ABSTRACT:Suppression of plasminogen activation and/or plasmin activity (PA) reduces blood loss and decreases hemorrhage-related death. However, whether the endogenous PA system is a biological mechanism to prevent intravascular thrombus formation is debated, and the potential that reduced PA may increase venous thrombosis/thromboembolism (VTE) risk cautions against the use of antifibrinolytic agents. We aimed to determine the contribution of PA to VTE. Type 1 plasminogen-deficient humans enrolled in the Hypoplasminogenemia International Retrospective and Prospective Cohort Study registry reported pathologic pseudomembrane formation, but not unprovoked VTE. When subjected to an experimental model of venous thrombosis, compared with Plg+/+ mice, neither partial (Plg+/-) nor complete (Plg-/-) deletion of plasminogen altered thrombus mass or thrombus nucleated cell, platelet, or fibrin(ogen) content at 24 or 6 hours after thrombus induction. Administration of tranexamic acid (TXA) to mouse plasma in vitro or healthy mice in vivo dose-dependently delayed and suppressed plasma plasmin generation for up to 3 hours. However, mice administered TXA did not have significantly altered thrombus mass or thrombus composition at 24 or 6 hours after thrombus induction, despite unexpectedly persistent TXA in plasma. In a genome-wide association study, variants in gene regions encoding PA pathway proteins were not significantly associated with VTE risk. In the UK Biobank repository, plasminogen protein levels were not significantly associated with VTE risk. These data from genetic, pharmacologic, and proteomic analyses of mice and humans indicate that perturbations in PA do not increase VTE risk. Collectively, these results suggest PA is not a molecular regulatory mechanism to protect against VTE. This trial was registered at www.clinicaltrials.gov as #NCT03797495).

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

Introduction: Arterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3-0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma.Materials and methods: Platelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured.Results: APAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition.Conclusions: Intravenous APAC targets arterial injury sites to exert local dual antiplatelet , anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, high-lighting APAC as a novel antithrombotic to reduce cardiovascular complications.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Pathological blood clotting, or thrombosis, limits vital blood flow to organs; such deprivation can lead to catastrophic events including myocardial infarction, pulmonary embolism, and ischemic stroke. Prompt restoration of blood flow greatly improves outcomes. We explored whether aptamers could serve as molecular imaging probes to rapidly detect thrombi. An aptamer targeting thrombin, Tog25t, was found to rapidly localize to and visualize pre-existing clots in the femoral and jugular veins of mice using fluorescence imaging and, when circulating, was able to image clots as they form. Since free aptamer is quickly cleared from circulation, contrast is rapidly developed, allowing clot visualization within minutes. Moreover, administration of an antidote oligonucleotide further enhanced contrast development, causing the unbound aptamer to clear within 5min while impacting the clot-bound aptamer more slowly. These findings suggest that aptamers can serve as imaging agents for rapid detection of thrombi in acute care and perioperative settings.

Platelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein-coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet-leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet-neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress.

Pancreatic cancer patients have a high risk of venous thromboembolism (VTE). Plasminogen activator inhibitor 1 (PAI-1) inhibits plasminogen activators and increases the risk of thrombosis. PAI-1 is expressed by pancreatic tumors and human pancreatic cell lines. However, to date, there are no studies analyzing the association of active PAI-1 and VTE in pancreatic cancer patients. We investigated the association of active PAI-1 in plasma and VTE in pancreatic cancer patients. In addition, we determined if the presence of human pancreatic tumors expressing PAI-1 impairs venous thrombus resolution in mice. Plasma levels of active PAI-1 in patients with pancreatic cancer and mice bearing human tumors were determined by enzyme-linked immunosorbent assay. We measured PAI-1 expression in 5 different human pancreatic cancer cell lines and found that PANC-1 cells expressed the highest level. PANC-1 tumors were grown in nude mice. Venous thrombosis was induced by complete ligation of the inferior vena cava (IVC). Levels of active PAI-1 were independently associated with increased risk of VTE in patients with pancreatic cancer (subdistribution hazard ratio per doubling of levels: 1.39 [95% confidence interval, 1.09-1.78], P = .007). Mice bearing PANC-1 tumors had increased levels of both active human and active mouse PAI-1 and decreased levels of plasmin activity. Importantly, mice bearing PANC-1 tumors exhibited impaired venous thrombus resolution 8 days after IVC stasis compared with nontumor controls. Our results suggest that PAI-1 contributes to VTE in pancreatic cancer.

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice.

Background: Venous thrombosis (VT) and pulmonary embolism (PE), collectively venous thromboembolism (VIE), cause high mortality and morbidity. Factor XIII (FXIII) crosslinks fibrin to enhance thrombus stability and consequently may influence PE risk. Elucidating mechanisms contributing to PE is limited by a lack of models that recapitulate human PE characteristics. Objective: We aimed to develop a mouse model that permits embolization of red blood cell (RBC)- and fibrin-rich VT and determine the contribution of FXIII to PE risk. Methods and Results: In a thrombin-infusion PE model, F13a(+/+), F13a(+/-), and F13a(-/-) mice had similar incidence of microthrombi in the lungs; however, thrombi were small, with low RBC content (<= 7%), unlike human PEs (similar to 70%). To identify a model producing PE consistent with histological characteristics of human PE, we compared mouse femoral vein electrolytic injury, femoral vein FeCl3 injury, and infrarenal vena cava (IVC) stasis models of VT. Electrolytic and FeCl3 models produced small thrombi with few RBCs (5% and 4%, respectively), whereas IVC stasis produced large thrombi with higher RBC content (68%) that was similar to human PEs. After IVC stasis and ligature removal (de-ligation) to permit thrombus embolization, compared to F13a(+/+) mice, F13a(+/-) and F13a(-/-) mice had similar and increased PE incidence, respectively. Conclusions: Compared to thrombin infusion-, electrolytic injury-, and FeCl3-based models. IVC stasis produces thrombi that are more histologically similar to human thrombi. IVC stasis followed by de-ligation permits embolization of existing RBC- and fibrin-rich thrombi. Complete FXIII deficiency increases PE incidence, but partial deficiency does not.

Lack of long-term patency has hindered the clinical use of small-diameter prosthetic vascular grafts with the majority of these failures due to the development of neointimal hyperplasia. Previous studies by our laboratory revealed that small-diameter expanded polytetrafluoroethylene (ePTFE) grafts coated with antioxidant elastomers are a promising localized therapy to inhibit neointimal hyperplasia. This work is focused on the development of poly(diol-co-citrate-co-ascorbate) (POCA) elastomers with tunable properties for coating ePTFE vascular grafts. A bioactive POCA elastomer (@20 : 20 : 8, [citrate] : [diol] : [ascorbate]) coating was applied on a 1.5 mm diameter ePTFE vascular graft as the most promising therapeutic candidate for reducing neointimal hyperplasia. Surface ascorbate density on the POCA elastomer was increased to 67.5 ± 7.3 ng mg-1 cm-2. The mechanical, antioxidant, biodegradable, and biocompatible properties of POCA demonstrated desirable performance for in vivo use, inhibiting human aortic smooth muscle cell proliferation, while supporting human aortic endothelial cells. POCA elastomer coating number was adjusted by a modified spin-coating method to prepare small-diameter ePTFE vascular grafts similar to natural vessels. A significant reduction in neointimal hyperplasia was observed after implanting POCA-coated ePTFE vascular grafts in a guinea pig aortic interposition bypass graft model. POCA elastomer thus offers a new avenue that shows promise for use in vascular engineering to improve long-term patency rates by coating small-diameter ePTFE vascular grafts.

Targeted drug delivery for maintaining blood fluidity can reduce the risks associated with systemic anticoagulants that can lead to off-target bleeding. Recently, there has been much interest in targeted delivery of tissue-type plasminogen activator (tPA) for treating thrombotic complications. The work presented here characterizes a fibrin-specific nanogel (FSN) design for targeted delivery of tPA to treat thrombotic complications. Fibrin binding and clot degradation were characterized in vitro, and animal models of thrombosis were used to examine nanogel effects on coagulation parameters. In vitro assays showed tPA-FSNs attach to fibrin in a dose-dependent manner independent of tPA loading. In animal models of thrombosis, including an electrolytic injury to monitor clot properties in real time, and a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) animal model, tPA-FSNs modulated fibrin/fibrinogen and platelet incorporation into clots and at optimized dosing could recover consumptive coagulopathy in DIC. Distribution of unloaded and tPA-loaded FSNs showed potential clearance of tPA-FSNs after 24 h, although unloaded FSNs may be retained at sites of fibrin deposits. Maximum tolerated dose studies showed tPA-FSNs have minimal toxicity up to 20 times the optimized therapeutic dose. Overall, these studies demonstrate the therapeutic efficacy of targeted fibrinolysis for systemic microthrombi and begin to evaluate key translational parameters for tPA-FSN therapeutics, including optimal tPA-FSN dosage in a DIC rodent model and safety of intravenous tPA-FSN therapeutics.

Background The intrinsic pathway factors (F) XII and FXI have been shown to contribute to thrombosis in animal models. We assessed the role of FXII and FXI in venous thrombosis in three distinct mouse models. Methods Venous thrombosis was assessed in mice genetically deficient for either FXII or FXI. Three models were used: the inferior vena cava (IVC) stasis, IVC stenosis, and femoral vein electrolytic injury models. Results In the IVC stasis model, FXII and FXI deficiency did not affect the size of thrombi but their absence was associated with decreased levels of fibrin(ogen) and an increased level of the neutrophil extracellular trap marker citrullinated histone H3. In contrast, a deficiency of either FXII or FXI resulted in a significant and equivalent reduction in thrombus weight and incidence of thrombus formation in the IVC stenosis model. Thrombi formed in the IVC stenosis model contained significantly higher levels of citrullinated histone H3 compared with the thrombi formed in the IVC stasis model. Deletion of either FXII or FXI also resulted in a significant and equivalent reduction in both fibrin and platelet accumulation in the femoral vein electrolytic injury model. Conclusions Collectively, these data indicate that FXII and FXI contribute to the size of venous thrombosis in models with blood flow and thrombus composition in a stasis model. This study also demonstrates the importance of using multiple mouse models to assess the role of a given protein in venous thrombosis.

Background: Plasma coagulation Factor XII (FXII) plays a crucial role in contact activation, ultimately regulating both the kinin-kallikrein system and the intrinsic pathway of coagulation. A growing body of evidence suggests that inhibition of FXII can prevent thrombosis. Given FXII does not appear to modulate hemostasis, targeting FXII is a promising strategy for the prevention of pathological thrombus formation without the hemostatic risks typically associated with anticoagulants. To this end, a subcutaneously administered investigational RNAi therapeutic targeting liver F12 mRNA (ALN-F12) was developed. Aim: To investigate the thrombo-protective and hemostatic effects of FXII reduction by ALN-F12 in rodent thrombosis and hemostasis models. Methods: A single dose of ALN-F12 was subcutaneously administered to C57Bl/6 mice. After reaching steady state FXII reduction, the impact on thrombosis (ferric chloride arterial thrombosis and electrolytic injury induced venous thrombosis models) and hemostasis (saphenous vein injury and tail tip transection bleeding models) was evaluated. Result: Administration of ALN-F12 resulted in dose-dependent reductions of both liver F12 mRNA and plasma FXII protein. In mice, ALN-F12 led to dose-dependent reductions in platelet and fibrin accumulation in the venous electrolytic-injury model and in the time to occlusion in the ferric chloride arterial thrombosis model. At 10 mg/kg ALN-F12, the top dose level evaluated, this resulted in > 95% reduction of FXII and similar to 10 fold reduction in fibrin deposition. Finally, hemostasis models showed that > 95% reduction of FXII had no impact on bleeding time or blood loss. Conclusion: Our findings support that reduction of plasma Factor XII by ALN-F12 provided thrombo-protective effects with no increased bleeding risk in rodent models of thrombosis and hemostasis.

BACKGROUND:The compositions of venous (red blood cell-rich) and arterial (platelet-rich) thrombi are mediated by distinct pathophysiologic processes; however, fibrin is a major structural component of both. The transglutaminase factor XIII (FXIII) stabilizes fibrin against mechanical and biochemical disruption and promotes red blood cell retention in contracted venous thrombi. Previous studies have shown factor XIII (FXIII) inhibition decreases whole blood clot mass and therefore, may be a therapeutic target for reducing venous thrombosis. The role of FXIII in arterial thrombogenesis is less studied, and the particular contribution of platelet FXIII remains unresolved.OBJECTIVE:To determine whether FXIII reduction prevents experimental arterial thrombogenesis.METHODS:Using wild-type mice and mice with genetically imposed deficiency in FXIII, we measured thrombus formation and stability following ferric chloride-induced arterial thrombosis. We also determined the impact of FXIII on the mass of contracted platelet-rich plasma clots.RESULTS:Following vessel injury, F13a+/+ , F13a+/- , and F13a-/- mice developed occlusive arterial thrombi. FXIII deficiency did not significantly reduce the incidence or prolong the time to occlusion. FXIII deficiency also did not alter the timing of reflow events or decrease platelet-rich clot mass.CONCLUSIONS:FXIII does not significantly alter the underlying pathophysiology of experimental arterial thrombus formation.

BACKGROUND:The timing for initiation of effective antithrombotic therapy relative to the onset of arterial thrombosis may influence outcomes. This report investigates the hypothesis that early administration of heparin anticoagulation relative to the onset of thrombotic occlusion will effect a reduction in occlusion. METHODS:A standard rat model of experimental thrombosis induction was used, injuring the carotid artery exposure with FeCl3-saturated filter paper, followed by flow monitoring for onset of occlusion and subsequent embolization events. Intravenous heparin administration (200 units/mL) was timed relative to the initiation of injury or onset of near occlusion, compared with controls (no heparin administration). RESULTS:No occlusion was found for delivery of heparin 5 min prior to thrombus induction, whereas all vessels occluded without heparin. Unstable (embolic) thrombi were seen with heparin given at or shortly after initial occlusion. Only 9% (1/11) of the vessels had permanent occlusion when heparin was given at the time of thrombotic onset (p < 0.0001 vs. unheparinized), while 50% occluded when heparin was delayed by 5 min (p > 0.05). CONCLUSIONS:These findings provide evidence that antithrombotic therapy may need to be administered prior to the onset of anticipated loss of patency, with less effectiveness when given after occlusion has occurred.

Heparan sulfate (HS) is a sulfated glycosaminoglycan abundant on the cell surface and in the extracellular matrix and has several biological activities including anticoagulation and anti-inflammation. Liver ischemia reperfusion injury is associated with coagulation and inflammatory responses. Here, we synthesized HS oligosaccharides with defined sulfation patterns and show that synthetic anticoagulant HS oligosaccharides limit liver ischemia reperfusion injury in a mouse model. Using a small targeted HS library, we demonstrate that an oligosaccharide that possesses both anticoagulant activity and binding affinity to HMGB1, the inflammatory target, decreases injury greater than oligosaccharides that only bind to HMGB1 or only have anticoagulant activity. HS oligosaccharides may represent a potential new therapeutic option for decreasing liver damage resulting from ischemia reperfusion injury.