Cytoprotective 3K3A-activated protein C and plasma: A comparison of therapeutics for the endotheliopathy of trauma.

BACKGROUND: Both healthy plasma and cytoprotective aPC (3K3A-aPC) have been shown to mitigate the endotheliopathy of trauma (EoT), but optimal therapeutics remain unknown. Our aim was therefore to determine optimal therapies to mitigate EoT by investigating the effectiveness of 3K3A-aPC with and without plasma-based resuscitation strategies. METHODS: Electric cell-substrate impedance sensing (ECIS) was used to measure real-time permeability changes in endothelial cells. Cells were treated with a 2 µg/mL solution of aPC 30 minutes prior to stimulation with plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Healthy plasma, or plasma frozen within 24 hours (FP24), was added concomitantly with TP. Cells treated with thrombin and untreated cells were included in this study as control groups. RESULTS: A dose-dependent difference was found between the 5% and 10% plasma-treated groups when HUVECs were simultaneously stimulated with TP (µd 7.346 95%CI 4.574 to 10.12). There was no difference when compared to TP alone in the 5% (µd 5.713 95%CI -1.751 to 13.18) or 10% group (µd -1.633 95%CI -9.097 to 5.832). When 3K3A-aPC was added to plasma and TP, the 5% group showed improvement in permeability compared to TP alone (µd 10.11 95%CI 2.642 to 17.57), but there was no difference in the 10% group (µd -1.394 95%CI -8.859 to 6.070). The combination of 3K3A-aPC, plasma, and TP at both the 5% plasma (µd -28.52 95%CI-34.72 to -22.32) and 10% plasma concentrations (µd -40.02 95%CI -46.22 to -33.82) had higher inter-cellular permeability than the 3K3A-aPC pre-incubation group. CONCLUSION: Our data shows that FP24, in a post-trauma environment, pre-treatment with 3K3A-aPC can potentially mitigate the EoT to a greater degree than FP24 with or without 3K3A-aPC. Although further exploration is needed, this represents a potentially ideal and perhaps superior therapeutic treatment for the dysregulated thromboinflammation of injured patients. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Therapeutic/Care Management, Level III.

Biased agonism of protease-activated receptor-1 regulates thromboinflammation in murine sickle cell disease.

ABSTRACT: Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein-coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease-activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C.

BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.

Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature.

Type 1 diabetes (T1D) is a prototypic T cell-mediated autoimmune disease. Because the islets of Langerhans are insulated from blood vessels by a double basement membrane and lack detectable lymphatic drainage, interactions between endocrine and circulating T cells are not permitted. Thus, we hypothesized that initiation and progression of anti-islet immunity required islet neolymphangiogenesis to allow T cell access to the islet. Combining microscopy and single cell approaches, the timing of this phenomenon in mice was situated between 5 and 8 wk of age when activated anti-insulin CD4 T cells became detectable in peripheral blood while peri-islet pathology developed. This "peri-insulitis," dominated by CD4 T cells, respected the islet basement membrane and was limited on the outside by lymphatic endothelial cells that gave it the attributes of a tertiary lymphoid structure. As in most tissues, lymphangiogenesis seemed to be secondary to local segmental endothelial inflammation at the collecting postcapillary venule. In addition to classic markers of inflammation such as CD29, V-CAM, and NOS, MHC class II molecules were expressed by nonhematopoietic cells in the same location both in mouse and human islets. This CD45- MHC class II+ cell population was capable of spontaneously presenting islet Ags to CD4 T cells. Altogether, these observations favor an alternative model for the initiation of T1D, outside of the islet, in which a vascular-associated cell appears to be an important MHC class II-expressing and -presenting cell.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

BACKGROUND: Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS: We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 µg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS: Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Tumor-expressed factor VII is associated with survival and regulates tumor progression in breast cancer.

Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

Selective modulation of activated protein C activities by a nonactive site-targeting nanobody library.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.

Understanding COVID-19-associated coagulopathy.

COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured ECs. Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice.

Acute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

Activated protein C and PAR1-derived and PAR3-derived peptides are anti-inflammatory by suppressing macrophage NLRP3 inflammasomes.

Essentials Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective effects. We tested whether APC or non-canonical PAR-derived peptides suppress inflammasome activity. APC or PAR1- and PAR3-derived peptides restrict inflammasome-dependent caspase-1 activity. Combined PAR1-derived and PAR3-derived peptides synergistically suppress caspase-1 activity. ABSTRACT: Background Activated protein C (APC) has been shown to restrict murine inflammasome activity. However, whether APC can exert anti-inflammatory activity in part through suppression of inflammasome activation in human systems is unknown. Objectives Studies were made to determine whether either APC or protease activated receptor (PAR)-derived peptides can reduce NLRP3 inflammasome activity in differentiated human THP-1 macrophage-like cells or in primary human monocytes stimulated to activate the inflammasome. Methods Human THP-1 cells or primary human monocytes were differentiated, treated with APC or PAR-derived peptides, and then stimulated with lipopolysaccharide and ATP to induce caspase-1 activity, a product of inflammasome activation. Results Activated protein C or noncanonical PAR1-derived or PAR3-derived peptides significantly reduced caspase-1 activity, detection of fluorescent NLRP3, and IL-1ß release from THP-1 cells. At low concentrations where no effect was observed for each individual peptide, combinations of the PAR1-derived peptide and the PAR3-derived peptide resulted in a significant synergistic decrease in caspase-1 and IL-1ß release. Caspase-1 activity was also reduced in primary human monocytes. Studies using blocking antibodies and small molecule PAR1 inhibitors suggest that EPCR, PAR1, and PAR3 each play roles in the observed anti-inflammatory effects. Several shortened versions of the PAR1- and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. Conclusions The results indicate that both APC and certain PAR1- and PAR3-derived peptides, which are biased agonists for PAR1 or PAR3, can reduce inflammasome activity in stimulated human monocytes as measured by caspase-1 activity and IL-1ß release and that PAR-derived biased peptide agonist combinations are synergistically anti-inflammatory.

An engineered factor Va prevents bleeding induced by direct-acting oral anticoagulants by different mechanisms.

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.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo.

OBJECTIVE: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). CONCLUSIONS: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.

C-terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities.

BACKGROUND: Activated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3. OBJECTIVE: To date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C-terminus of the light chain have not been studied in detail. METHODS: Site-directed Ala-scanning mutagenesis of six positively charged residues within G142-L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities. RESULTS AND CONCLUSIONS: K151 was involved in protein S dependent-anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S-dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C-terminal region of APC's light chain overlap.

Role of thrombomodulin expression on hematopoietic stem cells.

BACKGROUND: Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co-express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi-cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR-bound protein C by the thrombin-THBD-complex, and subsequent activation of PAR1 by the aPC-EPCR complex. OBJECTIVE: To determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions. METHODS: Hematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD-deficiency by flow cytometry, functional assays, and single cell RNA profiling. RESULTS: THBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD-deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities. CONCLUSION: THBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.

Mechanisms of vascular permeability and remodeling associated with hemarthrosis in factor VIII-deficient mice.

BACKGROUND: Vascular remodeling associated with hemophilic arthropathy (HA) may contribute to bleed propagation, but the mechanisms remain poorly understood. OBJECTIVES: To explore molecular mechanisms of HA and the effects of hemostasis correction on synovial vascular remodeling after joint injury in hypocoagulable mice. METHODS: Factor VIII (FVIII)-deficient mice +/- FVIII treatment and hypocoagulable wild-type mice (Hypo BALB/c) were subjected to subpatellar puncture. Hypo BALB/c mice were treated with warfarin and anti-FVIII before injury, after which warfarin was continued for 2 weeks or reversed +/- continuous anti-FVIII until harvest. Synovial vascularity was analyzed at baseline and 2 to 4 weeks post injury by histology, musculoskeletal ultrasound with power Doppler (microvascular flow), and Evans blue extravasation (vascular permeability). Synovial gene expression and systemic markers of vascular collagen turnover were studied in FVIII-deficient mice by RNA sequencing and enzyme-linked immunosorbent assay. RESULTS: Vascular changes occurred in FVIII-deficient and Hypo BALB/c mice after injury with minimal effect of hemostasis correction. Increased vascular permeability was only significant in FVIII-deficient mice, who exhibited more pronounced vascular remodeling than Hypo BALB/c mice despite similar bleed volumes. FVIII-deficient mice exhibited a strong transcriptional response in synovium that was only partially affected by FVIII treatment and involved genes relating to angiogenesis and extracellular matrix remodeling, with vascular collagen turnover markers detected systemically. CONCLUSIONS: Intact hemostasis at the time of hemarthrosis and during healing are both critical to prevent vascular remodeling, which appears worse with severe and prolonged FVIII deficiency. Unbiased RNA sequencing revealed potential targets for intervention and biomarker development to improve management of HA.