Heparin-induced Thrombocytopenia Plasma Research Articles

Cleavage of Anti-PF4/H IgG Antibodies By Ides, and Potential Benefit in the Treatment of Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe drug-adverse event due to platelet-activating antibodies (Abs) directed against platelet factor 4 (PF4)/heparin (H) complexes. In most patients, HIT Abs are IgG that directly activate platelets and monocytes in the presence of heparin via FcγRIIA receptors. The interaction between the Fc fragment of anti-PF4/H IgG and FcγRIIA is thus a key step for cellular activation in HIT. Several bacterial proteases such as IdeS (IgG-degrading enzyme of Streptococcus pyogenes) are cleaving IgG in the lower hinge region of heavy chain leading to the formation of single cleaved IgG (scIgG) and then of Fab'2. Importantly, cleavage of IgG by IdeS can abolish their ability to bind FcγR and suppress the cellular effects resulting from this interaction.The aim of this study was therefore to evaluate whether anti-PF4/H IgG cleavage by IdeS could inhibit cell activation induced by HIT antibodies, and their pathogenicity. To achieve this objective, we studied the effects of IdeS on platelet responses to 5B9, a monoclonal chimeric anti-PF4/H IgG1 recently developed in our laboratory, and which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017).IdeS was demonstrated to quickly (6 minutes) cleave purified 5B9 IgG, leading to the formation of sc5B9, without any reduction in its binding ability to PF4/H complex. However, flow cytometry experiments showed that heparin-dependent binding of sc5B9 to platelets and FcγRIIA was dramatically reduced compared to those of uncleaved 5B9. In addition, functional assays (serotonin release assays and platelet aggregation tests) also confirmed that sc5B9 was unable to induce platelet activation and aggregation in the presence of heparin. Incubation of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood containing 5B9 IgG or HIT plasma samples also lead in every sample tested to the cleavage of anti-PF4/H Abs, which fully abolished their capacity to induce heparin-dependent platelet aggregation, as demonstrated by impedance aggregometry (Multiplate analyzer). As expected, no effect of IdeS was observed on platelet aggregation induced by collagen (1 µg/mL), or ADP (10 µM). Moreover, tissue factor (TF) gene expression induced in monocytes by 5B9 in the presence of heparin was also completely abolished after addition of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood, whereas no inhibitory effect of this protease on TF expression induced by LPS was evidenced. We also showed that platelet aggregation and fibrin formation induced by 5B9 with heparin was completely inhibited after IdeS treatment when whole blood was perfused in vWF-coated microfluidic channels with shear rates similar to those of venous flow (500s-1). Finally, IdeS was also showed to prevent efficiently thrombocytopenia and hypercoagulability (with no increase in thrombin/anti-thrombin plasma levels) induced by 5B9 in transgenic mice expressing human PF4 and FcγRIIA receptors, when previously treated by this protease (0.5 µg/g) before IV injection of heparin.In conclusion, the cleavage of anti-PF4/H IgG by IdeS prevents heparin-dependent cellular activation induced by HIT antibodies, thereby reducing their pathogenicity. Therefore, injection of IdeS could be considered as a potential treatment in patients with severe HIT, particularly in those who necessitate emergent cardiac surgery with cardiopulmonary bypass and thus anticoagulation with unfractionated heparin, which remains the safest and easiest anticoagulant to be used in this specific surgical procedure. DisclosuresNo relevant conflicts of interest to declare.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin‐induced thrombocytopenia antibodies

Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

Characterization of 5B9, a Chimeric Monoclonal Anti-PF4/H Antibody with a Human Fc Fragment and Which Mimics the Effects of HIT Human Antibodies

Heparin-induced thrombocytopenia (HIT) is a frequent drug-adverse event caused in the majority of patients by platelet-activating antibodies (Abs) directed against complexes of heparin (H) bound to platelet factor 4 (PF4). In most cases, HIT Abs are IgG which are potentially pathogenic as they are able to activate platelets directly in the presence of heparin via FcgRIIA receptors. The diagnosis of HIT is based on both clinical and biological criteria. However, despite recent improvements in HIT laboratory assays, a standard is always lacking for both immunological and functional assays. First, platelets from healthy donors exhibit wide variability in their response to HIT antibodies. Second, many patients who synthesize significant levels of antibodies to PF4/H while being treated with heparin do not develop HIT and the mechanisms that regulate the pathogenicity of HIT antibodies have not been fully defined. However, several studies suggested that epitope specificity of IgG antibodies is critical for the pathophysiology of HIT, especially by influencing the stability of PF4 tetramers.We therefore aimed to develop a monoclonal anti-PF4/H antibody with a human Fc fragment using transgenic mice homozygous for Cg gene of a G-class human immunoglobulin and that directly produce chimeric IgG antibodies.After immunization, several clones were found to synthesize anti-PF4 IgG antibodies but only one (5B9) produced an IgG1 that specifically bind PF4/H complexes without reactivity against PF4 alone. 5B9 was able to induce dose-dependent platelet activation and aggregation in the presence of low concentrations of UFH (0.1 and 0.5 IU/mL), and this effect was not observed without UFH or with high concentration of UFH (10 IU/ml), and was fully inhibited by IV.3 antibody. In addition, 5B9 with UFH (0.5 IU/mL) induced strong TF mRNA synthesis in isolated monocytes. Injection of 5B9 with UFH to transgenic mice expressing human PF4 and FcgRIIA receptors was followed by significant thrombocytopenia, similar to that observed with KKO, a murine IgG2b anti-PF4/H antibody. Plasma levels of thrombin/anti-thrombin (TAT) also significantly increased after injection of heparin in all mice having received 5B9 or KKO, but this effect was more pronounced on day 1 in 5B9-treated mice (mean value= 47 vs. 5 ng/mL at day 0) than in mice injected with KKO (33 vs. 11 ng/mL, respectively).Competitive immunoassays were also developed and 15 of 25 plasma samples (60%) collected in HIT patients were shown to inhibit (by at least 20%) the binding of 5B9 to PF4/H complexes compared to 3/25 (12%) samples containing non pathogenic anti-PF4/H antibodies (obtained in patients without HIT). Similar experiments were performed with KKO and its binding to PF4/H was also inhibited by 32% of HIT plasma samples (8/25), and none of the non-HIT plasmas. However, the levels of 5B9 and KKO binding inhibition to PF4/H were highly correlated (R2=0.85), thereby suggesting that the epitopes recognized by the antibodies are similar. Noticeably, KKO was also shown to inhibit in a concentration-dependent manner the binding of 5B9 to PF4/H complexes, further supporting this hypothesis.5B9 was then sequenced and a docking model was elaborated based on VH and VL sequences of 5B9 obtained and a recently described crystal structure of KKO/PF4 tetramer complex. According to this model, 5B9 Fab likely interacts with the B and D monomers of PF4, thus possibly contributing to the stability of tetramers. In addition, the binding of 5B9 to PF4 involves 28 aa, 16 in the B monomer including Asp-7, Gln-9, Pro-34 and Pro-37 and 12 in the D monomer including Gln-9. Importantly, 13 of these 28 aa have also been identified as critical in the formation of PF4/KKO complex (Cai et al, Nat Commun. 2015;6:8277). Three regions (Asp-7 to Thr-15 and Ala-32 to Lys-41 in the B monomer and Gln-9 to Asp-18 in the D monomer) therefore appear particularly important in the binding of both 5B9 and KKO on PF4 tetramers.In conclusion, 5B9 is the first anti-PF4/H monoclonal antibody that has a human Fc fragment, and fully mimics the effect of HIT human antibodies. 5B9 could therefore be used as a standard in HIT laboratory assays. Moreover, our results support that three regions both recognized by 5B9 and KKO within PF4 tetramers are critical for the binding and pathogenicity of HIT antibodies. DisclosuresNo relevant conflicts of interest to declare.

Increased risk of thrombosis in FcγRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2

AbstractThrombosis results in heparin-induced thrombocytopenia (HIT) from cellular activation involving Fc receptors. In this study, the FcγRIIA 131RR genotype was found to increase the risk of thrombosis in HIT patients (odds ratio: 5.9; 95% confidence interval: 1.7-20). When platelet aggregation tests (PATs) were performed with platelet-rich plasma (PRP), a shorter lag time was measured in 131RR donors compared to individuals with the HR and HH genotypes in response to HIT plasma or 5B9, a recently developed humanized monoclonal antibody to PF4/heparin. Importantly, this difference was no longer detectable when PATs were performed with washed platelets or immunoglobulin (Ig)G-depleted PRP. Moreover, polyclonal IgG or monoclonal IgG1 added to IgG-depleted PRP increased the lag time in response to 5B9. HH platelets were also sensitive to IgG2, which in contrast, failed to inhibit the response of 131RR platelets to 5B9. Finally, higher tissue factor messenger RNA levels were measured in the whole blood of 131RR donors after activation by HIT antibodies, with increased phospholipid procoagulant activity. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to increased cell activation by antibodies to PF4/heparin, with a lower inhibitory effect of endogenous IgG, especially from the IgG2 subclass.

Influence of FcγRIIA H131R Polymorphism on the Risk of Thrombosis in HIT and Variable Effect of Normal IgG on Platelet Activation

Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of IgG antibodies that activate platelets via FcγRIIA receptors. HIT is associated with thrombotic complications occurring in about 50% of affected patients.Moreover, the response of platelets to anti-PF4/H IgG varies from one subject to another. This variability, partly explained by gene polymorphisms affecting FcγRIIA signaling (Rollin et al, Blood 2012), might also depend on variations in the binding of HIT antibodies to FcγRIIA receptors.Objective. The influence of FcγRIIA H131R polymorphism on the risk of HIT and thrombosis was investigated in parallel with the effect of normal IgG on the platelet response to HIT antibodies.Patients and Methods. A cohort of 89 patients with definite HIT (PF4-specific ELISA and serotonin release assay both positive) and two control groups with (Abpos ; n=160) or without (Abneg ; n=179) significant levels of PF4-specific antibodies after cardiac surgery were studied. Platelet aggregation tests (PAT) were performed in 59 healthy donors using HIT plasma samples or 5B9, a humanized chimeric monoclonal antibody specific to PF4/H complexes that we have recently developed which fully mimics the effects of human HIT antibodies. The influence of normal IgG and IgG subclasses on platelet aggregation was also evaluated according to the FcγRIIA H131R polymorphism. Finally, tissue factor mRNA levels and plasma phospholipid-dependent procoagulant activity were measured after addition of HIT antibodies and heparin in the whole blood collected from healthy donors with different FCGR2A genotypes.Results. Genotypes and allele frequencies were similar in the HIT patients and both Abneg and Abpos controls. However, interestingly, homozygous FcγRIIA 131RR subjects were more frequent among the HIT patients who had developed thrombotic complications compared to those without thrombosis (34.5% vs. 8.5%, respectively, p=0.008). The FcγRIIA 131RR genotype therefore appeared to increase the risk of thrombosis in HIT significantly (OR = 5.9; 95% CI 1.7-20). This association was not found in two other cohorts of patients who had developed ischemic stroke or venous thromboembolism in a medical context other than HIT, thus indicating that the impact of FcγRIIA H131R polymorphism on the risk of thrombosis was specific to HIT. PAT performed with platelet-rich plasma (PRP) showed that the lag time measured in response to 5B9 with heparin (5B9/H) was significantly longer with FcγRIIA 131HH platelets compared to RR platelets (p=0.03). Importantly, this variable platelet response to 5B9/H related to the FcγRIIA polymorphism was no longer detectable when PAT were performed with washed platelets, when the IgG fraction had been removed from the plasma, and when collagen was used as inducer. On the other hand, the addition of human polyvalent IgG to IgG-depleted PRP inhibited the aggregation induced by 5B9/H, but lower concentrations of IgG were required to achieve this effect in FcγRIIA 131HH subjects compared to RR donors. Importantly, this variable effect of normal IgG on platelet aggregation induced by HIT antibodies was shown to depend mainly on the IgG2 subclass, which binds to the H131 isoform more effectively, whereas IgG1 exerted a similar inhibitory effect in RR and HH homozygous donors. Finally, a stronger increase in TF mRNA levels was also evidenced in homozygous FcγRIIARR donors compared to homozygous FcγRIIAHH donors after incubation of 5B9 and heparin in whole blood. Moreover, this effect was associated with a greater relative shortening of plasma clotting time indicating that the release of procoagulant phospholipid microparticles was more pronounced in RR subjects in response to HIT antibodies (p=0.04).Conclusion. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to an increased cell activation induced by antibodies to PF4/H with a lower inhibitory effect of endogenous IgG, especially of the IgG2subclass. DisclosuresNo relevant conflicts of interest to declare.

Binding of heparin‐dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay

The minimal structural requirements of low-molecular-weight heparins that determine the risk of developing heparin-induced thrombocytopenia (HIT) are not fully defined. The ability of enoxaparin-derived oligosaccharides (OS) to induce platelet activation and exposure of platelet-factor 4 (PF4) epitopes recognized by antibodies developed in HIT was studied by surface plasmon resonance (SPR) and serotonin release assay. Decasaccharides with ≥ 11 sulfate groups induced platelet activation in the presence of plasma from patients with confirmed HIT. Serotonin release of > 80% without full inhibition at 100 μg mL(-1) was achieved with decasaccharides containing 14 or 15 sulfate groups, 2 dodecasaccharides and 2 tetradecasaccharides. An SPR method was developed using purified PF4 immobilized on carboxymethylated dextran. Antibodies from all HIT samples bound to PF4/heparin in SPR assays with resonance units (RU) ratio of 109-173 with HIT plasma vs. 88-93 with control plasma. RU ratios > 100 were measured when PF4 was pre-incubated with OS with ≥ 10 saccharide units and one octasaccharide containing 10 sulfate groups. RU ratios > 140, similar to those measured when PF4 was pre-incubated with unfractionated heparin or enoxaparin, were obtained with purified dodeca- and tetradecasaccharides. RU values strongly correlated with the number of sulfate groups in the decasaccharides tested (r = 0.93, P = 0.02). LMWHs with fragments > 10 saccharides and a large number of sulfate groups are more likely to be associated with a higher risk of HIT. These structure-activity relationships were independent of the ability of the OS to bind antithrombin.

Platelet Microparticle Generation Assay: a Valuable and Simpler Alternative to 14C-SRA for Type-II HIT Diagnosis.

AbstractAbstract 1442 Introduction:Type II heparin-induced thrombocytopenia (HIT) is a serious immune-mediated adverse effect of heparin treatment. The release of procoagulant platelet microparticles (PMPs) is considered as major component of this thrombogenic disease. Early diagnosis of HIT is essential in order to improve clinical outcomes. However, such a diagnosis is challenging. The current diagnostic tests consist of the combination of the clinical scoring system (“4T's rule”) with serological or functional tests such as the gold standard 14C-serotonin release assay (14C-SRA). Serological tests are acceptable for ruling out type II HIT but lack still specificity and are also not standardised. 14C-SRA is time-consuming and is only available in highly specialized laboratories and not in all countries. Aims:The aim of our study is to develop a PMPs generation assay (PMPGA) in whole blood that could be routinely used to diagnose HIT. The ultimate goal is to provide a more specific functional test than the existing serological assays. In addition, this test should be easy to use,faster and as accurate as 14C-SRA. Method:The platelet-poor plasma of HIT-suspected patients (n=71) was first incubated 20 minutes at 37°C with citrated 109 mM whole blood from a healthy donor containing 0, 1 or 500 IU heparin/ml. Those three concentrations aimed at determining a basal PMP concentration, the PMP concentration generated by the HIT antibodies and checking the specificity, respectively. In the second step, PMPs positive or negative for annexin-V FITC (phosphatidylserine (PS)) and anti-CD62P-PE (P-selectin) were quantified on a BDIS FACS Aria® flow cytometer (BD Biosciences).The size of the PMPs was defined using a blend of monodisperse fluorescent beads (Megamix, BioCytex, Marseille, France) of three diameters (0.5, 0.9 and 3 μm) according to a previously described protocol. ROC Curves were performed to determine the optimal cut-offs for MPs concentrations and ratios. 4T's rule and clinical follow-up were registered. Results for ELISA, Light Transmission Aggregometry (LTA), PMPGA, and gold standard14C-Serotonin Release Assay (SRA) were compared by Chi-Square test. Results:During the incubation at low heparin concentration, PMPs expressing PS are generated due to the formation of immune complexes whereas PMPs rate decreases in presence of higher heparin concentration, allowing the definition of positive HIT. The determination of an optimal cut-off for MP concentrations requires standardization of many preanalytical and analytical conditions. However, ratios are much more convenient and the optimal cut-off for the ratio of PMP at low and high heparin concentration was calculated as 2. The relation between PMPGA and SRA was much more pronounced (p<0.0005, n=56 whose 9 positive SRA) than with LTA (p=0.0098, n=39) and ELISA (p=0.0374, n=53). In comparison to SRA, sensitivity and specificity of PMPGA were 78 and 98%, respectively. However, the 2 patients negative with PMPGA and positive with SRA were defined as HIT-negative afterwards. Indeed, the first patient had a 4T rule of 3 with negative serology and LTA. The second patient had a 4T rule of 6 with negative serology and positive LTA. She received low-molecular-weight heparin since more than 3 months and her thrombocytopenia increased after switch to danaparoid and hirudin. On preliminary data (n=19), circulating PMPs determination without heparin in the milieu allows positive and negative HIT plasma discrimination. Conclusion:PMPGA is a rapid and reliable test which reproduces the in vivo type II HIT reaction. It improves performances of LTA and is applicable in labs without LTA. It shows similar results than 14C-SRA with a slightly higher specificity. Finally, a screening test by circulating PMPs determination followed by a confirmation test with PMPGA could be an interesting and a simpler strategy improving HIT diagnosis. Disclosures:No relevant conflicts of interest to declare.

A Unique Biomarker Is Associated with the Prevalence of Functional Heparin Induced Thrombocytopenia (HIT) Associated Antibodies: Results from Proteomic (ProteinChip Array) Profiling of ELISA Positive HIT Plasma Samples.

Heparin induced thrombocytopenia (HIT) represents a complex immunopathologic syndrome resulting in the activation of coagulation, inflammatory and cellular processes, which contribute to the overall pathogenesis. Protease mediated transformation of endogenous proteins resulting in the generation of certain mediators and specific cleavage products may be used to understand the pathogenesis of this syndrome. It was hypothesized that unique biomarkers may be generated in patients who develop HIT antibodies upon exposure to heparins. The identification of unique biomarkers and their prevalence in heparin exposed patients may provide an additional parameter in the prognosis of this syndrome. To profile the proteomic biomarkers in serum, specimens were selected from archived sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). Eleven of these specimens were positive in the SRA. All of these samples were proteomic profiled utilizing a ProteinChip Array method using a Gold (AU) Chip in the molecular weight range of 0 – 150 kDa. The normal sera samples (n = 40) were also analyzed simultaneously in a crossover fashion along with the patients' specimens. Surface enhanced laser desorption/ionization (SELDI) technique utilizing Ciphergen PBS II (Fremont, CA) was used for mass profiling. Proteomic profiling is widely used to identify unique biomarkers in various hemotologic disorders. Although electrophoretic methods identify biomarkers at molecular weights ≥ 25 kDa, the SELDI technique provides a complete spectrum in the lower molecular weight range. The ProteinChip Software for mass profiling was used to analyze the data. Proteins such as albumin were used as control proteins throughout the study. Of the 54 HIT samples profiled, 36 showed a unique biomarker peak at 11.9 kDa (67%) whereas none of the normal sera (n = 40) showed this peak. All of the SRA positive samples also showed the 11.9 kDa biomarker peak. Several biomarker peaks in the range of 5 – 10 kDa were present in the HIT positive sera suggesting increased proteolysis. While no other unique biomarkers were obvious in the HIT samples, the relative proportion of some of the biomarker peaks in the range of 15 – 20 kDa were differentiable in the HIT group. These studies demonstrate that HIT patients' sera exhibit a unique biomarker profile, which is distinguishable from normal serum samples. Combined usage of the ProteinChip Array analysis of the HIT patients' sera with other diagnostic methods may further enhance the diagnostic process for this syndrome. Additional molecular characterization of these biomarkers will also help in the understanding of the pathogenesis of HIT with particular reference to the protease activation processes.

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

AbstractThe 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.

A Flow Cytometric Assay of Platelet Activation Marker P-Selectin (CD62P) Distinguishes Heparin-induced Thrombocytopenia (HIT) from HIT with Thrombosis (HITT)

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.