Protein S antibody as an adjunct therapy for hemophilia B.

ABSTRACT: Hemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Systemic Review of Clot Retraction Modulators.

Through a process termed clot retraction, platelets cause thrombi to shrink and become more stable. After platelets are activated via inside-out signaling, glycoprotein αIIbßIII binds to fibrinogen and initiates a cascade of intracellular signaling that ends in actin remodeling, which causes the platelet to change its shape. Clot retraction is also important for wound healing. Although the detailed molecular biology of clot retraction is only partially understood, various substances and physiological conditions modulate clot retraction. In this review, we describe some of the current literature pertaining to clot retraction modulators. In addition, we discuss compounds from Cudrania trucuspidata, Arctium lappa, and Panax ginseng that diminish clot retraction and have numerous other health benefits. Caffeic acid and diindolylmethane, both common in plants and vegetables, likewise reduce clot retraction, as do all-trans retinoic acid (a vitamin A derivative), two MAP4K inhibitors, and the chemotherapeutic drug Dasatinib. Conversely, the endogenous anticoagulant Protein S (PS) and the matricellular protein secreted modular calcium-binding protein 1 (SMOC1) both enhance clot retraction. Most studies aiming to identify mechanisms of clot retraction modulators have focused on the increased phosphorylation of vasodilator-stimulated phosphoprotein and inositol 1,4,5-triphosphate receptor I and the decreased phosphorylation of various phospholipases (e.g., phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2), c-Jun N-terminal kinase, and (PI3Ks). One study focused on the decreased phosphorylation of Sarcoma Family Kinases (SFK), and others have focused on increased cAMP levels and the downregulation of inflammatory markers such as thromboxanes, including thromboxane A2 (TXA2) and thromboxane B2 (TXB2); prostaglandin A2 (PGE2); reactive oxygen species (ROS); and cyclooxygenase (COX) enzyme activity. Additionally, pregnancy, fibrinolysis, and the autoimmune condition systemic lupus erythematosus all seem to affect, or at least have some relation with, clot retraction. All the clot retraction modulators need in-depth study to explain these effects.

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa.

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review.

Severe COVID-19 Is Characterized by an Impaired Type I Interferon Response and Elevated Levels of Arginase Producing Granulocytic Myeloid Derived Suppressor Cells.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Protein S: function, regulation, and clinical perspectives.

PURPOSE OF REVIEW: Protein S (PS) is an essential natural anticoagulant. PS deficiency is a major contributor to acquired hypercoagulability. Acquired hypercoagulability causes myocardial infarction, stroke, and deep vein thrombosis in millions of individuals. Yet, despite its importance in hemostasis, PS is the least understood anticoagulant. Even after 40 years since PS was first described, we are still uncovering information about how PS functions. The purpose of this review is to highlight recent findings that advance our understanding of the functions of PS and explain hypercoagulability caused by severe PS deficiency. RECENT FINDINGS: PS has long been described as a cofactor for Activated Protein C (APC) and Tissue Factor Pathway Inhibitor (TFPI). However, a recent report describes direct inhibition of Factor IXa (FIXa) by PS, an activity of PS that had been completely overlooked. Thrombophilia is becoming a more frequently reported disorder. Hereditary PS deficiency is an anticoagulant deficiency that results eventually in thrombophilia. In addition, PS deficiency is a predisposing factor for venous thromboembolism (VTE), but an effect of PS deficiency in arterial thrombosis, such as arterial ischemic stroke, is uncertain. Plasma PS concentration decreases in pregnant women. Inherited thrombophilias are important etiologies for recurrent pregnancy loss, and anticoagulation therapy is of benefit to women with recurrent pregnancy loss who had documented only PS deficiency.Hypoxia is a risk factor for VTE, and hypoxia downregulates plasma PS level. Importantly, COVID-19 can lead to hypoxemia because of lung damage from IL6-driven inflammatory responses to the viral infection. Because hypoxia decreases the abundance of the key anticoagulant PS, we surmise that the IL6-induced cytokine explosion combined with hypoxemia causes a drop in PS level that exacerbates the thrombotic risk in COVID-19 patients. SUMMARY: This review is intended to advance understanding of the anticoagulant function of an important plasma protein, PS. Despite 40+ years of research, we have not had a complete description of PS biology as it pertains to control of blood coagulation. However, the picture of PS function has become sharper with the recent discovery of FIXa inhibition by PS. Hemostasis mediated by PS now includes regulation of FIXa activity alongside the cofactor activities of PS in the TFPI/APC pathways. In addition, the direct inhibition of FIXa by PS suggests that PS, particularly a small derivative of PS, could be used to treat individuals with PS deficiencies or abnormalities that cause thrombotic complications.

Transcriptome and Functions of Granulocytic Myeloid-Derived Suppressor Cells Determine their Association with Disease Severity of COVID-19.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19, that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of Granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1 + G-MDSC (Arg + G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg + G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Role of vitamin D in treating COVID-19-associated coagulopathy: problems and perspectives.

Aggressive inflammatory response leading to hypercoagulability has been found to be associated with disease severity in COVID-19 patients and portends bad treatment outcome. A state of acute disseminated intravascular coagulation (DIC), along with pulmonary embolism and/or deep vein thrombosis, has been observed in critically ill ICU patients. Autopsy reports of COVID-19 patients demonstrated microthrombi in lungs and in other organs, as well as marked inflammatory changes, characteristic clinicopathological features that exacerbate disease severity. Vitamin D supplementation was recommended by many clinicians across the globe to improve clinical symptoms of COVID-19 patients, mainly because of its immunomodulatory roles on immune cells. Furthermore, vitamin D and its associated molecules are also known to directly or indirectly regulate various thrombotic pathways. We propose that vitamin D supplementation not only attenuates the risk of Acute Respiratory Disease Syndrome (ARDS) but it also may have a role in reducing coagulation abnormalities in critically ill COVID-19 patients. The overarching goal of this review is to discuss the effects of vitamin D on coagulation pathways and other intertwined processes leading to thrombosis. Many clinical trials are currently investigating the efficacy of vitamin D supplementation in reducing the risk of COVID-19 infection. However, randomized placebo control clinical trials are also necessary to ascertain the effect of vitamin D supplementation on reducing the risk of coagulopathy in COVID-19 patients.

Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor.

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ∼ 150-160 µM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd∼70-80 µM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50-100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

Modulation of protein S and growth arrest specific 6 protein signaling inhibits pancreatic cancer cell survival and proliferation.

Thrombotic complications and hypercoagulopathies are commonly associated with the progression of pancreatic ductal adenocarcinoma (PDAC). Although the mechanistic link between the two phenomena is uncertain, there is evidently an increase in procoagulant proteins and a decrease in anticoagulants in PDAC patients. For example, the anticoagulant protein S (PS) is decreased during the progression of PDAC, a condition that possibly contributes to the hypercoagulopathies. PS is also an important signaling molecule that binds a family of tyrosine kinase receptors known as TAM (Tyro3, Axl and Mer) receptors; TAM receptors are often upregulated in different cancers. Growth Arrest Specific 6 or GAS6 protein, a homolog of PS, is also a TAM receptor family ligand. The downstream signaling pathways triggered by this ligand­receptor interaction perform diverse functions, such as cell survival, proliferation, efferocytosis, and apoptosis. Targeting the TAM receptors to treat cancer has had limited success; side effects are a significant obstacle due to the widespread numerous functions of TAM receptors. In the present study, it was revealed that PS­TAM interaction was pro­apoptotic, whereas GAS6­mediated TAM signaling promoted proliferation and survival in select PDAC cell lines. Furthermore, by regulating the balance between these two signaling pathways (by overexpressing PS or knocking down GAS6), the proliferative potential of the cells was decreased. Both long­term and short­term effects of natural PS overexpression were comparable to the treatment of the cells with the drug UNC2025, which inhibits the Mer­receptor. The present study lays the foundation for investigation of PS as a therapeutic agent to control cancer progression and to concurrently arrest thrombotic events.

COVID-19: a probable role of the anticoagulant Protein S in managing COVID-19-associated coagulopathy.

The COVID-19 pandemic has caused monumental mortality, and there are still no adequate therapies. Most severely ill COVID-19 patients manifest a hyperactivated immune response, instigated by interleukin 6 (IL6) that triggers a so called "cytokine storm" and coagulopathy. Hypoxia is also associated with COVID-19. So far overlooked is the fact that both IL6 and hypoxia depress the abundance of a key anticoagulant, Protein S. We speculate that the IL6-driven cytokine explosion plus hypoxemia causes a severe drop in Protein S level that exacerbates the thrombotic risk in COVID-19 patients. Here we highlight a mechanism by which the IL6-hypoxia curse causes a deadly hypercoagulable state in COVID-19 patients, and we suggest a path to therapy.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.

We previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5'-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12-/- and CalDAG GEFI-/- mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Padua FIXa resistance to Protein S and a potential therapy for hyperactive FIXa.

INTRODUCTION: Abnormalities in the levels and functions of proteins that maintain hemostasis can cause thrombosis. Factor IX (FIX) R338L, i.e., Factor IX Padua, is a hyperactive clotting factor that promotes thrombosis. The R338L mutation increases the clotting rate by 8-fold despite increasing the Factor IXa enzymatic activity by only 2-fold. Protein S (PS) is a natural anticoagulant that directly inhibits FIXa. Because individuals affected by the R338L mutation have normal concentrations of PS, we speculated that the Padua hypercoagulation phenotype is due to decreased inhibition of FIXa R338L by PS. METHODS: We measured the ability of PS to inhibit FIX R338L, and we assessed the ability of PS to mitigate the prothrombotic effect FIX R338L. RESULTS: Plasma clotting assays demonstrated that 3-fold more PS was required to inhibit FIXa R338L compared with inhibition of wild type FIXa. Thrombin generation assays with Padua patient plasma recapitulated this biochemical consequence of the R338L mutation. Importantly, the less efficient inhibition of FIXa R338L was reversed by increasing PS concentration. Binding and co-immunoprecipitation studies revealed that the decrease in the inhibition of FIXa R338L by PS was caused by a 3- to 4-fold reduction in FIXa R338L affinity for PS. CONCLUSION: In summary, the resistance of FIXa R338L to inhibition by PS likely contributes to the unexpectedly high clotting rate in Padua individuals. Moreover, PS-mediated reversal of the pathological properties of FIXa R338L suggests that PS administration may be a novel and effective means to mitigate thrombophilia caused by any source of elevated FIXa activity.

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.