Summary Background Platelet transfusion applications face severe challenges due to the limited availability and portability, high risk of contamination and short shelf-life of platelets. Therefore there is significant interest in synthetic platelet substitutes that can render hemostasis while avoiding these issues. Platelets promote hemostasis by injury site-selective adhesion and aggregation, and propagation of coagulation reactions on their membrane. Based on these mechanisms, we have developed a synthetic platelet technology (SynthoPlateTM) that integrates platelet-mimetic site-selective ‘adhesion’ and ‘aggregation’ functionalities via heteromultivalent surface-decoration of lipid vesicles with Von Willebrand Factor-binding, collagen-binding and active platelet integrin GPIIb-IIIa-binding peptides. Objective SynthoPlateTM was evaluated for its effects on platelets and plasma in vitro, and for systemic safety and hemostatic efficacy in severely thrombocytopenic mice in vivo. Methods In vitro, SynthoPlateTM was evaluated using aggregometry, fluorescence microscopy, microfluidics, and thrombin and fibrin generation assays. In vivo, SynthoPlateTM was evaluated for systemic safety using prothrombin and fibrin assays on plasma and for hemostatic effect on tail-transection bleeding time in severely thrombocytopenic (TCP) mice. Results SynthoPlateTM did not aggregate resting platelets or spontaneously promote coagulation in plasma, but could amplify recruitment and aggregation of active platelets at the bleeding site and thereby site-selectively enhance fibrin generation. SynthoPlateTM dose-dependently reduced bleeding time in TCP mice, to levels comparable to normal mice. SynthoPlateTM has a reasonable circulation residence time and is cleared mostly by the liver and spleen. Conclusion The results demonstrate the promise of SynthoPlateTM as a synthetic platelet substitute in transfusion treatment of platelet-related bleeding complications.

Objective: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM’s ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM’s in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM’s procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). Conclusions: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM’s procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM’s pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Summary Exomic rare variant polymorphisms (c. 300 000) were analysed in the Scripps Venous Thrombosis (VTE) registry (subjects aged <55 years). Besides coagulation factor V (F5) single nucleotide polymorphisms (SNPs), family with sequence similarity 134, member B (FAM134B; rs78314670, Arg127Cys) and myosin heavy chain 8 (MYH8; rs111567318, Glu1838Ala) SNPs were associated with recurrent VTE (n = 34 cases) (false discovery rate‐adjusted P < 0·05). FAM134B (rs78314670) was associated with low plasma levels of anticoagulant glucosylceramide. Analysis of 50 chr17p13.1 MYH rare SNPs (clustered skeletal myosin heavy chain genes) using collapsing methods was associated with recurrent VTE (P = 2·70 ×10−16). When intravenously injected, skeletal muscle myosin was pro‐coagulant in a haemophilia mouse tail bleeding model. Thus, FAM134B and MYH genetic variants are plausibly linked to VTE risk.

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25-30% of that in the corresponding Ctrl ASO group both before and after infection. KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites. KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels but did not impact neutrophil influx, lung pathology, activation of the vascular endothelium, activation of the coagulation system, or the extent of distant organ injury. These results were corroborated by studies in mice with a genetic deficiency of KNG, which were indistinguishable from wild-type mice during Klebsiella-induced sepsis. Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels, indicating the carrier function of HK for this zymogen. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

Our data demonstrate that light exposure during postnatal development is required for rod photoreceptor development and that this effect could be mediated by thyroid hormone signaling.

Previous studies have demonstrated that cleaved high-molecular-weight kininogen (HKa) induces endothelial apoptosis and inhibits angiogenesis and have suggested that this occurs through inhibition of Src family kinases. This study assessed the role of tyrosine-protein kinase Lck (p56/Lck) in this pathway. We analyzed early events leading to apoptosis of human endothelial cells exposed to HKa. The role of p56/Lck was investigated using short interfering (si) RNA knockdown and lentivirus expression in assays of endothelial tube formation, sprouting of neovessels from murine aorta, and angiogenesis in Matrigel plugs. HKa stimulated expression and phosphorylation of p56/Lck. siRNA knockdown of p56/Lck promoted endothelial proliferation and blocked HKa-induced apoptosis and activation of p53, Bax, and Bak. Lentivirus expression of p56/Lck in endothelial cells induced apoptosis and blocked tube formation. Expression of p56/Lck in murine aortic rings blocked sprouting angiogenesis. Lentivirus expressing p56/Lck blocked angiogenesis in Matrigel plugs, while p56/Lck short hairpin RNA inhibited the antiangiogenic effect of HKa. Scrambled siRNAs and empty lentiviral vectors were used in all experiments. Apoptosis of proliferating endothelial cells and inhibition of angiogenesis by HKa requires p56/Lck. This suggests a novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.-Betapudi, V., Shukla, M., Alluri, R., Merkulov, S., McCrae, K. R. Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.

Introduction High molecular weight kininogen (HK) is a central component of the contact activation and kallikrein-kinin systems. We have previously reported that the cleaved, bradykinin-free form of HK (HKa) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis, and that tumors grow larger and more quickly in kininogen deficient (mKng1-/-) mice. However, HK is known to interact with many different cell types, and the mechanism by which it inhibits tumor growth in vivo is uncertain. Here, we address this question by comparing protein expression and localization in wild type and mKng1-/- mice harboring experimental tumors. Methods A syngeneic tumor model in which 1 x 106 B16F10 melanoma cells cultured in log-growth phase were implanted subcutaneously in the flanks of wild-type and mKng1-/- mice was used in these studies. Tumor growth and animal condition were monitored daily. Parallel studies were performed in tumors from mKng1-/- mice treated immediately prior to tumor implantation with a lentiviral vector that was either empty (control) or contained cDNA for murine Kng1. Mice were sacrificed approximately 17 days after tumor cell implantation, at which time the size of tumors in mKng1-/- mice were ~ 1.5 cm, substantially greater than that in wild-type mice. Tumors were harvested at the time of sacrifice and processed for flow cytometry, immunohistochemistry, immunoblot and antibody array (Proteome Profiler, Angiogenesis Protein Array, RND Systems). Changes in protein levels seen on arrays were confirmed by immunoblotting. Results Tumors in mKng1-/- mice grew more rapidly and had a volume 2-3-fold greater than those in wild-type mice by day 17; these tumors also demonstrated increased vascular density. Antibody array and immunoblot analysis demonstrated increased expression of several tumor and stroma associated proteins including MMP3, MMP9, VEGF, PlGF2, CD44 and MCP1 in tumors from mKng1-/- mice; the same differences were observed between mKng1-/- mice treated with an empty lentivirus when compared to mKng1-/- mice in which kininogen expression was restored using lentivirus-HK. Cellular localization by immunohistochemistry and immunofluorescence staining further demonstrated expression of MMP3 and MMP9 primarily in the tumor compartment, while expression of VEGF was most prominent in the stroma. PLGF2 expression was highly localized to regions immediately surrounding tumor vasculature. Increased, diffuse expression of CD44, a stem cell marker, was evident in tumors from mKng1-/- mice, localized primarily to the tumor compartment; increased CD44 expression was confirmed by flow cytometry and cell sorting, and may suggest increased numbers of tumor stem cells in tumors from mKng1-/- mice, though expression of CD34+, FLK1+, and CD133+ was unaltered. Tumor associated macrophages were significantly reduced in tumors from mKng1-/- mice as demonstrated by F4/80 immunostaining. Conclusions While the anti-angiogenic activity of HKa likely contributes to the increased growth of tumors in mKng1-/- mice, these findings suggest that HK/HKa may also have profound effects on multiple cell types in the tumor microenvironment, including suppression of MMP, VEGF and PLGF2 expression. HKa may also inhibit the tumor stem cell compartment, though further characterization of the CD44 positive cell population in these tumors is needed. Additional studies are needed to better define the interactions of HK/HKa with these diverse cell types. Disclosures No relevant conflicts of interest to declare.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the non-active site of APC was generated with llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared to the protein C (PC) zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in bio-layer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and PAR1 cleavage, were employed to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential of regulating specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, LP20) inhibited only 2 of the 3 functions. Mono-function inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9, LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41 that results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.