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Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs., (© 2020 by The American Society of Hematology.)

Hemophilic arthropathy from joint bleeding remains a complication with major morbidity in the increasingly aging patients with hemophilia. Prophylactic clotting factor infusions, based on pharmacokinetic dosing to reduce bleeding rates, are being explored more and more. However, there is little evidence on the benefits of pharmacokinetic dosing in direct association with bleeding events. Here, we prospectively followed a cohort of adult patients with hemophilia A and B (n = 26) and arthropathic joints on various clotting factor products over a period of 2 years with clinical and radiographic joint health assessments, frequent joint ultrasound, and pharmacokinetic studies. Joint bleeds and synovitis with synovial vascularity changes were objectively diagnosed by musculoskeletal ultrasound and power Doppler and analyzed in relation to pharmacokinetic, joint- and patient-specific parameters. Results revealed that, contrary to common beliefs, bleeding episodes were not readily explained by pharmacokinetic features, as they were not associated with more time spent below certain clotting factor thresholds. Joint bleeding was found to be associated with prominent vascularity changes, suggesting that vascular remodeling and leakiness may contribute to joint bleeding that cannot be prevented by clotting factor replacement alone.

Objective: Hemophilic arthropathy is associated with pronounced vascular joint remodeling. Also, compared to the general population, PWH have a higher prevalence of hypertension not explained by usual risk factors. As vascular remodeling in various vascular beds is a hallmark of hypertension, we hypothesized that vascular joint remodeling is associated with elevated blood pressures and hypertension., Methods: Elbows, knees, and ankles of 28 adult PWH were evaluated for vascular abnormalities with MSKUS/PD, as well as for radiographic and clinical status and pain. Logistic and linear regression models were fitted to examine associations between hypertension, blood pressure, and PD score., Results: The extent of vascular abnormalities was associated with hypertension and blood pressures. Hypertensive patients had a higher PD score compared to nonhypertensive patients, and the risk of hypertension increased steeply with PD score. SBP was also strongly associated with PD score, while DBP was only weakly associated., Conclusions: Vascular remodeling in hemophilic joints is associated with hypertension and elevated blood pressures. As hypertension is a grave risk factor for intracranial hemorrhage, a prominent cause of mortality in hemophilia patients, future studies are needed to address the causal pathways between vascular joint remodeling and blood pressure., (© 2017 John Wiley & Sons Ltd.)

Background: In congenital Factor (F) VII deficiency bleeding phenotype and intrinsic FVII activity levels don't always correlate. Patients with FVII activity levels <30% appear to have a higher bleeding propensity, but bleeding can also occur at higher FVII activity levels. Reasons for bleeding at higher FVII activity levels are unknown, and it remains challenging to manage such patients clinically., Case: A 19year old male with spontaneous intracranial hemorrhage and FVII activity levels of 44%, requiring emergent surgical intervention and a strategy for FVII replacement. Genotyping showed the rare heterozygous FVII 9729del4 mutation. Bleed evacuation was complicated by epidural abscess requiring craniectomy, bone graft procedures, and prolonged administration of recombinant human (rh) activated FVII (FVIIa). The patient recovered without neurological deficits, and remains on prophylactic low dose treatment with rhFVIIa in relation to risky athletic activities., Conclusion: For clinicians, it is important to recognize that effects of rhFVIIa within these pathways are independent of its contribution to blood clot formation and cannot be assessed by clotting assays. Reduced FVII levels should therefore not be dismissed, as even a mild reduction may result in spontaneous bleeding. Treatment of mild FVII deficiency requires a careful case-by-case approach, based on the clinical scenario., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., Competing Interests: Doris V. K. Quon is a member of speakers' bureaux for Baxalta, Biogen, CSL, Grifols, and Novo Nordisk and also participates in advisory boards for Baxalta, Bayer, Biogen, and Novo Nordisk. Rebecca Kruse-Jarres is a paid consultant and participates in advisory boards for Baxalta, Bayer, Grifols, Novo Nordisk, Pfizer, and Roche and also receives research funding from Baxalta, Pfizer, and Roche. Annette von Drygalski has received honoraria for participating in scientific advisory board panels and for consulting and speaking engagements for Baxalta, Bayer, Biogen, CSL-Behring, Novo Nordisk, Grifols, and Pfizer and is also a cofounder and member of the Board of Directors of Hematherix LLC., a biotech company that is developing superFVa therapy for bleeding complications. The remaining authors declare no competing financial interests.

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 and osteoarthritis 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., (© 2015 Wiley Periodicals, Inc.)

Objective: An increased risk of bleeding is observed in patients receiving activated protein C (APC), which may be a limiting factor for the application of novel APC therapies. Since APC's therapeutic effects often require its cytoprotective activities on cells but not APC's anticoagulant activities, an agent that specifically antagonizes APC's anticoagulant effects but not its cytoprotective effects could provide an effective means to control concerns for risk of bleeding. We hypothesized that superFVa, an engineered activated FVa-variant that restores hemostasis in hemophilia could reduce APC-induced bleeding., Approach and Results: SuperFVa was engineered with mutations of the APC cleavage sites (Arg506/306/679Gln) and a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains, which augment its biological activity and cause high resistance to APC. SuperFVa normalized APC-prolonged clotting times and restored APC-suppressed thrombin generation in human and murine plasma at concentrations where wild-type (wt) FVa did not show effects. Following intravenous injection of APC into BALB/c mice, addition to whole blood ex vivo of superFVa but not wt-FVa significantly normalized whole blood clotting. Blood loss following tail clip or liver laceration was significantly reduced when superFVa was administered intravenously to BALB/c mice prior to intravenous APC-treatment. Furthermore, superFVa abolished mortality (∼50%) associated with excessive bleeding following liver laceration in mice treated with APC., Conclusions: Our results provide proof of concept that superFVa is effective in preventing APC-induced bleeding and may provide therapeutic benefits as a prohemostatic agent in various situations where bleeding is a serious risk.

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). '(super) FVa,' 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, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, 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, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia., (© 2013 International Society on Thrombosis and Haemostasis.)

Almost 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 carriers. Characterisation of the FV anticoagulant effect has elucidated multiple properties of the anticoagulant function of FV: 1) Cleavage of FV at position 506 by APC is required for anticoagulant function. 2) The C-terminal part of the FV B domain is required and the B domain must have an intact connection with the A3 domain of FV. 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.

Activated protein C (APC) binds to its substrates activated factor V (FVa) and activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma serpins. Charged residues of the autolysis loop were individually mutated to alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations. Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the autolysis loop. Together, these results confirmed that the autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa., (© 2011 Blackwell Publishing Ltd.)

Coagulation factor V (FV) promotes inactivation of activated factor VIII (FVIIIa) by activated protein C (APC) and protein S. Loss of this APC cofactor activity is proposed to be partially responsible for the APC resistance phenotype of FV(Leiden). However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. APC/protein S also readily inactivates activated FV (FVa). We therefore hypothesized that FV can function as an anticoagulant cofactor for APC/protein S in the inactivation of FVa. FV was titrated into FV-deficient plasma, and the APC sensitivity ratio (APCsr; a measure of APC activity) was measured in a clotting assay that was not sensitive to FVIII. Our results showed an increase in APCsr as the FV concentration increased, suggesting an anticoagulant function for FV in this assay. FV(Leiden) showed APC resistance with an APCsr of 1.0. Therefore, under our experimental conditions, FV acted as an anticoagulant cofactor for APC in the inactivation of FVa., (Copyright © 2010 S. Karger AG, Basel.)

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. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.

The aim of the study was to assess the potential application of teleradiology in the neonatal intensive care unit (NICU) by ascertaining whether any decrease in conspicuity of anatomic detail or interventional devices in the chest radiographs of premature infants is caused by picture archiving and communication system (PACS)-based soft copy interpretation of 10 : 1 compressed images. One hundred digital chest radiographs of low-birthweight infants were obtained in the NICU using a storage phosphor system. Laser-printed images were interpreted and the data set for each radiograph was then irreversibly compressed by a 10 : 1 ratio. Four radiologists with extensive PACS experience used a five-point grading system to score laser-printed hard copy images for the visibility of six parameters of anatomic landmarks and interventional devices in the chest. Compressed soft copy images displayed on 2K PACS workstation were subsequently scored using the same approach. Statistical manipulation demonstrated no loss of anatomic detail in five of the six parameters scored, with minimal difference in one landmark, the retrocardiac lung assessment. While further study is required to assess the clinical impact of the variance noted when evaluating lung parameters, the preservation or improvement of information in the remaining parameters following irreversible compression and soft copy interpretation is promising for the potential use of teleradiology in this population.

Objective: For the last several years the U.S. Department of Defense (DoD) has operated a telemedicine test bed at the U.S. Army Medical Research and Material Command's Medical Advanced Technology Management Office. The goal of this test bed is to reengineer the military health service system from the most forward deployed forces to tertiary care teaching medical centers within the United States by exploiting emerging telemedicine technologies., Methods: The test bed has conducted numerous proof-of-concept telemedicine demonstrations as part of military exercises and in support of real-world troop deployments. The most ambitious of those demonstrations is Primetime III, an ongoing effort to provide telemedicine and other advanced technology support to medical units supporting Operation Joint Endeavor in Bosnia., Results: Several of the first instances of the clinical use of the Primetime III systems are presented as case reports in this paper. These reports demonstrate capabilities and limitations of telemedicine., Conclusion: The Primetime III system demonstrates the technical ability to provide current telecommunications capabilities to medical units stationed in the remote, austere, difficult-to-serve environment of Bosnia. Telemedicine capabilities cannot be used without adequate training, operations, and sustainment support. Video consultations have eliminated the need for some evacuations. The system has successfully augmented the clinical capability of physicians assigned to these medical units. Fullest clinical utilization of telemedicine technologies requires adjustment of conventional clinical practice patterns.

Ten healthy male cardiopulmonary technicians, 10 aircrew members, and 10 patients performed a series of pulmonary function studies to compare the Cavitron Spirometric Computer to the conventional water-sealed Collins 13.5-/spirometer. A total of 50 studies were performed on the spirometers individually and then with the spirometers linked in-line. The Cavitron FVC averaged 6% less, the FEV1--7% less, and the FEF 25-75%--11.6% less than comparable measurements from the Collins spirometer. Since these difference are within the suggested guidelines for screening studies, the Cavitron Spirometric Computer is acceptable for use in clinical screening spirometry.