Summary
Background
Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis.
Objective
To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties
Patients/Methods
In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy.
Results
FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls.
Conclusions
Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.
Turbidimetry is used to characterize fibrin clot properties. In purified systems, maximum absorbance (MA) directly relates to fibrin fiber cross-sectional area. However, in plasma samples there are discrepancies in the relationships between MA and fibrinogen concentration, fiber diameter, other clot properties, and cardiovascular disease outcomes, which complicate data interpretation. This study aims to advance understanding of MA of plasma clots through testing how well it relates to fundamental dependence on fibrinogen concentration and fiber diameter as predicted by light scattering theory, other clot properties and lifestyle, and biochemical variables. Plasma samples from 30 apparently healthy individuals with a fibrinogen concentration from 2.4 to 6.4 g/L were included. We performed turbidimetry, permeability, scanning electron microscopy, and rheometry on in vitro formed plasma clots. MA correlated more strongly with fibrinogen concentration (r = 0.65; p < 0.001) than with fiber diameter (r = 0.47; p = 0.01), which combined explained only 46% of the MA variance. Of additional variables measured, only low-density lipoprotein cholesterol correlated with MA (r = 0.46; p = 0.01) and clot lysis (r = 0.62; p < 0.0001) but not with fiber diameter or fibrinogen concentration. MA correlated with clot lysis time (r = 0.59; p = 0.001), storage modulus (r = 0.61; p = 0.001), and loss modulus (r = 0.59; p = 0.001), and negatively with clot permeability (r = –0.60; p = 0.001) also after adjustment for fibrinogen concentration and fiber diameter. Increased MA is indicative of a prothrombotic clot phenotype irrespective of fibrinogen concentration. MA is more indicative of overall clot density than of fiber diameter. Other plasma components can alter internal fiber density without altering fiber diameter and should be considered when interpreting MA of plasma samples.
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