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.
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