FV) And VIII Research Articles

Mice with LMAN1 Deficiency Exhibit Thrombocytopenia and Reduced Serum Thrombopoietin Level

LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s).To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/- mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry.In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/- mice was indistinguishable from livers of WT littermate controls.In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Additional Targeted Lman1 Alleles in the Mouse Suggests a Dose Dependence for FV/FVIII Secretion and Novel Role for an LMAN1-Dependent Cargo in Thrombopoiesis

Combined deficiency of coagulation factors V and VIII (F5F8D) is a human autosomal recessive bleeding disorder caused by mutations in LMAN1 or MCFD2, which encode the components of the only known cargo-receptor complex in the mammalian secretory pathway. The LMAN1/MCFD2 complex facilitates secretion of coagulation factors V (FV) and VIII (FVIII) to the plasma. F5F8D patients exhibit FV and FVIII levels that are ~10-15% of normal. The relationship between Lman1 expression levels and the corresponding secretion of LMAN1-dependent cargos has not been characterized, and additional functions of LMAN1 remain to be determined. We previously described Lman1 mutant mice carrying a gene-trap insertion in intron 10 (Lman1gt1). Lman1gt1/gt1 mice have ~50% of normal FV and FVIII activity levels, and are therefore less severely affected than human F5F8D patients. This difference could be explained by low levels of residual LMAN1 expression, or by differences in FV and FVIII secretion between mice and humans. Lman1gt1/gt1 mice also exhibit a partially penetrant, perinatal lethality that is restricted to certain mouse genetic strains. We generated a second, independent LMAN1-deficient mouse with no Lman1 gene expression (Lman1-/-), as well as hypomorphic LMAN1-deficient mice (Lman1cgt/cgt) that retain ~7% of wildtype Lman1 expression levels on a C57BL/6J genetic background. Plasma FV and FVIII levels in Lman1-/- and Lman1cgt/cgtmice were measured by functional coagulation assays and compared to wildtype (WT) and Lman1gt1/gt1 mice. A dose-response relationship was observed, with Lman1-/- mice exhibiting FV and FVIII levels that are ~50% of normal (compared to ~10-15% in human patients), while hypomorphic Lman1cgt/cgtmice demonstrate an intermediate reduction in FV and FVIII (~70% of WT levels). A similar result was demonstrated for alpha-1 antitrypsin, another known LMAN1-dependent cargo. Taken together with the presence of two null LMAN1 alleles in nearly all reported LMAN1 F5F8D patients, these results suggest that hypomorphic LMAN1 mutations in humans may also result in milder reductions in plasma FV and FVIII that may have been missed clinically, though still contributing to FV and FVIII level variation and to bleeding susceptibility. The partially penetrant, strain-specific, perinatal lethality previously reported in Lman1gt1/gt1 mice was also confirmed in mice homozygous for the independently generated Lman1- allele on a pure C57BL/6J genetic background (p &lt; 0.007), thereby ruling out a passenger gene effect as the cause of the originally reported Lman1gt1/gt1 lethality. In contrast, Lman1cgt/cgt mice on the same background are viable and observed in the expected Mendelian ratios (p &gt; 0.19). Lman1-/- lethality was not observed on a mixed C57BL/6J and 129S1/SvImJ background (p &gt; 0.79). F5F8D is not associated with fetal loss in humans, nor would fetal loss be expected from moderate deficiency of FV and FVIII. These results thus suggest a critical role for additional, as yet unknown, LMAN1 cargo(s). Finally, adult Lman1-/- mice exhibit a mild thrombocytopenia compared to WT controls (9.3 x 105 vs. 12.3 x 105 cells/uL, p &lt; 0.004). In contrast, Lman1+/-, Lman1cgt/cgt, and Mcfd2-/- mice all exhibit normal platelet counts indistinguishable from WT controls. No Lman1-/- platelet morphologic abnormalities are evident by standard or transmission electron microscopy, with normal appearance of megakaryocyte number and morphology on bone marrow examination. Although thrombocytopenia has not been noted previously in human F5F8D patients, these results suggest a novel role for an LMAN1-dependent secretory cargo in thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Evaluation and performance characteristics of the Q Hemostasis Analyzer, an automated coagulation analyzer

The Q Hemostasis Analyzer (Grifols, Barcelona, Spain) is a fully-automated random-access multiparameter analyzer, designed to perform coagulation, chromogenic and immunologic assays. It is equipped with a cap-piercing system. The instrument was evaluated in a hemostasis laboratory of a University Hospital with respect to its technical features in the determination of coagulation i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen and single coagulation factors V (FV) and VIII (FVIII), chromogenic [antithrombin (AT) and protein C activity] and immunologic assays [von Willebrand factor antigen (vWF:Ag) concentration], using reagents from the analyzer manufacturer. Total precision (evaluated as the coefficient of variation) was below 6% for most parameters both in normal and in pathological ranges, except for FV, FVIII, AT and vWF:Ag both in the normal and pathological samples. No carryover was detected in alternating aPTT measurement in a pool of normal plasma samples and in the same pool spiked with unfractionated heparin (>1.5 IU/mL). The effective throughput was 154 PT, 66 PT/aPTT, 42 PT/aPTT/fibrinogen, and 38 PT/aPTT/AT per hour, leading to 154 to 114 tests performed per hour, depending of the tested panel. Test results obtained on the Q Hemostasis Analyzer were well correlated with those obtained on the ACL TOP analyzer (Instrumentation Laboratory), with r between 0.862 and 0.989. In conclusion, routine coagulation testing can be performed on the Q Hemostasis Analyzer with satisfactory precision and the same apply to more specialized and specific tests.

Factor V and VIII combined deficiency: Clinical perioperative management for tonsillectomy in a child

Combined factors V (FV) and VIII (FVIII) deficiency is a rarely seen hereditary coagulation disease. Experience of its management in surgery with a high-risk of bleeding is rare. The interest of this case report is to propose a strategy of perioperative management for such a deficit, but also to recall that a careful preoperative anesthetic evaluation with questioning and physical examination permits to detect unsuspected coagulation disorders and to schedule the preventive treatment. The protocol for the perioperative period consisted of the administration of desmopressin and fresh frozen plasma one hour before surgery. The administration of desmopressin was continued for 48hours. Fresh frozen plasma and tranexamic acid were administered during the first 9 postoperative days. A local bleeding occurred at 8 days (scab coming off) and required systematically a surgical hemostasis and an intensification of the therapeutic protocol. Recombinant plasmatic factor VIII was administered for 7 days together with a daily perfusion of fresh frozen plasma for a total treatment period of 14 days.

Rational design of small molecules targeting the C2 domain of coagulation factor VIII

AbstractThe C domains of coagulation factors V (FV) and VIII (FVIII) are structurally conserved domains and share a common and essential function in membrane binding. In vivo regulation of thrombin formation strongly depends on the expression and regulation of the cofactor activities of FVIII and FV. With this study, we explored the possibility of inhibition of thrombin formation in full blood with small druglike molecules. Such compounds may serve as lead molecules for the development of a new type of orally available coagulation inhibitors that act by blocking the interaction between the C domains of FVIII and the membrane surface. We identified 9 novel molecules that are able to inhibit binding of the FVIII C2 domain to a model membrane by application of a combined ligand-based and target structure–based virtual screening approach that took into account the knowledge of a set of previously identified low-molecular-weight FVIII binders that were, however, not active in full blood. The half-maximal inhibitory concentration values of our newly identified compounds varied from 2.1 to 19.9 µM, of which 7 of 9 molecules did not appreciably inhibit FV membrane binding and were thus specific for FVIII. The most active bioactive compound showed activity in both plasma and in full blood.

Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)

LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca(2+)-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca(2+) concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca(2+) concentrations. Our results suggest that compartmental changes in Ca(2+) concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.

Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

AbstractThe LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca2+- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.

Avaliação da anticoagulação natural em pacientes com diabetes mellitus tipo 2

INTRODUCAO: Alteracoes no mecanismo hemostatico tem sido associadas ao desenvolvimento de complicacoes vasculares em individuos com diabetes mellitus tipo 2 (DM2). A eficiencia dos mecanismos da anticoagulacao natural mediados pelas proteinas C (PC) e S (PS), bem como pela antitrombina (AT), depende da integridade da celula endotelial. Sendo assim, a avaliacao dos niveis plasmaticos dessas proteinas poderia fornecer importantes informacoes acerca da anticoagulacao natural que se contrapoe ao estado de hipercoagulabilidade no diabetes. OBJETIVO: O presente estudo teve como objetivo a determinacao dos niveis de AT, PC e PS e dos co-fatores fator V (FV) e fator VIII (FVIII) em individuos com e sem DM2 e hipertensao arterial sistemica (HAS). MATERIAL E METODOS: Individuos higidos (controles, n = 16), pacientes com DM2 (n = 7), com hipertensao (HAS, n = 12) e com DM2 associado a HAS (DM2 + HAS, n = 18) foram incluidos neste estudo para avaliacao da anticoagulacao natural. RESULTADOS: Para os niveis de AT o grupo HAS apresentou niveis aumentados em relacao ao controle. O grupo DM2 + HAS apresentou niveis aumentados de PC. Para a PS nao foi encontrada diferenca significativa entre os grupos. O aumento do FV ocorreu apenas no grupo DM2 + HAS; ja para o FVIII nao foi encontrada diferenca significativa entre os grupos. CONCLUSOES: O aumento da AT e ainda pouco conhecido no meio cientifico; outros estudos ja observaram aumento de PC em pacientes com DM2 e normoalbuminuria, entretanto o mecanismo permanece desconhecido. O aumento de FV observado no grupo DM2 + HAS e nao no grupo DM2 pode acontecer em decorrencia da HAS. Como o FVIII e uma proteina de fase aguda, a ausencia de diferenca significativa entre os grupos indica que os individuos avaliados nao apresentavam processos inflamatorios importantes.

Structural requirements for the activation of human factor VIII by thrombin

The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion-binding exosite-I (Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and anion-binding exosite-II (Arg98Ala), the Na(+)-binding site (Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the 50-insertion loop (Trp50Ala), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I appears to be important for cleavage at both sites, whereas the anion-binding exosite-II residue Arg98Ala is important for cleavage at Arg372 alone. The Glu229Ala mutant, which contributes to the Na(+)-binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at Arg372 and Arg1689. This suggests that the integrity of the active site and the Na(+)-bound form of thrombin are important for its procoagulant activity against FVIII. Detailed mutagenic analysis of thrombin can assist in understanding the pathogenesis of bleeding disorders and may lead to the rational design of selective thrombin inhibitors.

Homology Models of the C Domains of Blood Coagulation Factors V and VIII: A Proposed Membrane Binding Mode for FV and FVIII C2 Domains

ABSTRACT: We present homology models of the C domains of coagulation factors V (FV) and VIII (FVIII). Using a threading approach, we identified the binding domain of galactose oxidase as an appropriate template for each C domain. The C1 and C2 domains of FV associate to form an elongated cylinder of 80Å long and 30Å diameter. The folding unit is a β-sandwich with a long axis of 40Å and a diameter of 30Å. The current model allows us to propose a membrane binding mode for the C2 domains of FV and FVIII with three major characteristics: 1) solvent-exposed hydrophobic side chains from three loops at one end of the β-sandwich are buried in the hydrophobic layer of the outer phospholipid leaflet; 2) a crown of positively charged residues is located in the polar zone of the phospholipid head groups; and 3) the long axis of the β-sandwich of the C2 domain is perpendicular to the plane of the membrane. This proposal satisfies experimentally observed characteristics of membrane binding for the C2 domain and the light chain of FVa.

Molecular Models for the two Discoidin Domains of Human Blood Coagulation Factor V

Discoidin (DS) domains occur in a large variety of proteins. We have recently reported the D1 domain of galactose oxidase (GOase), a copper-containing enzyme whose structure has been determined at 1.7 A resolution, as distant member of the DS domain family. The D1 domain of GOase consists of a five-stranded antiparallel β-sheet packing against a three-stranded antiparallel β-sheet. We here show that it is possible to build 3D models for DS domains using GOase as initial template and propose a 3D structure for the C1 and C2 domains of factor V (residues 1879-2037 and 2038-2196). Factors V (FV) and VIII (FVIII) are essential and homologous non-enzymatic cofactors in the coagulation cascade. They share the domain organization A1-A2-B-A3-C1 and C2 and their C domains are members of the DS family. The C1 and C2 domains of FV are rich in positively charged residues. Several clusters of amino acids, most likely involved in inter-domain interactions, protein-protein interactions and/or phospholipid binding, are identified. Our report opens new avenues to study the structure-function relationships of DS domains.

The effect of hemorrhagic shock on the clotting cascade in injured patients.

The effects of injury with hemorrhagic shock on the clotting and fibrinolytic systems were studied serially in 22 patients receiving 21 +/- 13 transfusions and 1.26 +/- 0.58 L of fresh frozen plasma (FFP) during operation (OR). The PT, aPTT, thrombin time (TT), fibrinogen (FI), factors V (FV) and VIII (FVIII), fibrin(ogen) split products (FSP) and fibrin monomers were measured in OR and after OR at 6 and 15 hours, days 2 and 4, and at convalescence (25 days). The TT, PT, and aPTT were were prolonged in OR and reflected the low FI, FV, and FVIII, respectively. After OR, clotting times and factor levels returned toward normal. By day 4 and convalescence, FI, FV, and FVIII exceeded normal levels. FSP levels were normal in OR. After OR FSP rose progressively through day 4 when all patients had levels greater than 10 mcg/ml and most patients had levels above 40 mcg/ml. Fibrin monomers were absent until the 15-hour study after which a small number of patients had monomers through the convalescent study. The acute fall in clotting factors is likely due to increased hemostatic demands, plasma dilution from resuscitation, and extravascular relocation from shock-induced extravascular expansion. Later factor restoration likely reflects enhanced hepatic synthesis, factor half-life, capillary selectivity retaining large molecular weight factors, and intravascular relocation from abundant extravascular stores. Throughout this biphasic response, the clotting times reflect factor levels. Fibrinolysis contributes little to these changes.