in a healthy volunteer during a drug study. The thrombocytopenia developed within two weeks of initiation of quinidine therapy. During the thrombocytopenic episode, but not before receiving the drug, the patient had an IgG antiplatelet antibody that bound to control platelets in the absence of the drug. This antibody was absent when the drug was discontinued and the platelet count rose. The patient's acute serum also induced the release of serotonin from control platelets, and the reaction was enhanced by quinidine. This indicates that drug-dependent antiplatelet antibodies can develop rapidly and supports the hypothesis that quinidine-induced thrombocytopenia is due to a quinidine-dependent platelet-specific IgG.     

A diagnostic test for heparin-induced thrombocytopenia: detection of platelet microparticles using flow cytometry

Based on our previous observation that heparin-induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin-dependent fashion, we developed a test for HIT using flow-cytometry to measure heparin-dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen's kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non-radioactive test for HIT.     

Delayed-onset heparin-induced thrombocytopenia and thrombosis

BACKGROUND: Heparin-induced thrombocytopenia is a prothrombotic drug reaction caused by platelet-activating antibodies that recognize complexes of platelet factor 4 and heparin. OBJECTIVE: To describe a syndrome termed delayed-onset heparin-induced thrombocytopenia, in which thrombocytopenia and thrombotic events begin 5 or more days after withdrawal of heparin. DESIGN: Case series. SETTING: Secondary and tertiary care hospitals. PATIENTS: 12 patients who presented with serologically confirmed, delayed-onset heparin-induced thrombocytopenia, including 6 outpatients presenting after hospital discharge. MEASUREMENTS: The platelet serotonin-release assay was used to measure IgG-induced heparin-dependent and heparin-independent platelet activation; an enzyme immunoassay that detects IgG against platelet factor 4-heparin complexes was also used. RESULTS: Patients with delayed-onset heparin-induced thrombocytopenia presented with thrombocytopenia and associated thrombosis a mean of 9.2 days (range, 5 to 19 days) after stopping heparin therapy. Nine patients received additional heparin, with further decrease in platelet counts. Compared with controls, patients with delayed-onset heparin-induced thrombocytopenia had higher titers of IgG antibodies to platelet factor 4-heparin and greater IgG-induced heparin-dependent and heparin-independent platelet activation. CONCLUSIONS: Delayed-onset heparin-induced thrombocytopenia should be suspected when patients present with thrombocytopenia and thrombosis up to 3 weeks after exposure to heparin. This syndrome could be caused by high titers of platelet-activating IgG induced by heparin.     

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.     

Heparin-induced thrombocytopenia and thrombosis: clinical and laboratory studies

Summary. Heparin-induced thrombocytopenia is one of the most common and important immunological complications of drug therapy. Most patients with heparin-induced thrombocytopenia have isolated thrombocytopenia, which by itself seldom causes serious morbidity. However, a small proportion of patients also develop an acute arterial thrombotic episode which can be fatal. It remains uncertain why some patients have only isolated thrombocytopenia, whereas others have thrombotic complications. In this report we describe 53 patients with heparin-induced thrombocytopenia in whom the diagnosis was confirmed using the platelet ¹⁴C-serotonin release assay. The intent of the study was to look for laboratory or clinical characteristics that could be used to predict which patients will have the less serious thrombocytopenia and which patients will have thrombocytopenia plus thrombotic complications. The laboratory markers included AT-III, protein C, protein S and heparin cofactor II. No serological result identified whether a patient was at risk of having isolated thrombocytopenia or an acute thrombotic event. However, during the acute thrombocytopenic episode, there was evidence of global activation of the coagulation cascade as evidenced by reductions in the level of protein C, heparin cofactor II and antithrombin III. Following resolution of the thrombocytopenia, these inhibitory factors returned to normal indicating that the thrombotic complications were not caused by a familial deficiency.
We did observe a highly significant association ( P < 0·001) between concomitant cardiovascular complications and the occurrence of an arterial thrombosis in patients with heparin-induced thrombocytopenia. Recent surgery of any type was strongly associated with venous thrombi ( P < 0·001). Our data suggest that heparin-induced thrombocytopenia is a procoagulant disorder with thrombosis tending to occur at sites of pre-existing pathology.     

Drug-mediated thrombocytopenia

Thrombocytopenia due to drug-dependent antibodies most frequently occurs with quinine/quinidine and with heparin. Considerable evidence has accumulated about the mechanism of action of quinine/quinidine-induced antibodies but less is known about the effect of heparin. Although there is controversy, it is likely that the action of quinine/quinidine-induced antibodies follows a loose association between drug and platelet with antibodies acting independently of the Fc receptor. There is strong evidence that the complex of glycoprotein Ib and glycoprotein IX, absent in the Bernard-Soulier syndrome, provides the binding site for quinine/quinidine-dependent antibodies. It also appears that the two glycoproteins must be present in complex form for antibody binding to occur. There is some heterogeneity of quinine/quinidine-dependent antibodies since there are reports of a proportion of patient antibodies reacting with other membrane determinants or acting independently of the drug. Heparin-induced thrombocytopenia may be the consequence of a direct effect, or a more serious condition associated with thrombosis may occur when heparin-dependent antibodies are formed. The mode of action of these antibodies and the nature of their antigenic determinants remain unclear. Recognition of heparin-associated thrombocytopenia is important so that serious bleeding or thrombotic sequelae can be forestalled.     

Drug-induced Immune Thrombocytopenia

SUMMARY.

Immune thrombocytopenia is a relatively common problem associated with the clinical usage of drugs. Drugs frequently implicated include quinine, quinidine, heparin, penicillins, cephalosporins, co-trimoxazole, gold and D-penicillamine. Bleeding including bruising and purpura is the usual clinical manifestation except in immune heparin-induced thrombocytopenia in which thrombosis occurs more frequently than bleeding. Cessation of the offending drug is the important step in the treatment but other measures may also be required such as platelet transfusion and steroid therapy for patients with clinical bleeding or antithrombotic therapy with warfarin and dextran or low molecular weight heparin/heparinoid for patients with heparin-induced thrombocytopenia and thrombosis. Idiosyncratic drug-induced thrombocytopenia is mediated by an antibody which binds to platelets only in the presence of the drug resulting in the clearance of sensitised platelets by the reticuloendothelial system. In quinine/quinidine-induced thrombocytopenia, the antibodies recognise drug-dependent epitopes on platelet membrane glycoproteins Ib-IX and/or glycoproteins IIb-IIIa. In immune heparin-induced thrombocytopenia the current data suggest a mechanism which probably involves the binding of heparin-antibody complexes to the platelet Fc receptors but the precise mechanism is yet to be fully characterised. The associated thrombosis in this condition is likely to be due to platelet activation and possibly endothelial cell damage induced by the heparin-related antibody.     

A diagnostic test for heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia can be a serious and difficult-to- diagnose complication of heparin therapy. Serum from patients with heparin-induced thrombocytopenia can cause heparin-dependent platelet aggregation, but the low sensitivity and specificity of this test limit its clinical usefulness. In this report we describe an assay for heparin-induced thrombocytopenia that is both sensitive and specific. The improvement in the assay was accomplished by measuring platelet release instead of aggregation and by measuring platelet release at two heparin concentrations. The rationale for the use of two heparin concentrations was that sera from patients with heparin-induced thrombocytopenia caused release at therapeutic but not at high concentrations of heparin. Twenty-eight sera samples from patients suspected of having heparin-induced thrombocytopenia and 573 controls were coded and tested in the assay. The patients with possible heparin- induced thrombocytopenia were ranked according to the likelihood of having this disorder by using prospectively defined criteria. The test had a high specificity (99%); only one of 573 controls showed a positive result. The test was also very sensitive, and the likelihood of a positive test result was directly correlated with the clinical likelihood of the patient having heparin-induced thrombocytopenia. Six of six patients with definitive heparin-induced thrombocytopenia had positive test results, whereas zero of four patients in whom the diagnosis was unlikely had positive test results. The two-point test for heparin-induced thrombocytopenia represents a sensitive and specific test for this disorder. This test may be useful not only in confirming the diagnosis of this disorder but also may provide information about its pathogenesis.     

Induction of monocyte tissue factor expression by antibodies to heparin-platelet factor 4 complexes developed in heparin-induced thrombocytopenia

The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xa-based chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients.     

Drug-dependent and non-drug-dependent antiplatelet antibody in drug-induced immunologic thrombocytopenic purpura

The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid-phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug-dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.     

Endothelial cell damage in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia type II is a severe complication of heparin treatment that may result in thrombosis. When thrombosis occurs it carries a 50% mortality rate. The exact pathophysiology is not fully understood but in the majority of cases it is associated with the production of heparin/platelet factor 4 antibodies. The endothelium provides a protective anticoagulant surface over which blood flows. Perturbation of the endothelial cells causes a reversal of the anticoagulant properties of the cells to that of a procoagulant surface. This is often due to release or down-regulation of the anticoagulant membrane proteins such as thrombomodulin and up-regulation of procoagulant factors such as tissue factor. We studied 10 patients in our cardiothoracic institute with clinically and laboratory-confirmed heparin-induced thrombocytopenia type II for evidence of endothelial cell damage. There was a statistically significant rise in the concentrations of von Willebrand factor (P < 0.0001) and soluble thrombomodulin (P = 0.004) when patients with heparin-induced thrombocytopenia type II were compared with healthy laboratory controls and patients having had cardiopulmonary bypass surgery (von Willebrand factor 324 versus 103 versus 108 U/dl and soluble thrombomodulin 9.5 versus 2.3 versus 1.2 ng/ml, respectively). Our findings suggest that endothelial cell damage is a major factor in the pathophysiology of heparin-induced thrombocytopenia.     

Mechanisms of venous and arterial thrombosis in heparin-induced thrombocytopenia

Since the reports by Weismann and Tobin in 1958 and Roberts et al. in 1964 called attention to paradoxical thrombosis in patients treated with heparin, the thrombotic aspect of the heparin-induced thrombocytopenia syndrome (HIT) has been emphasized. Yet to this day, the mechanism of thrombosis associated with HIT (HITT) is unclear. It is important to understand the etiology of HITT because of its devastating clinical consequences. We believe one rational approach to understand the mechanism underlying HITTS is to invoke Virchow's triad: stasis, vascular injury and a hypercoagulable state. A hypercoagulable state exists in all HIT patients due to platelet activation by heparin antibody binding. Thrombin generation from platelet microparticles and exposed platelet phospholipid, coupled with stasis (elderly bedridden or otherwise sedentary ill patients who comprise the majority of the HIT population), provide two risk factors that can lead to venous thrombosis. A hypercoagulable state coupled with endothelial cell dysfunction due to injury from heparin antibody, activated platelets, leukocytes, platelet microparticles, complement, atherosclerosis or medical intervention can lead to arterial thrombosis. Of patients with HIT, HITT occurs in about 25%, suggesting that a second set of patient specific risk factors, in addition to the generation of pathological heparin antibodies, determine whether HITT will develop. Interaction between activated platelets and other platelets, and with endothelial cells, leukocytes, neutrophils, monocytes and cytokines are areas of research that may provide more specific characterization of the hypercoagulable state and vascular damage. Nuances involving genetic variation in platelets, endothelial cells and immune function are also likely to be a major component of the observed variability of this disease spectrum. Virchow's triad may explain the different manifestations of HITTS.     

Heparin-induced thrombocytopenia: an overview

Heparin-induced thrombocytopenia (HIT) is the most important immunological drug reaction that patients face today. HIT typically develops in patients 5 days after starting heparin therapy, but can occur sooner with recent heparin exposure or rarely have a delayed onset. The platelet count typically drops below 150 x 10(9)/L (average 60 x 10(9)/L), and patients may experience a thrombotic episode simultaneously or shortly after the onset of thrombocytopenia. The thrombocytopenia and the associated thrombotic episodes are now considered to be overlapping outcomes of the same syndrome. The pathophysiology of HIT has been characterized: immune complexes of IgG and heparin in association with a small platelet peptide, platelet factor 4 (PF4), activate platelets by binding to the Fc receptors (FcR) and releasing procoagulant-active, platelet-derived microparticles. The recognition that HIT is characterized by intense thrombin generation dictates the use of antithrombin agents in HIT therapy. Therapeutic approaches that are currently prevalent in the management of HIT will be discussed.     

Heparin-induced skin lesions

Six patients who developed heparin-induced skin lesions were tested for the presence of heparin-dependent, platelet-activating IgG irrespective of whether they developed thrombocytopenia. All six patients' sera contained potent heparin-dependent, platelet-activating IgG. However, only two of the six patients developed thrombocytopenia, and both of these patients developed thrombotic complications. One of the four patients who developed skin lesions without thrombocytopenia also developed a characteristic complication of heparin-induced thrombocytopenia (adrenal haemorrhagic infarction). These observations indicate that formation of heparin-dependent, platelet-activating IgG is a consistent feature of patients who develop heparin-induced skin lesions irrespective of whether thrombocytopenia occurs.     

Qualitative platelet defect in a case of heparin-induced thrombocytopenia

A qualitative platelet defect was demonstrated in a patient with heparin-induced thrombocytopenia. His platelets showed impaired aggregation to collagen and arachidonate and absent second wave aggregation to adenosine diphosphate and epinephrine but normal response to ristocetin. These aggregation abnormalities were similar to those reported in idiopathic thrombocytopenia and systemic lupus erythematosus. This platelet function defect may contribute to haemorrhagic complications which appear to be common in heparin-induced thrombocytopenia.     

Low molecular weight heparin fractions as an alternative therapy in heparin-induced thrombocytopenia

Eight patients with a delayed-onset heparin-induced thrombocytopenia, with thrombotic complications requiring immediate anticoagulation in 7 of them, were given low molecular weight heparin (LMWH) fractions as alternative therapy. This treatment led to normalization of platelet count within 3-5 days in 6 patients with clinical recovery in 5. In 2 patients, thrombocytopenia persisted despite LMWH therapy. In vitro platelet aggregation tests performed in all patients gave evidence of a relationship between the presence (or absence) of a LMWH-dependent platelet-aggregating factor in the patients' plasma and the persistence (or correction) of the thrombocytopenia with LMWH therapy. Although positive in vitro tests may not necessarily be associated with thrombocytopenia, in vitro testing may prove to be a useful guide before giving LMWH fractions as an alternative therapy in patients with heparin-induced thrombocytopenia requiring immediate anticoagulation.     

Immune heparin-induced thrombocytopenia can occur in patients receiving clopidogrel and aspirin

Platelet adenosine diphosphate (ADP) receptors may play a role in potentiating platelet activation induced by IgG from patients with immune heparin-induced thrombocytopenia (HIT), as shown by previous studies using the ADP receptor antagonists AR-C66096 and ticlopidine. Consistent with these observations, we found that platelet activation by HIT sera is also significantly reduced in patients receiving clopidogrel, an ADP receptor antagonist prodrug now in wide clinical use. Despite these in vitro and ex vivo findings, we observed two patients develop acute HIT while receiving both clopidogrel and aspirin: both patients' sera tested strongly positive in a heparin-dependent washed platelet activation assay (100% serotonin release) and PF4/heparin-enzyme-immunoassay (2.594 and 2.190 absorbance units). Both patients also developed HIT-associated clinical sequelae (acute systemic reaction postintravenous heparin bolus; thrombotic stroke) in association with their episode of HIT. We conclude that combined therapy with aspirin and clopidogrel does not necessarily protect against clinical HIT, at least in patients with HIT antibodies that have strong platelet-activating characteristics.