Diagnosis and management of heparin-induced thrombocytopenia: third edition

This guideline updates and widens the scope of the previ-ous British Society for Haematology (BSH) Clinical guide-lines for Diagnosis and Management of Heparin-Induced Thrombocytopenia: Second Edition1 to include functional assays in the diagnosis of heparin-induced thrombocytope-nia (HIT), when to use direct-acting oral anti-coagulants, and the role of intravenous (IV) immunoglobulins and plasma exchange in the management of HIT and spontane-ous HIT.HIT is an immune-mediated, highly pro-thrombotic dis-order of platelet activation caused by pathogenic antibodies against a platelet factor 4 (PF4)­heparin complex. It is the most frequent drug-induced immune thrombocytopenia and may lead to life-threatening thrombosis. There are two distinct forms of HIT: type I, also known as heparin-asso-ciated thrombocytopenia, which is a non-immunological response to heparin treatment, mediated by a direct interac-tion between heparin and circulating platelets causing plate-let clumping or sequestration, and type II, which is immune mediated.

P-selectin expression assay in a repeatedly serotonin-release assay-negative patient with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Pathogenic antibodies to PF4/heparin bind and activate platelets to propagate a hypercoagulable state culminating in life-threatening thrombosis. The serotonin-release assay (SRA) is considered the gold-standard test to diagnose HIT. However, the sensitivity of the SRA was questioned with reported cases of clinical diagnosis of HIT and negative SRA. Herein, we present the utility of platelet factor 4-dependent P-selectin expression assay (PEA) in diagnosing HIT in a patient with thrombocytopenia and recurrent thrombosis who repeatedly tested negative with SRA.

Long-acting PGE2 and Lisinopril Mitigate H-ARS.

Thrombocytopenia is a major complication in hematopoietic-acute radiation syndrome (H-ARS) that increases the risk of mortality from uncontrolled hemorrhage. There is a great demand for new therapies to improve survival and mitigate bleeding in H-ARS. Thrombopoiesis requires interactions between megakaryocytes (MKs) and endothelial cells. 16, 16-dimethyl prostaglandin E2 (dmPGE2), a longer-acting analogue of PGE2, promotes hematopoietic recovery after total-body irradiation (TBI), and various angiotensin-converting enzyme (ACE) inhibitors mitigate endothelial injury after radiation exposure. Here, we tested a combination therapy of dmPGE2 and lisinopril to mitigate thrombocytopenia in murine models of H-ARS following TBI. After 7.75 Gy TBI, dmPGE2 and lisinopril each increased survival relative to vehicle controls. Importantly, combined dmPGE2 and lisinopril therapy enhanced survival greater than either individual agent. Studies performed after 4 Gy TBI revealed reduced numbers of marrow MKs and circulating platelets. In addition, sublethal TBI induced abnormalities both in MK maturation and in in vitro and in vivo platelet function. dmPGE2, alone and in combination with lisinopril, improved recovery of marrow MKs and peripheral platelets. Finally, sublethal TBI transiently reduced the number of marrow Lin-CD45-CD31+Sca-1- sinusoidal endothelial cells, while combined dmPGE2 and lisinopril treatment, but not single-agent treatment, accelerated their recovery. Taken together, these data support the concept that combined dmPGE2 and lisinopril therapy improves thrombocytopenia and survival by promoting recovery of the MK lineage, as well as the MK niche, in the setting of H-ARS.

Utility of the 4Ts score in excluding heparin-induced thrombocytopenia in lung transplant recipients.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic complication following heparin exposure. Data is limited on the incidence of HIT and validity of 4Ts score in the solid organ transplant population. This retrospective observational cohort included patients who underwent lung transplant between August 2015 and June 2018 and had a clinical suspicion of HIT with heparin-PF4 testing. The 4Ts score was correlated with the heparin-PF4 antibody and serotonin release assay (SRA) results, with positive SRA considered confirmed HIT. Of 146 patients evaluated, the overall incidence of HIT was low (2(1%)). Fifty-one patients had heparin-PF4 testing and were included in the cohort; 5 (10%) had positive heparin-PF4 and 1 (2%) had confirmed HIT. The median 4Ts score was 3 (3-4). Thirty (59%), 17 (33%), and 4 (8%) patients had low, intermediate, and high risk, respectively. The intermediate/high risk group compared to the low risk group had a higher use of alternative non-heparin anticoagulation [13 (62%) vs 7 (23%); p = 0.0086)] and a higher incidence of thrombosis [13 (62%) vs 1 (3%); p < 0.0001]. No patient with a low 4Ts score had confirmed HIT, supporting the utility of low 4Ts score to exclude HIT diagnosis in lung transplant recipients.

Platelet factor 4 regulates T cell effector functions in malignant pleural effusions.

Malignant pleural effusion (MPE) is defined as the presence of tumor cells in pleural fluid and it is a fatal complication of advanced lung adenocarcinoma (LAC). To understand the immune response to the tumor in MPE, we compared the concentration of immunomodulatory factors in MPE of LAC and pleural effusion of heart failure (HF) patients by ELISA, and the proliferation and cytotoxic phenotype of T cells stimulated in the presence of LAC and HF pleural fluids by cytometry. Platelet factor 4 (PF4), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß) and P-selectin levels were higher in LAC than in HF pleural fluids. However, plasmatic PF4 and P-selectin levels were similar in LAC and HF. VEGF positively correlated with TGF-ß and sPD-L1 in LAC but not in HF pleural fluids. LAC pleural fluids also inhibited T lymphocyte proliferation and cytotoxicity and reduced IL-17 production. PF4 levels inversely correlated with T cell function. The high content of PF4 in MPE was associated with poor prognosis. Our findings suggest that an impaired response of T lymphocytes induced by PF4 provides a significant advantage for tumor progression.

Development of a method to analyze the complexes of enoxaparin and platelet factor 4 with size-exclusion chromatography.

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. However, heparin-induced thrombocytopenia and thrombosis (HITT) is a serious side effect of heparin therapy, resulting in relatively high risk of amputation and even death. HITT is caused by forming of complexes between heparin and platelet factor 4 (PF4). Enoxaparin, one of the most commonly used low molecular weight heparin (LMWH), were developed in 1980's. The lower molecular weight of enoxaparin reduces the risk of HITT by binding to less PF4. To detect the binding capacity between enoxaparin and PF4 could be an effect way to control this risk before it goes to patients. In this work, a size exclusion chromatography (SEC) method was developed to analyze the patterns of complexes formed between PF4 and enoxaparin. The chromatographic condition was optimized to separate PF4, enoxaparin, ultra-large complexes and small complexes. The linearity and stability of this method were confirmed. The impacts of PF4/enoxaparin mixture ratios and incubation time on the forming complexes were investigated. Four enoxaparin samples were analyzed with this method to verify its practicability. It is a robust, accurate and practicable method, and provides an easy way to monitor the capacity of enoxaparin forming complexes with PF4, suggesting the HITT related quality of enoxaparin.

Chronic periodontitis is associated with platelet factor 4 (PF4) secretion: A pilot study.

AIM: Platelets contribute to chronic inflammation, but their role in periodontitis is not well understood. The aim of this study was to compare platelet recruitment and activation in healthy and inflamed periodontium. MATERIALS AND METHODS: Gingival crevicular fluid (GCF) samples were obtained from sites of healthy periodontium, gingivitis and periodontitis. Platelets were quantified in the GCF by staining and microscopy. GCF concentrations of platelet factor 4 (PF4) [PF4]GCF and glycoprotein IIbIIIa ([GPIIbIIIa]GCF ) were determined by ELISA. Blood samples were obtained from the three patient groups. Platelets were isolated from whole blood and stimulated with lipopolysaccharide (LPS) from Porphyromonas gingivalis to evaluate and compare the LPS-induced PF4 release. RESULTS: Compared to controls, platelet recruitment was increased at gingivitis and periodontitis sites, based on platelet counts and [GPIIbIIIa]GCF . [PF4]GCF was elevated in periodontal pockets but not at gingivitis or healthy sites. Circulating plasma levels of PF4 were higher in patients with generalized severe periodontitis (SP), compared to patients with gingivitis or healthy periodontium. Platelets isolated from SP patients contained and released more PF4 in response to P. gingivalis LPS than platelets from gingivitis or periodontally healthy patients. CONCLUSIONS: Periodontitis is associated with increased platelet activation and PF4 release, both locally and systemically.

Trombocitopenia inducida por heparina con trombosis aortoilíaca tratada con argatrobán / Heparin-induced thrombocytopenia with aortoiliac thrombosis treated with argatroban

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Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Elevated Release of Beta-thromboglobulin and Platelet Factor 4 in Cerebral Infarction Patients with Branch Atheromatous Disease: A Preliminary Report.

This study evaluated the levels of the platelet activation markers beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) in patients with branch atheromatous disease (BAD). Patients with newly diagnosed cerebral infarctions were recruited into the study; those with cardiogenic cerebral infarctions were excluded. Beta-TG and PF4 levels were measured before therapeutic intervention and compared between patients with and without BAD; Welch's t-test was used to determine significant differences between the groups. A total of 15 subjects were enrolled in the study, and 8 were diagnosed with BAD. Beta-TG (P = 0.031) and PF4 (P = 0.041) levels were significantly higher in the BAD patients than in the non-BAD patients. Platelet activity is normally elevated in patients with cerebral infarctions, but is elevated to an even greater extent in BAD patients. The evaluation of beta-TG and PF4 levels may be beneficial for the elucidation of BAD.

Automated multiplexed ECL Immunoarrays for cancer biomarker proteins.

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.

Mechanism of action of antiplatelet drugs on decompression sickness in rats: a protective effect of anti-GPIIbIIIa therapy.

Literature highlights the involvement of disseminated thrombosis in the pathophysiology of decompression sickness (DCS). We examined the effect of several antithrombotic treatments targeting various pathways on DCS outcome: acetyl salicylate, prasugrel, abciximab, and enoxaparin. Rats were randomly assigned to six groups. Groups 1 and 2 were a control nondiving group (C; n = 10) and a control diving group (CD; n = 30). Animals in Groups 3 to 6 were treated before hyperbaric exposure (HBE) with either prasugrel (n = 10), acetyl salicylate (n = 10), enoxaparin (n = 10), or abciximab (n = 10). Blood samples were taken for platelet factor 4 (PF4), thiobarbituric acid reactive substances (TBARS), and von Willebrand factor analysis. Onset of DCS symptoms and death were recorded during a 60-min observation period after HBE. Although we observed fewer outcomes of DCS in all treated groups compared with the CD, statistical significance was reached in abciximab only (20% vs. 73%, respectively, P = 0.007). We also observed significantly higher levels of plasmatic PF4 in abciximab (8.14 ± 1.40 ng/ml; P = 0.004) and enoxaparin groups (8.01 ± 0.80 ng/ml; P = 0.021) compared with the C group (6.45 ± 1.90 ng/ml) but not CD group (8.14 ± 1.40 ng/ml). Plasmatic levels of TBARS were significantly higher in the CD group than the C group (49.04 ± 11.20 µM vs. 34.44 ± 5.70 µM, P = 0.002). This effect was prevented by all treatments. Our results suggest that abciximab pretreatment, a powerful glycoprotein IIb/IIIa receptor antagonist, has a strong protective effect on decompression risk by significantly improving DCS outcome. Besides its powerful inhibitory action on platelet aggregation, we suggest that abciximab could also act through its effects on vascular function, oxidative stress, and/or inflammation.

Trombocitopenia inducida por heparina en hemodiálisis tratada con argatroban / Heparin-induced thrombocytopenia in haemodialysis treated with argatroban

Se presenta el caso de un paciente con un diagnóstico de probabilidad de trombocitopenia inmune inducida por heparina (TIH II) con base en criterios clínicos y test de ELISA positivo, en que la urgencia terapéutica fue determinante ante la gravedad del proceso. La suspensión de heparina y la administración de argatroban solventaron la ineficacia de la diálisis debida a coagulación repetida del circuito y catéter, permitiendo el paso a diálisis peritoneal sin mayores contratiempos. Consideramos prioritaria la toma de decisiones antes de certificar la seguridad diagnóstica (AU)

We present the case of a patient diagnosed with likely immune heparin-induced thrombocytopenia (HIT II) based on clinical criteria and a positive ELISA test, in which emergency treatment was crucial, given the seriousness of the process. The discontinuation of heparin and administration of argatroban resolved inefficiency of dialysis resulting from repeated coagulation of the circuit and catheter, allowing peritoneal dialysis without further setbacks. We consider decision-making to be a priority before certifying diagnostic confidence (AU)

New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells.

INTRODUCTION: The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required. MATERIALS AND METHODS: We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin. RESULTS: The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab. CONCLUSION: The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.

Antiplatelet drugs reduce the immunoinflammatory response in a rat model of periodontal disease.

BACKGROUND AND OBJECTIVE: After activation, platelets express mediators that modulate inflammation. We hypothesized that drug-induced platelet inactivation may interfere in the inflammatory process in experimental periodontal disease by suppressing the release of biological mediators to the injury site. MATERIAL AND METHODS: To evaluate the effects of antiplatelet drugs on experimental periodontal disease, 60 rats were randomly assigned to six groups (n = 10) and ligatures were placed around lower first molars in three groups. The other three groups were not subjected to the induction of periodontal disease and were used as negative controls. During the experimental period, animals were given aspirin (30 mg/kg) or clopidogrel (75 mg/kg) intragastrically once daily for 3 d. On day 3, they were killed and gingival tissue were used to evaluate myeloperoxidase activity and the expression of the chemokine CXCL4. Hemi-mandibles were used for microscopic evaluation. RESULTS: Clopidogrel significantly reduced the inflammatory infiltrate and increased the amount of collagen fibers. Histometric analysis showed that clopidogrel impaired alveolar bone loss. Expression of CXCL4 was significantly increased (p < 0.001) in rats subjected to periodontal disease. Systemic administration of aspirin and clopidogrel induced a significant decrease ( p < 0.05) in the expression of CXCL4. Treatment with antiplatelet drugs resulted in a significant reduction of myeloperoxidase activity when compared to saline-treated animals with periodontal disease. CONCLUSION: Clopidogrel but not aspirin showed the ability of preventing bone loss in experimental periodontitis.

Platelet activation in acute pancreatitis.

OBJECTIVES: The aim of this study was to assess the functional state of platelets in patients with mild acute pancreatitis and severe acute pancreatitis (S-AP). METHODS: The number of platelets and their morphological parameters were measured with Advia 2120. ß-Thromboglobulin and platelet factor 4 concentrations were determined by enzyme-linked immunosorbent assay method. To evaluate the expression of platelet glycoproteins, flow cytometry method was used. RESULTS: At the time of admission, a multiparameter evaluation of the platelets' function in AP patients showed enhanced platelet activation, which was reflected by an increase in the number of large platelets, concentration of degranulation markers (platelet factor 4 and ß-thromboglobulin), expression of glycoprotein (Gp) IIb/IIIa, and decreased mean platelet component. Only in S-AP patients at day 1 a decreased number of platelets and high expression of P-selectin and GpIa were observed, which may suggest their prognostic value. At day 30, the procoagulation state was still present in S-AP patients, because of increased platelets and number of large platelets as well as high GpIIb/IIIa expression. CONCLUSIONS: These results may indicate an important role of platelet activation in the pathogenesis of acute pancreatitis and the development of complications in S-AP.

Functional divergence between 2 chemokines is conferred by single amino acid change.

CXCL4 and CXCL4L1 are 2 closely related CXC chemokines that exhibit potent antiangiogenic activity. Because interactions with glycosaminoglycans play a crucial role in chemokines activity, we determined the binding parameters of CXCL4 and CXCL4L1 for heparin, heparan sulfate, and chondroitin sulfate B. We further demonstrated that the Leu67/His67 substitution is critical for the decrease in glycan binding of CXCL4L1 but also for the increase of its angiostatic activities. Using a set of mutants, we show that glycan affinity and angiostatic properties are not completely related. These data are reinforced using a monoclonal antibody that specifically recognizes structural modifications in CXCL4L1 due to the presence of His67 and that blocks its biologic activity. In vivo, half-life and diffusibility of CXCL4L1 compared with CXCL4 is strongly increased. As opposed to CXCL4L1, CXCL4 is preferentially retained at its site of expression. These findings establish that, despite small differences in the primary structure, CXCL4L1 is highly distinct from CXCL4. These observations are not only of great significance for the antiangiogenic activity of CXCL4L1 and for its potential use in clinical development but also for other biologic processes such as inflammation, thrombosis or tissue repair.

Normal ranges of angiogenesis regulatory proteins in human platelets.

Platelets sequester angiogenesis regulatory proteins early in tumor growth, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA-based method for accurate and reproducible measurement of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1), fibroblast growth factor, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 +/- 0.37 pg/10(6) platelets); PDGF (23 +/- 6 pg/10(6)); PF4 (12 +/- 5 ng/10(6)); TSP-1 (31 +/- 12 ng/10(6)); bFGF (0.44 +/- 0.15 pg/10(6)); and endostatin (5.6 +/- 3.0 pg/10(6)). There was an excellent correlation (R(2) = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet-poor plasma by factors of: VEGF (215-fold); PDGF (914-fold); PF-4 (516-fold); TSP-1 (813-fold); and bFGF (17-fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5-week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP-1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet-derived measurements were accurate and reproducible with minimal biovariability.