Pathologic Shear and Elongation Rates Do Not Cause Cleavage of Von Willebrand Factor by ADAMTS13 in a Purified System.

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-1), elongational rates (0-1000 s-1) 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.

A mathematical model of coagulation under flow identifies factor V as a modifier of thrombin generation in hemophilia A.

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.

FVIII/VWF ratio is not a reliable predictor of VWD in children.

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.

Monitoring hypercoagulability and hypofibrinolysis following acute venous Thromboembolism in children: application of the CloFAL assay in a prospective inception cohort study.

Although individual thrombophilia tests are frequently performed in children with venous thromboembolism (VTE), global assays provide the opportunity to fill the gap in knowledge regarding their net impact on overall coagulative (and in some cases fibrinolytic) function. We first evaluated analytic sensitivity of the Clot Formation and Lysis (CloFAL) global assay to hypercoagulability and alterations in fibrinolysis, and then characterized changes in plasma coagulative and fibrinolytic capacities over time in children with acute VTE. In plasma ex vivo and in vitro experiments, the CloFAL assay area-under-the-curve (AUC) was analytically sensitive to hypercoagulable states, and its modified fibrinolytic index (FI2) was sensitive to both hyper- and hypofibrinolytic conditions. Clinical data and plasma samples for assay were collected during follow-up of 50 children enrolled in a prospective inception cohort study of VTE from May 2006 to June 2010. Follow-up periods were designated as follows: acute (<1 month post-event), sub-acute (1-3 months), early chronic (3-12 months), and late chronic (>12 months). Since most children were sampled at fewer than three pre-defined follow-up periods, study population findings were grouped by timepoint. AUC was significantly increased, and FI(2) significantly decreased, in the acute period of VTE when compared to healthy controls, indicating hypercoagulability and hypofibrinolysis, respectively. One-third of patients were hypercoagulable, and 23% were hypofibrinolytic, in the late chronic phase. AUC and FI(2) were strongly correlated with functional fibrinogen levels. These findings indicate the utility of the CloFAL assay in monitoring plasma coagulative and fibrinolytic capacities in children with VTE. Studies of its potential role in outcome prediction are ongoing.