Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype.

Host defense and inflammation are regulated by the NF-κB essential modulator (NEMO), a scaffolding protein with a broad immune cell and tissue expression profile. Hypomorphic mutations in inhibitor of NF-κB kinase regulatory subunit gamma (IKBKG) encoding NEMO typically present with immunodeficiency. Here, we characterized a pediatric autoinflammatory syndrome in 3 unrelated male patients with distinct X-linked IKBKG germline mutations that led to overexpression of a NEMO protein isoform lacking the domain encoded by exon 5 (NEMO-Δex5). This isoform failed to associate with TANK binding kinase 1 (TBK1), and dermal fibroblasts from affected patients activated NF-κB in response to TNF but not TLR3 or RIG-I-like receptor (RLR) stimulation when isoform levels were high. By contrast, T cells, monocytes, and macrophages that expressed NEMO-Δex5 exhibited increased NF-κB activation and IFN production, and blood cells from these patients expressed a strong IFN and NF-κB transcriptional signature. Immune cells and TNF-stimulated dermal fibroblasts upregulated the inducible IKK protein (IKKi) that was stabilized by NEMO-Δex5, promoting type I IFN induction and antiviral responses. These data revealed how IKBKG mutations that lead to alternative splicing of skipping exon 5 cause a clinical phenotype we have named NEMO deleted exon 5 autoinflammatory syndrome (NDAS), distinct from the immune deficiency syndrome resulting from loss-of-function IKBKG mutations.

Skeletal muscle myosin promotes coagulation by binding factor XI via its A3 domain and enhancing thrombin-induced factor XI activation.

Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (Kd = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.

Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs.

Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

Use of bilobed partial resuscitative endovascular balloon occlusion of the aorta is logistically superior in prolonged management of a highly lethal aortic injury.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a viable technique for management of noncompressible torso hemorrhage. The major limitation of the current unilobed fully occlusive REBOA catheters is below-the-balloon ischemia-reperfusion complications. We hypothesized that partial aortic occlusion with a novel bilobed partial (p)REBOA-PRO would result in the need for less intraaortic balloon adjustments to maintain a distal goal perfusion pressure as compared with currently available unilobed ER-REBOA. METHODS: Anesthetized (40-50 kg) swine randomized to control (no intervention), ER-REBOA, or pREBOA-PRO underwent supraceliac aortic injury. The REBOA groups underwent catheter placement into zone 1 with initial balloon inflation to full occlusion for 10 minutes followed by gradual deflation to achieve and subsequently maintain half of the baseline below-the-balloon mean arterial pressure (MAP). Physiologic data and blood samples were collected at baseline and then hourly. At 4 hours, the animals were euthanized, total blood loss and urine output were recorded, and tissue samples were collected. RESULTS: Baseline physiologic data and basic laboratories were similar between groups. Compared with control, interventions similarly prolonged survival from a median of 18 minutes to over 240 minutes with comparable mortality trends. Blood loss was similar between partial ER-REBOA (41%) and pREBOA-PRO (51%). Partial pREBOA-PRO required a significantly lower number of intraaortic balloon adjustments (10 ER-REBOA vs. 3 pREBOA-PRO, p < 0.05) to maintain the target below-the-balloon MAP. The partial ER-REBOA group developed significantly increased hypercapnia, fibrin clot formation on TEG, liver inflammation, and IL-10 expression compared with pREBOA-PRO. CONCLUSION: In this highly lethal aortic injury model, use of bilobed pREBOA-PRO for a 4-hour partial aortic occlusion was logistically superior to unilobed ER-REBOA. It required less intraaortic balloon adjustments to maintain target MAP and resulted in less inflammation.

Management of Anticoagulation in Pregnant Women With Mechanical Heart Valves.

IMPORTANCE: Mechanical heart valves (MHVs) pose significant thrombogenic risks to pregnant women and their fetuses, yet the choice of anticoagulation in this clinical setting remains unclear. Various therapeutic strategies carry distinct risk profiles that must be considered when making the decision about optimal anticoagulation. OBJECTIVE: We sought to review existing data and offer recommendations for the anticoagulation of pregnant women with MHVs, as well as management of anticoagulation in the peripartum period. EVIDENCE ACQUISITION: We performed a literature review of studies examining outcomes in pregnant women receiving systemic anticoagulation for mechanical valves, and also reviewed data on the safety profiles of various anticoagulant strategies in the setting of pregnancy. RESULTS: Warfarin has been shown to increase rates of embryopathy and fetal demise, although it has traditionally been the favored anticoagulant in this setting. Low-molecular-weight heparin, when dosed appropriately with close therapeutic monitoring, has been shown to be safe for both mother and fetus. CONCLUSIONS: We favor the use of low-molecular-weight heparin with appropriate dosing and monitoring for the anticoagulation of pregnant women with MHVs. Data suggest that this approach minimizes the thrombotic risk associated with the valve while also providing safe and effective anticoagulation that can be easily managed in the peripartum period.

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.

Long-term remission rates after splenectomy in adults with Evans syndrome compared to immune thrombocytopenia: A single-center retrospective study.

OBJECTIVE: Evans syndrome, the combination of immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) or autoimmune neutropenia, is associated with a high rate of relapsed/refractory disease. There are limited data on the efficacy of splenectomy for this condition. We reviewed patient outcomes after splenectomy for Evans syndrome compared to ITP at our institution. METHODS: We performed a retrospective analysis of patients who underwent splenectomy for autoimmune cytopenias over a 23-year period with the intention of comparing disease relapse rates after splenectomy in patients with Evans syndrome and in those with ITP. RESULTS: During the study period, 77 patients underwent splenectomy for ITP and seven underwent splenectomy for Evans syndrome. In the Evans cohort, splenectomy led to an 85.7% initial response rate with a 42.8% rate of relapse within one year and a long-term (one-year) response rate of 42.8%. In the ITP cohort, the initial response rate was 90.9% with a long-term response rate of 70.1%. CONCLUSION: Our data suggest that long-term remission rates after splenectomy are lower in adults with Evans syndrome compared to those with ITP, although splenectomy may still be an acceptable treatment for certain patients with Evans syndrome. Our findings underscore the need for further research and development of additional therapeutic strategies for this patient population.

Cardiac and Skeletal Muscle Myosin Exert Procoagulant Effects.

INTRODUCTION: Trauma-induced coagulopathy (TIC) and the tissue injury-provoked procoagulant profile are prevalent in severely injured patients, but their mechanisms remain unclear. Myosin, exposed by or released from tissue injury, may play a role in promoting thrombin generation and attenuating fibrinolysis. The objective of the study is to examine the effects of cardiac and skeletal muscle myosins on coagulation in whole blood using thrombelastography (TEG). MATERIALS AND METHODS: Whole blood was collected from healthy adult volunteers (n=8) and native TEGs were performed to evaluate the global coagulation response in the presence of cardiac or skeletal muscle myosin by measuring reaction (R) time (minutes), clot angle (), and maximum amplitude (MA, mm). TEG measurements were compared using paired t tests. RESULTS: Cardiac and skeletal muscle myosins decreased R, from 10.8 min to 8.0 min (P<0.0001) and 6.9 min (P =0.0007), respectively. There were no effects observed on clot propagation (angle) or clot strength (MA) with myosin addition. In the presence of tPA, both cardiac and skeletal muscle myosins shortened R from 11.1 min to 8.62 min (P=0.0245) and 7.75 min (P =0.0027), respectively), with no changes on angle or MA. CONCLUSIONS: Cardiac and skeletal muscle myosins exhibit procoagulant effects in TEG assays. These whole blood TEG results support the hypothesis that cardiac and skeletal muscle myosins may be either pro-hemostatic or prothrombotic depending on context.

The contact pathway and sepsis.

The contact pathway factors XI (FXI) and XII (FXII) have been demonstrated to be largely dispensable for hemostasis, as their absence results in a mild to absent bleeding diathesis. A growing body of literature, however, suggests that the contact pathway contributes to the pathologic host response to certain infectious organisms that produces the often-fatal syndrome known as sepsis. The contact pathway factors serve as a central node connecting inflammation to coagulation, and may offer a potentially safe therapeutic target to mitigate the morbidity and mortality associated with sepsis. Herein, we summarize published in vivo and in vitro data that have explored the roles of the contact pathway in sepsis, and discuss potential clinical applications of novel FXI- and FXII-inhibiting drugs currently under investigation.

The role of coagulation and platelets in colon cancer-associated thrombosis.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Effect of Pneumatic Tubing System Transport on Platelet Apheresis Units.

Platelet apheresis units are transfused into patients to mitigate or prevent bleeding. In a hospital, platelet apheresis units are transported from the transfusion service to the healthcare teams via two methods: a pneumatic tubing system (PTS) or ambulatory transport. Whether PTS transport affects the activity and utility of platelet apheresis units is unclear. We quantified the gravitational forces and transport time associated with PTS and ambulatory transport within our hospital. Washed platelets and supernatants were prepared from platelet apheresis units prior to transport as well as following ambulatory or PTS transport. For each group, we compared resting and agonist-induced platelet activity and platelet aggregate formation on collagen or von Willebrand factor (VWF) under shear, platelet VWF-receptor expression and VWF multimer levels. Subjection of platelet apheresis units to rapid acceleration/deceleration forces during PTS transport did not pre-activate platelets or their ability to activate in response to platelet agonists as compared to ambulatory transport. Platelets within platelet apheresis units transported via PTS retained their ability to adhere to surfaces of VWF and collagen under shear, although platelet aggregation on collagen and VWF was diminished as compared to ambulatory transport. VWF multimer levels and platelet GPIb receptor expression was unaffected by PTS transport as compared to ambulatory transport. Subjection of platelet apheresis units to PTS transport did not significantly affect the baseline or agonist-induced levels of platelet activation as compared to ambulatory transport. Our case study suggests that PTS transport may not significantly affect the hemostatic potential of platelets within platelet apheresis units.

Design and Utility of a Point-of-Care Microfluidic Platform to Assess Hematocrit and Blood Coagulation.

PURPOSE­: To develop a small volume whole blood analyzer capable of measuring the hematocrit and coagulation kinetics of whole blood. METHODS AND RESULTS­: A co-planar microfluidic chamber designed to facilitate self-driven capillary action across an internal electrical chip was developed and used to measure the electric parameters of whole human blood that had been anticoagulated or allowed to clot. To promote blood clotting, select chip surfaces were coated with a prothrombin time (PT) reagent containing lipidated tissue factor (TF), which activates the extrinsic pathway of coagulation to promote thrombin generation and fibrin formation. Whole human blood was added to the microfluidic device, and voltage changes within the platform were measured and interpreted using basic resistor-capacitor (RC) circuit and fluid dynamics theory. Upon wetting of the sensing zone, a circuit between two co-planar electrodes within the sensing zone was closed to generate a rapid voltage drop from baseline. The voltage then rose due to sedimentation of red blood cells (RBC) in the sensing zone. For anticoagulated blood samples, the time for the voltage to return to baseline was dependent on hematocrit. In the presence of coagulation, the initiation of fibrin formation in the presence of the PT reagent prevented the return of voltage to baseline due to the reduced packing of RBCs in the sensing zone. CONCLUSIONS­: The technology presented in this study has potential for monitoring the hematocrit and coagulation parameters of patient samples using a small volume of whole blood, suggesting it may hold clinical utility as a point-of-care test.

Dynamics of blood flow and thrombus formation in a multi-bypass microfluidic ladder network.

The reaction dynamics of a complex mixture of cells and proteins, such as blood, in branched circulatory networks within the human microvasculature or extravascular therapeutic devices such as extracorporeal oxygenation machine (ECMO) remains ill-defined. In this report we utilize a multi-bypass microfluidics ladder network design with dimensions mimicking venules to study patterns of blood platelet aggregation and fibrin formation under complex shear. Complex blood fluid dynamics within multi-bypass networks under flow were modeled using COMSOL. Red blood cells and platelets were assumed to be non-interacting spherical particles transported by the bulk fluid flow, and convection of the activated coagulation factor II, thrombin, was assumed to be governed by mass transfer. This model served as the basis for predicting formation of local shear rate gradients, stagnation points and recirculation zones as dictated by the bypass geometry. Based on the insights from these models, we were able to predict the patterns of blood clot formation at specific locations in the device. Our experimental data was then used to adjust the model to account for the dynamical presence of thrombus formation in the biorheology of blood flow. The model predictions were then compared to results from experiments using recalcified whole human blood. Microfluidic devices were coated with the extracellular matrix protein, fibrillar collagen, and the initiator of the extrinsic pathway of coagulation, tissue factor. Blood was perfused through the devices at a flow rate of 2 µL/min, translating to physiologically relevant initial shear rates of 300 and 700 s-1 for main channels and bypasses, respectively. Using fluorescent and light microscopy, we observed distinct flow and thrombus formation patterns near channel intersections at bypass points, within recirculation zones and at stagnation points. Findings from this proof-of-principle ladder network model suggest a specific correlation between microvascular geometry and thrombus formation dynamics under shear. This model holds potential for use as an integrative approach to identify regions susceptible to intravascular thrombus formation within the microvasculature as well as extravascular devices such as ECMO.

Utility of microfluidic devices to study the platelet-endothelium interface.

The integration of biomaterials and understanding of vascular biology has led to the development of perfusable endothelialized flow models, which have been used as valuable tools to study the platelet-endothelium interface under shear. In these models, the parameters of geometry, compliance, biorheology, and cellular complexity are varied to recapitulate the physical biology of platelet recruitment and activation under physiologically relevant conditions of blood flow. In this review, we summarize the mechanistic insights learned from perfusable microvessel models and discuss the potential utility as well as challenges of endothelialized microfluidic devices to study platelet function in the bloodstream in vitro.

Removal of the C-Terminal Domains of ADAMTS13 by Activated Coagulation Factor XI induces Platelet Adhesion on Endothelial Cells under Flow Conditions.

Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a "stringlike" conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.

Differential Roles for the Coagulation Factors XI and XII in Regulating the Physical Biology of Fibrin.

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.

Prothrombotic skeletal muscle myosin directly enhances prothrombin activation by binding factors Xa and Va.

To test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s-1 shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent Kd of myosin for factor Xa (Kd ∼0.48 nM), primarily by reducing koff, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas (P = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.