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Background: The variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown., Objective: To use a previously validated mathematical model of flow-mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency., Methods: We performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen-tissue factor (TF) surfaces at a shear rate of 100 per second., Results: Simulations identified low-normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high-normal prothrombin (150%). Low-normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high-normal prothrombin levels in both experimental models., Conclusions: These results show that low-normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII-deficient blood., (© 2019 International Society on Thrombosis and Haemostasis.)

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y 12 ., Summary: Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing α II b β 3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y 1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y 1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications., (© 2017 International Society on Thrombosis and Haemostasis.)

Sulfur mustard (SM) is a chemical warfare agent. When inhaled, SM causes significant injury to the respiratory tract. Although the mechanism involved in acute airway injury after SM inhalation has been well described previously, the mechanism of SM's contribution to distal lung vascular injury is not well understood. We hypothesized that acute inhalation of vaporized SM causes activated systemic coagulation with subsequent pulmonary vascular thrombi formation after SM inhalation exposure. Sprague Dawley rats inhaled SM ethanolic vapor (3.8 mg/kg). Barium/gelatin CT pulmonary angiograms were performed to assess for pulmonary vascular thrombi burden. Lung immunohistochemistry was performed for common procoagulant markers including fibrin(ogen), von Willebrand factor, and CD42d in control and SM-exposed lungs. Additionally, systemic levels of d-dimer and platelet aggregometry after adenosine diphosphate- and thrombin-stimulation were measured in plasma after SM exposure. In SM-exposed lungs, chest CT angiography demonstrated a significant decrease in the distal pulmonary vessel density assessed at 6 h postexposure. Immunohistochemistry also demonstrated increased intravascular fibrin(ogen), vascular von Willebrand factor, and platelet CD42d in the distal pulmonary vessels (<200 µm diameter). Circulating d-dimer levels were significantly increased (p < .001) at 6, 9, and 12 h after SM inhalation versus controls. Platelet aggregation was also increased in both adenosine diphosphate - (p < .01) and thrombin- (p < .001) stimulated platelet-rich plasma after SM inhalation. Significant pulmonary vascular thrombi formation was evident in distal pulmonary arterioles following SM inhalation in rats assessed by CT angiography and immunohistochemistry. Enhanced systemic platelet aggregation and activated systemic coagulation with subsequent thrombi formation likely contributed to pulmonary vessel occlusion., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The type of thrombus that forms depends on the nature of the injury and hemodynamics. There are many models of intravascular thrombus formation whereby blood is exposed to prothrombotic molecules on a solid substrate. However, there are few models of extravascular thrombus formation whereby blood escapes into the extravascular space through a hole in the vessel wall. Here, we describe a microfluidic model of hemostasis that includes vascular, vessel wall, and extravascular compartments. Type I collagen and tissue factor, which support platelet adhesion and initiate coagulation, respectively, were adsorbed to the wall of the injury channel and act synergistically to yield a stable thrombus that stops blood loss into the extravascular compartment in ~7.5 min. Inhibiting factor VIII to mimic hemophilia A results in an unstable thrombus that was unable to close the injury. Treatment with a P2Y12 antagonist to reduce platelet activation prolonged the closure time two-fold compared to controls. Taken together, these data demonstrate a hemostatic model that is sensitive to both coagulation and platelet function and can be used to study coagulopathies and platelet dysfunction that result in excessive blood loss.

Type 1 von Willebrand disease (VWD) is characterized by low plasma levels of von Willebrand factor (VWF) and clinical bleeding. Several mechanisms have been described that cause a decrease in plasma VWF levels in VWD, and the goal of this study was to elucidate the pathogenic origins of VWD for a group of mutations in the VWF D'D3 region traditionally associated with type 1 VWD. Varying ratios of mutant-to-wild-type VWF were expressed in two cell lines in order to study the intracellular location, multimer assembly, secretion and function of VWF. We identified four mutants (M771I, Y1146C, T1156M, R782Q) that caused defective intracellular packaging and markedly reduced VWF secretion. Consistent with previous reports, Y1146C and T1156M VWF led to a loss of high molecular weight multimers. In a functional analysis, Y1146C demonstrated a novel FVIII binding defect. Mutations R924W and I1094T were processed normally and did not show abnormal FVIII binding suggesting that other mechanisms such as plasma clearance or platelet binding defects may contribute to the pathogenicity of these mutants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)