Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation.
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ABSTRACT:
SUBMITTER: Maas C
PROVIDER: S-EPMC2518075 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Laminin is the most abundant non-collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis.To investigate the ability of laminin to promote coagulation and support thrombus formation under shear.Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI- and FXII-dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin-coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX.This study identifies a role for laminin in the initiation of coagulation and the formation of platelet-rich thrombi under shear conditions in a FXII-dependent manner.
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Project description: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.
| S-EPMC2118228 | biostudies-literature
Project description:Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI.To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain.A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to ?-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short ?-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates.These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation.
| S-EPMC4418343 | biostudies-literature
Project description:By inducing BK (bradykinin)-stimulated adrenomedullary catecholamine release, bolus injection of the ?-fragment of activated plasma coagulation Factor XII (?-FXIIa) transiently elevates BP (blood pressure) and HR (heart rate) of anaesthetized, vagotomized, ganglion-blocked, captopril-treated bioassay rats. We hypothesized that intravenous infusion of ?-FXIIa into intact untreated rats would elicit a qualitatively similar vasoconstrictor response. BN (Brown Norway) rats received for 60 min either: (i) saline (control; n=10); (ii) ?-FXIIa (85 ng/min per kg of body weight; n=9); or (iii) ?-FXIIa after 2ADX (bilateral adrenalectomy; n=9). LV (left ventricular) volume and aortic BP were recorded before (30 min baseline), during (60 min) and after (30 min recovery) the infusion. TPR (total peripheral resistance) was derived from MAP (mean arterial pressure), SV (stroke volume) and HR. Saline had no haemodynamic effects. ?-FXIIa infusion increased its plasma concentration 3-fold in both groups. In adrenally intact rats, ?-FXIIa infusion increased MAP by 6% (5±2 mmHg) and TPR by 45% (0.50±0.12 mmHg/ml per min), despite falls in SV (-38±8 ?l) and HR [-18±5 b.p.m. (beats/min)] (all P<0.05). In 2ADX rats, ?-FXIIa had no HR effect, but decreased SV (-89±9 ?l) and MAP (-4±1 mmHg), and increased TPR by 66% (0.59±0.15 mmHg/ml per min) (all P<0.05). After infusion, adrenally intact rats exhibited persistent vasoconstriction (MAP, 10±1 mmHg; TPR, 0.55±0.07 mmHg/ml per min; both P<0.05), whereas in 2ADX rats, MAP remained 5±1 mmHg below baseline (P<0.05) and TPR returned to baseline. End-study arterial adrenaline (epinephrine) concentrations in the three groups were 1.9±0.6, 9.8±4.1 and 0.6±0.2 nmol/l respectively. Thus, in neurally intact lightly anaesthetized untreated rats, ?-FXIIa infusion induces both adrenal catecholamine-mediated and adrenally independent increases in peripheral resistance.
| S-EPMC3294432 | biostudies-literature
Project description:The contributions of coagulation factor XI (FXI) and FXII to human clot formation is not fully known. Patients with deficiency in FXI have a variable mild bleeding risk, whereas FXII deficiency is not associated with bleeding. These phenotypes make FXII and FXI attractive target proteins in anticoagulant therapy. Here, we studied the mechanisms of fibrin clot formation, stability, and fibrinolytic degradation in patients with severe FXI or FXII deficiency. Thrombin generation was triggered in platelet-poor (PPP) and platelet-rich plasma (PRP) with the biological FXII trigger sulfatides. Intrinsic and extrinsic thrombus formation and degradation in whole blood were determined with rotational thromboelastometry (ROTEM). Clot formation under flow was assessed by perfusion of whole blood over collagen microspots with(out) tissue factor (TF). Thrombin generation and clot formation were delayed in FXII- and FXI-deficient patients triggered with sulfatides. In FXI-deficient plasma, this delay was more pronounced in PRP compared to PPP. In whole blood of FXII-deficient patients, clots were smaller but resistance to fibrinolysis was normal. In whole blood of FXI-deficient patients, clot formation was normal but the time to complete fibrinolysis was prolonged. In flow chamber experiments triggered with collagen/TF, platelet coverage was reduced in severe compared with moderate FXI deficiency, and fibrin formation was impaired. We conclude that quantitative defects in FXII and FXI have a substantial impact on contact activation-triggered coagulation. Furthermore, FXI deficiency has a dose-dependent suppressing effect on flow-mediated and platelet/TF-dependent clot formation. These last data highlight the contribution of particularly FXI to hemostasis.
| S-EPMC6736668 | biostudies-literature
Project description:Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.
| S-EPMC4563738 | biostudies-literature
Project description:BackgroundFactor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS.ObjectiveTo investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG).MethodsIn plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers.ResultsHigher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients.ConclusionIn MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression.
| S-EPMC5919941 | biostudies-literature
Project description:IntroductionVascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis.ObjectiveGenerate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity.MethodsMonoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation.ResultsA FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate.ConclusionSelective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.
| S-EPMC8010086 | biostudies-literature
Project description:Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.
| S-EPMC4331558 | biostudies-literature
Project description:Bleeding is a common contributor to death and morbidity in animals and provides strong selective pressure for the coagulation system to optimize hemostasis for diverse environments. Although coagulation factor XII (FXII) is activated by nonbiologic surfaces, such as silicates, which leads to blood clotting in vitro, it is unclear whether FXII contributes to hemostasis in vivo. Humans and mice lacking FXII do not appear to bleed more from clean wounds than their counterparts with normal FXII levels. We tested the hypothesis that soil, a silicate-rich material abundant in the environment and wounds of terrestrial mammals, is a normal and potent activator of FXII and coagulation. Blood loss was compared between wild-type (WT) and FXII-knocked out (FXII-/-) mice after soil or exogenous tissue factor was applied to transected tails. The activation of FXII and other components of the coagulation and contact system was assessed with in vitro coagulation and enzyme assays. Soils were analyzed by time-of-flight secondary ionization mass spectrometry and dynamic light scattering. Soil reduced blood loss in WT mice, but not FXII-/- mice. Soil accelerated clotting of blood plasma from humans and mice in a FXII-dependent manner, but not plasma from a cetacean or a bird, which lack FXII. The procoagulant activity of 13 soils strongly correlated with the surface concentration of silicon, but only moderately correlated with the ζ potential. FXII augments coagulation in soil-contaminated wounds of terrestrial mammals, perhaps explaining why this protein has a seemingly minor role in hemostasis in clean wounds.
| S-EPMC7189277 | biostudies-literature