During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or ␣-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX. (Blood. 2009;114:452-458) IntroductionThe plasmas of placental and marsupial mammals contain factor XI (fXI), 1 the zymogen of a protease (fXIa) that contributes to fibrin formation and stability through activation of factor IX (fIX). [2][3][4] Congenital fXI deficiency is associated with a variable traumainduced bleeding disorder in humans and other species. [5][6][7][8] The mechanism by which fXI is converted to fXIa during blood coagulation has been a topic of recent debate. 9,10 When blood is exposed to a surface in vitro, the process of contact activation converts factor XII (fXII) to the protease fXIIa, which then activates fXI. 3,4 Substances, such as RNA, 11 polyphosphates, 12 and collagen, 13 induce pathologic coagulation in mice in a fXIIdependent manner 13,14 and may represent physiologic surfaces for fXII activation. However, the contribution of fXIIa-mediated fXI activation to normal hemostasis is unclear, as fXII deficiency, unlike fXI deficiency, is not associated with abnormal bleeding in any species in which it has been identified. 4 This key observation supports hypotheses proposing that fXI is either activated during hemostasis by a protease other than fXIIa or that auxiliary mechanisms for fXI activation compensate in the absence of fXII. 3,[15][16][17] Candidates for fXI-activating proteases include ␣-thrombin, 15,16 meizothrombin, 18 and fXIa (autoactivation). 15,16 Thrombin has received much attention in this regard. Several laboratories have presented evidence suggesting that a protease generated early in coagulation, such as thrombin, converts fXI to fXIa. [19][20][21][22][23] This hypothesis has been challenged by a recent study that did not find evidence for fXI activation in thrombin or tissue facto...

The bleeding diathesis associated with hereditary factor XI (fXI) deficiency is prevalent in Ashkenazi Jews, in whom the disorder appears to be an autosomal recessive condition. The homodimeric structure of fXI implies that the product of a single mutant allele could confer disease in a dominant manner through formation of heterodimers with wild-type polypeptide. We studied 2 unrelated patients with fXI levels less than 20% of normal and family histories indicating dominant disease transmission. Both are heterozygous for single amino acid substitutions in the fXI catalytic domain (Gly400Val and Trp569Ser). Neither mutant is secreted by transfected fibroblasts. In cotransfection experiments with a wild-type fXI construct, constructs with mutations common in Ashkenazi Jews (Glu117Stop and Phe283Leu) and a variant with a severe defect in dimer formation (fXI-Gly350Glu) have little effect on wild-type fXI secretion. In contrast, cotransfection with fXI-Gly400Val or fXITrp569Ser reduces wild-type secretion about 50%, consistent with a dominant negative effect. Immunoprecipitation of cell lysates confirmed that fXI-Gly400Val forms intracellular dimers. The data support a model in which nonsecretable mutant fXI polypeptides trap wild-type polypeptides within cells through heterodimer formation, resulting in lower plasma fXI levels than in heterozygotes for mutations that cause autosomal recessive fXI deficiency. IntroductionCoagulation factor XI (fXI) is the zymogen of a plasma protease (fXIa) that contributes to hemostasis through activation of factor IX. 1 Hereditary fXI deficiency is an autosomal disorder characterized by trauma or surgery-induced hemorrhage and only rarely by "spontaneous" bleeding into soft tissues or joints. 2,3 The condition is prevalent in Ashkenazi Jews, in whom the heterozygote frequency may be as high as 10%. 4,5 Two mutations, Glu117Stop and Phe283Leu, account for most of the abnormal alleles in this population. 2,5 Reports on inheritance patterns for fXI deficiency have been conflicting. First described by Rosenthal and coworkers in 1953 as plasma thromboplastin antecedent (PTA) deficiency, 6 fXI deficiency was initially considered an autosomal dominant disorder with variable expressivity based on clinical symptoms and family histories. 7-9 However, Rapaport et al observed that PTA deficiency exists as major (homozygous) and minor (heterozygous) variants and is best described as following an incompletely recessive or "intermediate" mode of inheritance, with few symptoms occurring in heterozygotes. 10 These conclusions, based on measurements of plasma fXI activity primarily in Jewish kindreds, demonstrated distinct ranges for major (fXI level up to 20% of normal) and minor (fXI 30%-65% of normal) PTA deficiency. Subsequent studies supported this work 4,11 and led to the general opinion that fXI deficiency in Jewish patients is a recessive disorder. 12 Not all cases of fXI deficiency are consistent with a simple autosomal recessive model. Ragni and colleagues studied 25 fXI-deficient kindre...

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl Ϫ ) and sodium (Na ϩ ) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na ϩ ), CFTR (Cl Ϫ ), and SLC26A3 (DRA) (Cl Ϫ /HCO3 Ϫ ) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl Ϫ and Na ϩ stool loss in MVID diarrhea.CFTR; brush border; MVI; Myo5b; NHE3; MVID MICROVILLUS INCLUSION DISEASE (MVID) is a rare but life-threatening disease that affects newborns and children and leads to rapid death from severe secretory diarrhea. MVID clusters in the Middle East and Navajo Indian populations in the US and is associated with consanguinity (41,42,47,51,61). Stool volumes are greater than 125 ml·kg Ϫ1 ·day Ϫ1 with elevated levels of chloride (Cl Ϫ ) and sodium (Na

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.

The bleeding disorder associated with factor XI (fXI) deficiency is typically inherited as an autosomal recessive trait. However, some fXI mutations may be associated with dominant disease transmission. FXI is a homodimer, a feature that could allow certain mutations to exert a dominant-negative effect on wild-type fXI secretion through heterodimer formation. We describe 2 novel fXI mutations (Ser225Phe and Cys398Tyr) that form intracellular dimers, are secreted poorly, and exhibit dominant-negative effects on wild-type fXI secretion in cotransfection experiments. Available data now suggest that mutations associated with crossreactive material-negative fXI deficiency fall into 1 of 3 mechanistic categories: ( IntroductionThe homodimeric plasma protein factor XI (fXI) is the precursor of the coagulation protease fXIa. 1,2 FXI dimer formation, which is unique among coagulation proteases, 1-3 is a prerequisite for fXI secretion from cells. 4,5 The noncatalytic portion of the fXI polypeptide contains 4 repeats called apple domains (A1-A4 from the N-terminus). 2 The A4 domain is the primary mediator of dimer formation, 6 and mutations in A4 that interfere with dimerization are associated with retention of fXI monomer within cells and low plasma fXI levels. 4,5 The dimeric structure has implications for inheritance patterns in congenital fXI deficiency. This disorder is prevalent in Ashkenazi Jews, in whom 2 point mutations, Glu117Stop and Phe283Leu, predominate. 7,8 These mutations are associated with an apparently autosomal recessive bleeding disorder, as heterozygotes are generally asymptomatic. 7,9 The situation is not as clear for fXI-deficient patients not of Jewish ancestry, in whom no single mutation predominates. There are reports of heterozygotes for fXI mutations with plasma fXI levels of less than 0.2 U/mL, a range commonly seen in homozygous or compound heterozygous fXI-deficient Jewish patients. 5,[10][11][12] In 2 cases, mutations were identified (Gly400Val and Trp569Ser) that exert dominant-negative effects on fXI secretion in transfection experiments, probably through formation of nonsecretable heterodimers. 5 It is not clear whether these cases are unusual or represent a common mechanism in fXI deficiency. Prompted by these data, and personal observations of patients with fXI levels in an intermediate range (20%-30% of normal) between those typically seen in homozygous and heterozygous Jewish patients, we analyzed the effects of 2 novel fXI mutations on wild-type fXI secretion. The results, in conjunction with prior work, suggest a simple classification system for cross-reactive material-negative (CRM Ϫ ) fXI deficiency. Study design PatientsPatient 1 is a 34-year-old man with a history of left knee hemarthrosis and frequent epistaxis. His fXI levels range from 22% to 30% of normal. His 2 children have fXI levels of 25% to 37%. All 3 individuals are heterozygous for a CϾT change in fXI exon 7, resulting in a Ser225 to Phe substitution. Patient 2 is a 38-year-old woman with heavy postpartum bleeding,...

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a’s role in BBM localization of sucrase-isomaltase.

Summary. The coagulation protease zymogen factor (F)XI is a disul®de bond-linked homodimer, a con®guration that is necessary for protein secretion and function. The non-catalytic portion of the FXI polypeptide contains four repeats called apple domains (A1±A4). It is clear that FXI A4 plays a key role in dimer formation, however, the importance of other apple domains to this process has not been examined. We prepared recombinant FXI molecules in which apple domains were exchanged with those of the structurally homologous monomeric protein prekallikrein (PK). As expected, FXI/PK chimeras containing FXI A4 are dimers, while those with PK A4 are monomers. FXI A4 contains cysteine at position 321 that forms the interchain disul®de bond, while Cys321 in PK is unavailable for interchain bond formation because it is paired with Cys326. FXI/PK chimeras containing PK A4 were modi®ed by changing Cys326 to glycine, leaving Cys321 unpaired (PKA4-Gly326). FXI with a PK A4 domain is a monomer, however, introducing PKA4-Gly326 results in a disul®de bondlinked dimer. This indicates that dimer formation can occur in the absence of FXI A4. In proteins containing PKA4-Gly326, replacing FXI A3 with PK A3 partially interferes with dimer formation, while substitution of A2, or A2 and A3 prevents dimer formation. PKA4-Gly326 cannot induce the native PK molecule to dimerize. The data indicate that FXI A2 and A3 make contributions to dimer formation. As these domains are involved in activities that require dimeric protein, it seems reasonable that they stabilize this conformation.Keywords: apple domain, dimer, factor XI, prekallikrein. A major difference between FXI and PK is that the former is comprised of two identical polypeptides connected through a single disul®de bond, while the latter is a single chain protein.Two hypotheses have been proposed to explain the functional signi®cance of the FXI homodimer. Mutations associated with FXI de®ciency in humans that interfere with intracellular dimer formation result in poor secretion of FXI from transfected cells in culture [15], suggesting dimeric structure is necessary for intracellular processing and/or secretion from hepatocytes. The dimer may also be required for protease function on the surface of activated platelets, where one polypeptide binds to the platelet while the other interacts with the substrate FIX [16].It is clear that a major function of the FXI A4 domain is to facilitate dimer formation. The disul®de bond linking the two polypeptides involves cysteine 321 (Cys321), which resides in A4 [3,17]. In contrast, Cys321 in PK forms an intrachain bond with a cysteine unique to PK at position 326. Introducing FXI A4 into the single chain protein tissue plasminogen activator (tPA) causes the protein to dimerize [17]. While these observations demonstrate the importance of FXI A4 to dimerization, some evidence suggests that other FXI domains contribute. In an attempt, to generate single-chain FXI, we replaced FXI A4 with PK A4 [10,16]. The resulting chimera (FXI/PKA4) is a monomer ...

) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K D ‫؍‬ 1600 ؎ 300 M, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCIIdependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.The central coagulation proteinase, ␣-thrombin (T), 2 is covalently inactivated by the serpins antithrombin (AT) and heparin cofactor II (HCII), in reactions that are accelerated by sulfated glycosaminoglycans (GAGs) (1-6). Two electropositive sites on thrombin, exosites I and II, are differentially involved in its inactivation by HCII and AT (7,8). High molecular weight GAGs act as templates between thrombin exosite II and the GAG binding sites on AT and HCII (1, 2, 5, 9 -11), and an 18 saccharide unit length is required for template activity (12).The HCII mechanism also utilizes the allosteric interaction of thrombin exosite I with the Glu 53 -Asp 75 acidic sequence in the HCII NH 2 -terminal region that contains two hirudin-(54 -65)-like repeats (3,4,(13)(14)(15)(16)(17). This sequence, not present in AT, becomes available for thrombin interaction upon GAG binding of HCII (3,16). Direct evidence was provided by the crystal structure of the HCII⅐S195A-thrombin Michaelis complex, in which residues 56 -72 of HCII make contact with exosite I (16). Both repeats are required for heparin-and DS-catalyzed thrombin inactivation, as demonstrated by the decreased inhibitory potential of HCII NH 2 -terminal deletion mutants (3,14). Mutation of thrombin exosite I residues Arg 67 and Arg 73 resulted in significantly slower inactivation by native HCII (15). In reactions utilizing template-forming GAGs, both the template and allosteric interactions contribute to the mechanism by binding of GAG to thrombin exosite II and interaction of thrombin-complexed GAG with the heparin binding site in HCII, thereby triggering interaction of the HCII NH 2 -terminal sequence with exosite I. The intermediate T⅐GAG⅐HCII complexes, stabilized by two interactions, are significantly tighter than the T⅐GAG⅐AT complexes (9).Oligosaccharides shorter than 18 saccharide units, such as dermatan sulfate hexasaccharides, and sulfated bis-lactobionic and bis-maltobionic acid amides moderately accelerated inhibition by HCII but not [18][19][20]. These molecules are too small for template action, and it is unknown whether they bind to thrombin. Their mechanism of action is proposed to be solely allosteric, by binding to HCII and triggering interaction of the NH 2 -terminal sequence with exosite I. The sulfated disaccharide, sucrose octasulfate (SOS), a known anti-ulcer drug (21) recently identified as an antitumor...