John P. Sheehan

Solutions for a cultivated planet

Increasing population and consumption are placing unprecedented demands on agriculture and natural resources. Today, approximately a billion people are chronically malnourished while our agricultural systems are concurrently degrading land, water, biodiversity and climate on a global scale. To meet the world’s future food security and sustainability needs, food production must grow substantially while, at the same time, agriculture’s environmental footprint must shrink dramatically. Here we analyse solutions to this dilemma, showing that tremendous progress could be made by halting agricultural expansion, closing ‘yield gaps’ on underperforming lands, increasing cropping efficiency, shifting diets and reducing waste. Together, these strategies could double food production while greatly reducing the environmental impacts of agriculture.

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism

Summary.  Background:  Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined.

New developments in the management of moderate-to-severe hemophilia B

Hemophilia B is an X-linked genetic deficiency of coagulation factor IX (FIX) activity associated with recurrent deep tissue and joint bleeding that may lead to long-term disability. FIX replacement therapy using plasma-derived protein or recombinant protein has significantly reduced bleeding and disability from hemophilia B, particularly when used in a prophylactic fashion. Although modern factor replacement has excellent efficacy and safety, barriers to the broader use of prophylaxis remain, including the need for intravenous (IV) access, frequent dosing, variability in individual pharmacokinetics, and cost. To overcome the requirement for frequent factor dosing, novel forms of recombinant FIX have been developed that possess extended terminal half-lives. Two of these products (FIXFc and rIX-FP) represent fusion proteins with the immunoglobulin G1 (IgG1) Fc domain and albumin, respectively, resulting in proteins that are recycled in vivo by the neonatal Fc receptor. The third product has undergone site-specific PEGylation on the activation peptide of FIX, similarly resulting in a long-lived FIX form. Clinical trials in previously treated hemophilia B patients have demonstrated excellent efficacy and confirmed less-frequent dosing requirements for the extended half-life forms. However, gaps in knowledge remain with regard to the risk of inhibitor formation and allergic reactions in previously untreated patient populations, safety in elderly patients with hemophilia, effects on in vivo FIX distribution, and cost-effectiveness. Additional strategies designed to rebalance hemostasis in hemophilia patients include monoclonal-antibody-mediated inhibition of tissue factor pathway inhibitor activity and siRNA-mediated reduction in antithrombin expression by the liver. Both of these approaches are long acting and potentially involve subcutaneous administration of the drug. In this review, we will discuss the biology of FIX, the evolution of FIX replacement therapy, the emerging FIX products possessing extended half-lives, and novel “rebalancing” approaches to hemophilia therapy.

Increased Red Blood Cell Stiffness Increases Pulmonary Vascular Resistance and Pulmonary Arterial Pressure

Patients with sickle cell anemia (SCD) and pulmonary hypertension (PH) have a significantly increased risk of sudden death compared to patients with SCD alone. Sickled red blood cells (RBCs) are stiffer, more dense, more frequently undergo hemolysis, and have a sixfold shorter lifespan compared to normal RBCs. Here, we sought to investigate the impact of increased RBC stiffness, independent of other SCD-related biological and mechanical RBC abnormalities, on the hemodynamic changes that ultimately cause PH and increase mortality in SCD. To do so, pulmonary vascular impedance (PVZ) measures were recorded in control C57BL6 mice before and after ∼50 μl of blood (Hct = 45%) was extracted and replaced with an equal volume of blood containing either untreated RBCs or RBCs chemically stiffened with glutaraldehyde (Hct = 45%). Chemically stiffened RBCs increased mean pulmonary artery pressure (mPAP) (13.5 ± 0.6 mmHg at baseline to 23.2 ± 0.7 mmHg after the third injection), pulmonary vascular resistance (PVR) (1.23 ± 0.11 mmHg*min/ml at baseline to 2.24 ± 0.14 mmHg*min/ml after the third injection), and wave reflections (0.31 ± 0.02 at baseline to 0.43 ± 0.03 after the third injection). Chemically stiffened RBCs also decreased cardiac output, but did not change hematocrit, blood viscosity, pulmonary arterial compliance, or heart rate. The main finding of this study is that increased RBC stiffness alone affects pulmonary pulsatile hemodynamics, which suggests that RBC stiffness plays an important role in the development of PH in patients with SCD.

Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer

OBJECTIVE Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC. METHODS TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expressions were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists. RESULTS Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4-10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present. CONCLUSIONS Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation.

Selective disruption of heparin and antithrombin‐mediated regulation of human factor IX

Interaction with antithrombin and heparin regulates distribution, activity, and clearance of factor IXa (FIXa). Hemophilia B prophylaxis targets plasma FIX levels > 1% but neglects extravascular FIX, which colocalizes with antithrombin–heparan sulfate.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception

Objective— Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability. Approach and Results— A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-&agr; antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-&agr; compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-&agr; and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation. Conclusions— Elevation of plasma FIXa activity in association with reductions in TFPI-&agr; and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.

The Regulation of Factor IXa by Supersulfated Low Molecular Weight Heparin

Supersulfated low molecular weight heparin (ssLMWH) inhibits the intrinsic tenase (factor IXa-factor VIIIa) complex in an antithrombin-independent manner. Recombinant factor IXa with alanine substitutions in the protease domain (K126A, N129A, K132A, R165A, R170A, N178A, R233A) was assessed with regard to heparin affinity in solution and ability to regulate protease activity within the factor IXa-phospholipid (PL) and intrinsic tenase complexes. In a soluble binding assay, factor IXa K126A, K132A, and R233A dramatically (10-20-fold) reduced ssLMWH affinity, while factor IXa N129A and R165A moderately (5-fold) reduced affinity relative to wild type. In the factor IXa-PL complex, binding affinity for ssLMWH was increased 4-fold, and factor X activation was inhibited with a potency 7-fold higher than predicted for wild-type protease-ssLMWH affinity in solution. In the intrinsic tenase complex, ssLMWH inhibited factor X activation with a 4-fold decrease in potency relative to wild-type factor IXa-PL. The mutations increased resistance to inhibition by ssLMWH in a similar fashion for both enzyme complexes (R233A > K126A > K132A/R165A > N129A/N178A/wild type) except for factor IXa R170A. This protease had ssLMWH affinity and potency for the factor IXa-PL complex similar to wild-type protease but was moderately resistant (6-fold) to inhibition in the intrinsic tenase complex based on increased cofactor affinity. These results are consistent with conformational regulation of the heparin-binding exosite and macromolecular substrate catalysis by factor IXa. An extensive overlap exists between the heparin and factor VIIIa binding sites on the protease domain, with residues K126 and R233 dominating the heparin interaction and R165 dominating the cofactor interaction.

Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis

Objective— PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS–FIXa interaction in hemostasis. Approach and Results— We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein–protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa. Conclusions— Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.

Low-molecular-weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: a reply to rebuttal.

We thank Drs Hemker and Beguin for their comments on our recent paper [1]. In our introduction, we stated that “acceleration of factor (F)Xa inhibition via conformational activation of anthrombin is generally accepted as the predominant therapeutic mechanism for low-molecular-weight forms of heparin”. While our experiments primarily address the mechanisms of action for LMWH in plasma, the writer’s conclusion that the predominant activity of heparin is due to ‘increased thrombin inactivation and not to decreased prothrombin conversion’ is not supported by our data. Drs Hemker and Beguin extrapolate a pentasaccharide concentration for the unfractionated heparin (UFH) preparation in Table 1 of our paper to support their contention that the acceleration of thrombin inhibition is the primary mechanism of action for heparin. However, caution should be employed in comparing EC50 values between different heparin forms. The concentrations are expressed as molar values only to provide a common scale for these disparate forms of heparin. With the exception of Fondaparinux, these molar concentrations are based on an average molecular weight for each heparin preparation. They do not take into account the heterogeneity and relative polydispersity of UFH preparations. Thus, these molarity assumptions do not translate easily into pentasaccharide concentrations, particularly as the longer ‘high-affinity’ chains in UFH may contain more than one pentasaccharide sequence [2,3]. The more relevant and accurate comparisons reside within each heparin type based on the tissue factor concentration (Table 1), absence or presence of antithrombin, and their respective therapeutic ranges (Table 2). In contrast to LMWH, Fondaparinux and UFH demonstrate similar potency under FIX-dependent or independent conditions (Table 1), and are both dramatically (42to 62-fold) less potent in the absence of antithrombin (Table 2). These results suggest that the relevant mechanism(s) of action for these heparins in human plasma do not depend on inhibition of the intrinsic tenase complex, and as expected, are highly antithrombindependent. In our Western blot analysis of plasma thrombin generation, 0.012 lM or 0.04 U mL UFH (specific activity approximately 165 U mg) is present in Fig. 2E, which is approximately eight-fold below the therapeutic range. While this sub-therapeutic UFH concentration accelerated the appearance of the TAT complex, it did not prevent depletion of the prothrombin band, and only modestly reduced thrombin generation. In comparison, 0.31 lM or 0.92 U mL of UFH is present in Fig. 2F, which demonstrates essentially complete inhibition of prothrombin consumption. A similar, although less complete reduction of prothrombin consumption is observed with the low-molecular-weight forms of heparin at their EC50 values (Fig. 2B–D). Although 0.92 U mL UFH represents a 25-fold higher concentration than the EC50 value for inhibition of thrombin generation (not 1200to 1600-fold as stated in the letter), it is only modestly higher than the therapeutic range for UFH in venous thromboembolic disease (0.3–0.7 U mL), and substantially lower than levels expected during cardiopulmonary bypass (> 2.0 U mL) [4]. Thus, the findings in Fig. 2F are relevant to expected plasma concentrations during the clinical usage of UFH. While these data do not specifically address the potential effects of accelerated thrombin inhibition on cofactor activation in the lag (initiation) phase, they do suggest that inhibition of prothrombin consumption is a major effect of all therapeutic heparin forms. The complexity of heparin effects in plasma makes definitive identification of the critical antithrombotic mechanisms difficult, which was part of the motivation for our paper. The clinical efficacy of Fondaparinux See also Hemker HC, Béguin S. Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: a rebuttal. This issue, pp 564.