In vivo evaluation of the dual inhibition of TAFI and PAI-1: towards effective and safe thrombolysis

Thrombin activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are important regulators of fibrinolysis. Bothproteins attenuate the tissue-type plasminogen activator (tPA)-mediatedconversion of plasminogen into fibrin-degrading plasmin, however with distinct modes of action. Activated TAFI (TAFIa) removes C-terminal lysines from the fibrin surface, rendering fibrin less susceptible to fibrinolysis. PAI-1 neutralises tPA by forming a covalent complex and is considered as the main inhibitor of tPA. Elevated levels of TAFI and PAI-1 arelinked to an increased risk for thrombosis. There is an unmet clinical need to improve thrombolytic treatment, especially in acute ischemic stroke (AIS). The only drugs available for thrombolysis comprise plasminogen activators. However, plasminogen activators remain underutilised owing to significant drawbacks, e.g. a low recanalising rate, high bleeding risk and possible neurotoxicity. Therefore, we embarked on a novel thrombolytic strategy: dual targeting of TAFI and PAI-1 to effectively enhance endogenous fibrinolysis. In a previous study, a first heterodimer diabody against human TAFI and human PAI-1 was generated that exhibited a strong profibrinolytic capacity in lysing experimental human thrombi. A heterodimer diabody is a recombinant antibody derivative with dualspecificity and was used in this study to circumvent complications associated with the administration of two different inhibitors. In the present study, we evaluated the application of dual TAFI/PAI-1 inhibition through a diabody in vivo. In chapter 2, a recombinant bispecific diabody was generated from MA-RT36A3F5 directed towards rat TAFI (with cross-reactivity to mouse, but not human TAFI) and MA-33H1F7 directed towards human PAI-1 (with cross-reactivity to mouse and rat PAI-1). MA-RT36A3F5 inhibits TAFI by destabilising TAFIa. MA-33H1F7 converts active PAI-1 to a substrate of tPA resulting in the inactivation of PAI-1. Although the diabody was successfully expressed in bacteria and it preserved the inhibitory properties of the parental antibodies, the profibrinolytic effect was transient because of its low plasma stability. Through antibody engineering (e.g. CDR-grafting and linker addition), we obtained a stabilised version of this diabody with comparable profibrinolytic properties as those of the parental antibodies. Inchapter 3, a second candidate diabody was generated from MA-TCK26D6, directed towards a stable variant of human TAFI, and MA-33H1F7, also used in the previous diabody, against PAI-1. MA-TCK26D6 is a first-in-class antibody to have cross-reactivity towards human and rodent TAFI. A great asset of the corresponding diabody, Db-TCK26D6x33H1F7, is that it can beevaluated in both preclinical as clinical settings. The diabody retained the functional properties of the parental antibodies and did not encounter stability issues. This resulted in a strong profibrinolytic effect in vitro which exceeded that of the combination of parental antibodies. Moreover, the diabody did not impair the beneficial anti-inflammatory properties of TAFIa. Therefore, Db-TCK26D6x33H1F7 was selected for furtherevaluation in vivo. In chapter 4, the profibrinolytic properties of the parental antibodies or corresponding diabody (Db-TCK26D6x33H1F7) were first assessed in a mouse model of thromboembolism evoked bysystemic administration of tissue factor. Whereas the antibodies, administered separately or combined, caused a half-maximal clearance of fibrin from the lungs, the diabody, administered at a corresponding dose, resulted in a maximal effect. In a model of cerebral ischemia/reperfusion, whereas the administration of a single parental antibody at a certain dose caused no effect, the administration of the combination of antibodiesor the diabody resulted in a complementary effect in mitigating ischemia/reperfusion injury (reduced lesion volumes and improved neurological and motor outcomes). The diabody was slightly more effective than the combination of antibodies in improving functional outcome. The superior profibrinolytic capacity of the diabody is most likely ascribed to its relatively small (58 kDa) and flexible structure. In chapter 5, the thrombolytic capacity of Db-TCK26D6x33H1F7 was assessed by administering the diabody post thrombus formation in two types of thrombosis-induced stroke models. The effect of the diabody was compared to thatof tPA, the current treatment of AIS. In the thrombin-induced model, fibrin-rich clots are produced which are susceptible to tPA-mediated fibrinolysis. 24 hours post stroke onset, diabody administration caused a substantial reduction in lesion sizes which exceeded the effect of tPA administration. In the FeCl(3)-induced model, platelet-rich clots are produced which are resistant to tPA-mediated fibrinolysis. 24 hours post stroke onset, whereas tPA was non-effective, the diabody recanalised the artery, improved brain perfusion and reduced lesion sizes. Moreover, in contrast to tPA, the diabody did not prolong bleeding times. Inconclusion, this study clearly demonstrates the strong profibrinolytic properties of a dual targeting strategy against two antifibrinolytic proteins, TAFI and PAI-1, in vivo. The two main findings of this study are that (i) dual targeting of TAFI and PAI-1 is more effective with a diabody than with the combined use of parental antibodies and (ii) the diabody is more effective and potentially safer than tPA in the treatment of experimental stroke.nrpages: 140status: publishe

Development of an LC/MS assay for the bacterial transglycosylation reaction through measurement of Lipid II

Transglycosylation is the second to last step in the production of bacterial peptidoglycan. It is catalyzed by a transglycosylation site in class A penicillin binding proteins (PBP) or monofunctional glycosyl transferases. Several potential inhibitors have been suggested and need to be tested for activity. In this article, we describe the development and validation of an LC/MS assay for Lipid II, the substrate for transglycosylation. The developed assay can be used to monitor the transglycosylation activity of S. aureus PBP2. There was no need for modification of Lipid II with a fluorescent tag that could alter affinity of inhibitors towards Lipid II. Recombinant PBP2 was produced in E. coli and has been tested for activity. This LC/MS method is suitable for a transglycosylation assay for PBP2 and since it is relatively fast, it can be used to test inhibitors. This article is protected by copyright. All rights reserved.status: publishe

Defective TAFI activation in hemophilia a mice is a major contributor to joint bleeding

Joint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti–factor VIII [FVIII] antibody) and congenital HA (FVIII2/2) mice. Both aHA and FVIII2/2 mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII2/2 mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI2/2 mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII2/2 mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII2/2 mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)–induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator–mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII2/2 mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding. (Blood. 2018;132(15):1593-1603

Innovative thrombolytic strategy using a heterodimer diabody against TAFI and PAI-1 in mouse models of thrombosis and stroke

Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia/reperfusion, diabody administration (0.8mg/kg, 1h post occlusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion (MCAo), the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissue-type plasminogen activator (tPA). Early administration of diabody (0.8mg/kg, 20min post occlusion) caused a 2-fold decrease in brain lesion size, whereas that of tPA (10mg/kg) had a much smaller effect. Delayed administration of diabody or tPA (90min post occlusion) had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding.status: publishe

Defective TAFI activation in hemophilia A mice is a major contributor to joint bleeding

Joint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti-factor VIII [FVIII] antibody) and congenital HA (FVIII-/-) mice. Both aHA and FVIII-/- mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII-/- mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI-/- mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII-/- mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII-/- mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)-induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator-mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII-/- mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding.status: publishe

Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor and Plasminogen Activator Inhibitor-1 Reduces Ischemic Brain Damage in Mice

BACKGROUND AND PURPOSE: Cerebral ischemia and reperfusion is associated with activation of the coagulation cascade and fibrin deposition in cerebral microvessels. Both thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) attenuate fibrinolysis and are therefore attractive targets for the treatment of ischemic stroke. METHODS: TAFI and PAI-1 were inhibited by monoclonal antibodies in a mouse model of transient middle cerebral artery occlusion. Twenty-four hours after stroke, mice were neurologically scored, cerebral thrombotic burden was assessed, and brain infarct sizes were calculated. RESULTS: Inhibition of TAFI or PAI-1 significantly decreased cerebral infarct sizes by 50% 24 hours after stroke. This reduction in cerebral damage was associated with a significant decrease in fibrin(ogen) deposition in the ischemic brain. Concurrently, functional recovery of the animals was improved. Interestingly, combined targeting of TAFI and PAI-1 using low, and by themselves inactive, doses of antibodies improved cerebral blood flow and reduced cerebral fibrin(ogen) deposition and infarct sizes by 50%. When dual treatment was delayed to 1 hour after the start of reperfusion, it still reduced brain injury; however, this was not statistically significant. CONCLUSIONS: Targeting of PAI-1 and TAFI is protective in an ischemic stroke model by attenuating fibrin(ogen) deposition, thereby improving reperfusion. Combined inhibition has a co-operative effect that could become useful in ischemic stroke therapy.status: publishe

Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice

Cerebral cavernous malformations (CCM) are common brain vascular dysplasias prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here we show that central nervous system (CNS) hemorrhage in CCM is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions and in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO) or Pdcd10 (Pdcd10ECKO), which cause CCM formation, result in increased levels of vascular TM and EPCR, and in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of one or two copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial co-factors that generate anticoagulant APC can contribute to bleeding in CCMs and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs