scholarly journals Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marco Heestermans ◽  
Clément Naudin ◽  
Reiner K. Mailer ◽  
Sandra Konrath ◽  
Kristin Klaetschke ◽  
...  
Keyword(s):  
Partial Thromboplastin Time ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Coagulation Factors ◽  
Activated Partial Thromboplastin Time ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Contact Activation ◽  
Intrinsic Pathway ◽  
Surface Binding

AbstractContact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. 4134-4141 ◽  
Author(s):  
Jessica L. MacQuarrie ◽  
Alan R. Stafford ◽  
Jonathan W. Yau ◽  
Beverly A. Leslie ◽  
Trang T. Vu ◽  
...  
Keyword(s):  
Partial Thromboplastin Time ◽  
Activated Partial Thromboplastin Time ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
High Affinity ◽  
Factor Xiia ◽  
Platelet Release ◽  
Contact Pathway ◽  
Physiologic Role

Abstract Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2μM and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentration-dependent fashion but has no effect on tissue factor–induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn2+, but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa–mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn2+ will promote this interaction.

scholarly journals Discovery and Characterization of a Highly Specific Antibody Inhibitor of Factor XIIa, and the Subsequent Generation of a Factor XIIa/Plasma Kallikrein Bispecific Antibody

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2268-2268
Author(s):  
Shauna D Mason ◽  
Jon Kenniston ◽  
Stephen R Comeau ◽  
Gregory P Conley ◽  
Niksa Kastrapeli ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bispecific Antibody ◽  
Thrombus Formation ◽  
Human Antibody ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Contact Activation ◽  
Single Chain ◽  
Related Sequence ◽  
Factor Xiia

Abstract Factor XII (FXII), also called Hageman Factor, is a key component of the plasma contact system. When blood is exposed to artificial surfaces or polyanionic substances, the zymogens FXII and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and plasma kallikrein, respectively. FXIIa initiates coagulation through the intrinsic pathway by activating factor XI, while plasma kallikrein mediates generation of the potent vasodilator bradykinin. Patients lacking FXII or PK do not experience abnormal bleeding, indicating these proteins are not required for hemostasis. However, FXIIa and plasma kallikrein are required for formation of occlusive clots in animal thrombosis models, and FXIIa likely contributes to thrombus formation in humans when blood is passed through extracorporeal circuits (e.g. cardiopulmonary bypass). These observations suggest that FXIIa inhibition could be an effective antithrombotic strategy that would not have bleeding side effects associated with current approved anticoagulants. We used our human antibody phage display library to identify a highly selective and potent monoclonal antibody inhibitor of FXIIa, DX-4012. DX-4012 inhibits the proteolytic activity of FXIIa with an apparent Ki of ~15 pM, and does not inhibit closely related sequence homologs or other coagulation factors at concentrations up to 1 µM. When tested at 1 µM in human plasma, DX-4012 prolonged the activated partial thromboplastin time (aPTT) 3.4-fold, with no effect on the prothrombin time (PT). In a non-human primate pharmacokinetic study, an intravenous infusion of 10 mg/kg DX-4012 prolonged the aPTT 2.4-fold but had no effect on the PT. Given the importance of plasma kallikrein to FXII activation, we reasoned that the antithrombotic effect of DX-4012 could be augmented by combination with a kallikrein inhibitor. To test this, a variant of DX-4012 was converted into a single chain variable fragment (scFv) and combined with DX-2930, a potent and specific monoclonal antibody inhibitor of plasma kallikrein, to generate a "Morrison format" bispecific antibody. Enzyme inhibition assays determined that the apparent Ki values of the individual anti-kallikrein and anti-FXIIa components of the bispecific antibody were similar to the parent molecules (apparent Ki 389 pM and 73 pM, respectively). In contact-activated dilute plasma, the bispecific antibody was > 5 times more effective at preventing kallikrein generation than a 1:1 combination of DX-4012 and DX-2930, and more than 20-fold more effective than either DX-4012 or DX-2930 alone. Our data indicate that DX-4012, either as a monoclonal antibody or as a component of a bispecific antibody, shows potential as a novel antithrombotic therapy. Simultaneous inhibition of FXIIa and plasma kallikrein may be a uniquely potent method of blocking FXIIa activity through inhibition of the positive feedback loop during contact activation. Disclosures Mason: Dyax Corp: Employment. Kenniston:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Kastrapeli:Dyax Corp: Employment. Kopacz:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Kivaa:Dyax Corp: Employment. Qiu:Dyax Corp: Employment. Faucette:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Tenhoor:Dyax Corp: Employment. Wallisch:Aronora: Employment. Gruber:Aronora, Inc.: Employment, Equity Ownership, Patents & Royalties, Research Funding. Adelman:Dyax Corp: Employment. Nixon:Dyax Corp: Employment.

scholarly journals A Rare Cause of Isolated Prolonged Activated Partial Thromboplastin Time: An Overview of Prekallikrein Deficiency and the Contact System

2021 ◽  
Vol 9 ◽  
pp. 232470962110121
Author(s):  
Ivy Riano ◽  
Klaorat Prasongdee
Keyword(s):  
Partial Thromboplastin Time ◽  
Normal Activity ◽  
Fresh Frozen Plasma ◽  
Activated Partial Thromboplastin Time ◽  
Recessive Trait ◽  
Bleeding Tendency ◽  
Contact Activation ◽  
Asymptomatic Patients ◽  
Prolonged Aptt ◽  
Fresh Frozen

Prekallikrein (PK) deficiency, also known as Fletcher factor deficiency, is a very rare disorder inherited as an autosomal recessive trait. It is usually identified incidentally in asymptomatic patients with a prolonged activated partial thromboplastin time (aPTT). In this article, we present the case of a 52-year-old woman, with no prior personal or family history of thrombotic or hemorrhagic disorders, who was noted to have substantial protracted aPTT through the routine coagulation assessment before a kidney biopsy. The patient had an uneventful biopsy course after receiving fresh frozen plasma (FFP). Laboratory investigations performed before the biopsy indicated normal activity for factors VIII, IX, XI, XII, and von Willebrand factor (vWF) as well as negative lupus anticoagulant (LA) screen. The plasma PK assay revealed low activity at 15% consistent with mild PK deficiency. The deficit of PK is characterized by a severely prolonged aPTT and normal prothrombin time (PT) in the absence of bleeding tendency. PK plays a role in the contact-activated coagulation pathway and the inflammatory response. Thus, other differential diagnoses of isolated prolonged aPTT include intrinsic pathway factor deficiencies and nonspecific inhibitors such as LA. We concluded that the initial evaluation of a prolonged aPTT with normal PT should appraise the measurement of contact activation factors and factor inhibitors. PK deficiency should be considered in asymptomatic patients with isolated aPTT prolongation, which corrects on incubation, with normal levels of the contact activation factors and factor inhibitors.

scholarly journals Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.M Engelen ◽  
C Van Laer ◽  
M Jacquemin ◽  
C Vandenbriele ◽  
K Peerlinck ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Heart Valves ◽  
Thrombus Formation ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Mechanical Heart Valves ◽  
Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

scholarly journals Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

2006 ◽  
Vol 203 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Christoph Kleinschnitz ◽  
Guido Stoll ◽  
Martin Bendszus ◽  
Kai Schuh ◽  
Hans-Ulrich Pauer ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Coagulation Factor ◽  
Artery Occlusion ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
Fibrin Formation ◽  
Vessel Injury ◽  
Normal Hemostasis ◽  
A Site

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor–treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

How to Optimize Activated Partial Thromboplastin Time (APTT) Testing: Solutions to Establishing and Verifying Normal Reference Intervals and Assessing APTT Reagents for Sensitivity to Heparin, Lupus Anticoagulant, and Clotting Factors

2019 ◽  
Vol 45 (01) ◽  
pp. 022-035 ◽  
Author(s):  
Geoffrey Kershaw ◽  
Soma Mohammed ◽  
Giuseppe Lippi ◽  
Emmanuel Favaloro
Keyword(s):  
Molecular Weight ◽  
Partial Thromboplastin Time ◽  
Lupus Anticoagulant ◽  
High Sensitivity ◽  
Activated Partial Thromboplastin Time ◽  
Factor Xii ◽  
Reference Ranges ◽  
Clotting Factors ◽  
Hospital System ◽  
Normal Reference

AbstractThe activated partial thromboplastin time (APTT) assay is a very common coagulation test, used for several reasons. The test is conventionally used for assessing the contact factor (intrinsic) pathway of blood coagulation, and thus for screening deficiencies in this pathway, most typically factors VIII, IX, and XI. The APTT is also sensitive to contact factor deficiencies, including factor XII, prekallikrein, and high-molecular-weight kininogen. The APTT may also be elevated in a variety of conditions, including liver disease, vitamin K deficiency, and disseminated intravascular coagulation. The APTT can also be used for monitoring unfractionated heparin (UFH) therapy, as well as for screening lupus anticoagulant (LA) or for assessing thrombosis risk. Which of these separate uses is important to a given laboratory or clinician depends on the laboratory and the clinical context. For example, UFH sensitivity is important in hospital-based laboratories, where UFH therapy is used, but not in hospital-based laboratories where low-molecular-weight heparin (LMWH) is largely employed or where UFH may be assessed by anti-factor Xa testing, or in private/community laboratories not associated with a hospital system. High sensitivity to (low levels of) factors VIII, IX, and XI is generally preferred, as their deficiencies are clinically significant. Also preferred, but not usually achieved, is low sensitivity to factor XII and other contact factors, as these deficiencies are usually asymptomatic. Nevertheless, a good knowledge of factor sensitivity is usually needed, if only to help explain the reasons for a prolonged APTT in a given patient, or whether factor testing or other investigation is required. A good working knowledge of reagents sensitivity to LA is also advisable, especially when the reagent is used as part of a LA test panel, or else as a “general-purpose screening reagent.” The current report is aimed at providing some guidance around these questions, and is intended as a kind of “how to” guide, that will enable laboratories to assess APTT reagents in regard to their sensitivity to heparin, LA, and clotting factors. The report also provides some advice on generation of normal reference ranges, as well as solutions for troubleshooting prolonged APTTs, when performing factor testing or searching for inhibitors.

scholarly journals Inherited Factor XII Deficiency—What Is the Real Concern for Neuroanesthesiologist: Bleeding or Clotting

2020 ◽  
Author(s):  
Mouleeswaran Sundaram ◽  
Sonia Bansal ◽  
Rohini M. Surve
Keyword(s):  
Perioperative Management ◽  
Activated Partial Thromboplastin Time ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
Vertebral Fusion ◽  
Factor Xii Deficiency ◽  
Risk Of Bleeding ◽  
Increased Risk ◽  
Patient Factor

AbstractFactor XII deficiency is a rare disorder that can complicate the perioperative management of a patient. Factor XII plays an important role in the activation of intrinsic pathway of coagulation; the deficiency, therefore, results in prolongation of activated partial thromboplastin time (aPTT). This aPTT prolongation is expected to cause increased bleeding during surgery. However, on the contrary, in vivo isolated factor XII deficiency is associated with increased risk of thromboembolism (this risk being higher than the risk of bleeding). We report the perioperative management of a patient with factor XII deficiency who underwent cervical vertebral fusion (C1–C2) for atlantoaxial dislocation.

The Fibronectin Type II Domain of Factor XII Ensures Zymogen Quiescence

2020 ◽  
Vol 120 (03) ◽  
pp. 400-411 ◽  
Author(s):  
Chantal C. Clark ◽  
Zonne L. M. Hofman ◽  
Wariya Sanrattana ◽  
Lyanne den Braven ◽  
Steven de Maat ◽  
...  
Keyword(s):  
Full Length ◽  
Activation Loop ◽  
Factor Xii ◽  
Type Ii ◽  
Contact Activation ◽  
Zymogen Activation ◽  
Amidolytic Activity ◽  
Surface Binding ◽  
Epidermal Growth ◽  
Fibronectin Type

AbstractFactor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation), plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation loop of FXII is shielded to regulate FXII activation and subsequent contact activation. In this study, we aimed to elucidate this mechanism by expressing and characterizing seven consecutive N-terminally truncated FXII variants as well as full-length wild-type (WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1–71), FXII cleavage products appear on Western blot. These fragments display spontaneous amidolytic activity, indicating that FXII without the fibronectin type II domain is susceptible to autoactivation. Additionally, truncated FXII Δ1–71 is more easily activated by PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation, we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A Δ1–71 is highly susceptible to cleavage by PKa, indicating exposure of the activation loop. In surface binding experiments, we found that the fibronectin type II domain is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation of full-length WT FXII by PKa increases approximately threefold in the presence of kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined, these findings suggest that the fibronectin type II domain shields the activation loop of FXII, ensuring zymogen quiescence.

Dual role of collagen in factor XII–dependent thrombus formation

Blood ◽  
2009 ◽  
Vol 114 (4) ◽  
pp. 881-890 ◽  
Author(s):  
Paola E. J. van der Meijden ◽  
Imke C. A. Munnix ◽  
Jocelyn M. Auger ◽  
José W. P. Govers-Riemslag ◽  
Judith M. E. M. Cosemans ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Type I Collagen ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Dual Role ◽  
Type I ◽  
Factor Xii ◽  
Glycoprotein Vi

Abstract In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

A shortened activated partial thromboplastin time is associated with the risk of venous thromboembolism

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3631-3634 ◽  
Author(s):  
Armando Tripodi ◽  
Veena Chantarangkul ◽  
Ida Martinelli ◽  
Paolo Bucciarelli ◽  
Pier Mannuccio Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Partial Thromboplastin Time ◽  
Odds Ratio ◽  
Case Control Study ◽  
Coagulation Factors ◽  
Case Control ◽  
Activated Partial Thromboplastin Time ◽  
Simple Test ◽  
Coagulation Time ◽  
Control Study

Hypercoagulability due to high coagulation factors XI, VIII, IX, II, and fibrinogen is recognized as a risk factor of venous thromboembolism (VTE). These factors are cumulatively explored by the activated partial thromboplastin time (APTT). To test the hypothesis that a short APTT increases the risk of VTE, a case-control study was carried out in 605 patients referred for thrombophilia testing after documented VTE and in 1290 controls. Median APTT ratio (coagulation time of test-to-reference plasma) values were 0.97 (range: 0.75-1.41) for patients and 1.00 (range: 0.72-1.33) for controls (P < .001). In patients who had an APTT ratio smaller than the fifth percentile of the distribution in controls, the odds ratio (OR) for VTE was 2.4 (95% confidence interval [CI]: 1.7-3.6) and was independent of inherited thrombophilic abnormalities. Further statistical analyses in 193 patients and 259 controls for whom factor VIII (FVIII) levels were available showed a decrease of the OR from 2.7 (95% CI: 1.4-5.3) to 2.1 (95% CI: 1.0-4.2), indicating that the risk was only partially mediated by high FVIII levels. In conclusion, hypercoagulability detected by a shortened APTT is independently associated with VTE. This inexpensive and simple test should be considered in the evaluation of the risk of VTE.

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