Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105) and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis (RA) and osteoarthritis (OA) and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg 336 in the A1 subunit and Arg 562 in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. Hemophilia A is an X-linked bleeding disorder, affecting 1 in 5000 males. It is caused by deficiency of blood coagulation factor (F) VIII and can cause severe bleeding after e.g. surgery and trauma or chronic bleeding into muscles and joints (1). Upon initial production of small amounts of thrombin, the intrinsic coagulation pathway is initiated by a positive feedback loop (2) in which thrombin activates factors V, VIII, and XI. Activated FXI (FXIa) activates FIX to FIXa, which in turn activates FX to FXa. FXa then converts prothrombin to thrombin, leading to fibrin clot formation. FVa and FVIIIa are cofactors for FXa and FIXa, respectively, and enhance the proteolytic function of these enzymes considerably, resulting in rapid production of thrombin.FVIII is a 2332-amino-acid protein with the domain structure A1-A2-B-A3-C1-C2 (3), which is cleaved within the B domain during secretion and circulates in the blood as a heterodimer bound to von Willebrand factor. Thrombin converts FVIII to its activated form (FVIIIa) (5, 6). The cleavage at Arg 740 leads to dissociation of the B domain from the rest of the molecule, but this cleavage is not required for FVIIIa activation (5). After cleavage and dissociation of the B domain, the remaining subunits form a non-covalently linked heterotrimer, consisting of the A1 subunit (1-372), the A2 subunit (373-740), and a light chain containing the A3, C1, and C2 domains (1690 -2332) (8, 9).In FVIIIa, the A2 subunit spontaneously dissociates, which inactivates FVIIIa with a half-life of about 2 min (10 -12). FVIIIa can also be inactivated through proteolysis by activated protein C (APC), 2 which cleaves FVIIIa at Arg 336 in the A1 domain and at Arg 562 in the A2 domain. The proteolytic activity of APC is enhanced by its non-enzymatic cofactors protein S (13) and factor V (FV) (14). Protein S enhances both FVa (15, 16) and FVIIIa (13, 15) inactivation by APC. FV enhances cleavage of FVIIIa by APC in the presence of protein S in purified systems and prolongs clotting time in plasma-based clotting assays measuring FVIIIa activity (14,17). This APC cofactor effect of FV was discovered as a result of the thrombosis risk factor FV Leiden (18,19). FV Leiden has Arg 506 mutated to Gln and therefore cannot be cleaved by APC at this position (20,21). This cleavage is required...

Summary Background Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia. Objectives To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A. Methods Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice. Results and conclusions FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). ‘superFVa,’ a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVLeiden(Arg506Gln), or FVaLeiden(A2-SS-A3). Furthermore, superFVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, superFVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose superFVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, superFVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that superFVa enhances not only clot formation but also clot stabilization. Thus, superFVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

Background. The etiology of the high prevalence of hypertension among patients with hemophilia (PWH) remains unknown. Methods. We compared 469 PWH in the United States with males from the National Health and Nutrition Examination Survey (NHANES) to determine whether differences in cardiovascular risk factors can account for the hypertension in hemophilia. Results. Median systolic and diastolic BP were higher in PWH than NHANES (P < 0.001) for subjects not taking antihypertensives. Those taking antihypertensives showed similar differences. Differences in both systolic and diastolic BP were especially marked among adults <30 years old. Differences between PWH and NHANES persisted after adjusting for age and risk factors (body mass index, renal function, cholesterol, smoking, diabetes, Hepatitis C, and race). Conclusions. Systolic and diastolic BP are higher in PWH than in the general male population and especially among PWH < 30 years old. The usual cardiovascular risk factors do not account for the etiology of the higher prevalence of hypertension in hemophilia. New investigations into the missing link between hemophilia and hypertension should include age of onset of hypertension and hemophilia-specific morbidities such as the role of inflammatory joint disease.

SummaryAlmost two decades ago an anticoagulant function of factor V (FV) was discovered, as an anticoagulant cofactor for activated protein C (APC). A natural mutant of FV in which the R506 inactivation site was mutated to Gln (FV Leiden ) was inactivated slower by APC, but also could not function as anticoagulant cofactor for APC in the inactivation of activated factor VIII (FVIIIa). This mutation is prevalent in populations of Caucasian descent, and increases the chance of thrombotic events in car 3) FV must be bound to a negatively charged phospholipid membrane. 4) Protein S also needs to be present. 5) FV acts as a cofactor for inactivation of both FVa and FVIIIa. 6) The prothrombotic function of FV Leiden is a function of both reduced APC cofactor activity and resistance of FVa to APC inactivation. However, detailed structural and mechanistic properties remain to be further explored.