Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets (original) (raw)
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British Journal of Haematology, 1990
Previously described platelet-aggregating antibodies associated with thrombosis and thrombocytopenia required heparin for their in vivo and in vitro expression. We have observed a patient with thrombosis who became thrombocytopenic during heparin treatment, but who suffered further thrombotic events and continued thrombocytopenia for 3 months after heparin withdrawal. The patient's plasma contained a potent platelet aggregating factor reactive with both his own and normal platelets in the absence of heparin. It also caused ['*C]serotonin secretion from labelled platelets from normal donors and patients with either Glanzmann's thrombasthenia or Bernard-Soulier syndrome.
Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia
Journal of Clinical Medicine, 2020
Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and sero...
The mechanism of heparin-induced platelet aggregation
European Journal of Haematology, 2009
When heparin was added to platelet-rich plasma, mild but irreversible platelet aggregation was demonstrated. This platelet response was not accompanied by release of a-granules and dense body constituents, nor by prostaglandin biosynthesis. It did, however, require metabolic energy and divalent cations as metabolic inhibitors (antimycin A and 6-deoxyglucose) and EDTA blocked the reaction. Bernard-Soulier syndrome platelets, which lack glycoprotein (GP) Ib, but not Glanzmann's Thrombasthenia platelets, which lack GP IIb/IIIa, were aggregated by heparin. Monoclonal antibody (mAb) against GP IIb/IIIa, but not mAb against GP Ib, strongly inhibited
Increased expression of platelet IgG Fc receptors in immune heparin-induced thrombocytopenia
Blood, 1993
Our previous finding that heparin-dependent antibodies in heparin-induced thrombocytopenia (HIT) bind to platelets via platelet IgG Fc receptors (FcRs) prompted this study. Platelet FcRs in 16 patients with HIT, 23 control patients, and 42 normal subjects were studied. Patients with HIT had substantially increased platelet FcRs during the acute illness. Those who suffered serious thrombotic complications or died shortly after diagnosis had significantly more FcRs per platelet than those with milder disease. Consistent with their increased FcRs, platelets of patients with HIT showed increased aggregation reactivity to aggregated IgG and heparin-dependent antibodies. Platelet FcRs in patients with HIT remained elevated for 1 to 3 months after the acute illness then stabilized to a mean value not significantly different from either control group. The increased expression of FcRs on HIT platelets and their increased reactivity to heparin-dependent antibodies may contribute to the pathog...
Platelet GPIIb/IIIa antagonist, XV459, in heparin-induced thrombocytopenia
American Journal of Hematology, 2007
Heparin-induced thrombocytopenia (HIT) is a serious, immune-related complication of heparin therapy. One of the most severe manifestations of HIT is the development of thromboembolic events, which is based on platelet activation and aggregation caused by HIT-associated antibodies. Therapeutic options for patients with HIT are limited despite advancement toward the development of alternative (nonheparin) anticoagulants, such as direct thrombin inhibitors and indirect anti-factor Xa agents. Platelet GPIIb/IIIa receptor antagonists have been shown to be the final common pathway for platelet aggregation regardless of the use of activator or anticoagulant. In this study, the ability of a novel platelet GPIIb/IIIa antagonist, a free acid form of roxifiban (XV459), to block platelet activation/aggregation in response to highly characterized heparin-PF4 antibody-positive plasma/heparin was examined using light transmittance aggregometry, serotonin release, and 125 I-fibrinogen binding assays to human platelets. XV459 at 20 nM maximally inhibited (P < 0.001) the platelet-activation/aggregation responses as mediated by the HIT antibody-positive plasma (in the presence of therapeutic heparin concentrations). Compared with controls, both HIT antibodies/heparin and TEAC (a mixture of thrombin [0.1 IU/ml], epinephrine [1 mg/ml], arachidonate [0.1 mM], and collagen [10 mg/ml]) resulted in significantly higher levels of fibrinogen binding to human platelets (5-7-fold increase; P < 0.001). Concentration-dependent profiles of XV459 on the mean percent inhibition of 125 I-fibrinogen binding in the presence of HIT antibodies and TEAC were achieved (*50% inhibition at 10 nM XV459). The platelet GPIIb/IIIa receptor antagonist (XV459) might be of potential benefit in the management of thrombotic thrombocytopenia produced by heparin and/or related glycosaminoglycans. Am. J. Hematol. 82:276-282, 2007. V V C 2006 Wiley-Liss, Inc.
Platelet binding of IgG from patients with heparininduced thrombocytopenia
Journal of Laboratory and Clinical Medicine, 1996
Although heparin induces immune-mediated thrombocytopenia, it has been difficult to demonstrate heparin specificity of the putative immunoglobin. Recently, however, a body of data has indicated that platelet factor 4 (PF4) is required for heparin-induced thrombocytopenia (HIT) antibody to bind to heparin. Using viable platelets in a physiologic buffer, we have now documented specific and reversible platelet binding of iodine 125-1abeled IgG from 5 patients with HIT and binding of 1251-1abeled F(ab')2 from 2 of them. The binding requires the presence of both heparin and PF4 in a molar ratio of-1:1. We have also shown that platelet activation increases HIT antibody binding. Our data suggest that the F(ab')2 domain of HIT antibody binds to heparin-PF4 complexes that accumulate on the platelet surface when equimolar concentrations of heparin and PF4 are present.
Journal of Clinical Investigation, 1994
Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (. 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alphagranules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of -1:2 (fresh heparin) and -1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the FcyRII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc'yRII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and 1gM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin. (J. Clin. Invest. 1994. 93:81-88.)
American Journal of Hematology, 1979
Eleven patients with heparin-induced thrombocytopenia were studied. Thrombocytopenia appeared 3-16 days following the initiation of prophylactic or therapeutic doses of heparin. The mean lowest platelet count recorded was 48,000/mm3. When heparin was stopped, recovery from thrombocytopenia began within 24 hours and was complete by ten days. Two patients developed fatal thromboses, and two others had myocardial infarctions while thrombocytopenic. In the serum of seven patients, including three of the four with arterial thrombosis, a heparin-dependent platelet aggregating factor was present. The factor caused release of platelet 14C serotonin but did not lyse platelets. It was present in the globulin fraction of all positive sera, and in one serum studied it was isolated in the IgG/IgA immunoglobulin fraction. The factor was not present in 16 normal sera or in the sera of 1 5 nonthrombocytopenic patients receiving heparin. Our observations suggest that heparin-induced thrombocytopenia is common and that, in some patients it may be accompanied by severe arterial thrombosis. In vivo platelet aggregation is a possible explanation for the thrombocytopenia and the thrombosis in this disorder.
…, 1994
Background: As clinical diagnosis of heparin-associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable. Stud Design and Methods: The sensitivity of the heparin-induced platelet activation (LIPA) test and the platelet aggregation test (PAT) was prospectively compared by using the sera of 209 patients with the putative diagnosis of HAT. Both assays were performed concomitantly with platelets of the same four donors using a different combination of donors from day to day. Further, all sera were assessed with a platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Results: Positive results were obtained with 33 percent of sera in the PFWheparin ELISA, with 33.5 percent of sera in the HlPA test, and with 11.5 percent of sera in the PAT. The PF4/heparin ELISA and the HlPA test showed no difference in sensitivity (p = 0.27 by McNemar's test) and were more sensitive than PAT (~4 0 " by McNemar's test). However, they recognized different patient cohorts. Nine HIPAindeterminate and 12 HIPA-negative sera were positive in the PFWheparin ELISA. Eight of the nine indeterminate sera caused platelet activation with high he arin concentrations in the HlPA test. Eleven of the 12 negative sera contained no LG, but 9 contained I M and 2 contained IgA HAT antibodies. Four sera that were indeterminate in the bF4,heparin ELISA and 18 sera that were negative were positive in the HlPA test. None of the sera that were positive in the PAT was missed in the HlPA test, but two of those were negative in the PFUheparin ELISA. All sera were assessed with four low-molecular-weight heparins and a low-molecular-weight heparinoid in the HlPA test with platelets from the same four donors. Low-molecular-weight heparin caused platelet activation with positive sera in 98 percent of tests, and the heparinoid did so in 10 percent; in a further 12.8 percent, crossreactivity to the low-molecular-weight heparinoid could not be excluded. Conclusion: The majority of HAT antibodies react with a PFWheparin complex, but there is stron evidence that other antigens are involved in some patients.The HlPA test and the jF4iheparin ELSA are sensitive for diagnosing HAT, and they complement one another. TRANSFUSION 1994;34:381-385. Abbreviations: ELSA = enzyme-linked lmmunosorbent assay; HAT = heparln-assoclated thrombocytopenia; HIPA = heparln-induced platelet activation (test); LMW = low molecular weight; PAT = platelet aggregation test; PF4 = platelet factor 4; SRA = serotonin-release assay.
Thrombosis Research, 2002
Antibodies to heparin -platelet factor 4 (PF4) complexes have been observed in patients with heparin-induced thrombocytopenia (HIT) syndrome. These antibodies may be of various isotypes and differ with respect to their ability to activate platelets/endothelial cells. This study determined the isotypes and functionality of antiheparin -platelet factor 4 (AHPF4) antibodies in 111 patients treated with heparin and clinically suspected for HIT. In this patient population, 50% had detectable AHPF4 cumulative IgA, IgG, and IgM (determined by enzyme-linked immunosorbent assay, ELISA), but only 35% was positive when tested with the 14 C-serotonin release assay (SRA). Using antihuman Ig specific for different isotypes, we found that 50% of the 111 samples was positive for IgG, 45% for IgM, and 37% for IgA. In 50 normal human serum (NHS) samples, only two were positive for IgG, but 33 were positive for IgM, indicating a potential humoral response to the heparin -PF4 complex prior to heparin administration. Patients that were ELISA + for AHPF4 antibody titer were subdivided into SRA-positive (+) and SRA-negative ( À ) groups. The SRA + group had a mean ELISA optical density (OD) for AHPF4 IgA/IgG/IgM of 2.1, while the SRA À group had a mean OD of 0.8 ( P < .001). The SRA + group had greater mean OD values for all three individual isotypes. Using flow cytometry, we determined the ability of different patient samples to activate platelets. Samples that contained IgG and were SRA + activated platelets (as measured by microparticle generation and P-selectin expression) in the presence of therapeutic concentrations of heparin. NHS and samples containing IgA and/or IgM that were SRA À were not able to produce microparticles nor were they able to increase expression of P-selectin. Together, these data indicate that IgG is the principal mediator of platelet activation in patients with HIT, with IgA and IgM playing a less significant role in the pathophysiology of this syndrome. D