Pathogenicity of human anti-platelet factor 4 (PF4)/heparin <i>in vivo</i>: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin

Blank, Miri; Cines, Douglas B.; Arepally, Gowthami M.; Eldor, Amiram; Afek, A.; Shoenfeld, Yehuda

doi:10.1046/j.1365-2249.1997.d01-1008.x

Cited by 35 publications

(12 citation statements)

References 42 publications

(61 reference statements)

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“…31 Mice immunized with HIT antibodies develop anti-PF4/ heparin antibodies as part of the anti-idiotypic response and develop thrombocytopenia when exposed to heparin. 32 Passive transfer of a murine monoclonal anti-PF4/heparin antibody into mice transgenic for human PF4 and platelet Fc␥RIIA leads to severe thrombocytopenia and disseminated thrombosis, the salient clinical features of the human disease. 28 These findings indicate the need to understand how such self-reactive antibodies arise once a complex is formed between PF4 and heparin.…”

Section: Discussionmentioning

confidence: 99%

“…diseased tissue become important scientific issues. Third, although anti-PF4/heparin antibodies can be demonstrated in a significant proportion (15% to 70%) of asymptomatic patients repetitively exposed to heparin, [32][33][34][35] the reason only a subset of immunized patients develop symptomatic disease remains unknown. Clinical variables such as atherosclerosis, surgery, and vascular trauma may contribute to the risk, [45][46][47] while some researchers have implicated differences in antibody titer, 48 affinity, 49 isotype, 50 subclass, 51,52 and platelet Fc receptor polymorphism (Fc␥RIIA-H/R 131 ).…”

Section: Defining Hit/t Antigenic Sitementioning

confidence: 99%

See 1 more Smart Citation

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Liu

Park

et al. 2002

Blood

116

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Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/ human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant.The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin. IntroductionHeparin is the most common known cause of drug-induced immune thrombocytopenia, occurring in 1% to 3% of patients receiving unfractionated heparin. 1-3 A significant fraction of these patients develop limb-or life-threatening thromboses. 4,5 Heparininduced thrombocytopenia/thrombosis (HIT/T) is mediated by antibodies directed at complexes that form between heparin or other anionic mucopolysaccharides and platelet factor 4 (PF4) in plasma and on the surface of vascular cells. [6][7][8][9][10] Immune complexes composed of HIT/T antibodies and PF4/heparin bind to the surface of platelets and induce their activation by cross-linking Fc␥IIA receptors, [11][12][13] and bind to the surface of the endothelium and monocytes, 14-16 inducing procoagulant activity. 11,14 PF4 is a 70-amino acid, platelet-specific CXC chemokine in which the first 2 of the 4 conserved cysteine residues are separated by 1 amino acid residue. 17 PF4 has been sequenced 18 and cloned, 19 and its x-ray crystallographic structure has been defined ( Figure 1A). [20][21][22] PF4 exists in many biologic fluids primarily in the form of a tetramer with the 3 ␤-sheets of each subunit facing inwards, and the N and C termini lying on the surface of the molecule. The C termini are rich in lysines, which contribute to the circumferential ring of positive charges that form the interface between the PF4 tetramer and heparin. [23][24][25] The mechanism by which PF4/heparin complexes become antigenic is unknown. We have previously defined an ant...

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Section: Discussionmentioning

confidence: 99%

Section: Defining Hit/t Antigenic Sitementioning

confidence: 99%

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Liu

Park

et al. 2002

Blood

116

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“…24,25 However, the severity of the resultant thrombocytopenia was only modest; thrombosis was not detected; and the contribution of other autoantibodies that the mice developed during the course of epitope spread was unclear. 25 A mouse model of HIT/HITT that recapitulates the disease process in humans would help clarify the factors that predispose some patients to develop thrombocytopenia or thrombosis and to investigate novel therapeutic approaches.…”

Section: Introductionmentioning

confidence: 99%

Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcγRIIA

Reilly

Taylor

Hartman

et al. 2001

Blood

200

196

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Heparin-induced thrombocytopenia/thrombosis (HIT/HITT) is a severe, life-threatening complication that occurs in 1% to 3% of patients exposed to heparin. Interactions between heparin, human platelet factor 4 (hPF4), antibodies to the hPF4/heparin complex, and the platelet Fc receptor (FcR) for immunoglobulin G, Fc␥RIIA, are the proposed primary determinants of the disease on the basis of in vitro studies. The goal of this study was to create a mouse model that recapitulates the disease process in humans in order to understand the factors that predispose some patients to develop thrombocytopenia and thrombosis and to investigate new therapeutic approaches. Mice that express both human platelet Fc␥RIIA and hPF4 were generated. The Fc␥RIIA/hPF4 mice and controls, transgenic for either Fc␥RIIA or hPF4, were injected with KKO, a mouse monoclonal antibody specific for hPF4/heparin complexes, and then received heparin (20 U/d). Nadir platelet counts for KKO/heparin-treated Fc␥RIIA/hPF4 mice were 80% below baseline values, significantly different (P < .001) from similarly treated controls. Fc␥RIIA/hPF4 mice injected with KKO IntroductionHeparin is one of the most widely used anticoagulants during invasive vascular procedures and to treat thromboembolic diseases. Among patients who receive therapeutic courses of heparin, 1% to 3% will develop an antibody-mediated thrombocytopenia, 1-3 and 30% to 70% of these patients will develop potentially lifethreatening thrombosis. There is abundant evidence that more than 95% of patients with heparin-induced thrombocytopenia (HIT) alone and those with thrombocytopenia and thrombosis (HITT) develop antibodies that recognize complexes between platelet factor 4 (PF4) and heparin. 4-7 PF4, a major component of platelet ␣-granules, is released when platelets are activated. 8,9 It is currently believed that complexes between PF4 and heparin form on the surface of activated platelets, where they are positioned to be recognized by anti-PF4/heparin antibodies. [10][11][12] Antibodies to the PF4/heparin complex have been shown to activate human platelets in vitro via the platelet Fc receptor (FcR) for immunoglobuin (Ig)-G, Fc␥RIIA. [13][14][15][16] The binding of HIT antibodies to activated platelets probably promotes microparticle release as well as platelet-platelet and platelet-vessel wall interactions, predisposing to thrombosis. 15,17 However, the mechanism by which HIT antibodies activate platelets and promote thrombosis is uncertain. It has been proposed that HIT antibodies bind to cell-surface-associated PF4/heparin complexes via the Fab end of the molecule, providing an opportunity to transduce platelet-activating signals through the interaction between the Fc portion of the bound IgG and Fc␥RIIA. 12,13 Fc␥RIIA, the sole Fc␥ receptor expressed on platelets, 18 has been shown to transduce signals without the requirement for another transmembrane partner. 19 A monoclonal antibody to Fc␥RIIA blocks platelet aggregation and secretion induced by HIT antibodies. 12,13 Although increased...

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“…This disorder causes a ‘moderate’ thrombocytopenia (in the range of 20–150×10 9 platelets/l), and can be accompanied by thrombosis [16]. Many studies have been performed to elucidate the possible mechanism of HIT, which involves the binding of platelet factor–4/heparin complex to the heparin receptor on the platelet, and is dependent on heparin concentration [17, 18]. An autoantibody then triggers an immune response in some (but not all) patients [19].…”

Section: Discussionmentioning

confidence: 99%

Collection of Heparinized Plasma by Plasmapheresis

Meer¹,

Vrielink²,

Pietersz³

et al. 1999

Vox Sanguinis

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Background and Objectives: Heparinized plasma can be used for exchange transfusions in neonates and is usually collected by drawing whole blood using heparin as anticoagulant. The heparinized red blood cells and buffy coat cannot be used and are therefore discarded. To collect heparinized plasma more efficiently, a method was developed using an apheresis machine. Materials and Methods: With an MCS3p apheresis machine (Haemonetics), plasma was collected from volunteer donors as anticoagulant, heparin in saline (30,000 IU/l) was added in a 1:9 ratio. The activated partial thromboplastin time (APTT) of the donors was measured before and immediately after the procedure, and various parameters were determined in the collected plasma. Results: In 2 collection cycles, an average of 456±52 ml (mean ± SD; n = 20) of heparinized plasma was collected, and 504±57 ml (n = 2; donors with a high hemoglobin level) when 3 cycles were performed. The leukocyte and platelet contamination in the plasma (n = 22) was 1.11±0.92×10⁶ and 0.05±0.22×10⁹ per unit, respectively, which conformed to national specifications. Sodium levels were normal, but due to dilution of the plasma with heparin solution, potassium and calcium levels were about 20% lower than the serum levels in the donors. The donor APTT values were slightly longer after the procedure than before, but remained all within normal values. Conclusion: For the collection of heparinized plasma, apheresis has the advantage that (1) high–quality heparinized plasma can be harvested; (2) no blood components need to be discarded; (3) more plasma can be harvested with each donation, and (4) these procedures can be performed more often than whole blood donations.

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Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin

Cited by 35 publications

References 42 publications

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcγRIIA

Collection of Heparinized Plasma by Plasmapheresis

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