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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.

Introduction-Pathological flows in patients with severe aortic stenosis are associated with acquired von Willebrand syndrome. This syndrome is characterized by excessive cleavage of von Willebrand factor by its main protease, A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13) leading to decreased VWF function and mucocutaneous bleeding. Aortic valve replacement and correction of the flow behavior to physiological levels reverses the syndrome, supporting the association between pathological flow and acquired von Willebrand syndrome. We investigated the effects of shear and elongational rates on von Willebrand factor cleavage in the presence of ADAMTS13. Methods-We identified acquired von Willebrand syndrome in five patients with severe aortic stenosis. Doppler echography values from these patients were used to develop three computational fluid dynamic (CFD) aortic valve models (normal, mild and severe stenosis). Shear, elongational rates and exposure times identified in the CFD simulations were used as parameters for the design of microfluidic devices to test the effects of pathologic shear and elongational rates on the structure and function of von Willebrand factor. Results-The shear rates (0-10,000s À1), elongational rates (0-1000 s À1) and exposure times (1-180 ms) tested in our microfluidic designs mimicked the flow features identified in patients with aortic stenosis. The shear and elongational rates tested in vitro did not lead to excessive cleavage or decreased function of von Willebrand factor in the presence of the protease. Conclusions-High shear and elongational rates in the presence of ADAMTS13 are not sufficient for excessive cleavage of von Willebrand Factor.

Adults with von Willebrand Disease (VWD) are known to have a ratio of factor VIII activity (FVIII:C) to von Willebrand factor antigen (VWF:Ag) greater than 1. We, however, noted healthy children with ratios that are unexpectedly high. Though the FVIII:C/VWF:Ag ratio differs significantly between healthy children and VWD patients in some age groups, the substantial overlap of observed ranges suggests that a ratio threshold-based screening approach alone cannot reliably discriminate between these groups. The diagnostic performance of this ratio is poor for VWD in children, which may decrease its value as a screening tool in the pediatric population.

INTRODUCTION The unpredictable clinical response of patients to bypassing therapy and the lack of a proper laboratory tools to measure clot formation and stability renders prophylaxis and surgery on these patients a huge challenge. These patients are at a risk for bleeding or thromboembolic complications. AIMS In this study we introduce a novel plasma based microfluidic assay that can qualitatively and quantitatively measure fibrin deposition, thrombin and plasmin generation, and fibrinolysis under flow. We then examined the dynamics of thrombus formation in patients with hemophilia and their response to replacement and bypass therapies under flow conditions. METHODS Coagulation in the plasma based assay was initiated by spherical 1µm lipidated- Tissue Factor biomimetic silica beads which were patterned into 200µm circles on a substrate surface. Plasma samples were obtained from Hemophilia patients and inhibitor patients, before and after replacement or bypassing treatment and perfused over the tissue factor rich surfaces at a sheer rate of 100 s-1 Fibrinolysis was initiated with the addition of tPAto the plasma samples before perfusion. RESULTS The microfluidic assay was sensitive enough to measure the activation of coagulation triggered by the bypassing agents. Fibrin generation and thrombin generation were measurable both qualitatively and quantitatively using three metrics; lag time, rate of production and maximum quantity produced. Individuals on replacement therapy showed normalized fibrin formation with a 69% increase in fibrin formation, a decrease in lag time and an overall increase in maximum fibrin and thrombin production (See attached Figure). The microfluidic assay was also able to show an increase in overall fibrin generation in certain Individuals that were given more bypassing treatment than needed. Compared to healthy controls the rate of fibrin generation and maximum fibrin was greater, thereby identifying a risk for prothrombotic state. (See attached Figure). Finally, using the microfluidic assay we were able to observe both clot formation and lysis and asses the the stability of the fibrin clot produced when these inhibitor patients were on and off treatments, which reflects a more complete picture of the coagulation process. CONCLUSION We are able to show that individuals, treated by replacement therapies showed normalized clot formation. Individuals with hemophilia treated by bypassing therapies also showed normalized clot formation. Sometimes however, the fibrin production is more than a normal control, which could lead to a risk of prothrombosis. By using microfluidic assay, the treatment can be given in doses and fibrin production observed, to decrease the overall fibrin formation from a hypocoagulable to a hemostatic state, avoiding hypercoagulability. Figure Figure. Disclosures No relevant conflicts of interest to declare.