Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature.

Type 1 diabetes (T1D) is a prototypic T cell-mediated autoimmune disease. Because the islets of Langerhans are insulated from blood vessels by a double basement membrane and lack detectable lymphatic drainage, interactions between endocrine and circulating T cells are not permitted. Thus, we hypothesized that initiation and progression of anti-islet immunity required islet neolymphangiogenesis to allow T cell access to the islet. Combining microscopy and single cell approaches, the timing of this phenomenon in mice was situated between 5 and 8 wk of age when activated anti-insulin CD4 T cells became detectable in peripheral blood while peri-islet pathology developed. This "peri-insulitis," dominated by CD4 T cells, respected the islet basement membrane and was limited on the outside by lymphatic endothelial cells that gave it the attributes of a tertiary lymphoid structure. As in most tissues, lymphangiogenesis seemed to be secondary to local segmental endothelial inflammation at the collecting postcapillary venule. In addition to classic markers of inflammation such as CD29, V-CAM, and NOS, MHC class II molecules were expressed by nonhematopoietic cells in the same location both in mouse and human islets. This CD45- MHC class II+ cell population was capable of spontaneously presenting islet Ags to CD4 T cells. Altogether, these observations favor an alternative model for the initiation of T1D, outside of the islet, in which a vascular-associated cell appears to be an important MHC class II-expressing and -presenting cell.

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

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are 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 hemorrhage in CCMs 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, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1 ECKO ) or Pdcd10 (Pdcd10 ECKO ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as 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 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10 ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10 ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo.

OBJECTIVE: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). CONCLUSIONS: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Activated protein C, protease activated receptor 1, and neuroprotection.

Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role as a plasma protease, pharmacologic APC is a pleiotropic protease that activates diverse homeostatic cell signaling pathways via multiple receptors on many cells. Engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks >90% anticoagulant activity but retains normal cell signaling activities. This 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein-coupled receptor, protease activated receptor 1. Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC-induced signaling that arises due to APC's cleavage in protease activated receptor 1 at a noncanonical Arg46 site. This cleavage causes biased signaling that provides a major explanation for APC's in vivo mechanism of action for neuroprotective activities. 3K3A-APC appeared to be safe in ischemic stroke patients and reduced bleeding in the brain after tissue plasminogen activator therapy in a recent phase 2 clinical trial. Hence, it merits further clinical testing for its efficacy in ischemic stroke patients. Recent studies using human fetal neural stem and progenitor cells show that 3K3A-APC promotes neurogenesis in vitro as well as in vivo in the murine middle cerebral artery occlusion stroke model. These recent advances should encourage translational research centered on signaling selective APC's for both single-agent therapies and multiagent combination therapies for ischemic stroke and other neuropathologies.

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.

PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke.

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli-induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.

Activated protein C in neuroprotection and malaria.

PURPOSE OF REVIEW: Activated protein C (APC) is a homeostatic coagulation protease with anticoagulant and cytoprotective activities. Focusing on APC's effects in the brain, this review discusses three different scenarios that illustrate how APC functions are intimately affecting the physiology and pathophysiology of the brain. RECENT FINDINGS: Cytoprotective APC therapy holds promise for the treatment of ischemic stroke, and a recently completed trial suggested that cytoprotective-selective 3K3A-APC reduced bleeding in ischemic stroke patients. In contrast, APC's anticoagulant activity contributes to brain bleeding as shown by the disproportional upregulation of APC generation in cerebral cavernous malformations lesions in mice. However, too little APC generation also contributes to maladies of the brain, such as in case of cerebral malaria where the binding of infected erythrocytes to the endothelial protein C receptor (EPCR) may interfere with the EPCR-dependent functions of the protein C pathway. Furthermore, discoveries of new activities of APC such as the inhibition of the NLRP3-mediated inflammasome and of new applications of APC therapy such as in Alzheimer's disease and graft-versus-host disease continue to advance our knowledge of this important proteolytic regulatory system. SUMMARY: APC's many activities or lack thereof are intimately involved in multiple neuropathologies, providing abundant opportunities for translational research.

Activated protein C: biased for translation.

The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC's activation of these G protein-coupled receptors differs completely from thrombin's activation mechanism due to biased signaling via either G proteins or ß-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies.

2016 Scientific Sessions Sol Sherry Distinguished Lecturer in Thrombosis: Thrombotic Stroke: Neuroprotective Therapy by Recombinant-Activated Protein C.

APC (activated protein C), derived from the plasma protease zymogen, is antithrombotic and anti-inflammatory. In preclinical injury models, recombinant APC provides neuroprotection for multiple injuries, including ischemic stroke. APC acts directly on brain endothelial cells and neurons by initiating cell signaling that requires multiple receptors. Two or more major APC receptors mediate APC's neuroprotective cell signaling. When bound to endothelial cell protein C receptor, APC can cleave protease-activated receptor 1, causing biased cytoprotective signaling that reduces ischemia-induced injury. Pharmacological APC alleviates bleeding induced by tissue-type plasminogen activator in murine ischemic stroke studies. Remarkably, APC's signaling promotes neurogenesis. The signaling-selective recombinant variant of APC, 3K3A-APC, was engineered to lack most of the APC's anticoagulant activity but retain APC's cell signaling actions. Recombinant 3K3A-APC is in ongoing National Institutes of Health (NIH)-funded clinical trials for ischemic stroke.

Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization.

OBJECTIVE: Activated protein C (APC), a plasma serine protease, initiates cell signaling that protects endothelial cells from apoptosis and endothelial barrier disruption. Apolipoprotein E receptor 2 (ApoER2; LRP8) is a receptor known for mediating signaling initiated by reelin in neurons. ApoER2 contributes to APC-initiated signaling in monocytic U937 cells. The objective was to determine whether ApoER2 is required for APC's beneficial signaling in the endothelial cell surrogate EA.hy926 line. APPROACH AND RESULTS: We used small interfering RNA and inhibitors to probe requirements for specific receptors for APC's antiapoptotic activity and for phosphorylation of disabled-1 by Src family kinases and of Akt. When small interfering RNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was used, APC's antiapoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2- to 3-fold increased phosphorylation of Ser473-Akt and Tyr232-disabled-1, a phosphorylation known to trigger disabled-1-mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 small interfering RNA or by inhibitors of Src (PP2), phosphatidylinositol-3 kinase (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 small interfering RNA blocked the ability of APC to prevent thrombin-induced endothelial barrier disruption in TransEndothelial Resistance assays. Binding studies using purified APC and purified immobilized wild-type and mutated ApoER2 ectodomains suggested that APC binding involves Lys49, Asp50, and Trp64 on the surface of the N-terminal LA1 domain of ApoER2. CONCLUSIONS: ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial antiapoptotic and barrier protective signaling.

Heterozygous congenital Factor VII deficiency with the 9729del4 mutation, associated with severe spontaneous intracranial bleeding in an adolescent male.

BACKGROUND: In congenital Factor (F) VII deficiency bleeding phenotype and intrinsic FVII activity levels don't always correlate. Patients with FVII activity levels <30% appear to have a higher bleeding propensity, but bleeding can also occur at higher FVII activity levels. Reasons for bleeding at higher FVII activity levels are unknown, and it remains challenging to manage such patients clinically. CASE: A 19year old male with spontaneous intracranial hemorrhage and FVII activity levels of 44%, requiring emergent surgical intervention and a strategy for FVII replacement. Genotyping showed the rare heterozygous FVII 9729del4 mutation. Bleed evacuation was complicated by epidural abscess requiring craniectomy, bone graft procedures, and prolonged administration of recombinant human (rh) activated FVII (FVIIa). The patient recovered without neurological deficits, and remains on prophylactic low dose treatment with rhFVIIa in relation to risky athletic activities. CONCLUSION: For clinicians, it is important to recognize that effects of rhFVIIa within these pathways are independent of its contribution to blood clot formation and cannot be assessed by clotting assays. Reduced FVII levels should therefore not be dismissed, as even a mild reduction may result in spontaneous bleeding. Treatment of mild FVII deficiency requires a careful case-by-case approach, based on the clinical scenario.

Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity.

Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease-activated receptor (PAR) 1, and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Noncanonical PAR1 and PAR3 activation by APC vs canonical activation by thrombin provides an explanation for the functional selectivity of these proteases. Here we found that factor Xa (FXa) activated PAR1 at canonical Arg41 similar to thrombin but cleaved PAR3 at noncanonical Arg41 similar to APC. This unique PAR1-PAR3 activation profile permitted the identification of noncanonical PAR3 activation as a novel activation pathway for barrier protective tunica intima endothelial receptor tyrosine kinase 2 (Tie2). APC, FXa, and the noncanonical PAR3 tethered-ligand peptide induced prolonged activation of Tie2, whereas thrombin and the canonical PAR3 tethered-ligand peptide did not. Tie2 activation by FXa required PAR3 and EPCR. FXa and the noncanonical PAR3 tethered-ligand peptide induced Tie2- and PAR3-dependent upregulation of tight-junction-associated protein zona occludens 1 (ZO-1), translocation of ZO-1 to cell-cell borders, and the formation of typical ZO-1 honeycomb patterns that are indicative of tight-junction stabilization. These data provide intriguing novel insights into the diversification of functional selectivity of protease signaling achievable by canonical and noncanonical PAR activation, such as the activation of vascular-protective Tie2 by noncanonical PAR3 activation.

Plasmodium falciparum picks (on) EPCR.

Of all the outcomes of Plasmodium falciparum infection, the coma of cerebral malaria (CM) is particularly deadly. Malariologists have long wondered how some patients develop this organ-specific syndrome. Data from two recent publications support a novel mechanism of CM pathogenesis in which infected erythrocytes (IEs) express specific virulence proteins that mediate IE binding to the endothelial protein C receptor (EPCR). Malaria-associated depletion of EPCR, with subsequent impairment of the protein C system promotes a proinflammatory, procoagulant state in brain microvessels.

Safety, Stability and Pharmacokinetic Properties of (super)Factor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding.

PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 µg/mL), (super)FVa did not (6.7 µg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 µg/mL) or saline (5.6 µg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3.

The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC's cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC's cytoprotective versus thrombin's proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects.

Vascular remodeling underlies rebleeding in hemophilic arthropathy.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46.

Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas ß-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3ß phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1.