In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Surgeon-Reported Needs for Improved Training in Identifying and Managing Free Flap Compromise.

Background This study examined the need for improved training in the identification and management of free flap (FF) compromise and assessed a potential role for simulated scenario training. Methods Online needs assessment surveys were completed by plastic surgeons and a subsample with expertise in microsurgery education participated in focus groups. Data were analyzed using descriptive statistics and mixed qualitative methods. Results In this study, 77 surgeons completed surveys and 11 experts participated in one of two focus groups. Forty-nine (64%) participants were educators, 65 and 45% of which reported having an insufficient volume of FF cases to adequately teach the management and identification of compromise, respectively. Forty-three percent of educators felt that graduating residents are not adequately prepared to manage FF compromise independently. Exposure to normal and abnormal FF cases was felt to be critical for effective training by focus group participants. Experts identified low failure rates, communication issues, and challenging teaching conditions as current barriers to training. Most educators (74%) felt that simulated scenario training would be "very useful" or "extremely useful" to current residents. Focus groups highlighted the need for a widely accepted algorithm for re-exploration and salvage on which to base the development of a training adjunct consisting of simulated scenarios. Conclusion Trainee exposure to FF compromise is inadequate in existing plastic surgery programs. Early exposure, high case volume, and a standardized algorithmic approach to management with a focus on decision making may improve training. Simulated scenario training may be valuable in addressing current barriers.

Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond.

Platelets are small anucleate blood cells generated from megakaryocytes in the bone marrow and cleared in the reticuloendothelial system. At the site of vascular injury, platelet adhesion, activation and aggregation constitute the first wave of hemostasis. Blood coagulation, which is initiated by the intrinsic or extrinsic coagulation cascades, is the second wave of hemostasis. Activated platelets can also provide negatively-charged surfaces that harbor coagulation factors and markedly potentiate cell-based thrombin generation. Recently, deposition of plasma fibronectin, and likely other plasma proteins, onto the injured vessel wall has been identified as a new "protein wave of hemostasis" that may occur even earlier than the first wave of hemostasis, platelet accumulation. Although no experimental evidence currently exists, it is conceivable that platelets may also contribute to this protein wave of hemostasis by releasing their granule fibronectin and other proteins that may facilitate fibronectin self- and non-self-assembly on the vessel wall. Thus, platelets may contribute to all three waves of hemostasis and are central players in this critical physiological process to prevent bleeding. Low platelet counts in blood caused by enhanced platelet clearance and/or impaired platelet production are usually associated with hemorrhage. Auto- and allo-immune thrombocytopenias such as idiopathic thrombocytopenic purpura and fetal and neonatal alloimmune thrombocytopenia may cause life-threatening bleeding such as intracranial hemorrhage. When triggered under pathological conditions such as rupture of an atherosclerotic plaque, excessive platelet activation and aggregation may result in thrombosis and vessel occlusion. This may lead to myocardial infarction or ischemic stroke, the major causes of mortality and morbidity worldwide. Platelets are also involved in deep vein thrombosis and thromboembolism, another leading cause of mortality. Although fibrinogen has been documented for more than half a century as essential for platelet aggregation, recent studies demonstrated that fibrinogen-independent platelet aggregation occurs in both gene deficient animals and human patients under physiological and pathological conditions (non-anti-coagulated blood). This indicates that other unidentified platelet ligands may play important roles in thrombosis and might be novel antithrombotic targets. In addition to their critical roles in hemostasis and thrombosis, emerging evidence indicates that platelets are versatile cells involved in many other pathophysiological processes such as innate and adaptive immune responses, atherosclerosis, angiogenesis, lymphatic vessel development, liver regeneration and tumor metastasis. This review summarizes the current knowledge of platelet biology, highlights recent advances in the understanding of platelet production and clearance, molecular and cellular events of thrombosis and hemostasis, and introduces the emerging roles of platelets in the immune system, vascular biology and tumorigenesis. The clinical implications of these basic science and translational research findings will also be discussed.

Fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is an alloimmune disorder resulting from platelet opsonization by maternal antibodies that destroy fetal platelets. The major risk of FNAIT is severe bleeding, particularly intracranial hemorrhage. Miscarriage has also been reported but the incidence requires further study. Analogous to adult autoimmune thrombocytopenia (ITP), the major target antigen in FNAIT is the platelet membrane glycoprotein (GP)IIbIIIa. FNAIT caused by antibodies against platelet GPIbα or other antigens has also been reported, but the reported incidence of the anti-GPIbα-mediated FNAIT is far lower than in ITP. To date, the maternal immune response to fetal platelet antigens is still not well understood and it is unclear why bleeding is more severe in FNAIT than in ITP. In this review, we introduce the pathogenesis of FNAIT, particularly those new discoveries from animal models, and discuss possible improvements for the diagnosis, therapy, and prevention of this devastating disease.

Plasma fibronectin supports hemostasis and regulates thrombosis.

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, ß3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet ß3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Burn plus lipopolysaccharide augments endoplasmic reticulum stress and NLRP3 inflammasome activation and reduces PGC-1α in liver.

Extensively burned patients often suffer from sepsis (especially caused by Pseudomonas aeruginosa), which may prolong metabolic derangement, contribute to multiple organ failure, and increase mortality. The molecular and cellular mechanisms of such infection-related metabolic derangement and organ dysfunction are unclear. We have previously shown that severely burned patients have significant and persisting hepatic endoplasmic reticulum (ER) stress. We hypothesized that ER stress and the unfolded protein response correlate with NOD-like receptor, pyrin domain containing 3 (NLRP3) inflammasome activation in burn. These may trigger profound metabolic changes in the liver, which form the pathological basis of liver damage and liver dysfunction after burn injury. A two-hit rat model was established by a 60% total body surface area scald burn and intraperitoneal injection of P. aeruginosa-derived lipopolysaccharide (LPS) 3 days after burn. One day later, animals were killed, and liver tissue samples were collected for gene expression and protein analysis of NLRP3 inflammasome activation, ER stress, and glucose and lipid metabolism. Liver damage was assessed by plasma markers (alanine aminotransferase and aspartate aminotransferase) and liver immunohistochemical analysis. Our results showed that burn injury and LPS injection induced inflammasome activation in liver and augmented hepatic ER stress and liver damage. Although there was an increased metabolic demand after burn, hepatic NLRP3 inflammasome activation corresponded to inhibition of PGC-1α (peroxisome proliferator-activated receptor γ-coactivator 1α) and its upstream regulators protein kinase A catalyst unit, AMP-activated protein kinase α, and sirtuin-1 may provide a mechanism for the enhanced metabolic derangement after major burn injury plus sepsis. In conclusion, burn + LPS augments inflammasome activation and ER stress in liver, which in turn contribute to postburn metabolic derangement.

Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis.

Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. It has been well demonstrated that interaction between glycoprotein (GP) Ibα and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. GPIb has long been suggested as a desirable antithrombotic target, but anti-GPIb therapy has never been successfully developed. Here, we evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist.We found Anfibatide inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF. It also blocked botrocetin-induced binding of murine plasma VWF to recombinant human GPIbα. Interestingly, Anfibatide did not inhibit botrocetin-induced aggregation of platelet-rich plasma, indicating that its binding site may differ from other snake venom-derived GPIb antagonists. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation in perfusion chambers at high shear conditions and efficiently dissolved preformed thrombi. Anfibatide also inhibited thrombus growth at low shear conditions, though less than at high shear. Using intravital microscopy, we found that Anfibatide markedly inhibited thrombosis in laser-injured cremaster vessels and prevented vessel occlusion in FeCl3-injured mesenteric vessels. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice, suggesting that Anfibatide has additional antithrombotic effect beyond its inhibitory role in GPIb-VWF interaction. Anfibatide did not significantly cause platelet activation, prolong tail bleeding time, or cause bleeding diathesis in mice. Thus, consistent with the data from an ongoing clinical trial, the data from this study suggests that Anfibatide is a potent and safe antithrombotic agent.

Fenofibrate does not affect burn-induced hepatic endoplasmic reticulum stress.

BACKGROUND: Burn injury causes major metabolic derangements such as hypermetabolism, hyperlipidemia, and insulin resistance and is associated with liver damage, hepatomegaly, and hepatic endoplasmic reticulum (ER) stress. Although the physiological consequences of such derangements have been delineated, the underlying molecular mechanisms remain unknown. Previously, it was shown that fenofibrate improves patient outcome by attenuating postburn stress responses. METHODS: Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, regulates liver lipid metabolism and has been used to treat hypertriglyceridemia and hypercholesterolemia for many years. The aim of the present study is to determine the effects of fenofibrate on burn-induced hepatic morphologic and metabolic changes. We randomized rats to sham, burn injury, and burn injury plus fenofibrate. Animals were sacrificed and livers were assessed at 24 or 48 h post burn. RESULTS: Burn injury decreased albumin and increased alanine transaminase (P = 0.1 versus sham), indicating liver injury. Fenofibrate administration did not restore albumin or decrease alanine transaminase. In addition, ER stress was significantly increased after burn injury both with and without fenofibrate (P < 0.05 versus sham). Burn injury increased fatty acid metabolism gene expression (P < 0.05 versus sham), downstream of peroxisome proliferator-activated receptor alpha. Fenofibrate treatment increased fatty acid metabolism further, which reduced postburn hepatic steatosis (burn versus sham P < 0.05, burn + fenofibrate versus sham not significant). CONCLUSIONS: Fenofibrate did not alleviate thermal injury-induced hepatic ER stress and dysfunction, but it reduced hepatic steatosis by modulating hepatic genes related to fat metabolism.

XBP-1s is linked to suppressed gluconeogenesis in the Ebb phase of burn injury.

The first 24 h following burn injury is known as the ebb phase and is characterized by a depressed metabolic rate. While the postburn ebb phase has been well described, the molecular mechanisms underlying this response are poorly understood. The endoplasmic reticulum (ER) regulates metabolic rate by maintaining glucose homeostasis through the hepatic ER stress response. We have shown that burn injury leads to ER stress in the liver during the first 24 h following thermal injury. However, whether ER stress is linked to the metabolic responses during the ebb phase of burn injury is poorly understood. Here, we show in an animal model that burn induces activation of activating transcription factor 6 (ATF6) and inositol requiring enzyme-1 (IRE-1) and this leads to increased expression of spliced X-box binding protein-1 (XBP-1s) messenger ribonucleic acid (mRNA) during the ebb phase. This is associated with increased expression of XBP-1 target genes and downregulation of the key gluconeogenic enzyme glucose-6-phosphatase (G6Pase). We conclude that upregulation of the ER stress response after burn injury is linked to attenuated gluconeogenesis and sustained glucose tolerance in the postburn ebb phase.

Hepatic apoptosis postburn is mediated by c-Jun N-terminal kinase 2.

The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance after burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-Jun N-terminal kinase (JNK) 2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (-/-) with wild-type mice after inducing a 30% total body surface area thermal injury. Animals were killed after 1, 3, and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western blotting, and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Liver function was quantified by aspartate aminotransferase and alanine aminotransferase activity assays. Endoplasmic reticulum stress increased after burn in both JNK2 and wild-type mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in interleukin 6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2 group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2. As expected, apoptosis in the liver increased after burn injury in wild-type mice but not in JNK2. Aspartate aminotransferase/alanine aminotransferase activity revealed that liver function recovered more quickly in JNK2. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn.

Effects of metformin on burn-induced hepatic endoplasmic reticulum stress in male rats.

Severe burn injury causes hepatic dysfunction that results in major metabolic derangements including insulin resistance and hyperglycemia and is associated with hepatic endoplasmic reticulum (ER) stress. We have recently shown that insulin reduces ER stress and improves liver function and morphology; however, it is not clear whether these changes are directly insulin mediated or are due to glucose alterations. Metformin is an antidiabetic agent that decreases hyperglycemia by different pathways than insulin; therefore, we asked whether metformin affects postburn ER stress and hepatic metabolism. The aim of the present study is to determine the effects of metformin on postburn hepatic ER stress and metabolic markers. Male rats were randomized to sham, burn injury and burn injury plus metformin and were sacrificed at various time points. Outcomes measured were hepatic damage, function, metabolism and ER stress. Burn-induced decrease in albumin mRNA and increase in alanine transaminase (p < 0.01 versus sham) were not normalized by metformin treatment. In addition, ER stress markers were similarly increased in burn injury with or without metformin compared with sham (p < 0.05). We also found that gluconeogenesis and fatty acid metabolism gene expressions were upregulated with or without metformin compared with sham (p < 0.05). Our results indicate that, whereas thermal injury results in hepatic ER stress, metformin does not ameliorate postburn stress responses by correcting hepatic ER stress.