Identifying patients at high risk of heparin-induced thrombocytopenia-associated thrombosis with a platelet activation assay using flow cytometry

2017 ◽  
Vol 117 (01) ◽  
pp. 127-138 ◽  
Author(s):  
Takuma Maeda ◽  
Katsura Nakagawa ◽  
Kuniko Murata ◽  
Yoshiaki Kanaumi ◽  
Shu Seguchi ◽  
...  
Keyword(s):  
High Risk ◽  
Platelet Activation ◽  
Heparin Induced Thrombocytopenia ◽  
Activated Platelets ◽  
Activation Assay ◽  
Significant Difference ◽  
Increased Sensitivity ◽  
Supplementary Material ◽  
Clinical Profiles ◽  
Heparin Exposure

SummaryTo diagnose heparin-induced thrombocytopenia (HIT), detection of platelet-activating antibodies (HIT antibodies) is crucial. However, serum platelet activation profiles vary across patients and depend on test conditions. We evaluated the association between clinical outcomes and platelet-activating profiles assessed by a platelet microparticle assay (PMA), which detects activation of washed platelets induced by HIT antibodies, in 401 consecutive patients clinically suspected of having HIT. We made modifications to the assay, such as donor selection for washed platelets that increased sensitivity. Serum that activated platelets at a therapeutic (but not high) heparin concentration was defined as positive. Of these, serum that activated platelets within 30 minutes or in the absence of heparin was defined as strongly positive. The remaining samples were considered weakly positive. As a result, 97 % and 93 % of patients who tested strongly and weakly positive had clinical profiles consistent with HIT, respectively. The incidence of thromboembolic events (TEEs) after heparin exposure in patients who tested strongly positive, weakly positive, and negative was 61 %, 40 %, and 29 %, respectively. Among patients who did not experience a TEE on the day HIT was suspected, there was no significant difference in the cumulative incidence of subsequent TEEs between patients who tested strongly and weakly positive when argatroban was initiated on the same day (19.0 % vs 7.1 %, p=0.313), but there was a significant difference when argatroban therapy was delayed by one or more days (61.1 % vs 17.6 %, p=0.007). The modified PMA is effective in diagnosing HIT and identifying patients at high risk for HIT-associated TEEs.Supplementary Material to this article is available online at www.thrombosis-online.com.

Spontaneous Heparin-Induced Thrombocytopenia Syndrome: Two New Cases and a Proposal For Defining This Disorder

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2328-2328 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Paul Andrew Basciano ◽  
Richard A. Bernstein
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Research Funding ◽  
Left Vertebral Artery ◽  
Platelet Factor ◽  
Shoulder Hemiarthroplasty ◽  
Heparin Induced Thrombocytopenia ◽  
Activation Assay ◽  
Count Recovery ◽  
Heparin Exposure

Abstract Introduction Heparin-induced thrombocytopenia (HIT) is a transient, autoimmune-like, prothrombotic disorder caused by heparin-dependent, platelet-activating IgG reactive against platelet factor 4/heparin (PF4/H). There is an emerging literature (Am J Med 2008;121:632-6. J Thromb Haemost 2008;6:1598-1600; Thromb Haemost 2013;109:669-75) pointing to rare instances of “spontaneous” HIT in patients without preceding heparin. We report 2 new cases and propose a definition for this controversial disorder. CASE #1. A 62-y.o. man presented with left middle cerebral artery stroke and thrombocytopenia (platelet count, 65×109/L). There was no previous history of thrombocytopenia, surgery, hospitalization, or heparin exposure. Clot extraction performed with heparin was complicated by further platelet count decline to 27 (nadir) and progressive thrombosis of the carotid artery. Aspirin was started, and the platelets recovered to >150 by day 13. CASE #2. A 54-y.o. female developed right leg swelling, left-upper extremity weakness/paresthesias, and thrombocytopenia (61×109/L) 15 days post-shoulder hemiarthroplasty; no intra-/postoperative heparin had been given. Brain MRI demonstrated acute infarct in the left posterior inferior cerebellar artery territory; angiography showed non-visualization of the left vertebral artery. Ultrasound revealed right lower-limb deep-vein thrombosis. Heparin treatment resulted in further platelet count fall to 37 (nadir). Treatment with argatroban, followed by fondaparinux, was associated with platelet count recovery to >150 by day 39. Methods Testing for HIT antibodies was performed by commercial EIA-IgG/A/M (Immucor GTI Diagnostics), in-house EIA-IgG (McMaster), and serotonin-release assay (SRA). Results Both patients’ sera (obtained before any heparin administration) tested strongly positive for HIT antibodies (Table), including strong platelet activation at 0.1 and 0.3 IU/mL heparin, as well as at 0 U/mL heparin, with no platelet activation at 100 IU/mL heparin: these serological features are characteristic of “delayed-onset HIT” (Ann Intern Med 2001;135:502-6). Antibody reactivity declined markedly by 2 to 4 weeks (including loss of platelet-activating properties at 0 IU/mL heparin), in keeping with the usual transience of HIT antibodies (N Engl J Med 2001;344:1286-92), and paralleling both patients’ platelet count recovery. Discussion These cases further support spontaneous HIT as an unusual explanation for acute arterial stroke and thrombocytopenia. One patient had preceding orthopedic surgery, an event previously reported with spontaneous HIT (Thromb Haemost 2013;109:669-75). The strong serum-dependent platelet activation at 0 IU/mL heparin helps to explain how thrombocytopenia and thrombosis can occur in a patient not receiving heparin. RECOMMENDATION. Based on the serological findings of these and previous cases, we propose that a definitive diagnosis of spontaneous HIT syndrome should be based upon all of the following criteria: thrombocytopenia, thrombosis, lack of proximate heparin exposure, strong-positive PF4-dependent immunoassay(s), and a strong-positive platelet activation assay featuring both heparin-dependent (e.g., high heparin neutralization) and heparin-independent platelet activation (at 0 IU/mL heparin). Disclosures: Warkentin: Pfizer Canada: Honoraria; Paringenix: Consultancy; Immucor GTI Diagnostics: Research Funding; WL Gore: Consultancy; GSK: Research Funding.

scholarly journals Antibody Cloning Identifies Pathogenic and Non-Pathogenic Antibodies in Heparin-Induced Thrombocytopenia and Defines the Molecular Signatures That Differentiate the Two Types of Antibodies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 439-439
Author(s):  
Wen Zhu ◽  
Yongwei Zheng ◽  
Mei Yu ◽  
Yaling Wu ◽  
Jianhui Wei ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Platelet Activation ◽  
Solid Phase ◽  
Variable Regions ◽  
Heparin Induced Thrombocytopenia ◽  
Tyrosine Residues ◽  
Healthy Donors ◽  
Activation Assay ◽  
Positively Charged

Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction associated with frequent life-threatening thrombotic complications. The hallmark of HIT is polyclonal antibodies (Abs) that recognize platelet alpha granule chemokine PF4 when it binds to heparin (PF4/H). These Abs can be detected in solid phase assays that use PF4/H as a target (PF4 ELISA), but only a minority of patients testing positive actually have HIT, i.e., most heparin-induced Abs are non-pathogenic. In patients who have clinical HIT, Abs that activate platelets can be detected using a platelet-activation assay such as the serotonin release assay, or the PF4-dependent p-selectin expression assay (PEA) (Chest 2016; 150:506). Thus, there are at least two distinct types of heparin-induced Abs - those that react only in PF4 ELISA and are seemingly "non-platelet-activating" and "non-pathogenic" and those that are "platelet-activating" and "pathogenic". To date, the molecular basis for the differing clinical and serologic behaviors of pathogenic and non-pathogenic Abs is uncertain. To address this issue, we performed single cell cloning to clone B cell receptors from IgG1+ B cells from HIT patients. We deposited single B cells (CD19+IgG1+) from 6 patients with "classical" and 2 patients with "spontaneous" HIT into 96 well plates containing feeder cells (from G Kelsoe, Duke U) that support B cell proliferation and Ab secretion (Immunity 2018;48:174). Clones secreting IgG were first screened in PF4 ELISA and positive results were obtained with 55 clones from 6 patients. Further screening showed that 7 of these clones (from 4 patients) were also PEA-positive (platelet-activating). Clones positive only in PF4 ELISA, positive in both PF4 ELISA and PEA, or negative in PF4 ELISA were designated NP (non-pathogenic), PA (platelet-activating) and NB (non-binding), respectively. H and L chain variable regions were defined in 7 PA, 42 NP and 34 NB clones. The following findings were made when sequences in the 3 clonal groups were compared: PA clones preferentially used JH6 (p=0.002) and the VH3/JH6 combination (p=0.0003)The PA and NP Abs all employed κ chains, whereas κ chain usage for NB clones was 61% (p<0.0001).No preferred signatures were identified in κ chain complementarity determining regions (LCDR3) of PA clones that differentiate them from NP and NB Abs.PA Abs had longer heavy chain CDR3s (HCDR3) than NP (p<0.001) or NB (p=0.0001) AbsPA Abs contained more positively charged amino acid residues compared to NP (p=0.058) or NB (p=0.002) Abs.PA Abs contained more tyrosine residues compared to NP (p=0.067) or NB (p<0.0001) AbsFive of 7 PA clones contained an RX1-2K/RX1-2R/H (RKH) motif in HCDR3; the remaining 2 PA clones contained a string of at least 5 tyrosines (Y5 motif) in HCDR3. The RKH and Y5 motifs were not found in any of the 76 NP and NB clones. Substitution of alanine for positively charged residues of the RKH motif or of tyrosine residues in the Y5 motif in PA clones reduced PF4/H binding and platelet activation, arguing for functional significance of both motifs. Utilization of nearly identical H and L chains within 3 groups of clones and of shared H chains within 3 groups of clones (both PA and NP) was observed in multiple patients. Moreover, utilization of a shared H chain was observed within 3 NP clones from two unrelated patients. These findings indicate clonal amplification and convergence of the B cell (both PA and NP) response, likely in response to a common antigen. High throughput sequencing of IgG H chains were performed on peripheral blood mononuclear cells (PBMC) from 7 HIT patients and 3 healthy donors. Eleven of 1585 H chain sequences (0.69%) from HIT patients contained the RKH and 18 (1.1%) contained the Y5 motif. In 3 healthy donors, 4 of 1418 H chain sequences (0.28%) contained RKH and none (0%) contained Y5. The findings reflect amplification of B cells with receptors containing RKH and Y5 motifs in HIT patients (p=0.1 for excess RKH and p<0.0001 for Y5 in HIT). These observations provide the first characterization of Ig structural motifs that are favored for selection in the humoral immune response leading to HIT and suggest that the RKH and Y5 CDR3 motifs in particular may contribute importantly to Ab pathogenicity. Findings made are expected to facilitate further work to define features specific to "pathogenic" HIT Abs and, possibly, to identify genetic variants that predispose individuals to experience HIT. Disclosures Padmanabhan: Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Wisconsin: Patents & Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Janssen R&D: Consultancy.

Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 182-187 ◽  
Author(s):  
Peter M. Newman ◽  
Beng H. Chong
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Surface Expression ◽  
Dynamic Binding ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Activated Platelets ◽  
Igg Binding ◽  
Iodine 125 ◽  
First Time

Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3683-3683
Author(s):  
Jerôme Rollin ◽  
Claire Pouplard ◽  
Dorothee Leroux ◽  
Marc-Antoine May ◽  
Yves Gruel
Keyword(s):  
Immune Response ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Platelet Factor 4 ◽  
Control Group ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Protein Tyrosine ◽  
Significant Difference

Abstract Abstract 3683 Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study. To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods. A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results. The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion. Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures: No relevant conflicts of interest to declare.

Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: A survey of North American specialized coagulation laboratories

2007 ◽  
Vol 98 (12) ◽  
pp. 1357-1361 ◽  
Author(s):  
Catherine Hayward ◽  
Karen Moffat ◽  
Jane Moore ◽  
Theodore Warkentin ◽  
James Zehnder ◽  
...  
Keyword(s):  
North America ◽  
Platelet Activation ◽  
North American ◽  
Laboratory Testing ◽  
Online Survey ◽  
Current Status ◽  
Heparin Induced Thrombocytopenia ◽  
Reporting Practices ◽  
Activation Assay ◽  
Result Interpretation

SummaryHeparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. As HIT is considered a clinico-pathologic entity, laboratory practices have an important role in diagnosing or excluding HIT. It was the objective of this study to assess the current status of laboratory testing for HIT in North America. An online survey consisting of 67 questions related to laboratory testing for HIT was developed by the North American Specialized Coagulation Laboratory Association (NASCOLA), and distributed to its 59 members. The survey included queries about HIT test ordering practices, HIT immunoassay and activation assays performed, and reporting practices. Data was collected from the 44 NASCOLA laboratories who responded. Of these sites, 88% performed immunoassays for HIT, commonly using commercial assays. However, sites varied in practices related to use of controls, immunoglobulin class of antibody detected, and in result interpretation and reporting. Platelet activation assays for HIT were performed by 36% of sites, commonly using assays of serotonin release (50%) or heparin- induced platelet aggregation (43%). Sites varied in the use of washed platelets versus platelet-rich plasma, controls, and heparin concentrations. This survey is the first comprehensive assessment of patterns of practice in HIT testing among diagnostic coagulation laboratories in North America. We observed sitespecific variability of testing methods encompassing all stages of testing, including pre-analytical handling, testing methodologies, and result interpretation and reporting. The variability in HIT platelet activation assay methods among institutions indicates a need for proficiency testing to assess assay performance,and for consensus guidelines on HIT laboratory testing.

scholarly journals Clinical Features of Heparin-Induced Thrombocytopenia Diagnosed By Lumi-Aggregometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4185-4185
Author(s):  
Elona Turley ◽  
Artur J. Szkotak ◽  
Irwindeep Sandhu ◽  
Cynthia M Wu
Keyword(s):  
Platelet Activation ◽  
Light Flash ◽  
Patient Characteristics ◽  
Serotonin Release ◽  
Radioactive Isotopes ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Counts ◽  
Dense Granules ◽  
Heparin Exposure

Abstract Background: Heparin-induced thrombocytopenia (HIT) is associated with anti-platelet factor-4 (PF4)/heparin antibodies that activate platelets, resulting in thrombocytopenia and a pro-thrombotic state. At our institution antibody-mediated platelet activation is demonstrated by lumi-aggregometry, which is a method previously validated against the gold standard serotonin-release assay (SRA). Lumi-aggregometry does not involve radioactive isotopes, which is its major advantage over the SRA. The clinical course of HIT diagnosed via SRA and ELISA has been previously described, and clinical prediction tools such as the 4-T score were validated using these diagnostic tests. However, the clinical picture of HIT diagnosed by lumi-aggregometry has not been previously described. Aims: The objective of this study is to describe the clinical and laboratory presentation of patients diagnosed with HIT by lumi-aggregometry. Methods: Patients with clinically suspected HIT and quantitative anti-PF4 IgG-specific ELISA OD ≥0.400 (Gen-Probe, San Diego) received confirmatory HIT testing by lumi-aggregometry. Briefly, HIT antibody-induced activation of washed healthy donor platelets was tested at therapeutic (0.1U/mL and 0.5U/mL) and high (100U/mL) porcine heparin concentration. The degree of platelet activation was quantitated luminographically based on the light flash reaction of ATP (released from platelet dense-granules) with luciferin luciferase reagent. A ratio of therapeutic to high heparin luminescence amplitude of >5.0 and platelet aggregation at therapeutic, but not high, concentrations was considered a positive result. The results of assays performed by our regional HIT testing referral laboratory from June 2009 to July 2012 were reviewed to identify patients with positive HIT testing by lumi-aggregometry. Patient records were retrospectively reviewed to obtain predefined data on baseline patient characteristics, heparin exposure, platelet counts, and thrombotic events occurring in the 5 days preceding or the 30 days following the date of positive HIT testing. Results: We identified 43 patients diagnosed with HIT by lumi-aggregometry (median age 68.0, 49% male) while under the care of local academic (46%) or urban community hospitals (37.2% medical; 53.5% surgical; 9.3% intensive care). Median baseline platelet count was 187 (14-349). Median date of platelet drop post-heparin exposure was 6 days (range 3-14) in patients without prior heparin exposure or platelet transfusions (Figure 1). Platelet drop >50% and platelet nadir ≥20x109/L were present in the majority of patients (Table 1). Thrombocytopenia occurred prior to (70.5%) or the same day (23.5%) as thrombosis in 16/17 patients with serial platelet counts who developed HIT-associated thromboembolism. Conclusion: Patients diagnosed with HIT by lumi-aggregometry present with similar findings to those described in SRA-confirmed HIT. These findings lend support to the use of lumi-aggregometry as an accurate diagnostic assay for the clinico-pathologic syndrome of HIT. Figure 1 Figure 1. Table 1. Percentage platelet drop from baseline and platelet nadir Percent platelet drop Platelet nadir >50% 30-50% <30% ≥20 x 109/L 28 3 2 10-19 x 109/L 5 1 0 <10 x 109/L 1 1 0 Disclosures Szkotak: Alexion Pharmaceuticals: Research Funding. Sandhu:Celgene: Honoraria; Jansen: Honoraria; Novartis: Honoraria.

Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 182-187 ◽  
Author(s):  
Peter M. Newman ◽  
Beng H. Chong
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Surface Expression ◽  
Dynamic Binding ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Activated Platelets ◽  
Igg Binding ◽  
Iodine 125 ◽  
First Time

Abstract Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.

scholarly journals Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2540-2540
Author(s):  
Michelle Sung ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
Omer Iqbal ◽  
Mamdouh Bakhos
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Test Group ◽  
The United States ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Aggregation Response ◽  
Significant Difference

Abstract Background Heparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin. Materials and Methods Seven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software. Results In the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT. Conclusion In these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. Figure. Figure. Disclosures Walenga: Eurofarma: Research Funding.

scholarly journals A Prothrombotic Thrombocytopenic Disorder Resembling Heparin-Induced Thrombocytopenia Following Coronavirus-19 Vaccination

2021 ◽  
Author(s):  
Andreas Greinacher ◽  
Thomas Thiele ◽  
Theodore E. Warkentin ◽  
Karin Weisser ◽  
Paul Kyrle ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Adenoviral Vector ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Post Vaccination ◽  
Receptor Blocking ◽  
Vein Thrombosis ◽  
Activation Assay ◽  
Laboratory Features ◽  
High Concentrations

Abstract Background. Vaccines are important for managing the COVID-19 pandemic caused by SARS-CoV-2. However, following widespread vaccination using a recombinant adenoviral vector encoding the spike protein antigen of SARS-CoV-2 (AZD1222, AstraZeneca), reports have emerged of some vaccine recipients developing unusual thrombotic events and thrombocytopenia. We investigated whether such patients could have a prothrombotic disorder caused by platelet-activating antibodies directed against platelet factor 4 (PF4), as is known to be caused by heparin and sometimes other environmental triggers.Methods. We summarized the clinical and laboratory features of 9 patients in Germany and Austria who developed thrombosis and thrombocytopenia events following AZD1222 vaccination. Serum from four patients was used to test for anti-PF4/heparin antibodies, both by immunoassay and by platelet activation assays performed in the presence of heparin, PF4, or both.Results. The 9 patients (8 female; median age, 36 [range, 22—49) presented with thrombosis beginning 4 to 16 days post-vaccination: 7 patients had cerebral venous thrombosis (CVT), 1 had pulmonary embolism, and 1 had splanchnic vein thrombosis and CVT; 4 patients died. None had received heparin prior to symptom onset. All four patients tested strongly positive for anti-PF4/heparin antibodies by immunoassay; all 4 patients tested strongly positive in the platelet activation assay in the presence of PF4 independently of heparin. Platelet activation was inhibited by high concentrations of heparin, Fc receptor-blocking monoclonal antibody, and intravenous immunoglobulin.Conclusions. The AZD1222 vaccine is associated with development of a prothrombotic disorder that clinically resembles heparin-induced thrombocytopenia but which shows a different serological profile.

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