Targeting factor XI and factor XIa to prevent thrombosis.

ABSTRACT: Direct oral anticoagulants (DOACs) that inhibit the coagulation proteases thrombin or factor Xa (FXa) have replaced warfarin and other vitamin K antagonists (VKAs) for most indications requiring long-term anticoagulation. In many clinical situations, DOACs are as effective as VKAs, cause less bleeding, and do not require laboratory monitoring. However, because DOACs target proteases that are required for hemostasis, their use increases the risk of serious bleeding. Concerns over therapy-related bleeding undoubtedly contribute to undertreatment of many patients who would benefit from anticoagulation therapy. There is considerable interest in the plasma zymogen factor XI (FXI) and its protease form factor XIa (FXIa) as drug targets for treating and preventing thrombosis. Laboratory and epidemiologic studies support the conclusion that FXI contributes to venous and arterial thrombosis. Based on 70 years of clinical observations of patients lacking FXI, it is anticipated that drugs targeting this protein will cause less severe bleeding than warfarin or DOACs. In phase 2 studies, drugs that inhibit FXI or FXIa prevent venous thromboembolism after total knee arthroplasty as well as, or better than, low molecular weight heparin. Patients with heart disease on FXI or FXIa inhibitors experienced less bleeding than patients taking DOACs. Based on these early results, phase 3 trials have been initiated that compare drugs targeting FXI and FXIa to standard treatments or placebo. Here, we review the contributions of FXI to normal and abnormal coagulation and discuss results from preclinical, nonclinical, and clinical studies of FXI and FXIa inhibitors.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

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 αMß2 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.

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.

The contact activation inhibitor AB023 in heparin-free hemodialysis: results of a randomized phase 2 clinical trial.

End-stage renal disease (ESRD) patients on chronic hemodialysis have repeated blood exposure to artificial surfaces that can trigger clot formation within the hemodialysis circuit. Dialyzer clotting can lead to anemia despite erythropoietin and iron supplementation. Unfractionated heparin prevents clotting during hemodialysis, but it is not tolerated by all patients. Although heparin-free dialysis is performed, intradialytic blood entrapment can be problematic. To address this issue, we performed a randomized, double-blind, phase 2 study comparing AB023, a unique antibody that binds factor XI (FXI) and blocks its activation by activated FXII, but not by thrombin, to placebo in 24 patients with ESRD undergoing heparin-free hemodialysis. Patients were randomized to receive a single predialysis dose of AB023 (0.25 or 0.5 mg/kg) or placebo in a 2:1 ratio, and safety and preliminary efficacy were compared with placebo and observations made prior to dosing within each treatment arm. AB023 administration was not associated with impaired hemostasis or other drug-related adverse events. Occlusive events requiring hemodialysis circuit exchange were less frequent and levels of thrombin-antithrombin complexes and C-reactive protein were lower after AB023 administration compared with data collected prior to dosing. AB023 also reduced potassium and iron entrapment in the dialyzers, consistent with less blood accumulation within the dialyzers. We conclude that despite the small sample size, inhibition of contact activation-induced coagulation with AB023 was well tolerated and reduced clotting within the dialyzer. This trial was registered at www.clinicaltrials.gov as #NCT03612856.

Factor XII plays a pathogenic role in organ failure and death in baboons challenged with Staphylococcus aureus.

Activation of coagulation factor (F) XI promotes multiorgan failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus aureus. Here we used the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared with untreated control animals, repeated 5C12 administration before and at 8 and 24 hours after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa, and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes was also prevented. D-dimer levels exhibited a profound increase in the untreated animals but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1ß, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress, and multiorgan failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, whereas untreated control animals suffered irreversible multiorgan failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least 2 enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S aureus.

Cross-Talk between the Complement Pathway and the Contact Activation System of Coagulation: Activated Factor XI Neutralizes Complement Factor H.

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.

The protein C activator AB002 rapidly interrupts thrombus development in baboons.

Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.

Role of platelets in regulating activated coagulation factor XI activity.

Factor XI (FXI) has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, whereas FXI is not essential for hemostasis but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in a purified system and increased coagulation Factor IX (FIX) activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by activated coagulation factor XII (FXIIa) and the activation of FXII by kallikrein (PKa). Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets, the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa [by FXIa-platelet membrane immunoprecipitation] showed that the interaction with platelets is zinc dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib. FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.

A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Thrombin generation and activity in multiple sclerosis.

The coagulation cascade and immune system are intricately linked, highly regulated and respond cooperatively in response to injury and infection. Increasingly, evidence of hyper-coagulation has been associated with autoimmune disorders, including multiple sclerosis (MS). The pathophysiology of MS includes immune cell activation and recruitment to the central nervous system (CNS) where they degrade myelin sheaths, leaving neuronal axons exposed to damaging inflammatory mediators. Breakdown of the blood-brain barrier (BBB) facilitates the entry of peripheral immune cells. Evidence of thrombin activity has been identified within the CNS of MS patients and studies using animal models of experimental autoimmune encephalomyelitis (EAE), suggest increased thrombin generation and activity may play a role in the pathogenesis of MS as well as inhibit remyelination processes. Thrombin is a serine protease capable of cleaving multiple substrates, including protease activated receptors (PARs), fibrinogen, and protein C. Cleavage of all three of these substrates represent pathways through which thrombin activity may exert immuno-regulatory effects and regulate permeability of the BBB during MS and EAE. In this review, we summarize evidence that thrombin activity directly, through PARs, and indirectly, through fibrin formation and activation of protein C influences neuro-immune responses associated with MS and EAE pathology.

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways.

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI).

Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results- Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa-PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa-PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions- PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa-PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview- An online visual overview is available for this article.

Contact Activation Inhibitor and Factor XI Antibody, AB023, Produces Safe, Dose-Dependent Anticoagulation in a Phase 1 First-In-Human Trial.

Objective- Factor XI (FXI) contributes to thrombotic disease while playing a limited role in normal hemostasis. We generated a unique, humanized anti-FXI antibody, AB023, which blocks factor XIIa-mediated FXI activation without inhibiting FXI activation by thrombin or the procoagulant function of FXIa. We sought to confirm the antithrombotic activity of AB023 in a baboon thrombosis model and to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adult subjects. Approach and Results- In a primate model of acute vascular graft thrombosis, AB023 reduced platelet and fibrin accumulation within the grafts by >75%. To evaluate the safety of AB023, we performed a first-in-human study in healthy adult volunteers without any serious adverse events. Overall, 10 of 21 (48%) subjects experienced 20 treatment-emergent adverse events, with 7 of 16 (44%) subjects following active treatment and 3 of 5 (60%) subjects following placebo. AB023 did not increase bleeding or prothrombin times. Anticoagulation was verified by a saturable ≈2-fold prolongation of the partial thromboplastin time for over 1 month after the highest dose. Conclusions- AB023, which inhibits contact activation-initiated blood coagulation in vitro and experimental thrombus formation in primates, produced a dose-dependent duration of limited anticoagulation without drug-related adverse effects in a phase 1 trial. When put in context with earlier observations suggesting that FXI contributes to venous thromboembolism and cardiovascular disease, although contributing minimally to hemostasis, our data further justify clinical evaluation of AB023 in conditions where contact-initiated FXI activation is suspected to have a pathogenic role. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT03097341.

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.

Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex.

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.

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.

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.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor.

Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI.

Factor XI as a Therapeutic Target.

Factor XIa is a plasma serine protease that contributes to thrombin generation primarily through proteolytic activation of factor IX. Traditionally considered part of the intrinsic pathway of coagulation, several lines of evidence now suggest that factor XIa serves as an interface between the vitamin-K-dependent thrombin generation mechanism and the proinflammatory kallikrein-kinin system, allowing the 2 systems to influence each other. Work with animal models and results from epidemiological surveys of human populations support a role for factor XIa in thromboembolic disease. These data and the clinical observation that deficiency of factor XI, the zymogen of factor XIa, produces a relatively mild bleeding disorder suggest that drugs targeting factor XI or XIa could produce an antithrombotic effect while leaving hemostasis largely intact. Results of a recent trial comparing antisense-induced factor XI reduction to standard-dose low molecular-weight heparin as prophylaxis for venous thrombosis during knee replacement are encouraging in this regard. Here, we discuss recent findings on the biochemistry, physiology, and pathology of factor XI as they relate to thromboembolic disease.