ALBERT

Description: The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks vβ3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with vβ3. We found the following: (1) Binding of monoclonal antibodies against GPIb to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an vβ3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbβ/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIb was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbβ/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified vβ3 receptor did not interact with the alboaggregins, thereby excluding the contribution of vβ3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.

Description: Our previous studies have demonstrated that platelet-released PF4 (chemokine CXCL4) promotes survival in a murine LPS-induced endotoxicity model, although the molecular basis for PF4’s protective effects was not fully defined. We hypothesized that enhanced generation of cytoprotective activated protein C (APC) by PF4 might contribute to the molecular mechanism of PF4’s beneficial effects in vivo, based on the observation that PF4 stimulates protein C (PC) activation by the thrombin/thrombomodulin complex both in vitro and in vivo. Here we show that PF4 in vitro affects human (h) PC activation in the presence of human thrombomodulin (hTM) in a bell-shaped concentration curve, i.e. stimulation at low, but inhibition at high PF4 concentrations with a peak around 3 μM. This curve is similar to that seen with PF4 for surface-bound heparin-induced thrombocytopenia (HIT) antigenicity, suggesting that similar complexes of PF4 with glycosaminoglycans (GAGs) that occur in HIT (termed ultralarge complexes (ULC)) are relevant to PF4’s interaction with the hPC/hTM. Addition of heparin blocks PF4 increase of APC generation in a similar fashion as it does for surface-bound ULC. A PF4 variant PF4K50E that poorly forms PF4 tetramers requires 8-fold higher concentrations to enhance APC generation, supporting that PF4 tetramers are central for APC generation as they are for formation of ULC. Neither PF4 nor PF4K50E accelerated in vitro generation of APC in the presence of hTM that was depleted of its chondroitin sulfate chain, suggesting that PF4 binds to this domain on hTM. In vivo studies involving simultaneous infusions of PF4 and thrombin into PF4 knock out (mPF4−/−) mice showed that PF4 leads to enhanced mouse (m) APC generation not seen with infused PF4K50E, consistent with our in vitro studies. We then asked if surface heparan sulfate on the endothelial lining was necessary for the observed PF4 effect on in vivo mAPC formation. We studied mice with a Tie2-Cre conditional knock out of N-deacetylase-N-sulfotransferase-1 activity (NDST-1−/−) that have only 15% of normal endothelial cell surface heparan sulfate content using a similar thrombin/PF4 infusion model. We found that mAPC generation was accelerated by PF4 to the same extent both in NDST1−/−/mPF4−/− and mPF4−/− mice, suggesting that surface GAGs are not involved in the PF4 effect. We have also tested the in vivo effect of PF4 on mAPC formation in TM mutant (TMpro/pro) mice that have impaired capacity for APC formation to further demonstrate that PF4’s positive effect in LPS endotoxic shock survival involves enhanced mAPC generation. Upon injection of high doses of thrombin (40 U/kg), mAPC levels are increased to the same extent in WT and TMpro/pro mice. After injection of low amounts of thrombin (8 U/kg), generation of mAPC was impaired in TMpro/pro as compared to WT mice. Concurrent infusion of PF4 increased mAPC formation in TMpro/pro mice after injection of low doses of thrombin, approximately equal to that seen in WT mice with no PF4 injected. As previously described, TMpro/pro mice had increased mortality after injection of LPS as compared to WT mice; however, with concurrent platelet PF4 overexpression, mortality decreased to that seen in WT mice, suggesting that the biological value of PF4 in LPS endotoxicity is related to its effect on the generation of APC. Thus, these studies support enhanced APC generation as the basis for the positive effect of PF4 on LPS endotoxicity and further define the molecular basis for increased APC generation by PF4 by forming ULC with the chondroitin sulfate domain of TM, but not with heparan sulfate on the vascular surface.

Description: The platelet-specific chemokine platelet factor 4 (PF4) is released in large amounts at sites of vascular injury. PF4 binds to heparin with high affinity, but its in vivo biologic role has not been defined. We studied the role of PF4 in thrombosis using heterozygote and homozygote PF4 knock-out mice (mPF4+/– and mPF4–/–, respectively) and transgenic mice overexpressing human PF4 (hPF4+). None of these lines had an overt bleeding diathesis, but in a FeCl3 carotid artery thrombosis model, all showed impaired thrombus formation. This defect in thrombus formation in the mPF4–/– animals was corrected by infusing hPF4 over a narrow concentration range. The thrombotic defect in the mPF4+/– and mPF4–/– animals was particularly sensitive to infusions of the negatively charged anticoagulant heparin. However, the same amount of heparin paradoxically normalized thrombus formation in the hPF4+ animals, although these animals were anticoagulated systemically. Upon infusion of the positively charged protein, protamine sulfate, the reverse was observed with mPF4+/– and mPF4–/– animals having improved thrombosis, with the hPF4+ animals having worsened thrombus formation. These studies support an important role for PF4 in thrombosis, and show that neutralization of PF4 is an important component of heparin's anticoagulant effect. The mechanisms underlying these observations of PF4 biology and their clinical implications remain to be determined.

Description: The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34+cells and ex vivo expanded early colony-forming unit–granulocyte-macrophage (CFU-GM)–derived, burst forming unit–erythroid (BFU-E)–derived, and CFU–megakaryocyte (CFU-Meg)–derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor–2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor–1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor–α, Fas ligand, interferon α, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein–1α [MIP-1α], MIP-1β, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein–3 [MCP-3], MCP-4, IL-8, interferon-inducible protein–10, macrophage-derived chemokine [MDC], and platelet factor–4 [PF-4]) to be expressed by CD34+ cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor–β1 (TGF-β1), TGF-β2, RANTES, MIP-1α, MIP-1β, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34+ cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34+ cells; chemo-attract CFU-GM– and CFU-Meg–derived cells as well as other CD34+ cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM–, CFU-Meg–, and BFU-E–derived cells. It is concluded that normal human CD34+ cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.

Description: Heparin-induced thrombocytopenia/thrombosis (HIT/T), the most frequent drug-induced immune thrombocytopenia, is a common cause of life- and limb-threatening thrombosis. Although heparin/platelet factor 4 (PF4) antibodies are detected in many patients treated with heparin, there is little understanding of why only a subset of patients develops thrombosis. We recently produced and characterized the first mouse model that recapitulates the salient features of the disease. A second generation model, designated IIA/hPF4-mPF4KO, expresses human FcγRIIA and PF4 but lacks endogenous mouse PF4. These models allow systematic investigations of factors that contribute to pathogenic consequences of HIT/T antibodies. In the current study, we hypothesize that hypercholesterolemia, a known stimulus for atherosclerosis, endothelial dysfunction, and platelet hyperreactivity, would augment thrombosis in our mouse model of HIT/T. Age and sex-matched IIA/hPF4-mPF4KO mice were fed an atherogenic diet (Paigen diet) (AD; 15% cocoa butter, 1% cholesterol, 0.5% cholate) (n=10) or maintained on standard diet (SD; 4.5% fat, no cholate) (n=10). Mice fed the AD for only 4 weeks had significantly increased cholesterol levels (173 ± 29 mg/dl vs. 50 ± 18 mg/dl for SD-fed; p 〈 0.0001). Mice were then injected with 30 U heparin and KKO, a mouse monoclonal heparin/PF4 antibody. The mean nadir platelet count in AD-fed mice was 34.6% ± 9.1 lower than that in the SD-fed mice (p〈 0.0001). Thrombin-anti-thrombin III (TAT) levels, which reflect thrombin generation in vivo, in the AD-fed mice increased from 32 ± 5 μg/at baseline to 79 ± 16 μg/l (p 〈 0.0001) coincident with the platelet nadir. In contrast, SD-fed mice, with a less profound fall in platelets, showed no increase in TAT. Histological examination showed multiple platelet-fibrin thrombi in lungs and livers of AD-fed mice, whereas the SD-fed mice showed no histological evidence of thrombosis. Thus, in our mouse model, short-term diet-induced hyperlipidemia significantly increases the severity of heparin/PF4 antibody-mediated thrombocytopenia and thrombosis. Our studies provide evidence that a specific host factor can enhance the pathologic effects of heparin/PF4 antibodies in vivo and contribute to thrombotic risk in patients with HIT/T. An increased understanding of the contribution of prothrombotic factors not only will facilitate identification of patients with heparin/PF4 antibodies who are at increased risk of thrombosis, but also provide novel approaches to treatment of HIT/T.

Description: Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

Description: Abstract 3339 Use of plasminogen activators (PAs) is restricted to life-threatening thrombotic conditions because high concentrations are required to diffuse into clots, overcome PA inhibitors and compensate for rapid clearance, thereby predisposing to bleeding. We hypothesized that targeting pro-PAs to platelets would circumvent these obstacles and preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots. To test this concept, we expressed a chimeric protein (anti-PLT scFv/uPA-T) composed of an N-terminal scFv generated from a monoclonal antibody MoAb 312.8 directed to human αIIb integrin chain linked via a serine-rich linker peptide to human low molecular weight thrombin activatable pro-urokinase (uPA-T) wherein the plasmin cleavage site was replaced with a thrombin cleavage site, which we reasoned would be activated preferentially at sites of active clot propagation. Anti-PLT scFv/uPA-T expressed in Drosophila S2 insect cells bound specifically to human platelets and to transgenic mouse platelets that contain only human αIIb/mouse β3 on their cell surface (HαIIb+ Tg), but not to wild type mouse platelets. The anti-PLT scFv/uPA-T bound specifically to immobilized human αIIbβ3 with a Kd of ∼80 nM. The anti-PLT scFv/uPA-T did not interfere with either ADP-induced platelet aggregation or activation as demonstrated by expression of P-selectin by flow cytometry, and retained its zymogenic properties until activated specifically by thrombin. The fibrinolytic activity of anti-PLT scFv/uPA-T and uPA-T were compared using the FeCl3 carotid artery injury model in HαIIb+ Tg mice. The mice were protected from forming occlusive thrombi for at least 10 hrs post injection of anti-PLT scFv/uPA-T whereas even five-fold higher concentrations of uPA-T were effective for only 2–5 min. These studies support a novel approach for prophylactic targeting drug delivery by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombotic vascular occlusion. Disclosures: No relevant conflicts of interest to declare.

Description: Sepsis is a high-risk clinical setting often resulting in multi-organ failure and death. Release of chromatin NETS (neutrophil extracellular traps) from neutrophils and the toxic role of highly-positively charged histones in late sepsis have been noted previously. Also, for NET formation to occur, peptidylarginine deiminase 4 activity must be present in the neutrophils, leading to citrullinated (cit) histones formation and loss of a portion of the positive charge. The four histones (H2A, H2B, H3 and H4) alone and as octamers of the four units tightly bind DNA. H3 and H4 histones as well as mixed octameric histones can induce a sepsis-like state in mice. One feature previously noted was that histones could inhibit activated protein C (aPC) production in the presence of thrombomodulin (TM). Since aPC generation is felt to protect against vascular damage, it was felt that this might - in part - account for the deleterious effects of histones in sepsis. We have shown that another highly-positive, small molecule, platelet factor 4 (PF4, CXCL4), which exists as a tetramer and which is stored in high concentrations in platelet alpha granules to be released in large amounts post-platelet activation, binds to the chondroitin sulfate (CS) side-chain of TM (TMCS) and enhanced aPC production along a bell-shaped curve with a peak effect around 25 µg/ml. Non-modified mixed histones had a similar bell-shaped effect on aPC generation and [histones + PF4] are additive on affecting aPC generation via TMCS. We wondered, because of this overlapping biology and the fact that significant levels of free PF4 are available in late sepsis, whether PF4 might affect other histone pathobiological pathways in late sepsis focusing on PF4’s interactions with non-modified and cit-histones. We first asked whether released PF4 might affect the binding of histones to DNA within NETS. We found that PF4 binds to DNA with greater affinity than histones in a competitive binding assay and that this effect was more marked for cit-histone consistent with its decreased positive charge. We then studied PF4 biology in three known targets of histone in sepsis. (1) In aPC generation, we examined cit-histones (either mixed, H3 or H4) relative to non-modified histones in stimulating aPC generation and found that they had a more limited effect on aPC generation with TMCS, but that again, PF4 cooperated in inducing aPC generation along a bell-shaped curve. (2) Histones are known to activate platelets (known to involve the toll-like receptor 4), likely contributing to the observed thrombocytopenia in late sepsis. We affirmed this affect with mixed histones and H4. Cit-mixed histones and cit-H4 also activated platelets in a platelet aggregation system, but much more weakly. PF4 had no effect on platelet activation by non-modified histones, but enhanced platelet activation by both cit-mixed histones and cit-H4. This was especially true for platelet activation studies with cit-H4 which on its own had nearly no affect on platelet activation though in the presence of moderate levels of PF4 (25 µg/ml), cit-H4 activated platelets as well as non-modified histones. (3) Finally, both non-modified and cit-histones activate endothelial cells (EC) by binding to their cell surfaces and likely contribute to the vascular damage of late sepsis. Using a microfluidic system involving controlled photochemical injury of the EC lining we found that PF4 enhanced the observed damage after cit-H4 exposure, but not notably after a comparable H4 exposure so that peak damage (as detected by propidium iodide staining) after cit-H4 approached that seen after H4 alone. In conclusion, NET formation involves citrullination of histones, and these modified histones likely contribute significantly to pathobiology in late sepsis. We now propose that in late sepsis, free histones, especially cit-histones, are mobilized out of NETs by PF4 because the PF4 binds DNA with higher affinity. After the histones and cit-histones are released from DNA, PF4 modifies the biology of these histones, especially the cit-histones enhancing their effects on aPC generation, platelet activation and EC injury. These studies provide additional insights of how histones achieve their pathobiological effects in sepsis. Such new insights may be critical for both understanding and monitoring clinical outcome and may lead to new therapeutic targets in sepsis. Disclosures No relevant conflicts of interest to declare.

Description: Platelets play roles in both thrombosis and inflammation, and chemokines that are released at sites of inflammation could potentially activate platelets. Among the chemokine receptors expressed on platelets, the CXCR4 is the receptor for chemokine stromal cell-derived factor-1 (SDF-1), and the CCR4 is the receptor for macrophage-derived chemokine (MDC). Of the chemokines tested, SDF-1 and MDC were the only 2 that activated platelets. Both are weak agonists, but they enhanced response to low-dose adenosine 5′-diphosphate (ADP), epinephrine, or serotonin. When SDF-1 and MDC were added together, full and brisk platelet aggregation occurred. Platelet activation by these 2 chemokines appears to involve distinct pathways: SDF-1 inhibited an increase in cyclic adenosine monophosphate (cAMP) following prostaglandin (PG) I2, while MDC had no effect. In contrast, MDC, but not SDF-1, lead to Ca++mobilization by platelets. Further, second-wave aggregation induced by MDC in platelet-rich plasma was inhibited by aspirin, ADP scavenger creatine phosphate/creative phosphokinase (CP/CPK), and ARL-66096, an antagonist of the ADP P2TAC receptor involved in adenylyl cyclase inhibition. But the aggregation was not affected by A3P5PS, an inhibitor of the ADP P2Y receptor. SDF-1–induced aggregation was inhibited by aspirin, but it was only slightly affected by CP/CPK, ARL-66096, or A3P5PS. Finally, the presence of chemokines in platelets was determined. Reverse transcriptase–polymerase chain reaction studies with platelet RNA did not detect the presence of SDF-1 or MDC. In summary, SDF-1 and MDC are platelet agonists that activate distinct intracellular pathways. Their importance in the development of thrombosis at sites of inflammation needs to be further evaluated.