Increased Factor VIII Levels Have a Thrombogenic Effect In Vitro and In Vivo.

Abstract The procoagulant co-factor, factor VIII (FVIII), plays a crucial role in the intrinsic blood coagulation cascade. Epidemiologic studies have established a causal association between elevated FVIII levels and venous thrombosis incidence, whereas no such association has been confirmed with arterial thrombosis. Importantly, no objective in vitro or in vivo examinations of this association have previously been performed to establish a mechanistic role for elevated FVIII levels and thrombogenicity. To establish the in vitro thrombogenic effect of elevated FVIII activity, platelet-poor C57Bl/6 hemophilia A mouse plasma was spiked with recombinant human FVIII (r-huFVIII) to FVIII levels 100% and 250% that of normal plasma. Physiological concentrations of tissue factor and calcium chloride were added to the plasma to stimulate the formation of thrombin-antithrombin (TAT) complexes. Over 25 minutes, at 5-minute intervals, the plasma was sub-sampled, and the TAT complex concentration measured using an ELISA. The results showed that as FVIII concentration was increased, the rate of formation of TAT complexes was increased and the maximum concentration of complex was increased. As a second in vitro test of thrombogenicity, FVIII deficient human plasma was spiked with r-huFVIII to FVIII levels similar to those in the TAT experiments. Thromboelastography (TEG) was performed on the samples following the addition of physiological concentrations of tissue factor and calcium chloride. As FVIII levels were increased, the time to initial fibrin formation decreased significantly, in a negative logarithmic manner. As well, the speed with which the clot formed increased significantly, in a logarithmic manner. Although the clot strength (MA) of the FVIII spiked samples differed significantly to the 0% FVIII samples, there was no significant change in MA as FVIII levels were elevated to levels greater than 100%, likely as a result of the platelet poor nature of the samples. To examine the effect of increasing FVIII levels in vivo, fluorescence intravital microscopy was used to visualize the arterioles of the cremaster muscle. Circulating platelets were labeled in vivo with rhodamine 6G (200ng/mL). C57Bl/6 normal mice (n=5), C57Bl/6 hemophillia A mice (n=5), and C57Bl/6 mice hemophilia A mice whose FVIII levels were elevated to 200% (n=3) through an intravenous infusion of r-huFVIII were examined. Arterioles were injured for 3 minutes with 10% ferric chloride soaked filter paper and observed for 40 minutes. None of the hemophilic mice arterioles occluded in the observation period, whereas the normal mice occluded at 25 minutes and the mice with 200% FVIII activity occluded at 20 minutes. Fluorescence analysis revealed that the normal mice and those with 200% FVIII activity had stable platelet accumulation, as there was little variation in the fluorescence intensity over time, but a gradual, and persistent, increase in overall intensity. In contrast, there was strong variation in the accumulation of platelets within the injured arterioles of the hemophilic mice and no persistence in the intensity of fluorescence throughout the observation period. Together, these in vitro and in vivo data indicate that elevated FVIII levels produce a thrombogenic effect that increases with FVIII elevations. However, it is necessary to further examine this relationship to determine whether the thrombogenicity of FVIII is proportional to the FVIII increase and whether the thrombogenicity is affected by the duration of FVIII elevation.

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