FVIII-associated LDL circulates longer and participates in blood clot formation Free

Background and rationale. Hemophilia A is a bleeding disorder caused by deficient or absent in coagulation factor VIII (FVIII). Several clinical studies reported overall lower prevalence of atherosclerotic cardiovascular diseases in hemophilia patients, but the underlying mechanism is unknown. Low-density lipoprotein (LDL) is well known for its contribution to the development of astherosclerosis, a chronic condition which can further develop to its advanced stages of vulnerable plaques, prone to rupture or to erode and trigger lethal arterial thrombotic events, including myocardial infacrtion and ischemic stroke. Interestingly, high LDL cholesterol is also an independent risk factor of venous thrombosis, which occurs at vascular locations without lipid-rich atheroslcerotic plaques, suggesting LDL plays a role in exacerbating thrombosis independent of its pro-atherogenic functions. A potential pro-coagulant effect of LDL has been speculated, but has not been investigated in detail for causality and the underlying mechanism. In this study, we aim to understand the mechanism underlying the reduced cardiovascular risk in hemophilia A and to provide insights into the role of LDL in coagulation.

Objectives. To test the hypotheses that 1) FVIII directly interacts with apolipoproteinB (apoB) at its binding site to the LDL receptor (LDLR), therefore blocking LDL clearance. 2) The LDL–FVIII complex accelerates blood clotting time in FVIII deficiency by delivering FVIII to the site of coagulation.

Methods. Plasma level of apoB, which reflects LDL particle numbers, as well as plasma choelsterol, were tested in healthy blood donors and hemophilia A patients. FVIII-deficient mice, LDL KO mice, mouse primary hepatocytes, FVIII-and von Willebrand factor (VWF)-double-depleted human plasma, and recombinant human FVIII (rFVIII) were used in mechanistic studies.

Results. Hemophilia A patients are categorized as mild, moderate and severe based on their residual FVIII activity. Compared to age- and gender-matched healthy donors (n=45), hemophilia A patients (n=46, including 17 mild, 8 moderate and 19 severe) had lower plasma total cholesterol, LDL cholesterol and apoB, with the loweset in the severe group, and no difference in HDL cholesterol. To further verify the phenotype and explore the mechanism leading to low LDL in FVIII deficiency, FVIII-deficient mice and WT littermates were fed a high-fat, high-cholesterol diet for 14 weeks. Compared to their WT littermates, FVIII-deficient mice had lower total cholesterol, LDL cholesterol, and apoB. As each LDL particle has only one copy of apoB, the concentration of apoB serves as a surrogate for the number of LDL particles. FVIII-deficient mice had lower plasma apoB than WT littermates, indicating fewer circulating LDL particle numbers. LDL clearance assay showed that FVIII-free LDL was cleared faster in FVIII deficient mice than their WT littermates, but there was no difference in the liver LDL receptor levels between the groups. Interestingly, knocking out LDLR in FVIII-deficient mice diminished the lower plasma cholesterol and apoB phenotype. In fluorescence-activated cell sorting assay, adding rFVIII reduced the binding of LDL to WT mouse primary hepatocytes, but not to LDLR KO mouse primary hepatocytes. Inhibition assay using surface plasmon resonance (SPR) also demonstrated that rFVIII inhibits LDL binding to LDLR. Direct interaction measured by SPR showed a Kd of 2.59 ± 0.42 nM between rFVIII and LDL.

To investigate the functional consequences of FVIII-LDL interaction, Rotational Thromboelastometry (ROTEM) and Activated Partial Thromboplastin Time (aPTT) assays were used and showed that FVIII-associated LDL shortens clotting time in blood from FVIII deficient mice. By confocal microscopy immunofluorescence imaging, abundant apoB was observed in the cross-sections of both human and mouse clots. In these clot cross-sections, about 70% of fibrin, the final product of coagulation, was colocalized with apoB.

Conclusion. Our study demonstrates that FVIII directly interacts with apoB and inhibits LDL binding to LDLR. In the absence of FVIII, hemophilia A has lower LDL, resulted from a faster LDL clearance rate through hepatocytes. Furthermore, LDL transports FVIII to the site of blood clotting, shortens clotting time, and contributes to fibrin formation, suggesting a pro-coagulant effect of LDL.