Localization of distal regulatory domains in the megakaryocyte-specific platelet basic protein/platelet factor 4 gene locus

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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...

Section: Generation Of Double-transgenic Fc␥riia/hpf4 Micementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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

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196

“…17,18 PC ϩ/Ϫ mice were supplied by Dr Frank Castellino. 19 All mice were on a common C57Bl6 background, and littermate wild-type (WT) controls were used.…”

Section: Methodsmentioning

confidence: 99%

Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge

Kowalska

Mahmud

Lambert

et al. 2007

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Pharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4 ؊/؊ ) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C-deficient (PC ؉/؊ ) mice. Transgenic mice overexpressing human PF4 (hPF4 ؉ ) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC ؉/؊ mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4 ؉ and PC ؉/؊ /hPF4 ؉ mice had improved survival. Further, infusion of hPF4 ؉ platelets improved survival of wild-type mice after an LPS challenge. IntroductionIn addition to its anticoagulant function, activated protein C (APC) has been shown to exert an antiinflammatory influence 1,2 and inhibit apoptosis. [3][4][5] All of these effects may contribute to the efficacy of APC infusions in improving outcome in various animal models of inflammation 6-9 and in reducing mortality in patients with sepsis. [10][11][12] In a lipopolysaccharide (LPS)-induced survival model of sepsis, heterozygous protein C-deficient (PC ϩ/Ϫ ) mice have enhanced vulnerability to death, 6,8 reinforcing the crucial role played by endogenously generated APC in this setting. Recent clinical studies demonstrate that basal PC levels are an independent predictor of sepsis outcome 13 and that endogenous vascular generation of APC is a crucial determinant of survival in sepsis. 14 We have shown previously that platelet factor 4 (PF4, CXCL4) can enhance APC generation in vitro 15 and in vivo 16 ; however, the biologic impact of endogenous, platelet-derived PF4 in regulating systemic APC generation has not been tested. In this study, we examined whether endogenously released PF4 from activated platelets stimulates APC generation in response to thrombin infusion and improves outcome in thrombosis/inflammatory models. These studies were done in mice with targeted disruption of the murine Pf4 gene (mPf4 Ϫ/Ϫ ) and transgenic mice that overexpressed human PF4 (hPF4 ϩ ). 17 We found that platelet PF4 content correlated with APC generation after thrombin infusion and survival after thrombin or LPS challenge and that released PF4 can compensate for the lower PC levels in PC ϩ/Ϫ mice. 6,8 Further, infusion of platelets with high PF4 content is protective in the LPS challenge model, supporting endogenous PF4 as an important variable in outcome from sepsis and suggesting a novel therapy for sepsis. Materials and methodsThe mPf4 Ϫ/Ϫ and hPF4 ϩ mice have been previously described. 17,18 PC ϩ/Ϫ mice were supplied by Dr Frank Castellino. 19 All mice were on a common C57Bl6 background, and littermate wild-type (WT) controls were used. Murine studies were approved by the Children's Hospital of Philadelphia institutional animal care and use committee.Circulating plasma APC levels were mea...

“…8,31 These mice were crossbred with mouse PF4 knockout (mPF4 KO ) mice 32 and with mice expressing human Fc␥RIIA (generously provided by Steven McKenzie, Thomas Jefferson University). 33 All murine lines had been previously backcrossed onto the C57BL/6J background more than 12 times.…”

Section: Transgenic Micementioning

confidence: 99%

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

Rauova

Hirsch

Greene

et al. 2010

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160

Heparin-induced thrombocytopenia (HIT)is a life-and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation. IntroductionPlatelet factor 4 (PF4) is a cationic chemokine with high affinity for unfractionated heparin (UFH) and other large, negatively charged molecules. 1 PF4 is stored in platelet ␣-granules, released upon activation, when it then binds rapidly to glycosaminoglycan (GAG) side chains expressed on the surface of platelets 2 and other vascular cells, with little remaining free in the circulation. 3 Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize complexes of human (h) PF4 with heparin or other GAGs. 4,5 In solution, formation of antigenic complexes between PF4 and heparin is critically dependent on their molar ratio, with loss of antibody binding when the optimal ratio is disrupted by an excess of either component. 6,7 Antigen formation on the platelet surface also follows a bell-shaped curve as PF4 concentration is increased, with maximal binding of antibody seen at an exogenous PF4 concentration of 50 g/mL. 8 Chondroitin sulfates (CSs) are the predominant GAG side chains expressed on platelets. 9,10 We have shown that the binding of the HIT-like monoclonal antibody KKO 11 to platelets is abrogated by chondroitinase ABC, 8 indicating that HIT antibodies bind to PF4/CS complexes on this cell type. Therapeutic concentrations of UFH disrupt antibody binding in part by eluting PF4 from the platelet surface, which reduces formation of antigenic complexes and the potential for platelet activation 8 through platelet Fc␥RIIA. 12 Thus, variation in the expression of platelet-derived PF4 (or CS) might help to explain why only a small percentage of patients who generate antibodies to PF4/UFH develop HIT. 13 Although it has been generally accepte...