scholarly journals Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia

2016 ◽  
Vol 1 (1) ◽  
pp. 62-74 ◽  
Author(s):  
Douglas B. Cines ◽  
Serge V. Yarovoi ◽  
Sergei V. Zaitsev ◽  
Tatiana Lebedeva ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Platelet Activation ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Activated Platelets ◽  
Key Points

Key Points Polyphosphates form antigenic complexes with PF4 that are recognized by HIT antibodies. Polyphosphate/PF4 complexes released by activated platelets can mediate platelet aggregation by HIT antibodies in the absence of heparin or cell-surface chondroitin sulfate.

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.

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 Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbβ3, and platelet factor 4

Blood ◽  
2014 ◽  
Vol 123 (20) ◽  
pp. 3166-3174 ◽  
Author(s):  
Mònica Arman ◽  
Krystin Krauel ◽  
Dorothea O. Tilley ◽  
Claudia Weber ◽  
Dermot Cox ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Lag Time ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Integrin Αiibβ3 ◽  
Key Points

Key Points FcγRIIA activation is key for platelet aggregation in response to bacteria, and depends on IgG and αIIbβ3 engagement. PF4 binds to bacteria and reduces the lag time for platelet aggregation.

In Vitro Efficacy of Platelet Glycoprotein IIb/IIIa Antagonist in Blocking Platelet Function in Plasma of Patients with Heparin-induced Thrombocytopenia

1998 ◽  
Vol 80 (12) ◽  
pp. 989-993 ◽  
Author(s):  
Koon-Hou Mak ◽  
Linda Brooks ◽  
Eric Topol ◽  
Kandice Kottke-Marchant
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Underlying Mechanism ◽  
Platelet Factor 4 ◽  
Platelet Glycoprotein ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Microparticle Formation

SummaryHeparin-induced thrombocytopenia (HIT) is an important complication following administration of heparin. Platelet activation and aggregation induced by heparin/platelet factor 4/immunoglobulin complexes are thought to be the underlying mechanism for this condition, so it was hypothesized that abciximab (a humanized murine monoclonal antibody directed against the glycoprotein IIb/IIIa receptor) would prevent heparin-induced platelet aggregation and activation in plasma from patients with HIT. Platelet aggregation was tested in vitro with platelet-poor plasma (obtained from 23 patients with HIT), platelet-rich plasma (from normal donors with known reactivity), heparin (0.5 U/ml), and ascending doses of abciximab (0.07-0.56 μg/ml). The ability of abciximab to prevent platelet activation was also evaluated using flow cytometry (P selectin expression, mepacrine release, microparticle formation) and platelet factor 4 immunoassay. In vitro, abciximab inhibited heparin-induced platelet aggregation in a dose-dependent fashion (IC50 0.103 μg/ml) and inhibited microparticle formation, the expression of P-selectin, release of mepacrine and platelet factor 4. These findings suggest that abciximab may be useful in treatment of patients with HIT and warrants further clinical evaluation.

Pathophysiology of Heparin-Induced Thrombocytopenia

2000 ◽  
Vol 124 (11) ◽  
pp. 1657-1666 ◽  
Author(s):  
Fabrizio Fabris ◽  
Sarfraz Ahmad ◽  
Giuseppe Cella ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
New Information ◽  
Study Selection ◽  
Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

scholarly journals Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

2020 ◽  
Vol 21 (7) ◽  
pp. 2556
Author(s):  
Elmira R. Mordakhanova ◽  
Tatiana A. Nevzorova ◽  
Gulnaz E. Synbulatova ◽  
Lubica Rauova ◽  
John W. Weisel ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Gel Filtration ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Mitochondrial Membrane Depolarization ◽  
Low Platelet Count ◽  
Apoptotic Pathways

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

Functional Microparticles Are up Regulated in Patients with Anti-Heparin/Platelet Factor 4 Antibodies: A Potential Mechanism of Thrombogenesis in the Heparin-Induced Thrombocytopenia Syndrome.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2105-2105
Author(s):  
Josephine Cunanan ◽  
Michelle Kujawski ◽  
He Zhu ◽  
Margaret Prechel ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Medical Center ◽  
Annexin V ◽  
Platelet Factor 4 ◽  
Cellular Damage ◽  
Clinical Samples ◽  
Limiting Factor ◽  
Therapeutic Effects ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is one of the most catastrophic adverse effects of heparin therapy, representing a complex syndrome involving immunopathologic and hemostatic disorders. Vascular and blood cellular damage results in the generation of microparticles (MP). These MP are formed from stress conditions/cellular disruption and apoptosis. Cellular MP mediated pathophysiologic responses include platelet activation, up regulation of adhesion molecules, monocyte activation, up regulation of tissue factor and endothelial dysfunction. Several methods based on flow cytometric and other immunologic probes have been used to measure MP in the HIT syndrome. Recently, a functional method based on the complexation of MP with annexin V promoting the generation of factor Xa and thrombin has become available (Hyphen Biomedical, Neuville-Oise, France). To validate the hypothesis that functional MP are elevated in the HIT syndrome, this method was utilized for the quantitation of MP in sera ELISA positive for anti-heparin/platelet factor 4 (HIT) antibodies. Specimens (n = 53) were selected from archived samples that had been referred to Loyola University Medical Center for the laboratory diagnosis of HIT by quantitating anti-heparin/PF4 antibodies by ELISA and by evaluating HIT antibody induced platelet activation using the 14C Serotonin Release Assay (SRA). All selected specimens were positive for HIT antibodies in the GTI PF4 Enhanced ELISA with a broad range of antibody titers (absorbance range of 0.4 – 2.5). Eleven of these specimens were positive in the SRA. In addition, serial samples from HIT patients treated with argatroban (from the ARG-911 clinical study) were included (n = 23). The normal samples represented control sera obtained from healthy human volunteers (n = 25) and processed in the same manner as the clinical samples. Test samples were added to microtiter plates coated with streptavidin and biotinylated annexin V. MP present in the test sample bound to annexin V via exposed surface phospholipids. Following incubation and washing steps, a FXa – FVa mixture containing calcium and prothrombin was added. The assay was optimized so that MP associated phospholipid was the limiting factor for the generation of thrombin. In normal non-HIT sera, the MP levels ranged 5.6 – 10.1 nM (6.1 ± 2.8 nM). The pre-treatment, baseline levels of circulating MP in the suspected HIT patients ranged from 4.2 – 26.8 nM (15.8 ± 7.3 nM). Interestingly, SRA positive/ELISA positive samples had relatively higher levels of MP (19.9 ± 7.7 nM; range 11.5 – 29.8 nM) than SRA negative/ELISA positive samples (14.2± 4.6; range 6.8–21.2). In the ARG-911 study, sequential blood samples exhibited MP levels at the baseline ranging from 8.2 – 38.6 nM (21.8 ± 10.8 nM), whereas after 3 days of argatroban treatment were reduced to 5.1 – 19.2 nM (12.6 ± 6.3). The results of these studies suggest that circulating functional MP are increased in patients with ELISA positive HIT antibodies. Anticoagulation with such direct thrombin agents as argatroban effectively decreases the circulating functional MP levels. Since the elevated MP levels may mediate thrombin and FXa generation, the therapeutic effects of these drugs in HIT may be related to the decreased activation of coagulation and related thrombogenic processes.

Antibodies to Macromolecular Platelet Factor 4-Heparin Complexes in Heparin-induced Thrombocytopenia: a Study of 44 Cases

1995 ◽  
Vol 73 (01) ◽  
pp. 021-028 ◽  
Author(s):  
J Amiral ◽  
F Bridey ◽  
M Wolf ◽  
C Boyer-Neumann ◽  
E Fressinaud ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Early Diagnosis ◽  
Healthy Subjects ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Sulphated Polysaccharides ◽  
Major Interest ◽  
Heparin Complexes

SummaryAs heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 <0.3) and 35 patients with other causes of thrombocytopenia (A492 <0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 ± 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped ≤ 100 × 109/l only at days 11–12. Surprisingly, among 41 patients under heparin for >7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.

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.

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