HIT-like Monoclonal Antibody KKO Research Articles

Platelet Factor 4 Binds to Released Von Willebrand Factor Strands from Injured Endothelium and Forms a Prothrombotic HIT Antigenic Complex

Heparin-induced thrombocytopenia (HIT) is driven by the binding of pathogenic HIT antibodies to platelet factor 4 (PF4) complexed to heparin. We, and others, have shown that PF4 bound to a variety of other polyanionic molecules, like surface glycosaminoglycans, also form HIT antigenic complexes. This is likely due to the formation of high molecular weight aggregates by the PF4. While studying the pathogenesis of HIT in a microfluidic system lined with human umbilical endothelial cells (HUVEC), we noted that PF4 did not bind diffusely to the HUVECs after a hematoporphyrin-induced photochemical injury as one would expect if binding were to the injured surface GAGs, but rather in a linear pattern described previously for von Willebrand Factor (vWF) under flow. We confirmed this by concurrent staining using PF4 and vWF-specific labeled antibodies. We also demonstrated that the HIT-like monoclonal antibody KKO, as well as IgG isolated from patients with likely HIT as determined by clinical assessment and ELISA positivity and positive serotonin release assay, bound to these PF4-vWF complexes. The HIT-like complexes were not uniformly distributed along the vWF, but occurred in a periodic fashion along thicker vWF strands. This suggested that the complexes were composed of multiple molecules of PF4 and vWF forming cross-chained high molecular weight aggregates that induce or enhance binding of HIT antibodies as previously reported for PF4/heparin in solution. We then wanted to identify the potential for therapeutics to intervene with PF4-vWF-KKO complex formation or stability. We tested this by introducing heparin; 2-0,3-0 desulfated heparin (ODSH); ADAMTS13 or n-acetylcysteine into channels post-complex formation. All interventions at therapeutically relevant concentrations were able to significantly remove or disrupt complex formation. When whole human blood + PF4 + KKO were flowed through channels lined with injured endothelium, large platelet aggregates developed within 10 mins along the extended strands of vWF. Significantly fewer platelet aggregates formed in the absence of PF4 or with KKO alone or PF4 plus an isotype control TRA. After 1 hour of flow, aggregates in the PF4 + KKO channels had grown significantly, but had largely disappeared in the other settings. Furthermore, by adding the FcgammaRIIA blocking antibody, IV.3, platelet binding could be reduced to background levels. These studies identify large vWF polymeric strands as a binding site for PF4 and a HIT antigen target. The Fc portions of the bound HIT antibody then contribute to platelet binding to the vWF. We are presently examining whether PF4-vWF-KKO aggregates contribute to thrombotic complications in HIT and defining the vWF domain(s) involved as these targets offer a potential novel intervention target in HIT. [Display omitted] DisclosuresCines:Amgen: Consultancy; Ionic: Consultancy; Juno: Consultancy; Astellas: Consultancy; T2 Biosystems: Research Funding; Rigel: Consultancy; Syntimmune: Research Funding.

Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

Antibody Stabilization of Neutrophil Extracellular Trap-Platelet Factor 4 Complexes Is Therapeutic in a Murine Model of Endotoxemia

Sepsis is characterized by life-threatening organ dysfunction caused by a dysregulated response to infection. Neutrophils play a crucial role in sepsis during which they release neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively-charged histones that kill pathogens but also damage host tissue. While it has been proposed that NET digestion may be beneficial in the treatment of sepsis, it is also possible that this strategy leads to the release of harmful NET degradation products (NDPs), such as cfDNA, histones, and myeloperoxidase (MPO) that cause endothelial damage. An alternative approach would be treatment strategies that prevent NET lysis and the release of NDPs. We posited that NET-bound platelet factor 4 (PF4, CXCL4) may have this effect. PF4 is a highly-positively charged, platelet-specific chemokine released in high concentrations following platelet activation and aggregates polyanionic molecules like heparin, polyphosphates, and DNA. We have found that PF4 causes physical compaction of NETs without inducing histone release. We studied the effect of this compaction using an in vitro microfluidic assay in which NETs released from neutrophils stimulated by tissue-necrosis factor (TNF) α were infused over a layer of cultured human umbilical vein endothelial cells (HUVECs) ± PF4 (25 μg/ml) and incubated for 12 hours. Channels exposed to PF4 were “heathier” with significantly more residual attached endothelial cells (44±24 vs. 150±29 cells/hpf, n=6, p=0.0002). We have previously shown that mice that overexpress PF4 are protected from mortality in lipopolysaccharide (LPS) endotoxemia. Compared to wildtype animals, PF4 null mice have increased circulating levels of NET markers including cfDNA, citrullinated histones, and MPO. Treatment with exogenous PF4 leads to a decrease in plasma levels of NET components and a reduction in mortality. In a microfluidic assay in which channel-adherent NETs were treated with increasing concentrations of PF4 (0-25 µg/ml) and then infused with DNase I (100 U/ml), we found that compacted NET-PF4 complexes become resistant to DNase I digestion. Prior studies have revealed that when PF4 binds to polyanionic molecules, it exposes HIT-like antigenic sites. We confirmed that the NET-PF4 complexes similarly bind HIT IgG isolated from clinical samples and the HIT-like monoclonal antibody KKO. Interestingly, KKO binding further enhanced DNase I resistance, a phenomenon not seen with a polyclonal anti-PF4 antibody (abcam). We hypothesized that KKO which causes PF4 oligomerization unlike the polyclonal antibody, further stabilized the crosslinked NET-PF4 complexes, providing additional protection from DNase I digestion and preventing the release of NDPs. We then asked whether KKO may serve as a targeted therapy in the treatment of sepsis, enhancing NET-PF4 complex resistance to nuclease digestion and leading to the sequestration of NDPs. As KKO is an IgG2bk antibody, we were unable to use pepsin digestion to remove the Fc region that causes platelet activation. We therefore used an IgG-specific endoglycosidase to develop a deglycosylated version of KKO (DG-KKO) that retains the ability to bind to NET-PF4 complexes (data not shown), but has a reduced capacity to interact with hematopoietic cell Fc receptors. DG-KKO has a markedly decreased ability to activate human platelets compared to KKO in the presence of added PF4 as measured by P-selectin level (3710±140 control vs. 1580±300 treated MFI, n = 2). Compared to control, DG-KKO infusion in the murine endotoxemia model prevented thrombocytopenia (mean platelet count 286±16 control vs. 537±69 treated X103/µl, n=4-5, p=0.016), the release of cfDNA (8.5±0.3 vs. 3.7±0.7 µg/ml, n=4-5, p=0.016), and the emission of MPO-DNA complexes (234±14 vs. 99±5 % increase from non-LPS injected controls, n=4-5, p=0.016) As depicted in Figure 1, we propose that in sepsis, NETosis occurs (Step 1) causing the release of harmful NDPs (Step 2). PF4 expelled from activated platelets stabilizes PF4-NET complexes (Step 3). Infused DG-KKO enhances NET stability and decreases release of NDPs following DNase I digestion (Step 4). These studies provide mechanistic insights into the release of NDPs during endotoxemia and offer a targeted, novel therapeutic to prevent their contribution to inflammatory states such as sepsis. [Display omitted] DisclosuresPoncz:Incyte Corporation: Consultancy, Research Funding.

Neutrophil Activation and Netosis Are the Key Drivers of Thrombosis in Heparin-Induced Thrombocytopenia

Adverse drug effects are common in clinical practice and often have a negative impact on patient safety. Heparin and heparin-derived drugs may induce an immune reaction, termed heparin-induced thrombocytopenia (HIT). HIT is mediated by IgG antibodies with specificity for heparin/platelet factor 4 (PF4) antigenic complexes. HIT is a hypercoagulable state which often causes severe and extensive thrombosis that results in high morbidity and mortality. The prevailing view is that these immune complexes activate platelets via FcγRIIa receptors leading to thrombocytopenia and thrombosis.Neutrophil extracellular traps (NETs) are DNA-containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non-immune disorders. Here we show that HIT immune complexes directly activate neutrophils via FcγRIIa and induce NETs formation. In addition, NETosis is also induced by activated platelets/neutrophil interactions mediated by P-selecting and PSGL-1. Ex vivo reconstitution of the HIT condition in a microfluidics system demonstrated the formation of thrombi rich in platelets, neutrophils, extracellular DNA and citrullinated histone 3 (Figure 1A, left panels). Neutrophil depletion abolished thrombus formation. Conversely neutrophil reconstitution restored thrombus deposition (Figure 1A, middle panels and right panels). Moreover, neutrophils alone treated with HIT IgG plus heparin formed thrombi containing extracellular DNA networks and citrullinated histone 3 on P-selectin coated channels (Figure 1B).Establishment of HIT in hFcγRIIa+/hPF4+ transgenic mice (HIT mice) using HIT patient's IgG or the HIT-like monoclonal antibody KKO recapitulated the hallmarks of NETosis: citrullinated histone 3, cell free DNA and MPO were detected in plasma and the presence of neutrophils, extracellular DNA and citrullinated histone 3 was found in lung thrombi. Low density neutrophils were also present in HIT mice treated with HIT IgG plus heparin but not in animals treated with control IgG. Treatment of HIT mice with DNase I or NETs formation inhibition with the PAD4 inhibitor GSK484 led to a dramatic decrease in thrombosis. This was corroborated by deletion of PAD4 in HIT mice. No thrombi were detected in hFcγRIIa+/hPF4+/PAD4-/- mice treated with HIT IgG and heparin (Figure 1C), indicating that NETs formation is required for thrombosis. Unlike thrombosis, thrombocytopenia was not affected by the absence of NETs formation, suggesting that these are separable processes. However, they are FcγRIIa-mediated mechanisms as anti-FcγRIIa antibodies abolished both processes. Analyses of sera from HIT patients revealed the presence of NETs markers and a significant proportion of neutrophils from patients with active HIT were undergoing NETosis. Our observations demonstrate that NETs formation is present in HIT and that it is essential for the development of thrombosis. Thrombocytopenia is not affected by the absence of NETosis. These findings suggest a new concept of the pathogenesis of thrombosis in HIT and as such are of clinical significance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endothelial antigen assembly leads to thrombotic complications in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

Membrane Remodeling By Pathogenic Antibodies Underlies Monocyte Activation in Heparin-Induced Thrombocytopenia

Heparin induced thrombocytopenia (HIT) is an iatrogenic antibody-mediated disorder with a paradoxically high propensity for thrombosis. We have shown previously that human HIT IgGs and the HIT-like monoclonal antibody (MAb) KKO bind to platelet factor 4 (PF4) complexed with glycosaminoglycans (GAGs) on the surface of platelets and monocytes, initiating cell activation in vitro, thrombocytopenia in a transgenic mouse model, and thrombus formation in a laser microvascular injury model in vivo even in the absence of exogenous heparin. Monocytes bind PF4 and HIT Ab more readily than platelets because they express higher affinity GAGs, heparan sulfate and dermatan sulfate, in addition to chondroitin sulfate found on both cell types. To study changes in the structure of the monocytes that accompany HIT, we used scanning electron microscopy, confocal microscopy and flow cytometry to characterize the morphology and function of isolated human monocytes and mouse transgenic Fcg receptor IIA positive (FcγRIIA+) or wt (FcγRIIA-) monocytes in the absence or presence of platelets. We show by scanning electron microscopy that upon binding of pathogenic HIT Abs to PF4/GAG complexes on FcgRIIA expressing monocytes, they initiate profound remodeling of the cell membrane. Addition of 100 μg/ml recombinant human PF4 in the absence of HIT Abs initiates the activation process with the appearance of 177 ± 53 nm "knobs" on the surface of 70% of monocytes. Subsequent addition of the HIT-like monoclonal antibody KKO at 50 μg/ml dramatically alters the cellular surface with the appearance of large 701 ± 208 nm membrane "blebs" that were not seen on FcγRIIA-mouse monocytes. These large, membrane-associated structures likely engage FcγRIIA, clustering them in proximity to cell-bound immune complexes, which promotes cell activation that leads to thrombosis. These blebs increase in size over time and are then shed from the cells as monocyte-derived microparticles, which self-aggregate. As a result of shedding of these blebs, the monocytes lose much of their typical ruffled surface (only 67% of monocytes maintain ruffles in the presence of PF4 plus KKO, compared to 97% of control monocytes) and appear smoother, sometimes with pores indicating degranulation. In the presence of platelets, monocytes exposed to PF4 and KKO formed heterocellular aggregates in addition to these subcellular changes. In contrast to KKO, addition of the non-pathogenic MAb RTO not only did not induce blebbing, but largely inhibited PF4-induced changes in the monocyte surface. This suggests that RTO might prevent monocyte activation by interfering with PF4 tetramerization. Structural analysis of the shed microparticles by microscopy revealed that they had an average diameter of 356 ± 307 nm, with many larger particles and aggregates. Flow cytometry confirmed that the shed particles contain cell membrane lipids and receptors. Confocal microscopy showed uniform binding of labeled PF4 to the monocyte cell membrane followed by rapid clustering into large complexes after the addition of KKO, but not RTO. These studies affirm the centrality of cell surface PF4/GAG complexes in the pathogenesis of HIT and provide quantitative morphometric characteristics of the changes in the monocyte membrane structure. We propose that PF4 released from activated platelets binds to the surface of GAG-expressing monocytes in vivo, forming clusters of PF4/GAG complexes that likely promote antibody binding and cause monocyte activation through FcγRIIA along with large-scale remodeling of the cell membrane and shedding of procoagulant microparticles. DisclosuresNo relevant conflicts of interest to declare.

New Insights into the Prothrombotic State of Heparin-Induced Thrombocytopenia (HIT) Using a Novel Microfluidic System: The Endothelial Lining and “Rolling Barrage” of Activation

The most feared feature of HIT is antibody-mediated thrombosis. We have shown that this prothrombotic state is related to binding of platelet factor 4 (PF4), a chemokine densely packaged into platelet alpha-granules, to surface glycosaminoglycans (GAGs) expressed on hematopoietic and vascular cells. PF4/GAG surface complexes are recognized by HIT antibodies, activating the targeted cells. Unlike platelets that express only low-affinity chondroitin sulfate surface GAGs, endothelial cells (EC) express a glycocalyx enriched in heparan sulfate, which has higher affinity for PF4, potentially increasing their propensity to become a target for immune injury leading to thrombosis. We examined the details of the development of in situ thrombi using the cremaster arteriole laser injury model beginning with transgenic mice expressing only human PF4 (hPF4+), but lacking FcγRIIA. These mice do not develop thrombocytopenia or thrombosis when injected with the HIT-like monoclonal antibody KKO or IgGs isolated from patients with HIT. In these mice, antigenic PF4/GAG complexes were recognized by KKO at sites of vascular injury even in the absence of infused heparin. In fact, infusion of sufficient heparin dissociated PF4 from sites of injury, consistent with its higher affinity for PF4 than cell surface GAGs. This suggests that antigenic PF4/GAG complexes normally develop intravascularly whenever thrombus formation occurs, yet these complexes do not typically initiate antibody-mediated thrombosis. Real-time confocal imaging of injured vessels revealed that PF4 first bound almost exclusively to the peri-injury endothelium. This was especially evident immediately upstream of the thrombus where turbulent blood flow may lead to platelet degranulation and subsequent adherence of the released PF4 to the glycocalyx. Beginning approximately two minutes post-injury, binding of KKO, presumably to PF4/GAG complexes on platelets, is seen at the interface between the shell and core of the thrombus. We then repeated these same studies in hPF4+/FcγRIIA+ mice, where infused KKO or HIT IgGs leads to significant thrombocytopenia and widespread development of thrombi as in HIT. Similar adherence of PF4 to the peri-injury EC and then to the core/shell interface of the thrombus as seen in the hPF4+ mice, but the changes were more extensive in hPF4+/FcγRIIA+ mice after KKO infusion and often lead to vascular occlusion. To further define the basis of the prothrombotic state in HIT and to extend our studies to a human system, we examined thrombus formation in HIT in a novel microfluidic system in which vascular injury was induced in an upstream portion of a human umbilical vein EC-lined channel by reactive oxygen species generated through excitation of infused hematoporphyrin by blue light (490 nm). Following infusion of human blood, platelets accumulated and released PF4, which bound the injured endothelium, while the downstream endothelium remained quiescent. Addition of KKO to the infused whole blood lead to a HIT-like state with marked increase in platelet adhesion and binding of PF4 to the injured endothelium, but binding of PF4 now spread downstream of the boundary between injured and uninjured endothelium. This was followed by downstream platelet adhesion and often occlusion of the channel. We proposed that this spread in EC injury was a result of a “rolling barrage” of PF4 released from platelets binding to the injured patch of EC complexing to the downstream glycocalyx on the non-injured endothelium followed by KKO binding and subsequent endothelial activation. The newly activated ECs bound additional platelets and the process repeats, rolling downstream and extending thrombus growth. Thus, these studies provide important new insights into the local sequence of events that propagate clots in HIT: Targeting of the endothelial glycocalyx by HIT antibodies is a major contributor to the prothrombotic state. Platelets adherent to the site of original injury release PF4, which then binds to downstream EC glycocalyx and initiates repetitive cycles of PF4 binding, EC activation and platelet adherence, and further release of PF4 that propagates growth of thrombi to previously uninvolved vasculature. DisclosuresNo relevant conflicts of interest to declare.

Imaging Morphologic Changes On Platelet and Monocyte Surfaces In Heparin-Induced Thrombocytopenia (HIT)

HIT is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, platelet factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). Unlike most other immune thrombocytopenias, HIT is markedly prothrombotic. We have proposed that this prothrombotic tendency is due to binding of pathogenic antibodies to PF4 complexes attached to the surface GAGs expressed by all intravascular cells, including platelets with their relatively low affinity surface GAGs, chondroitin sulfates, and monocytes with their higher affinity membrane GAGs, heparan and dermatan sulfates. Using isolated monocytes from healthy volunteers, we show by scanning electron microscopy that the addition of 10-50 µg/ml of recombinant human PF4 causes the appearance of ∼200 nm “knobs” on the cell surface. Subsequent addition of a HIT-like monoclonal antibody KKO at 50 µg/ml to the PF4-coated cells markedly alters their surface with the appearance of larger, up to 1-2 µm, membrane “blebs”. These blebs increase in size over time (15-60 minutes) and are shed from the cells. After shedding of these blebs, the monocytes lose their typical ruffled surface and appear spherical. These surface changes in the presence of KKO and PF4 are not seen in the presence of PF4 and 50 µg/ml of the anti-PF4 monoclonal antibody RTO, which does not induce the prothrombotic state of HIT. Platelets in suspension exposed to PF4 and KKO show by scanning electron microscopy similar knobs on their surface, but only minimally form blebs or microvesiculate. Platelets spread on fibrinogen in culture medium stimulated with PF4 and KKO and observed by hopping probe ion conductance microscopy progressively developed surface protrusions over a period of an hour, becoming more spherical. This morphological change was also observed in platelets exposed to IgG purified from 5 patients with HIT, but not when the KKO Fab fragment was tested. Neither PF4 alone nor with RTO antibody induced this morphological transition. Exposure to KKO plus PF4 for an hour induced minimal microvesiculation of platelets as measured by flow cytometry. Platelets adherent to fibrinogen underwent a similar morphological transition and did not microvesiculate after adding 50 mM of a thrombin-receptor activating peptide, whereas ADP-stimulated platelets rapidly microvesiculated during the same period of time. We believe that the “knobs” observed on monocytes and platelets represent aggregates of PF4-GAG complexes that are the targets of HIT antibodies. Bleb formation on monocytes and morphological transition of platelets result from clustering of knobs caused by bivalent HIT antibodies which cross-link Fc receptors. These blebs are released from monocytes, potentially becoming the microparticles found in the plasma of patients with HIT. In contrast, platelets treated with KKO plus PF4 showed minimal microvesiculation. This finding differs from reported high platelet microparticle counts in the plasma of HIT patients, suggesting that additional factors may be required to induce platelets to microvesiculate. Our images represent the first visualization of surface events when platelets and monocytes assume an active prothrombotic state. Whether these are unique to HIT or have wider applicability to the changes that occur in other prothrombotic, proinflammatory states needs to be addressed. Disclosures:No relevant conflicts of interest to declare.

Novel diagnostic assays for heparin-induced thrombocytopenia

AbstractLaboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from nonpathogenic antibodies. Specific functional assays are impracticable due to the need for platelets and radioisotope. We describe 2 assays that may overcome these limitations. The KKO-inhibition test (KKO-I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to platelet factor 4 (PF4)/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcγRIIa coupled to a luciferase reporter. We compared these assays to polyspecific and immunoglobulin (Ig)G-specific PF4/heparin enzyme-linked immunosorbent assays (ELISAs) in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ≥ 4 and positive 14C-serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs 26.0%; P < .0001) and induced greater luciferase activity (3.14-fold basal vs 0.96-fold basal; P < .0001). The area under the receiver-operating characteristic curve was greater for KKO-I (0.93) than for the polyspecific (0.82; P = .020) and IgG-specific ELISA (0.76; P = .0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (P = .046). KKO-I and DT40-luc showed better discrimination than 2 commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing.

Platelet-Targeted, Thrombin-Activatable Fibrinolytic Pro-Drugs As Novel Therapies: Application to the Prothrombotic Disorder of Heparin-Induced Thrombocytopenia (HIT)

Abstract 1171Thrombolytic therapies are limited in their use to acute life- or limb-threatening thrombosis due to the high risk of significant bleeding. To overcome this limitation, we propose a novel strategy incorporating two targeting features: 1) an N-terminal single-chain antibody variable region (scFv) chain that binds with high affinity to human platelet alphaIIb, and 2) a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site. We anticipate that such pro-drugs would preferentially bind and become activated at sites of active clot propagation. Two distinct anti-alphaIIb scFvs were studied: one (312.8) derived from a monoclonal antibody that bound human activated or quiescent alphaIIb whether complexed to human or mouse beta3, and one (LIBS) that bound preferentially to activated human or mouse alphaIIb bound to either human or mouse beta3. We anticipated that the 312.8/uPA-T chimera would have a more prolonged half-life, but that the LIBS version would be more effective at the nascent clot where activated platelets predominate. All proteins studied (uPA-T, 312.8/uPA-T and LIBS/uPA-T) were expressed and isolated from Drosophila S2 insect cells. Flow cytometric studies confirmed the expected binding of the chimera proteins to resting and activated human platelets, wildtype (WT) mouse platelets and mouse platelets that expressed only human alphaIIb/mouse beta3 (haIIb/mb3) on their surface. Neither chimeric protein interfered with platelet aggregation stimulated by adenosine diphosphate (10 μM). Further studies using diverse serine proteases confirmed that induction of fibrinolytic activities of both chimeras was thrombin-specific, and both were activated by thrombin to the same extent as isolated uPA-T. In haIIb/mb3 mice, both flow cytometric studies and tracking of 125I-labeled chimeric proteins showed 312.8/uPA-T had a longer half-life than uPA-T or LIBS/uPA-T. Ex vivo studies of thromboprophylactic efficacy of the chimeric proteins relative to uPA-T were performed using a microfluidic system with the surfaces coated with species-specific von Willebrand Factor. Platelets were isolated from whole human or haIIb/mb3 murine blood, exposed to a fibrinolytic agent or control, gel filtered to remove unbound drug, and added back to reconstitute whole blood. 312.8/uPA-T decreased platelet aggregation and virtually eliminated fibrin formation at drug concentrations <0.5 μg/ml, while both uPA-T and LIBS/uPA-T were ineffective even at the highest concentration tested (50 μg/ml). This microfluidic system was then modified to simulate the prothrombotic state seen in HIT by adding the well-characterized HIT-like monoclonal antibody KKO plus recombinant platelet factor 4 (PF4), markedly enhancing fibrin accumulation in the model. Again, low doses of 312.8/uPA-T enhanced fibrinolysis, while neither uPA-T nor LIBS/uPA-T were effective at a 100-fold higher concentration, consistent with both uPA-T and LIBS/uPA-T not binding well to resting platelets. In vivo studies of cremaster laser arteriolar injury were performed in haIIb/mb3 mice that were also transgenic for human PF4 and FcgammaRIIa before and after inducing a HIT-like state by injecting KKO. In this prothrombotic setting, pre-infusions of either chimeric proteins prevented fibrin accumulation and decreased platelet accumulation for up to 1 hour (latest time point studied). This demonstrates that LIBS/uPA-T is effective in thrombolysis despite its shorter half-life than 312.8/uPA-T. In summary, these studies support a novel approach towards thromboprophylaxis by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombosis. The preferred epitope for platelet targeting needs further study, as targeting to all platelets or specifically to activated platelets each appears to have advantages depending on the setting. These chimeric pro-fibrinolytic agents may be useful in systemic prothrombotic states such as HIT, but whether targeting and safety are preserved in vivo in such a setting needs further examination. Disclosures:Cines:Amgen: Consultancy; Genzyme: Consultancy; Sanofi: Consultancy; Eisai: Consultancy.

The Two Phases of Heparin-Induced Thrombocytopenia (HIT): Early Monocyte/Tissue Factor (TF) Phase and Late Platelet Phase

Abstract 270HIT is an immune thrombocytopenic disorder associated with a high risk of thrombosis. Mechanistic studies have focused on circulating platelet factor 4 (PF4)/heparin complexes and on the subsequent activation of platelets. We and others have shown that binding of PF4 to cell surface glycosaminoglycans (GAGs) not only makes platelets critical targets in the pathogenesis of HIT, but monocytes and neutrophils as well. Our previous observations that monocyte depletion using clodronate-laden liposomes prior to induction of HIT in a passive immunization model mitigated the prothrombotic state, but paradoxically exacerbated initial thrombocytopenia, suggest that our understanding of the relationship between monocyte activation, platelet activation, thrombocytopenia and thrombosis is unsettled. Chondroitin sulfate is the predominant GAG expressed by platelets; therefore, they bind PF4 less avidly than monocytes, which have a cell surface rich in heparan and dermatan sulfates. Based on this cell-type difference in surface affinity for PF4, we hypothesized that: 1) monocytes and perhaps other vascular cells bind PF4 and form surface PF4/HIT antigenic complexes preferentially when compared with platelets, and 2) depletion of this high-affinity (monocyte) “sink” shifts PF4 binding to platelets making them more targeted. Flow cytometric analysis of whole mouse and human blood support these hypotheses as monocytes bind ∼100-fold more FITC-labeled human (h) PF4 than platelets or red blood cells and ∼10-fold more than neutrophils or lymphocytes. When isolated platelets and white blood cells are admixed, the amount of exogenously added hPF4 bound to platelets is inversely related to the leukocyte:platelet ratio. In vitro, exposure of whole blood to the HIT-like monoclonal antibody KKO plus recombinant hPF4 generated an intense platelet activation characterized by binding of annexin V and factor Xa, consistent with formation of coated platelets. Importantly, formation of coated platelets was attenuated by monocyte depletion from whole blood samples or by inhibition of thrombin by PPACK. We then infused KKO into transgenic mice expressing hPF4 and hFcgRIIA to induce HIT and followed the temporal profile of antibody binding to various cell types. Binding of KKO to monocytes was detected within 30 min of injection. These monocytes remained the predominant target over the first 4 hrs, after which binding decreased as the circulating monocytes were depleted. The mechanism and implications of this relatively late monocytopenia during HIT is under study. However, these data provide an explanation of how clodronate-laden monocyte depletion prior to inducing HIT exacerbated thrombocytopenia in the murine model of HIT, while decreasing the prothrombotic state: Early in the disease when PF4 is limited, monocytes selectively bind the PF4 and are targeted by HIT antibodies, which induces tissue factor and generates thrombin, but limits initial thrombocytopenia. Later, after induction of large amounts of TF, activated monocytes are cleared, shifting the target of antigen-antibody interactions to the surface of platelets, enhancing their response to available thrombin, thereby establishing a feed-forward prothrombotic cycle and more platelet clearance. In conclusion, we propose that HIT evolves from a monocyte-focused to a platelet-focused disease and that early intervention to prevent monocyte activation provides a new important potential therapeutic target for intervention at the earliest stages of disease recognition that may become less effective as time passes. Disclosures:Cines:Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Formation of Procoagulant Platelets in Heparin-Induced Thrombocytopenia (HIT) Follows a Unique Signaling Pathway

Abstract 197HIT is a drug-induced, immune-mediated thrombocytopenia and thrombosis disorder associated with considerable morbidity and mortality. HIT is associated with generation of procoagulant platelet microparticles (Warkentin et al., Blood 1994; 84:3691), one of the cardinal features of procoagulant platelets, along with a phosphatidylserine-positive (PS+) external plasma membrane and surface retention of procoagulant proteins from plasma or released from platelet γ-granules. Other names for procoagulant platelets include COAT, or coated, platelets (Dale, J Thromb Haemostas 2005; 3:2185). To date, virtually all of the work on procoagulant platelets has used collagen or convulxin signaling via ITAM-associated GPVI, combined with thrombin signaling via GPCRs PAR1/PAR4. Batar and Dale reported the formation of coated platelets as a result of dual platelet FcγRIIa and thrombin stimulation; they used crosslinking of an anti-FcγRIIa antibody or anti-platelet glycoprotein antibodies which also engaged FcγRIIa (J Lab Clin Med 2001; 138:393). We report here the procoagulant activation of human and FcγRIIa transgenic (tg) mouse platelets stimulated by the HIT immune complex (IC) and thrombin (thr). HIT IC consist of ultralarge complexes of heparin and platelet factor 4 (hep/PF4) bound by HIT-like monoclonal antibody KKO. Washed human platelets, wild-type mouse platelets, or FcγRIIa tg mouse platelets were stimulated with the HIT IC + thr. Controls include convulxin (100 ng/ml) + thr, HIT IC alone, or thr alone as the stimulus. Human platelets and FcγRIIa-tg platelets, but not wild-type mouse platelets, develop a 40 to 60% PS+ population in response to HIT IC (100 ug/ml KKO)+ thr (0.5 U/ml), but not to either alone, as assessed by flow cytometry using labeled Annexin V. Unlike cvx + thr, which consistently shows a broad distribution of PS+ platelets in our hands and in the literature, HIT IC-stimulated platelets reproducibly show one discrete PS+ subpopulation (n = 6 per group). Preincubation of platelets with novel specific Syk inhibitor PRT318 (Reilly et al., Blood 2011; 117:2241) completely prevented PS+ platelet formation. FcγRIIa-tg platelets null for Akt2 showed a ∼50% reduction in the formation of PS+ platelets. Studies with FcγRIIa-tg mice null for Cal-DAG GEFI are ongoing. Understanding the unique pattern of procoagulant platelets generated by HIT IC + thr and the signals which produce it may lead to new diagnostic tests and therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

“Rolling Recruitment” of Endothelial Cell (EC) Activation in the Prothrombotic Nature of Heparin-Induced Thrombocytopenia (HIT)

Abstract 536HIT is an iatrogenic, prothrombotic, immune-mediated disorder associated with antibodies directed against platelet factor 4 (PF4) and heparin complexes. Once these pathogenic anti-PF4/heparin antibodies develop, they recognize and bind to antigenic complexes formed on cell surfaces composed of PF4 released from platelets bound to cell membrane glycosaminoglycans (GAGs). Unlike platelets, which have only chondroitin sulfate on their surface, endothelial cells express mainly heparan sulfate resulting in a very high capacity to form PF4/GAG complexes and become targets for HIT antibodies. In fact, activation of endothelial cells by HIT antibodies was recognized almost 25 years ago (Cines et. al., 1987). We now show direct interaction of the HIT-like monoclonal antibody KKO with perithrombus endothelial cells, which propagates thrombosis following laser induced cremaster vessel injury in a passive immunization murine model of HIT. PF4 released from activated platelets at the site of laser injury binds rapidly and predominantly to the adjacent endothelium and is recognized by KKO in both transgenic mice expressing only human PF4 (hPF4+) and mice expressing hPF4 and human FcgRIIA on platelets (hPF4+/FcgRIIA+). These studies show that HIT-like antigenic complexes are present within thrombi prior to antibody exposure, predominantly localized to endothelial cells, which is consistent with their surface GAGs having higher affinity for PF4 than platelets. These endothelial cell surface PF4/GAG complexes, and with it KKO binding, are dissociated by high concentrations of intravenous heparin. Infusion of KKO in hPF4+/FcgRIIA+ mice but not in control mice, reinitiates growth of previously stable thrombi at sites of laser injury, leading to a significant increase in vascular occlusion (24/33 injuries, 73%, compared to 0/30 in control mice; P<0.0001). Clot extension is followed by dissemination of endothelial activation, demarcated by binding of annexin V and Factor Xa, and extension of the endothelial surface that binds KKO. Based on these studies, we propose a model of feed-forward rolling recruitment to explain how the endothelial lining contributes to the prothrombotic state in HIT: PF4 released from activated platelets at a site of injury or disease binds to the surface of perithrombus endothelial cells, which then binds HIT antibodies followed by endothelial cell activation. These activated endothelial cells then bind and activate additional platelets, leading to the next round of PF4 release, more extensive endothelial cell activation and platelet recruitment into thrombi. Disclosures:No relevant conflicts of interest to declare.

Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin

AbstractAbstract 721Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient's antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT. Disclosures:No relevant conflicts of interest to declare.

Visualizing the Molecular and Cellular Basis of Heparin Induced Thrombocytopenia.

Abstract 228▪▪This icon denotes an abstract that is clinically relevant.Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). The origin of the sentinel feature of HIT - high propensity for thrombosis in the setting of mild thrombocytopenia - remains undefined. We have shown previously that monocytes are particularly susceptible to activation by HIT antibody because unlike platelets, whose surface glycosaminoglycans (GAGs) consist almost entirely of chondroitin sulfates (CS), monocytes express heparan and dermatan sulfates that bind to PF4 with greater affinity. Therefore, antigenic complexes form at lower concentrations of PF4 on monocytes than on platelets and are more resistant to removal by exogenous heparin. We now provide 2 lines of visual evidence in support of this model of the prothrombotic state in HIT: 1) We demonstrate by scanning electron microscopy that spheroidal particles indicative of PF4/GAG complexes are present on the surface of human monocytes upon addition of PF4. 2) Thrombus formation has been visualized in a previously described murine HIT model involving the infusion of the HIT-like monoclonal antibody KKO into mice double-transgenic for human PF4 (hPF4+) and FcγRIIA receptor (FcγRIIA+) on their platelets. After KKO infusion, hPF4+/FcγRIIA+ mice developed vigorous and concurrent accumulation of HIT antigenic complexes and fibrin at sites of laser-induced endothelial injury, not present in similarly treated single transgenic mice. Accumulation of PF4 and fibrin at sites of injury was not increased by a polyclonal anti-hPF4 antibody that does not induce thrombocytopenia in hPF4+/FcγRIIA+ mice. Monocytes and/or monocyte microparticles accumulated at sites of laser-induced injury in KKO-infused hPF4+/FcγRIIA+ mice, but not in KKO-infused single transgenic mice or hPF4+/FcγRIIA+ mice infused with an isotype control, beginning 2.5 min after formation of HIT antigenic complexes and fibrin deposition, accumulating preferentially at the upstream end of developing thrombi. These results provide new insights into the potential role of monocytes in the prothrombotic sequelae of HIT. Antigenic complexes form on monocytes exposed to PF4 and accumulate in HIT antibody-induced thrombi. Monocytes appear to reinforce thrombus formation initiated by injured endothelial cells and activated platelets. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Pathogenic Role of Surface Platelet Factor 4 Complexes in Heparin-Induced Thrombocytopenia: Diagnostic and Therapeutic Implications.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between high molecular weight heparin and Platelet Factor 4 (PF4). Current therapy with direct thrombin inhibitors is not effective in all cases, likely because it acts downstream of antibody-induced platelet activation. More directed therapies to the underlying pathology in HIT may be more effective. Heparin and PF4 only bind HIT antibodies over a narrow molar ratio of reactants at which ultralarge soluble complexes are formed. We asked whether similar complexes form between PF4 and endogenous platelet glycosaminoglycans (GAG) and their pathogenic role in experimental HIT. Platelet surface GAG:PF4 complexes are indeed antigenic over a narrow molar range of reactants. Heparin is not required for either HIT-IgG or a HIT-like monoclonal antibody KKO to bind to PF4 on human or mouse platelet surfaces in vitro, but enhances antigenicity when very high levels of surface PF4 are present. Antigenicity is maximal at a PF4 concentration of 50 μg/mL (well within the range that can be achieved within a thrombus) and ~25 μg/mL heparin (~0.5 U/mL, which is within the therapeutic range) optimally enhances antigenicity when surface PF4 levels were increased 4-fold. Using transgenic mice lines each with platelets expressing a different level of hPF4, ranging from 0.5 – 6 X's human platelet levels and all expressing FcRγIIA, were given KKO. The different lines developed thrombocytopenia proportional in severity and duration to hPF4 expression. A standard subcutaneous (sq) heparininzing dose (20 U/kg, sq daily) prolonged the duration of severe thrombocytopenia in high hPF4 expressing mice. We reasoned that altering the ratio of PF4 to GAG in either direction would alter antigenicity and could block the development of thrombocytopenia. In accordance with this concept, both high concentrations of anionic heparin (100 U/kg, sq daily) and cationic protamine sulfate (2 mg/kg, sq daily) decreased KKO binding in vitro and prevented KKO-induced thrombocytopenia in vivo as a demonstration of successful therapeutic intervention. These studies affirm a central role of surface GAG:PF4 complexes in the development of HIT, suggest ways to identify patients at high risk to develop HIT even prior to heparin exposure, and offers a new and rationale therapeutic paradigm based on disrupting surface antigen formation.