Agnostic identification of plasma biomarkers for postpartum hemorrhage risk.

BACKGROUND: Postpartum hemorrhage (PPH) is difficult to predict, is associated with significant maternal morbidity, and is the leading cause of maternal mortality worldwide. The identification of maternal biomarkers that can predict increased PPH risk would enhance clinical care and may uncover mechanisms that lead to PPH. OBJECTIVE: This retrospective case-control study employed agnostic proteomic profiling of maternal plasma samples to identify differentially abundant proteins in controls and PPH cases. STUDY DESIGN: Maternal plasma samples were procured from a cohort of >60,000 participants in a single institution's perinatal repository. PPH was defined as a decrease in hematocrit of ≥10% or receipt of transfusion within 24 hours of delivery. PPH cases (N=30) were matched by maternal age and delivery mode (vaginal or cesarean) with controls (N=56). Mass spectrometry was used to identify differentially abundant proteins using integrated peptide peak areas. Statistically significant differences between groups were defined by a p-value of <0.05 after controlling for multiple comparisons. RESULTS: By study design, cases and controls did not differ in race, ethnicity, gestational age at delivery, blood type, or pre-delivery platelet count. Cases had slightly, but significantly lower pre- and post-delivery hematocrit and hemoglobin. Mass spectrometry detected 1140 proteins, including 77 proteins for which relative abundance differed significantly between cases and controls (fold change >1.15, P<0.05). Of these differentially abundant plasma proteins, most had likely liver or placental origins. Gene ontology term analysis mapped to protein clusters involved in responses to wound healing, stress response, and host immune defense. Significantly differentially abundant proteins with the highest fold change (prostaglandin D2 synthase, periostin, and several serine protease inhibitors) did not correlate with pre-delivery hematocrit or hemoglobin, but identified PPH cases with logistic regression modeling revealing good-to-excellent area under the operator receiver characteristic curves (AUROC 0.802-0.874). Incorporating pre-delivery hemoglobin with these candidate proteins further improved identification of PPH cases. CONCLUSION: Agnostic analysis of maternal plasma samples identified differentially abundant proteins in controls and PPH cases. Several of these proteins are known to participate in biologically plausible pathways for PPH risk and have potential value for predicting PPH. These findings identify candidate protein biomarkers for future validation and mechanistic studies.

Plasma kallikrein supports FXII-independent thrombin generation in mouse whole blood.

Plasma kallikrein (PKa) is an important activator of factor (F)XII of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway-initiated TG compared to wild-type controls and was comparable to that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared to wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway-initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway-initiated TG in the complex milieu of mouse WB and that a component of this contribution occurs in a FXII-independent manner.

Clot formation and fibrinolysis assays reveal functional differences among hemostatic agents in hemophilia A plasma.

Background: Measuring the activity of hemostatic agents used to treat hemophilia A often requires drug-specific assays. In vitro assays show hemophilic clots have abnormal characteristics, including prolonged clotting time and decreased resistance to fibrinolysis. The ability of certain agents to correct these parameters in vitro is associated with hemostatic efficacy in vivo. Objectives: To compare effects of established and emerging hemostatic agents on clot formation and fibrinolysis in hemophilia A plasma. Methods: Pooled and individual hemophilia A platelet-poor plasmas were spiked with replacement (recombinant factor VIII [rFVIII], PEGylated rFVIII, polysialylated rFVIII, and porcine rFVIII) or bypassing (emicizumab, rFVIIa, and activated prothrombin complex concentrate) products. Effects on tissue factor-initiated clot formation and fibrinolysis were measured by turbidity. Results: Compared to normal pooled plasma, hemophilia-pooled plasma showed reduced clot formation and increased fibrinolysis, and all replacement agents improved these characteristics. rFVIII and PEGylated rFVIII produced similar effects at similar concentrations, whereas polysialylated rFVIII produced slightly higher and porcine rFVIII slightly lower effects at these concentrations. Bypassing agents enhanced clot formation and stability, but patterns differed from replacement agents. The clotting rate showed a concentration-response relationship for all agents. High concentrations of all products produced effects that exceeded the normal range in at least some parameters. Responses of individual donors varied, but all agents improved clot formation and stability in all donors tested. Conclusion: Clotting and fibrinolysis assays reveal hemostatic effects of replacement and bypassing therapies at clinically relevant concentrations. These assays may help characterize hemostatic agents and optimize dosing.

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg- ) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Thromboembolic risk with gender-affirming hormone therapy: potential role of global coagulation and fibrinolysis assays.

Gender-affirming hormonal therapies are a critical component of the care of transgender individuals. Transgender people are commonly prescribed estrogen or testosterone to promote male-to-female or female-to-male transitions and to preserve gender-specific characteristics long-term. However, some exogenous hormones, especially certain estrogen preparations, are an established risk factor of thrombosis. As the number of individuals seeking gender-based care is rising, there is an urgent need to identify and characterize the mechanisms underlying hormone-associated thrombosis and incorporate this information into clinical algorithms for diagnosis and management. Herein, we discuss historical evidence on the incidence of thrombosis and changes in plasma composition in transgender and cisgender cohorts. We present 3 case studies to demonstrate knowledge gaps in thrombosis risk stratification and prediction tools. We also present data from in vitro coagulation and fibrinolysis assays and discuss how information from these kinds of assays may be used to help guide the clinical management of transgender individuals.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

INTRODUCTION AND AIM: Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5 IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. MATERIALS AND METHODS: Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. RESULTS AND CONCLUSION: We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

Fibrinogen and fibrin: synthesis, structure, and function in health and disease.

Fibrinogen is an extraordinary molecule by any estimation. It is large, structurally intricate, and circulates at high concentrations. Its biological end product, insoluble fibrin(ogen) or fibrin, can assume a diverse array of conformations with the ability to interact with numerous plasma proteins and cells and withstand biochemical and biomechanical disruption to facilitate wound healing. Quantitative and qualitative defects in fibrinogen or fibrin are associated with bleeding, thrombosis, inflammation, and diseases affected by these processes. Numerous studies investigating mechanisms by which fibrin(ogen) and fibrin contribute to health and disease have been published. This review for the 20th-anniversary series in the Journal of Thrombosis and Haemostasis summarizes interesting aspects of fibrin(ogen) biology, biochemistry, biophysics, and physiology and highlights exciting findings published in the past 2 decades.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γΔ5 mice drives acute liver injury.

BACKGROUND: Hepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. OBJECTIVES: We sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbß3 binding domain in APAP-induced liver injury. METHODS: Mice expressing mutant fibrinogen incapable of engaging integrin αIIbß3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). RESULTS: We observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbß3. CONCLUSION: The results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography.

BACKGROUND: Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported. OBJECTIVES: Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples. METHODS: Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer. RESULTS: We confirmed the requirement of platelets, platelet contraction, and αIIbß3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk. CONCLUSION: Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.

High risk oral contraceptive hormones do not directly enhance endothelial cell procoagulant activity in vitro.

BACKGROUND: Oral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs. OBJECTIVE: Characterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERß and inflammatory processes. METHODS: Human umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERß (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively. RESULTS: Neither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus. CONCLUSIONS: The OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro.

Robust coagulation activation and coagulopathy in mice with experimental acetaminophen-induced liver failure.

BACKGROUND: Patients with acetaminophen (APAP)-induced acute liver failure (ALF) display both hyper- and hypocoagulable changes not necessarily recapitulated by standard hepatotoxic doses of APAP used in mice (eg, 300 mg/kg). OBJECTIVES: We sought to examine coagulation activation in vivo and plasma coagulation potential ex vivo in experimental settings of APAP-induced hepatotoxicity and repair (300-450 mg/kg) and APAP-induced ALF (600 mg/kg) in mice. RESULTS: APAP-induced ALF was associated with increased plasma thrombin-antithrombin complexes, decreased plasma prothrombin, and a dramatic reduction in plasma fibrinogen compared with lower APAP doses. Hepatic fibrin(ogen) deposits increased independent of APAP dose, whereas plasma fibrin(ogen) degradation products markedly increased in mice with experimental ALF. Early pharmacologic anticoagulation (+2 hours after 600 mg/kg APAP) limited coagulation activation and reduced hepatic necrosis. The marked coagulation activation evident in mice with APAP-induced ALF was associated with a coagulopathy detectable ex vivo in plasma. Specifically, prolongation of the prothrombin time and inhibition of tissue factor-initiated clot formation were evident even after restoration of physiological fibrinogen concentrations. Plasma endogenous thrombin potential was similarly reduced at all APAP doses. Interestingly, in the presence of ample fibrinogen, ∼10 times more thrombin was required to clot plasma from mice with APAP-induced ALF compared with plasma from mice with simple hepatotoxicity. CONCLUSION: The results indicate that robust pathologic coagulation cascade activation in vivo and suppressed coagulation ex vivo are evident in mice with APAP-induced ALF. This unique experimental setting may fill an unmet need as a model to uncover mechanistic aspects of the complex coagulopathy of ALF.

High plasma levels of C1-inhibitor are associated with lower risk of future venous thromboembolism.

BACKGROUND: C1-inhibitor (C1INH) is a broad-acting serine protease inhibitor with anticoagulant activity. The impact of C1INH plasma levels within the normal physiological range on risk of venous thromboembolism (VTE) is unknown. We assessed the association of plasma C1INH levels and VTE risk and evaluated the impact of C1INH on thrombin and plasmin generation in ex vivo assays. METHODS: A nested case-control study with 405 patients with VTE and 829 age- and sex-matched controls was derived from the Tromsø Study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE were estimated across plasma C1INH quartiles. Genetic regulation of C1INH was explored using quantitative trait loci analysis of whole exome sequencing data. The effect of plasma C1INH levels on coagulation was evaluated ex vivo by calibrated automated thrombography. RESULTS: Individuals with C1INH levels in the highest quartile had a lower risk of VTE (OR 0.68, 95% CI: 0.49-0.96) compared with those with C1INH in the lowest quartile. In subgroup analysis, the corresponding ORs were 0.60 (95% CI: 0.39-0.89) for deep vein thrombosis and 0.85 (95% CI: 0.52-1.38) for pulmonary embolism, respectively. No significant genetic determinants of plasma C1INH levels were identified. Addition of exogenous C1INH to normal human plasma reduced thrombin generation triggered by an activator of the intrinsic coagulation pathway, but not when triggered by an activator of the extrinsic coagulation pathway. CONCLUSIONS: High plasma levels of C1INH were associated with lower risk of VTE, and C1INH inhibited thrombin generation initiated by the intrinsic coagulation pathway ex vivo.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities for ultra-rare inherited bleeding disorders.

BACKGROUND: Ultra-rare inherited bleeding disorders (BDs) present important challenges for generating a strong evidence foundation for optimal diagnosis and management. Without disorder-appropriate treatment, affected individuals potentially face life-threatening bleeding, delayed diagnosis, suboptimal management of invasive procedures, psychosocial distress, pain, and decreased quality-of-life. RESEARCH DESIGN AND METHODS: The National Hemophilia Foundation (NHF) and the American Thrombosis and Hemostasis Network identified the priorities of people with inherited BDs and their caregivers, through extensive inclusive community consultations, to inform a blueprint for future decades of research. Multidisciplinary expert Working Group (WG) 3 distilled highly feasible transformative ultra-rare inherited BD research opportunities from the community-identified priorities. RESULTS: WG3 identified three focus areas with the potential to advance the needs of all people with ultra-rare inherited BDs and scored the feasibility, impact, and risk of priority initiatives, including 13 in systems biology and mechanistic science; 2 in clinical research, data collection, and research infrastructure; and 5 in the regulatory process for novel therapeutics and required data collection. CONCLUSIONS: Centralization and expansion of expertise and resources, flexible innovative research and regulatory approaches, and inclusion of all people with ultra-rare inherited BDs and their health care professionals will be essential to capitalize on the opportunities outlined herein.

Living with an ultra-rare inherited bleeding disorder is challenging. Patients can feel alone and unsure of where to find support because their disorder is so rare. In this paper, a group of ultra-rare bleeding disorder experts, including doctors, researchers, regulators, patient advocates, and patients, identify the research that could best improve the lives of people with these disorders. They propose a national network of specialists who can help doctors, who may never have seen these disorders before, to find the right diagnosis faster. A centralized laboratory specialized in ultra-rare bleeding disorders could also improve diagnosis and do research studies. This would help us learn, for example, how symptoms change throughout a patient's life, how effective different treatments are, and what it is like for patients to live with these disorders. A second research priority is to better understand each individual disorder so that the best treatments can be chosen or developed. A pathway showing doctors which treatment options to try, in which order, would help them help their patients. The third research priority is to make it easier to study new treatments for ultra-rare bleeding disorders. This requires designing studies with very small numbers of participants, identifying meaningful outcomes to measure, and convincing pharmaceutical companies to invest in these studies. International agreement on these requirements would allow more patients to participate and benefit from the research. These top-priority research goals should greatly improve knowledge about, and diagnosis and treatment of, ultra-rare inherited bleeding disorders.

Assays to quantify fibrinolysis: strengths and limitations. Communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee on fibrinolysis.

Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease plasmin, which cleaves and solubilizes crosslinked fibrin clots into fibrin degradation products. The quantity and quality of fibrinolytic enzymes, their respective inhibitors, and clot structure determine overall fibrinolysis. The quantity of protein can be measured by antigen-based assays, and both quantity and quality can be assessed using functional assays. Furthermore, variations of commonly used assays have been reported, which are tailored to address the role(s) of specific fibrinolytic factors and cellular elements (eg, platelets, neutrophils, and red blood cells). Although the concentration and/or activity of a protein can be quantified, how these individual components contribute to the overall fibrinolysis outcome can be challenging to determine. This difficulty is due to temporal changes within and around the thrombi during the clot breakdown, particularly the fibrin matrix structure, and composition. Furthermore, terms such as "fibrinolytic activity/potential," "plasminogen activation," and "plasmin activity" are often used interchangeably despite having different definitions. The purpose of this review is to 1) summarize the assays measuring fibrinolysis activity and potential, 2) facilitate the interpretation of data generated by these assays, and 3) summarize the strengths and limitations of these assays.