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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.
© 2015 by The American Society of Hematology.

BACKGROUND - Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that specifically reduces factor XI levels. We compared the efficacy and safety of FXI-ASO with those of enoxaparin in patients undergoing total knee arthroplasty.
METHODS - In this open-label, parallel-group study, we randomly assigned 300 patients who were undergoing elective primary unilateral total knee arthroplasty to receive one of two doses of FXI-ASO (200 mg or 300 mg) or 40 mg of enoxaparin once daily. The primary efficacy outcome was the incidence of venous thromboembolism (assessed by mandatory bilateral venography or report of symptomatic events). The principal safety outcome was major or clinically relevant nonmajor bleeding.
RESULTS - Around the time of surgery, the mean (±SE) factor XI levels were 0.38±0.01 units per milliliter in the 200-mg FXI-ASO group, 0.20±0.01 units per milliliter in the 300-mg FXI-ASO group, and 0.93±0.02 units per milliliter in the enoxaparin group. The primary efficacy outcome occurred in 36 of 134 patients (27%) who received the 200-mg dose of FXI-ASO and in 3 of 71 patients (4%) who received the 300-mg dose of FXI-ASO, as compared with 21 of 69 patients (30%) who received enoxaparin. The 200-mg regimen was noninferior, and the 300-mg regimen was superior, to enoxaparin (P<0.001). Bleeding occurred in 3%, 3%, and 8% of the patients in the three study groups, respectively.
CONCLUSIONS - This study showed that factor XI contributes to postoperative venous thromboembolism; reducing factor XI levels in patients undergoing elective primary unilateral total knee arthroplasty was an effective method for its prevention and appeared to be safe with respect to the risk of bleeding. (Funded by Isis Pharmaceuticals; FXI-ASO TKA ClinicalTrials.gov number, NCT01713361.).

INTRODUCTION - The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition.
METHODS - MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition.
RESULTS AND CONCLUSIONS - MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.
Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation for bleeding disorders in the context of suspected non-accidental injury (NAI) is inconsistent. We reviewed the hematologic evaluation of children who presented with symptoms of bleeding and/or bruising suspicious for NAI to determine the frequency of hematologic tests, abnormal hematologic laboratory results, and hematologic diagnoses. A retrospective cohort study design was employed at two freestanding academic children's hospitals. ICD-9 codes for NAI were used to identify 427 evaluable patients. Medical records were queried for the details of clinical and laboratory evaluations at the initial presentation concerning for NAI. The median age for the population was 326 days (range 1 day-14 years), 58% were male. Primary bleeding symptoms included intracranial hemorrhage (31.8%) and bruising (68.2%). Hematologic laboratory tests performed included complete blood cell count in 62.3%, prothrombin time (PT) in 55.0%, and activated partial thromboplastin time (aPTT) in 53.6%; fibrinogen in 27.6%; factor activity in 17.1%; von Willebrand disease evaluation in 14.5%; and platelet function analyzer in 11.7%. Prolonged laboratory values were seen in 22.5% of PT and 17.4% of aPTT assays; 66.0% of abnormal PTs and 87.5% of abnormal aPTTs were repeated. In our cohort, 0.7% (3 of 427) of the population was diagnosed with a condition predisposing to bleeding. In children with bleeding symptoms concerning for NAI, hemostatic evaluation is inconsistent. Abnormal tests are not routinely repeated, and investigation for the most common bleeding disorder, von Willebrand disease, is rare. Further research into the extent and appropriate timing of the evaluation is warranted.
Copyright © 2014 Elsevier Ltd. All rights reserved.

Anticoagulants currently used in clinical practice to treat thromboembolic disorders are effective but increase the risk of severe bleeding because they target proteins that are essential for normal coagulation (hemostasis). Drugs with better safety profiles are required for prevention and treatment of thromboembolic disease. Coagulation factor XIa has emerged as a novel target for safer anticoagulant therapy because of its role in thrombosis and its relatively small contribution to hemostasis.
Copyright © 2014 Elsevier Ltd. All rights reserved.

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

BACKGROUND - Factor XIa is traditionally assigned a role in FIX activation during coagulation. However, recent evidence suggests this protease may have additional plasma substrates.
OBJECTIVE - To determine whether FXIa promotes thrombin generation and coagulation in plasma in the absence of FIX, and to determine whether FXI-deficiency produces an antithrombotic effect in mice independently of FIX.
METHODS - FXIa, FXIa variants and anti-FXIa antibodies were tested for their effects on plasma coagulation and thrombin generation in the absence of FIX, and for their effects on the activation of purified coagulation factors. Mice with combined FIX and FXI deficiency were compared with mice lacking either FIX or FXI in an arterial thrombosis model.
RESULTS - In FIX-deficient plasma, FXIa induced thrombin generation, and anti-FXIa antibodies prolonged clotting times. This process involved FXIa-mediated conversion of FX and FV to their active forms. Activation of FV by FXIa required the A3 domain on the FXIa heavy chain, whereas activation of FX did not. FX activation by FXIa, unlike FIX activation, was not a calcium-dependent process. Mice lacking both FIX and FXI were more resistant to ferric chloride-induced carotid artery occlusion than FXI-deficient or FIX-deficient mice.
CONCLUSION - In addition to its predominant role as an activator of FIX, FXIa may contribute to coagulation by activating FX and FV. As the latter reactions do not require calcium, they may make important contributions to in vitro clotting triggered by contact activation. The reactions may be relevant to FXIa's roles in hemostasis and in promoting thrombosis.
© 2013 International Society on Thrombosis and Haemostasis.

BACKGROUND - Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin.
AIMS - To determine the importance of FXI ABSs to polyP enhancement of FXI activation.
METHODS - Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model.
RESULTS - In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation.
CONCLUSIONS - The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes.
© 2013 International Society on Thrombosis and Haemostasis.