Perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis.

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.

Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes. The platelet factor 4 vector was the strongest but with activity in hematopoietic stem cells. SUMMARY: Background Lentiviral transduction and transplantation of hematopoietic stem cells (HSCs) can be utilized to modify the phenotype of megakaryocytes and platelets. As the genetic modification in HSCs is transmitted onto all hematopoietic progenies, transgene expression from the vector should be restricted to megakaryocytes to avoid un-physiologic effects by ectopic transgene expression. This can be achieved by lentiviral vectors that control expression by lineage-specific promoters. Methods In this study, we introduced promoters of megakaryocyte/platelet-specific genes, namely human glycoprotein 6 (hGP6) and hGP9, into third generation lentiviral vectors and analyzed their functionality in vitro and in vivo in bone marrow transplantation assays. Their specificity and efficiency of expression was compared with lentiviral vectors utilizing the promoters of murine platelet factor 4 (mPf4) and hGP1BA, both with strong activity in megakaryocytes (MKs) used in earlier studies, and the ubiquitously expressing phosphoglycerate kinase (hPGK) and spleen focus forming virus (SFFV) enhancer/promoters. Results Expression from the mPf4 vector in MKs and platelets was the strongest similar to expression from the viral SFFV promoter, however, the mPf4 vector, also exhibited considerable off-target expression in hematopoietic stem and progenitor cells. In contrast, the newly generated hGP6 vector was highly specific to megakaryocytes and platelets. The specificity was also retained when reducing the promoter size to 350 bp, making it a valuable new tool for lentiviral expression in MKs/platelets. Conclusion MK-specific vectors express preferentially in the megakaryocyte lineage. These vectors can be applied to develop murine models to study megakaryocyte and platelet function, or for gene therapy targeting proteins to platelets.

Targeting platelet receptors in thrombotic and thrombo-inflammatory disorders.

Platelet activation at sites of vascular injury is critical for the formation of a hemostatic plug which limits excessive blood loss, but also represents a major pathomechanism of ischemic cardio- and cerebrovascular diseases. Although currently available antiplatelet therapies have proved beneficial in preventing the recurrence of vascular events, their adverse effects on primary hemostasis emphasize the necessity to identify and characterize novel pharmacological targets for platelet inhibition. Increasing experimental evidence has suggested that several major platelet surface receptors which regulate initial steps of platelet adhesion and activation may become promising new targets for antiplatelet drugs due to their involvement in thrombotic and thrombo-inflammatory signaling cascades. This review summarizes recent developments in understanding the function of glycoprotein (GP)Ib, GPVI and the C-type lectin-like receptor 2 (CLEC-2) in hemostasis, arterial thrombosis and thrombo-inflammation and will discuss the suitability of the receptors as novel targets to treat these diseases in humans.

Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets.

BACKGROUND: Platelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested in vivo for a variety of disorders. Two small-molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively. OBJECTIVES: To analyze the specificity of NSC23766 and EHT1864. METHODS: Platelet function was assessed in mouse wild-type and Rac1-deficient platelets by the use of flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed with differential interference contrast microscopy, and activation of effector molecules was analyzed with biochemical approaches. RESULTS: NSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100 µm in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoprotein Ib-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors p21-activated kinase (PAK)1 and PAK2. CONCLUSIONS: Our results reveal critical off-target effects of NSC23766 and EHT1864 at 100 µm in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.

Phospholipase D is a central regulator of collagen I-induced cytoskeletal rearrangement and podosome formation in megakaryocytes.

BACKGROUND: Blood platelets are small anucleated cell fragments generated from bone marrow megakaryocytes (MKs) by a cytoskeleton-driven process. Thereby, mature MKs form long cytoplasmic protrusions (pro-platelets), which extend into the sinusoids within the bone marrow and finally release platelets. Podosomes are F-actin rich matrix contacts that have been suggested to play an important role in cell migration, but also in pro-platelet formation by MKs. Phospholipase D (PLD) has been proposed to contribute to the regulation of actin dynamics through the local generation of phosphatidic acid but its role in platelet formation is unknown. OBJECTIVE: We sought to investigate the significance of PLD in MK podosome formation and thrombocytopoiesis. METHODS: Podosome formation, spreading and ultra-structure of PLD single- and double-deficient MKs were analyzed using confocal and transmission electron microscopy. RESULTS: Phospholipase D-deficient MKs displayed a highly altered ultra-structure in vivo and abnormal actin rearrangement, with almost abolished formation of podosomes upon spreading on collagen I in vitro. However, MK endomitosis and platelet production were not altered by PLD deficiency. CONCLUSION: Together, our findings point to a specific function of PLD in actin dynamics as well as podosome formation and size determination in MKs on a collagen I matrix. The normal platelet number in PLD-deficient mice, however, suggests the existence of compensatory mechanisms in vivo that overcome the defective podosome formation observed in vitro.

Orai1-induced store-operated Ca(2+) entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets.

BACKGROUND: Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. OBJECTIVE: We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. METHODS: Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. RESULTS: Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. CONCLUSION: The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.

The SLAM family member CD84 is regulated by ADAM10 and calpain in platelets.

BACKGROUND AND OBJECTIVE: Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact-dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. METHODS: We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. RESULTS: We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain-shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain-mediated intracellular cleavage of the CD84 C-terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo. CONCLUSIONS: These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra- and intracellular cleavage that may modulate platelet-platelet and platelet-immune cell interactions.

Redundant functions of phospholipases D1 and D2 in platelet α-granule release.

BACKGROUND: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD). OBJECTIVE AND METHODS: The aim of this study was to determine a potential function of PLD2 in platelet physiology. Thus, we investigated the function of PLD2 in platelet signaling and thrombus formation, by generating mice lacking PLD2 or both PLD1 and PLD2. Adhesion, activation and aggregation of PLD-deficient platelets were analyzed in vitro and in vivo. RESULTS: Whereas the absence of PLD2 resulted in reduced PLD activity in platelets, it had no detectable effect on the function of the cells in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective α-granule release and protection in a model of FeCl3 -induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. CONCLUSION: These results reveal redundant roles of PLD1 and PLD2 in platelet α-granule secretion, and indicate that this may be relevant for pathologic thrombus formation.

Defective diacylglycerol-induced Ca2+ entry but normal agonist-induced activation responses in TRPC6-deficient mouse platelets.

BACKGROUND: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca(2+) concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca(2+) influx pathway in murine platelets, but there are additional non-store-operated Ca(2+) (non-SOC) and receptor operated Ca(2+) (ROC) channels expressed in the platelet plasma membrane. OBJECTIVE: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested. METHODS: To investigate the function of TRPC6 in platelet Ca(2+) signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays. RESULTS: In the mutant platelets, DAG activated Ca(2+) influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca(2+) responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6. CONCLUSION: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca(2+) influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.

[Acute ischemic stroke. New approaches to antithrombotic treatment]. / Akuter ischämischer Schlaganfall. Neue Ansätze in der Antithrombosetherapie.

The only recommended therapy in the acute phase of ischemic stroke is thrombolysis within 4.5-(6) h after symptom onset. For secondary stroke prevention platelet inhibitors or, in cases of cardiac embolism, anticoagulants are used. However, these substances bear significant limitations: either they show only moderate efficacy (platelet inhibitors), or they are associated with a considerable bleeding risk (rt-PA, anticoagulants). Although the majority of strokes are caused by embolic or thrombotic vessel occlusion, strikingly little is known about the pathophysiological role of platelets and their local function in the brain vasculature. The recent development of novel transgenic mouse lines paved the way for the in-depth analysis of the different molecular steps of thrombus formation involving platelets and the plasma coagulation cascade in models of acute ischemic stroke. It was demonstrated that prevention of early platelet adhesion to the damaged vessel wall by blocking the platelet surface receptors GPIbα or GPVI dramatically protects against experimental stroke without increasing the frequency of intracranial hemorrhage. Moreover, the critical involvement of the blood coagulation factor XII (FXII)-driven intrinsic coagulation cascade in thrombus formation during the course of ischemic brain damage could be unraveled thereby disproving established concepts of hemostasis. Based on these findings novel pharmacological blockers of GPIbα and FXIIa were designed that likewise proved to be safe and effective in animal stroke studies. Those compounds now lay the groundwork for a novel and intriguing concept in ischemic stroke and other thromboembolic diseases: antithrombosis devoid of any bleeding complications. Further preclinical testing is currently ongoing.

Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke.

Platelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding of the glycoprotein (GP)Ib-V-IX complex to immobilised von Willebrand factor (VWF) initiates adhesion of flowing platelets to the ECM, and thereby enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by locally produced thrombin and platelet-derived secondary mediators, such as adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Together, these events promote a shift of ß1 and ß3 integrins from a low to a high affinity state for their ligands through 'inside-out' signalling allowing firm platelet adhesion and aggregation. Formed platelet aggregates are stabilised by fibrin formation and signalling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C-type lectin-like receptor 2 (CLEC-2). While occlusive thrombus formation is the principal pathogenic event in myocardial infarction, the situation is more complex in ischaemic stroke where infarct development often progresses despite sustained early reperfusion of previously occluded major intracranial arteries, a process referred to as 'reperfusion injury'. Increasing experimental evidence now suggests that early platelet adhesion and activation events, orchestrate a 'thrombo-inflammatory' cascade in this setting, whereas platelet aggregation and thrombus formation are not required. This review summarises recent developments in understanding the principal platelet adhesion receptor systems with a focus on their involvement in arterial thrombosis and ischaemic stroke models.

EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

BACKGROUND: Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. OBJECTIVES: To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. METHODS AND RESULTS: Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. CONCLUSIONS: EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.

The role of glycoprotein Ibalpha and von Willebrand factor interaction in stroke development.

Ischaemic stroke is a devastating disease with limited treatment options due to numerous uncertainties regarding the underlying pathophysiology. The contribution of glycoprotein (GP)Ibalpha and von Willebrand factor (VWF) in stroke development has only recently been established in mice. Complete blockade of GPIbalpha led to a significant reduction of infarct volumes in mice undergoing one hour of transient middle cerebral artery occlusion (tMCAO). High shear-induced changes in VWF confirmation are a prerequisite for VWF binding to collagen and GPIbalpha expressed on platelets. Importantly, transgenic VWF-/- mice were similarly protected against ischemic stroke after tMCAO, and hydrodynamic injection of a VWF-encoding plasmid restored VWF serum levels and the susceptibility towards stroke. Secreted VWF is rapidly cleaved by ADAMTS13. Accordingly, ADAMTS13 deficient mice developed larger infarction after tMCAO, while infusion of recombinant ADAMTS13 into wild-type mice was stroke-protective. In conclusion, there is compelling evidence that GPIbalpha/VWF interactions and downstream signaling via phospholipase D1 (PLD1) provide new therapeutic targets in ischemic stroke.

Arterial thrombus formation. Novel mechanisms and targets.

Platelet and coagulation factor-dependent thrombus formation is critical to limit posttraumatic blood loss at sites of vascular injury. However, under pathological conditions like rupture of an atherosclerotic plaque, it may also lead to vessel occlusion causing myocardial infarction or stroke. Therefore, antithrombotic treatment is the prime therapeutic option in the prophylaxis and treatment of ischaemic cardio- and cerebrovascular diseases. The use of existing antithrombotic agents is, however, limited by their inherent effect on primary haemostasis. In recent years, major advances have been made in understanding the mechanisms of thrombus formation in haemostasis and thrombosis and some studies raised the interesting possibility that occlusive thrombus formation and haemostasis may involve partially different mechanisms. This review briefly summarizes these developments and highlights newly identified mechanisms involved in platelet adhesion and activation, intracellular calcium signaling, integrin activation and initiation of coagulation. The suitability of these pathways as novel targets for antithrombotic therapy is discussed.

Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain-of-function mutation in phospholipase Cgamma2.

BACKGROUND: Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) beta or PLCgamma2. Activated PLC produces inositol 1,4,5-trisphosphate and diacylglycerol, which trigger Ca(2+) mobilization and the activation of protein kinase C, respectively. PLCbeta is activated downstream of Gq-coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCgamma2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. OBJECTIVE AND METHODS: We speculated that PLCgamma2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain-of-function mutation in the Plcg2 gene (Plcg2(Ali5/+)). RESULTS: Plcg2(Ali5/+) platelets showed enhanced Ca(2+) mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C-type lectin-like receptor-2 stimulation. Furthermore, integrin alpha(IIb)beta(3) outside-in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. CONCLUSIONS: These results demonstrate that the enzymatic activity of PLCgamma2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.

Enhanced cortical reperfusion protects coagulation factor XII-deficient mice from ischemic stroke as revealed by high-field MRI.

Intrinsic coagulation factor XII deficient (FXII(-/-)) mice are protected from ischemic stroke. To elucidate underlying mechanisms we investigated the early ischemic period in vivo by multimodal magnetic resonance imaging (MRI) at 17.6 Tesla. Cerebral ischemia was induced by either transient (60 min) or permanent occlusion of the middle cerebral artery (t/pMCAO). 10 FXII(-/-) mice underwent t- , 10 FXII(-/-) mice p- and 10 Wildtype (Wt) mice tMCAO. Cerebral blood flow (CBF), diffusion-weighted-imaging (DWI) and T2-relaxometry were measured at 2 h and 24 h after MCAO. Outcome measures were evaluated after motion correction and normalization to atlas space. 2 h after tMCAO CBF reduction was similar in FXII(-/-) and Wt mice extending over cortical (CBF (ml/100 g/min) 33.6+/-6.9 vs. 35.3+/-4.6, p=0.42) and subcortical regions (25.7+/-4.5 vs. 31.6+/-4.0, p=0.17). At 24 h, recovery of cortical CBF by +36% was observed only in tMCAO FXII(-/-) mice contrasting a further decrease of -30% in Wt mice after tMCAO (p=0.02, F((1,18))=6.24). In FXII(-/-) mice in which patency of the MCA was not restored (pMCAO) a further decrease of -75% was observed. Cortical reperfusion in tMCAO FXII(-/-) mice was related to a lower risk of infarction of 59% vs. 93% in Wt mice (p=0.04). Subcortical CBF was similarly decreased in both tMCAO groups (Wt and FXII(-/-)) relating to a similar risk of infarction of 89% (Wt) vs. 99% (FXII(-/-), p=0.17). Deficiency of FXII allows neocortical reperfusion after tMCAO and rescues brain tissue by this mechanism. This study supports the concept of FXII as a promising new target for stroke prevention and therapy.

Integrins in platelet activation.

Heterodimeric receptors of the beta1 and beta3 integrin families mediate platelet adhesion and aggregation in hemostasis and thrombosis. In resting platelets, integrins are expressed in a low-affinity state but they shift to a high-affinity state and efficiently bind their ligands in response to cellular activation. This review summarizes recent advances in understanding the functional regulation and (patho-) physiological significance of individual platelet integrins with a special focus on studies in genetically modified mice. It is now recognized that beta1 and beta3 integrins have partially redundant roles in the adhesion process and that their activation is regulated by similar mechanisms, involving Ca2+-dependent and -independent signaling events and essential functions of talin-1 and kindlin-3 in the terminal activation step.