aPC Generation Research Articles

Hypercoagulability impairs plaque stability in diabetes-induced atherosclerosis

Abstract Introduction Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Methadology Female ApoE-/- or TMPro/Pro ApoE-/- mice (age 6 to 8 weeks) were fed a normal chow diet and were made diabetic (DM) by injecting streptozotocin, reflecting type 1 DM. After 22 weeks of age, the body weight of the mice was measured, and the mice were sacrificed. Plasma and tissue samples were analysed for different parameters. Results Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. Conclusion Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis.

ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis.

Diabetes mellitus is hallmarked by accelerated atherosclerosis, a major cause of mortality among patients with diabetes. Efficient therapies for diabetes-associated atherosclerosis are absent. Accelerated atherosclerosis in diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we directly compared the effects of high glucose and oxidized LDL, revealing that high glucose induced more pronounced responses in regard to maladaptive unfolded protein response (UPR), senescence, and vascular endothelial cell barrier disruption. Ex vivo, diabetic ApoE−/− mice displayed increased levels of senescence and UPR markers within atherosclerotic lesions compared with nondiabetic ApoE−/− mice. Activated protein C pretreatment maintained barrier permeability and prevented glucose-induced expression of senescence and UPR markers in vitro. These data suggest that high glucose-induced maladaptive UPR and associated senescence promote vascular endothelial cell dysfunction, which—however—can be reversed by aPC. Taken together, current data suggest that reversal of glucose-induced vascular endothelial cell dysfunction is feasible.

Investigations of the effectiveness of heparin variants as inhibitors of histones

BackgroundExtracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. ObjectivesTo compare the effectiveness of unfractionated heparin (UFH), low‐molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones MethodsBinding affinities between heparin variants and histone subunits were determined by Bio‐layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP‐1 cells with histones ± heparin variants. ResultsUnfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM–66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM–81.1 mM). UFH, LMWH, and Vasoflux neutralize histone‐mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone‐mediated impairment of APC generation in a dose‐dependent manner. ConclusionsThe ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin‐binding pentasaccharide. In contrast, the ability of heparin to neutralize histone‐mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.

Hypercoagulability Impairs Plaque Stability in Diabetes-Induced Atherosclerosis.

Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis.

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors. To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury. Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl3-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect. We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.

Podocyte Integrin-β3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. The zymogen protein C and aPC bind to podocyte integrin-β3, a subunit of integrin-αvβ3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvβ3 induces transient binding of integrin-β3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-β3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-β3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-β3 interaction by specifically deleting podocyte integrin-β3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvβ3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvβ3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Activated protein C reverses epigenetically sustained p66Shc expression in plaque-associated macrophages in diabetes

Impaired activated protein C (aPC) generation is associated with atherosclerosis and diabetes mellitus. Diabetes-associated atherosclerosis is characterized by the hyperglycaemic memory, e.g., failure of disease improvement despite attenuation of hyperglycaemia. Therapies reversing the hyperglycaemic memory are lacking. Here we demonstrate that hyperglycaemia, but not hyperlipidaemia, induces the redox-regulator p66Shc and reactive oxygen species (ROS) in macrophages. p66Shc expression, ROS generation, and a pro-atherogenic phenotype are sustained despite restoring normoglycemic conditions. Inhibition of p66Shc abolishes this sustained pro-atherogenic phenotype, identifying p66Shc-dependent ROS in macrophages as a key mechanism conveying the hyperglycaemic memory. The p66Shc-associated hyperglycaemic memory can be reversed by aPC via protease-activated receptor-1 signalling. aPC reverses glucose-induced CpG hypomethylation within the p66Shc promoter by induction of the DNA methyltransferase-1 (DNMT1). Thus, epigenetically sustained p66Shc expression in plaque macrophages drives the hyperglycaemic memory, which—however—can be reversed by aPC. This establishes that reversal of the hyperglycaemic memory in diabetic atherosclerosis is feasible.

Carbon and Mo transformations during the synthesis of mesoporous Mo2C/carbon catalysts by carbothermal hydrogen reduction

The synthesis of mesoporous Mo2C/carbon catalysts by carbothermal hydrogen reduction is reported. Petroleum coke (petcoke) was activated with KOH at 800°C to obtain high surface area microporous activated petcoke (APC; ~ 2000m2/g). The APC was wet impregnated with ammonium heptamolybdate (AHM: 10wt% Mo), dried and reduced in H2 at temperatures from 400 to 800°C, to yield Mo2C/APC catalysts. Increased reduction temperature increased the Mo2C yield and the mesoporous volume of the Mo2C/APC. At a reduction temperature of 750°C the mesopore volume of the catalyst doubled compared to the APC support and accounted for 37% of the total pore volume. Maintaining the final CHR temperature for 90min further increased the Mo2C yield and mesoporosity of the catalyst. The role of Mo2C in the catalytic hydrogenation of the APC and mesopore generation is demonstrated. The activity of the Mo2C/carbon catalysts in the hydrodeoxygenation of 4-methyl phenol increased with increased CHR temperature and catalyst mesoporosity.

Coupling Therapeutics to Human Erythrocytes Demonstrates Target-Dependent Effects on Red Cell Physiology While Preserving Efficacy

Delivery of bio-therapeutics by red blood cells (RBCs) can greatly enhance pharmacokinetics and pharmacodynamics of the appended or loaded agents, and may even potentiate induction of immunologic tolerance. Our group and others have successfully used fusion proteins, antibodies, and peptides to couple therapeutics to murine, but not human, RBCs. It is known that extracellular ligands have the potential to induce marked, epitope-dependent changes in red cell physiology, including changes in deformability, phosphatidyl-serine (PS) exposure, and reactive oxygen species (ROS) production, particularly for agents targeted to glycophorin A and Band 3, two highly-expressed membrane proteins. To produce clinically translatable strategies for human RBCs, it is critical to identify optimal red cell target epitopes, understand their effects on red cell physiology, and create humanized or human-like ligands to minimize immunogenicity.We constructed single chain antibodies (scFv) against antigenic determinants on Band 3 protein (Wrb) and RHCE protein (Rh17/Hr0) on human erythrocytes using phage display libraries prepared from immunized cynamolgous macaques (Macaca fascicularis). Both these antigens are present on essentially 100% of the human population. Unfused scFvs were produced in E.coli while fusions of scFv with the extracellular domain of human thrombomodulin (TM-scFv) were produced in Drosophila S2 cells. Binding of recombinant proteins to human RBCs was measured by radioimmunoassay and flow cytometry. Generation of activated protein (APC) by RBCs loaded with TM-scFv fusions was measured by colorimetric assay. RBCs pre-incubated with varying concentrations of anti-Band3 and anti-RHCE fusions were assessed for osmotic resistance and mechanical integrity by exposure to hypo-osmolar medium and rotation in the presence of glass beads, respectively. PS exposure was measured by annexin V binding, and ROS generation was measured by dihydrorhodamine-associated fluorescence. Effects on RBC rheology were measured by flowing through microfluidic channels under controlled shear rates. Efficacy of TM-scFv fusions in diseased micro-vessels was assessed using a TNF-alpha stimulated, endothelialized microfluidic model.Single-chain antibody fragments and TM fusion proteins targeted to conserved epitopes on Band 3 protein and RHCE protein bound to human, but not murine or porcine, RBCs with high specificity and affinity (~50 nM), and in numbers consistent with the expected level of target expression (105 and 106 copies/RBC for RHCE and Band3, respectively). Coating RBCs with proteins targeted to Band 3 lessened RBC hypo-osmolar hemolysis (20% reduction) but increased hemolysis (2-fold) under mechanical stress, changes compatible with decreased red cell deformability. Proteins targeted to RHCE did not induce significant changes in hemolysis of RBCs under either osmotic or mechanical stress. Targeting neither Band 3 nor RHCE induced significant exposure of PS or production of ROS. Target-dependent effects on RBC rheology were observed under varying shear stresses in a microfluidic system. Fusion proteins of TM targeted to both epitopes demonstrated dose- and surface-copy-number-dependent generation of APC in the presence of PC and thrombin. Both TM-scFv fusion proteins were efficacious in a microfluidic model of disseminated intravascular coagulation using whole human blood by demonstrating near complete abrogation of fibrin generation in response to endothelial activation with TNF-alpha.In summary, we designed human RBC-specific non-human primate single chain antibody fragments capable of fusion to therapeutic cargoes. The TM-scFv fusions maintained therapeutic activity when bound to human RBCs and showed effective thromboprophylaxis in a whole-blood model of vasculitic injury. These antibodies and fusion proteins bound to erythroid-specific epitopes, and demonstrated target-dependent effects on several aspects of red cell physiology. The non-human primate origin of the antibodies should minimize their potential immunogenicity and the findings provide a platform to translate red cell targeted drug delivery into the clinical realm. DisclosuresNo relevant conflicts of interest to declare.

Thrombomodulin‐dependent protein C activation is required for mitochondrial function and myelination in the central nervous system

Background Studies with human samples and in rodents established a function of coagulation proteases in neuro-inflammatory demyelinating diseases (e.g. in multiple sclerosis [MS] and experimental autoimmune encephalitis [EAE]). Surprisingly, approaches to increase activated protein C (aPC) plasma levels as well as antibody-mediated inhibition of PC/aPC ameliorated EAE in mice. Hence, the role of aPC generation in demyelinating diseases and potential mechanisms involved remain controversial. Furthermore, it is not known whether loss of aPC has pathological consequences at baseline (e.g. in the absence of disease). Objective To explore the role of thrombomodulin (TM)-dependent aPC generation at baseline and in immunological and non-immunological demyelinating disease models. Methods Myelination and reactive oxygen species (ROS) generation were evaluated in mice with genetically reduced TM-mediated protein C activation (TMPro/Pro ) and in wild-type (WT) mice under control conditions or following induction of EAE. Non-immunological demyelination was analyzed in the cuprizone-diet model. Results Impaired TM-dependent aPC generation already disturbs myelination and mitochondrial function at baseline. This basal phenotype is linked with increased mitochondrial ROS and aggravates EAE. Reducing mitochondrial ROS (p66Shc deficiency), restoring aPC plasma levels or injecting soluble TM (solulin) ameliorates EAE in TMPro/Pro mice. Soluble TM additionally conveyed protection in WT-EAE mice. Furthermore, soluble TM dampened demyelination in the cuprizone-diet model, demonstrating that its myelin-protective effect is partially independent of an immune-driven process. Conclusion These results uncover a novel physiological function of TM-dependent aPC generation within the CNS. Loss of TM-dependent aPC generation causes a neurological defect in healthy mice and aggravates EAE, which can be therapeutically corrected.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

Genetic Absence of Thrombin Receptor Par4 Overcomes the Obligate Requirement of EPCR in Mouse Placenta

Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical role in placentation and the maintenance of pregnancy. Severe Protein C deficiency in the mother results in pregnancy failure in early gestation. Thrombomodulin (Thbd) or the Endothelial Protein C Receptor (EPCR/ProcR) gene deletions result in embryonic death, secondary to developmental and functional abnormalities of the placenta. These molecules play multiple roles in coagulation and inflammation. The mechanisms governing their role in placental development and maintenance of placental function remain to be fully understood. The objective of this work is to identify the critical functions of EPCR and Thbd that are required for placental development. Both Thbd and EPCR augment activated protein C generation, albeit to different extents. We have examined if reduced activated Protein C generation mediates placental abnormalities of EPCR- and Thbd-null mice. Activation of thrombin receptors expressed on platelets and trophoblast cells can also contribute to placental failure. We examined the role of thrombin receptor Par4 in placental failure of EPCR-null mice.Methods: To assess the role of a PC generation in placental phenotype of Thbd- and EPCR-null mice, we used a transgene to express a hyperactivatable form of murine protein C (hMPC) under the control of transthyretin promoter. Thrombin cleaves this mutant form of Protein C 30-fold more efficiently than wild type protein C, without requiring the cofactor function of thrombomodulin. Wild type mice expressing hMPC show 2-fold increase in PC and 3-fold increase in aPC levels. hMPC expression in PC-null mice restores their ability to carry pregnancies. Breeding strategies were used to generate hMPCtg ProcR+/- or hMPCtg Thbd+/- female mice. These were mated to ProcR+/- or Thbd+/- males, respectively, and survival of ProcR-/- and Thbd-/- embryos was analyzed. Similar genetic strategy was used to analyze the role of thrombin receptor Par4 in the demise of EPCR-null embryos. Placental phenotypes and embryonic survival was compared with experiments in which the mother was continuously infused with LMWH using a subcutaneous osmotic pump.Results: As previously reported, EPCR-null mice die before 10.5 days post coitum (dpc) (ProcR+/- intercrosses, out of 41 live embryos none were ProcR-/-, 10 were expected, 21 aborted not genotyped, 7 pregnancies analyzed at 11.5 dpc) and none are found at wean (out of 30 live pups none were ProcR-/-, 8 were expected, 5 litters analyzed). Transgenic expression of hMPC in the mother resulted in some live ProcR-/- embryos at 11.5 dpc (4 ProcR-/- out of 41 live embryos, 10 were expected, 15 aborted not genotyped, 7 pregnancies at 11.5 dpc) and pups at wean (2 ProcR-/- out of 28 live, 7 litters analyzed). Despite transgenic hMPC expression ProcR-/- embryos and pups were underrepresented (P=0.007, chi square GOF test). Surviving ProcR-/- embryos showed normal placental histology grossly comparable to littermate controls. Expression of hMPC in the mother did not ameliorate fetal death of Thbd-null mice (out of 38 live embryos none were Thbd-/-, 10 expected, 16 aborted not genotyped, 7 pregnancies at 9.5 dpc). Continuous infusion of LMWH also resulted in some live ProcR-/- embryos at 11.5 dpc (3 ProcR-/- out of 19 live embryos, 5 expected, 11 aborted not genotyped, 3 pregnancies analyzed), but two were growth retarded and all 3 placentae showed markedly reduced placental labyrinth formation. In contrast to transgenic expression of hMPC and treatment with LMWH, when Par4-/- ProcR+/- animals were intercrossed, ProcR-/- animals were born at an expected Mendelian frequency (7 ProcR-/- out of 35 live pups, 9 expected, 7 litters analyzed).Conclusions: Our results show that transgenic expression of hMPC allows normal placental development and rescues a fraction of EPCR-null embryos. Thus, placental defect of EPCR-null mice is in part mediated by reduced generation of aPC on placental cells. In contrast to the transgenic expression of hMPC and LMWH treatment, genetic absence of Par4 completely overcame the placental defect and allowed development of EPCR-null embryos. Further studies will clarify contributions of maternal versus fetal Par4 in this phenomenon. DisclosuresNo relevant conflicts of interest to declare.

Effect of Five Therapeutic Strategies on the Coagulation Defect Induced By the Thrombomodulin C.1611C>a Mutation

A novel autosomal dominant bleeding disorder characterized by trauma- and surgery-induced severe bleeding despite normal coagulation factor levels, platelet functional tests and fibrinolysis was recently described by two independent groups (1, 2). The genetic cause of this bleeding disorder was identified as a nonsense mutation in the THBD gene (c.1611C>A,p.Cys537Stop), resulting in very highlevels of soluble thrombomodulin (TM) in plasma. Extremely elevated plasma TM levelsenhance APC generation and down-regulate factor (F)Va, FVIIIa and thrombin generation (TG). This rare bleeding disorder is caused by a gain-of-function mutation in an anticoagulant factor. The clinical management of bleeding episodes in patients with this disorder remains challenging. The aim of this in-vitro study was to evaluate the ability of five haemostatic agents to restore the coagulation capacity of patients with TMc.1611C>A mutation.We compared the in-vitro efficacy of five haemostatic agents to restore thrombin generation in plasma of a patient affected by the disease.The addition of APCC in patient's plasma showed a dose dependent improvement of TG. A full normalization of ETP (1698nM.min) and peak thrombin (270nM) was observed with the lowest concentration of FEIBA. There was over-correction of TG at higher concentrations of FEIBA, which may represent a risk of thrombosis. In contrast, the haemostatic effect of PCC evaluated in the same experimental conditions showed no significant modification of TG with any concentration of PCC tested. No improvement of TG was observed with any concentration of rFVIIa added.In our patient, life-threatening hemorrhage could be stopped by the combined administration of platelet concentrates and fresh frozen plasma (2). This can be explained by considering that platelets contain and release at the site of injury, a form of FV which is partially activated and resistant to APC-mediated inactivation and therefore relatively insensitive to the anticoagulant effects of soluble TM. Since soluble TM and APC target not only FVa generation but also FVIIIa generation, and since platelets do not contain FVIII, we speculated that using FVIII concentrates in combination with platelet concentrates might be clinically even more effective in preventing bleeding in the patient. Our in vitro results showed a partial correction of TG with increasing concentrations of rFVIII concentrate. After addition of control platelets (50 - 100-150 x109/L) to the patient's plasma, TG was dose-dependently improved and normalized. The presence of functionally active TM in platelets might explain the need for exogenous platelets to normalize TG.The association of rFVIII concentrate 50 IU/dL with fresh PRP containing 50 - 100-150 x109 platelets/L, improved further the coagulation capacity of the patient and fully normalized TG at all tested concentrations. No over correction of TG was observed with this strategy.Our in vitro data suggest that APCC, platelet transfusion or the combined use of FVIII and platelet concentrates might be helpful to restore haemostasis in patients presenting this rare bleeding disorder. The clinical haemostatic efficacy of these therapeutic strategies needs to be confirmed during acute bleeding episodes or surgery. 1.Langdown J, Luddington RJ, Huntington JA, Baglin TP. A hereditary bleeding disorder resulting from a premature stop codon in thrombomodulin (p.Cys537Stop). Blood 2014; 124:1951-19562.Dargaud Y, Scoazec JY, Wielders SJ, Trzeciak C, Hackeng TM, Négrier C, Hemker HC, Lindhout T, Castoldi E. Characterization of an autosomal dominant bleeding disorder caused by a thrombomodulin mutation. Blood 2015; 125 :1497-501 DisclosuresNegrier:CSL Behring: Speakers Bureau.

Activated Protein C Ameliorates Renal Ischemia-Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination.

Ischemia-reperfusion injury (IRI) is the leading cause of ARF. A pathophysiologic role of the coagulation system in renal IRI has been established, but the functional relevance of thrombomodulin (TM)-dependent activated protein C (aPC) generation and the intracellular targets of aPC remain undefined. Here, we investigated the role of TM-dependent aPC generation and therapeutic aPC application in a murine renal IRI model and in an in vitro hypoxia and reoxygenation (HR) model using proximal tubular cells. In renal IRI, endogenous aPC levels were reduced. Genetic or therapeutic reconstitution of aPC efficiently ameliorated renal IRI independently of its anticoagulant properties. In tubular cells, cytoprotective aPC signaling was mediated through protease activated receptor-1- and endothelial protein C receptor-dependent regulation of the cold-shock protein Y-box binding protein-1 (YB-1). The mature 50 kD form of YB-1 was required for the nephro- and cytoprotective effects of aPC in vivo and in vitro, respectively. Reduction of mature YB-1 and K48-linked ubiquitination of YB-1 was prevented by aPC after renal IRI or tubular HR injury. aPC preserved the interaction of YB-1 with the deubiquitinating enzyme otubain-1 and maintained expression of otubain-1, which was required to reduce K48-linked YB-1 ubiquitination and to stabilize the 50 kD form of YB-1 after renal IRI and tubular HR injury. These data link the cyto- and nephroprotective effects of aPC with the ubiquitin-proteasome system and identify YB-1 as a novel intracellular target of aPC. These insights may provide new impetus for translational efforts aiming to restrict renal IRI.

A Proposed Role for Platelet Factor 4 in Histone Pathobiology in Sepsis

Sepsis is a high-risk clinical setting often resulting in multi-organ failure and death. Release of chromatin NETS (neutrophil extracellular traps) from neutrophils and the toxic role of highly-positively charged histones in late sepsis have been noted previously. Also, for NET formation to occur, peptidylarginine deiminase 4 activity must be present in the neutrophils, leading to citrullinated (cit) histones formation and loss of a portion of the positive charge. The four histones (H2A, H2B, H3 and H4) alone and as octamers of the four units tightly bind DNA. H3 and H4 histones as well as mixed octameric histones can induce a sepsis-like state in mice. One feature previously noted was that histones could inhibit activated protein C (aPC) production in the presence of thrombomodulin (TM). Since aPC generation is felt to protect against vascular damage, it was felt that this might - in part - account for the deleterious effects of histones in sepsis. We have shown that another highly-positive, small molecule, platelet factor 4 (PF4, CXCL4), which exists as a tetramer and which is stored in high concentrations in platelet alpha granules to be released in large amounts post-platelet activation, binds to the chondroitin sulfate (CS) side-chain of TM (TMCS) and enhanced aPC production along a bell-shaped curve with a peak effect around 25 µg/ml. Non-modified mixed histones had a similar bell-shaped effect on aPC generation and [histones + PF4] are additive on affecting aPC generation via TMCS. We wondered, because of this overlapping biology and the fact that significant levels of free PF4 are available in late sepsis, whether PF4 might affect other histone pathobiological pathways in late sepsis focusing on PF4’s interactions with non-modified and cit-histones. We first asked whether released PF4 might affect the binding of histones to DNA within NETS. We found that PF4 binds to DNA with greater affinity than histones in a competitive binding assay and that this effect was more marked for cit-histone consistent with its decreased positive charge. We then studied PF4 biology in three known targets of histone in sepsis. (1) In aPC generation, we examined cit-histones (either mixed, H3 or H4) relative to non-modified histones in stimulating aPC generation and found that they had a more limited effect on aPC generation with TMCS, but that again, PF4 cooperated in inducing aPC generation along a bell-shaped curve. (2) Histones are known to activate platelets (known to involve the toll-like receptor 4), likely contributing to the observed thrombocytopenia in late sepsis. We affirmed this affect with mixed histones and H4. Cit-mixed histones and cit-H4 also activated platelets in a platelet aggregation system, but much more weakly. PF4 had no effect on platelet activation by non-modified histones, but enhanced platelet activation by both cit-mixed histones and cit-H4. This was especially true for platelet activation studies with cit-H4 which on its own had nearly no affect on platelet activation though in the presence of moderate levels of PF4 (25 µg/ml), cit-H4 activated platelets as well as non-modified histones. (3) Finally, both non-modified and cit-histones activate endothelial cells (EC) by binding to their cell surfaces and likely contribute to the vascular damage of late sepsis. Using a microfluidic system involving controlled photochemical injury of the EC lining we found that PF4 enhanced the observed damage after cit-H4 exposure, but not notably after a comparable H4 exposure so that peak damage (as detected by propidium iodide staining) after cit-H4 approached that seen after H4 alone. In conclusion, NET formation involves citrullination of histones, and these modified histones likely contribute significantly to pathobiology in late sepsis. We now propose that in late sepsis, free histones, especially cit-histones, are mobilized out of NETs by PF4 because the PF4 binds DNA with higher affinity. After the histones and cit-histones are released from DNA, PF4 modifies the biology of these histones, especially the cit-histones enhancing their effects on aPC generation, platelet activation and EC injury. These studies provide additional insights of how histones achieve their pathobiological effects in sepsis. Such new insights may be critical for both understanding and monitoring clinical outcome and may lead to new therapeutic targets in sepsis. DisclosuresNo relevant conflicts of interest to declare.

Group V secretory phospholipase A2 impairs endothelial protein C receptor‐dependent protein C activation and accelerates thrombosis in vivo

Endothelial protein C receptor (EPCR) must be bound to a molecule of phosphatidylcholine (PC) to be fully functional, i.e. to interact with protein C/activated protein C (APC) properly. PC can be replaced with other lipids, such as lysophosphatidylcholine or platelet-activating factor, by the action of group V secretory phospholipase A2 (sPLA2-V), an enzyme that is upregulated in a variety of inflammatory conditions. Studies in purified systems have demonstrated that the substitution of PC notably impairs EPCR function in a process called EPCR encryption. To analyze whether sPLA2-V was able to regulate EPCR-dependent protein C activation in vivo, and its impact on thrombosis and the hemostatic system. Mice were transfected with sPLA2-V by hydrodynamic gene delivery. The effects on thrombosis were studied with the laser carotid artery occlusion model, and APC generation capacity was measured with ELISA. Global hemostasis was analyzed with thromboelastometry. We found that sPLA2-V overexpression in mice significantly decreased their ability to generate APC. Furthermore, a murine carotid artery laser thrombosis model revealed that higher sPLA2-V levels were directly associated with faster artery thrombosis. sPLA2-V plays a thrombogenic role by impairing the ability of EPCR to promote protein C activation.

Modulation of Protein C Activation by Histones, Platelet Factor 4, and Heparinoids

Objective— Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. Approach and Results— We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of &gt;5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Conclusions— Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.

Factor V Inhibits PAR2-Mediated Lethal Inflammation

Abstract 3360The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioactivity of purified, plasma-derived or recombinant fV was not expressed by the fV Leiden variant. Thus, in heterozygous fV Leiden individuals, aPC levels are increased by fV Leiden, while wild type fV enhances aPC's beneficial actions on innate immune cells. This explains why heterozygous carriers are protected from sepsis mortality by aPC, whereas homozygous Leiden carriers do not respond to endogenous or therapeutically administered aPC. FV Leiden hence emerges as a unique example of a balancing and evolutionary selectable polymorphism, whose balanced nature is the consequence of beneficial, cooperative interactions of the variant fV Leiden allele that augments generation of aPC and the normal fV allele that is necessary for the protective effects of aPC. Disclosures:Weiler:BloodCenter of Wisconsin: Patents & Royalties.

Mice with Genetic Modifications in Prothrombin Limiting Activation Cleavage Events to Meizothrombin Survive to Adulthood, but Exhibit Alterations in Hemostasis and Thrombus Formation

AbstractAbstract 496Thrombin-mediated proteolysis is the central event not only of hemostasis and thrombosis but also in distinct physiological and pathological contexts such as development, inflammation, cancer biology, and cardiovascular disease. The proteolytic conversion of prothrombin to α-thrombin (fIIa) by the prothrombinase complex occurs through two possible pathways: i) the inactive intermediate termed prethrombin 2, or ii) the proteolytically active two-chain intermediate termed meizothrombin (fIIaMZ). fIIaMZ, unlike fIIa, retains the γ-carboxyglutamic acid-rich gla domain and two kringle domains of prothrombin and has distinct catalytic properties relative to fIIa that could be biologically meaningful in vivo, including a diminished capacity to cleave fibrinogen and a significantly increased capacity to activate protein C in the presence of thrombomodulin. Here, the endogenous prothrombin gene was modified in mice to better explore the properties of fIIaMZin vivo and to test the hypotheses that mice carrying a mutant form of prothrombin with a terminal activation product of fIIaMZ will: 1) develop to term and survive to adulthood despite an altered hemostatic profile; and 2) exhibit significantly dampened inflammatory responses due to enhanced protein C activation. In order to limit cleavage events to the single site yielding fIIaMZ, alterations were introduced into the endogenous prothrombin gene resulting in three amino acid substitutions (R157A, R268A, and K281A) located at the P1 positions of potential fXa or autocatalytic cleavage sites. Unlike prothrombin-null mice, mice homozygous for these mutations (hereafter referred to as fIIMZ mice) were found to be present at weaning age and viable into adulthood. Furthermore, unlike mice with a major deficit in either clotting function (e.g., fibrinogen-null) or platelet function (e.g., Gαq-null), fIIMZ females were capable of successfully carrying a litter to term. However, analysis of over 700 progeny generated from crosses of heterozygous fIIMZ/WT mice revealed that only about half of the number homozygous fIIMZ mice expected, based on Mendelian transmission rates, were observed in weaning-age offspring. Complementary studies suggest that the fraction of fIIMZ offspring that fail are lost primarily in utero, but occasional post-partum failures were observed associated with hemorrhagic events. Successful adult fIIMZ mice were found to have similar prothrombin mRNA levels and equivalent fII protein expression to fIIWT mice. Furthermore, fIIMZ animals exhibited normal complete blood cell counts and predictably normal thrombin times, but prolonged PTs and aPTTs. Thrombin generation assays revealed a prolonged time to peak thrombin production, but no significant difference in peak thrombin levels. Consistent with these findings, tail bleeding times in fIIMZ mice were significantly prolonged. None of the fIIMZ mice assayed achieved hemostasis within the 10 minute observation window, whereas fIIWT mice all stopped within 2 minutes of challenge. More sophisticated comparative studies of thrombus formation in mesenteric arterioles following FeCl3 injury using a real-time intravital microscopy approach established that the time to first thrombus formation in fIIMZ mice was almost twice that of wildtype animals. The majority of the fIIMZ mice failed to occlude the injured vessel within a 25 min observation window, whereas all fIIWT mice successfully formed occlusive thrombi with a mean time of 12.5 minutes. In summary, site-directed alterations of the endogenous prothrombin gene in mice leading to a terminal prothrombin activation product of meizothrombin were found to be compatible with development, growth to adulthood and reproductive success, albeit with modified hemostatic function. Based on the catalytic properties of fIIaMZ favoring protein C activation, studies are underway to compare APC generation in control and fIIMZ mice and explore the theory that physiological and pathological inflammatory responses will be dampened in fIIMZ mice. Disclosures:No relevant conflicts of interest to declare.

SPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove

AbstractThe endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity. Our objective was to identify this phospholipid and to explore the possibility of lipid exchange as a regulatory mechanism of EPCR activity driven by the endothelially expressed secretory group V phospholipase A2 (sPLA2-V). We identified phosphatidylcholine (PCh) as the major phospholipid bound to human soluble EPCR (sEPCR). PCh in EPCR could be exchanged for lysophosphatidylcholine (lysoPCh) and platelet activating factor (PAF). Remarkably, lysoPCh and PAF impaired the protein C binding ability of sEPCR. Inhibition of sPLA2-V, responsible for lysoPCh and PAF generation, improved APC binding to endothelial cells. EPCR-dependent protein C activation and APC antiapoptotic effect were thus significantly enhanced. In contrast, endothelial cell supplementation with sPLA2-V inhibited both APC generation and its antiapoptotic effects. We conclude that APC generation and function can be modulated by changes in phospholipid occupancy of its endothelial cell receptor.