[Partial research progress of GGCX pathogenic variation associated phenotypes].

γ-glutamyl carboxylase (GGCX), also known as vitamin K-dependent glutamyl carboxylase, catalyzes the posttranslational modification of specific glutamate residues in vitamin K-dependent proteins (VKDPs), and participates multiple biological functions including blood coagulation, bone metabolism, vascular calcification, and cell proliferation. It has been reported originally that GGCX pathogenic variation causes blood coagulation deficiency, which is called as vitamin K-dependent coagulation factor deficiency 1 (VKCFD1). Recently, it has been found that GGCX gene variation results in multiple clinical phenotypes, including dermatological, ophthalmological, skeletal or cardiac abnormalities. Among them, dermatological phenotype is the most common, which is known as pseudoxanthoma elasticum-like syndrome. This paper has reviewed the GGCX pathogenic variation associated phenotypes, in order to increase the recognition of GGCX-related genetic diseases and to help its diagnosis and treatment.

The Role of GRP and MGP in the Development of Non-Hemorrhagic VKCFD1 Phenotypes.

Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX) gene. The GGCX enzyme catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins, which have function in blood coagulation, calcification, and cell signaling. Therefore, in addition to bleedings, some VKCFD1 patients develop diverse non-hemorrhagic phenotypes such as skin hyper-laxity, skeletal dysmorphologies, and/or cardiac defects. Recent studies showed that GGCX mutations differentially effect γ-carboxylation of VKD proteins, where clotting factors are sufficiently γ-carboxylated, but not certain non-hemostatic VKD proteins. This could be one reason for the development of diverse phenotypes. The major manifestation of non-hemorrhagic phenotypes in VKCFD1 patients are mineralization defects. Therefore, the mechanism of regulation of calcification by specific VKD proteins as matrix Gla protein (MGP) and Gla-rich protein (GRP) in physiological and pathological conditions is of high interest. This will also help to understand the patho-mechanism of VKCFD1 phenotypes and to deduce new treatment strategies. In the present review article, we have summarized the recent findings on the function of GRP and MGP and how these proteins influence the development of non-hemorrhagic phenotypes in VKCFD1 patients.

Evaluation of Bleeding Phenotype of Inherited Factor VII Deficiency in Children With a Bleeding Assessment Tool and Global Assays.

INTRODUCTION: Inherited factor VII (FVII) deficiency is the most common of the rare bleeding disorders and shows a heterogenous distribution of bleeding phenotypes independent of factor activity level. The bleeding score (BS) evaluates the phenotype of patients with rare bleeding disorders. Thromboelastography (TEG) and thrombin generation assays (TGAs) are 2 methods to evaluate global hemostasis, and controversially both tests are useful for identifying different bleeding tendency phenotypes. The purpose of this study was to investigate the use of the BS and global assays (TEG and TGAs) to predict the bleeding phenotype of inherited FVII deficiency. MATERIALS AND METHODS: A total of 27 patients with FVII deficiency were evaluated with the BS and global hemostasis assays. RESULTS: The BS was compatible with disease severity according to the FVII activity level (P<0.05) but the BS and bleeding grade of patients did not show a statistically significant correlation with factor activity level (P>0.05). No significant correlation was observed between the factor activity level and any TEG parameter (P>0.05). The factor activity level was negatively correlated with the lag time of the TGA on the contrary positively correlated with the peak thrombin time of the TGA (P<0.05). CONCLUSIONS: The global assays do not successfully predict the bleeding phenotype. The BS is a more suitable tool than conventional and global assays for predicting the bleeding phenotype.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis.

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

Fibrinolysis and the control of blood coagulation.

Fibrin plays an essential role in hemostasis as both the primary product of the coagulation cascade and the ultimate substrate for fibrinolysis. Fibrinolysis efficiency is greatly influenced by clot structure, fibrinogen isoforms and polymorphisms, the rate of thrombin generation, the reactivity of thrombus-associated cells such as platelets, and the overall biochemical environment. Regulation of the fibrinolytic system, like that of the coagulation cascade, is accomplished by a wide array of cofactors, receptors, and inhibitors. Fibrinolytic activity can be generated either on the surface of a fibrin-containing thrombus, or on cells that express profibrinolytic receptors. In a widening spectrum of clinical disorders, acquired and congenital defects in fibrinolysis contribute to disease morbidity, and new assays of global fibrinolysis now have potential predictive value in multiple clinical settings. Here, we summarize the basic elements of the fibrinolytic system, points of interaction with the coagulation pathway, and some recent clinical advances.

A novel congenital dysprothrombinemia leading to defective prothrombin maturation.

INTRODUCTION: Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening. AIM: Here we characterize a patient with a novel missense mutation in F2, c.1090T/A (p.Val322Glu), that causes severe dysprothrombinemia. METHODS: Coagulation assays, prothrombin Western Blotting, FII activation by Ecarin, fibrinogen degradation products quantification and thrombin generation assay were carried out to assess prothrombin expression and function. PCR followed by direct sequencing was carried out to characterize the mutation. In silico analysis for missense variant and molecular modeling were applied to predict the mechanism that leads to dysprothrombinemia. RESULTS AND CONCLUSIONS: The homozygous patient had a markedly prolonged prothrombin time, strongly reduced FII activity (0.82%) but normal antigen levels. In the thrombin generation assay the lag time and the peak height were unmeasurable, suggesting that the Val322Glu mutation results in the inability of the mutant prothrombin to be fully activated to thrombin. In fact, prothrombin activation by ecarin was defective, with a massive accumulation of the meizothrombin intermediate. Molecular modeling and dynamic simulation studies showed that the Val322Glu mutation interferes with protein flexibility at Arg271 and Arg320. This impairs the switch of the protein from zymogen to proteinase, thus preventing the formation of thrombin. Accumulated meizothrombin, however, maintains some fibrinogen-degrading activity, as shown by the formation of FDPs, and this probably explains the patient's mild bleeding phenotype.

Bleeding and non-bleeding phenotypes in patients with GGCX gene mutations.

Functional limitations for the vitamin K cycle, caused either by mutations in gamma-glutamyl carboxylase or vitamin K epoxide reductase genes, result in hereditary deficiency of vitamin K-dependent coagulation factors (VKCFD1 and VKCFD2, respectively). Patients suffering from VKCFD often share several other anatomical irregularities which are not related to haemostasis. Here we report on nine patients, eight of them previously unreported, who presented with VKCFD1. All were examined with special attention to vitamin K-dependent coagulation factors as well as to bone and heart development and to other anatomical signs of embryonal vitamin K deficiency. In total, we detected ten mutations in the gamma-glutamyl carboxylase gene of which seven have not been previously reported. Most interestingly, additional non-bleeding phenotypes were observed in all patients including midfacial hypoplasia, premature osteoporosis, cochlear hearing loss, heart valve defects, pulmonary stenosis, or pseudoxanthoma elasticum-like phenotype. Undercarboxylated matrix Gla protein, osteocalcin, and periostin appear to be responsible for these defects which are also observed in cases of fetal warfarin syndrome.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbß3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.

Congenital platelet disorders and understanding of platelet function.

Genetic defects of platelets constitute rare diseases that include bleeding syndromes of autosomal dominant, recessive or X-linked inheritance. They affect platelet production, resulting in a low circulating platelet count and changes in platelet morphology, platelet function, or a combination of both with altered megakaryopoiesis and a defective platelet response. As a result, blood platelets fail to fulfil their haemostatic function. Most studied of the platelet function disorders are deficiencies of glycoprotein mediators of adhesion and aggregation while defects of primary receptors for stimuli include the P2Y12 ADP receptor. Studies on inherited defects of (i) secretion from storage organelles (dense granules, α-granules), (ii) the platelet cytoskeleton and (iii) the generation of pro-coagulant activity have identified genes indirectly controlling the functional response. Signalling pathway defects leading to agonist-specific modifications of platelet aggregation are the current target of exome-sequencing strategies. We now review recent advances in the molecular characterization of platelet function defects.

Type II antithrombin deficiency caused by a large in-frame insertion: structural, functional and pathological relevance.

BACKGROUND: The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences. OBJECTIVE: To characterize the first antithrombin deficiency caused by a large in-frame insertion. PATIENTS/METHODS: Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK-EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin. RESULTS: The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in-frame insertion of 24 bp in SERPINC1, affecting strand 3 of ß-sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N-terminal disulphide bonds, and was conformationally sensitive. The variant was non-inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into ß-sheet A as the fourth strand, and maintained a native RCL. CONCLUSIONS: This is the first case of a large in frame-insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non-inhibitory variant, which acquires a hyperstable conformation with a native RCL.

Thrombosis in rare bleeding disorders.

Inherited deficiencies of blood coagulation factors are usually associated with lifelong bleeding tendency. In addition to Haemophilias A and B and von Willebrand disease, congenital deficiencies of such factors as fibrinogen, prothrombin (FII)), FV, FVII, FX, FXI, FXIII, and combined deficiencies occur and can lead to a diversity of clinical conditions. Paradoxically, for some of these disorders associated with significant bleeding tendency there are reports of thrombotic events, both arterial and venous. Thrombosis in hemophilia patients has a multifactorial pathogenesis and the main conditions associated with this complication are the use of long-term central venous catheters, intensive replacement therapy usually in the setting of surgical procedures, the use of bypassing agents or the coexistence of acquired or inherited prothrombotic risk factors. Regarding other rare bleeding disorders, thrombotic phenomena has been described particularly in patients with afibrinogenemia, FXI and FVII deficiency and the events can occur even in young patients, in the presence of concomitant risk factors or spontaneously. Replacement therapy must be individualized and should take into account past history of haemostatic challenges, family history of bleeding and thrombosis, just like the level of factor. For mild deficiencies when patients are asymptomatic the use of antithrombotic prophylaxis must be considered with or without concomitant use of replacement therapy. In patients with history of thrombosis it may be helpful to perform a thrombophilia screening to exclude coexisting prothrombotic defects and for all patients it is recommended to control known cardiovascular disease risk factors.

Crystal structure of the LMAN1-CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII.

LMAN1 is a glycoprotein receptor, mediating transfer from the ER to the ER-Golgi intermediate compartment. Together with the co-receptor MCFD2, it transports coagulation factors V and VIII. Mutations in LMAN1 and MCFD2 can cause combined deficiency of factors V and VIII (F5F8D). We present the crystal structure of the LMAN1/MCFD2 complex and relate it to patient mutations. Circular dichroism data show that the majority of the substitution mutations give rise to a disordered or severely destabilized MCFD2 protein. The few stable mutation variants are found in the binding surface of the complex leading to impaired LMAN1 binding and F5F8D.

Hemospermia in patients with congenital coagulation disorders: a study of three cases.

Hemospermia is usually a symptom of urological relevance, however it may have also a medical and hematological significance and has been reported in congenital or acquired bleeding disorders. Because of this symptom's negative psychological impact on the patient, it is likely that the condition is underplayed and therefore underdiagnosed. During the years 1967-2003 we had the opportunity to see 3 patients with hemospermia on a congenital bleeding disorder: a patient with hemophilia A, another with prothrombin deficiency and finally a patient with von Willebrand disease type I. All patients were heterosexual. In all instances the course was benign since it required administration of substitution therapy on only 2 occasions. Rest and abstinence from sexual activity appeared to be helpful. The first patient had other signs and symptoms compatible with the diagnosis of urethritis due to Escherichia coli and he underwent a course of antibiotic therapy. The other 2 cases appeared to be idiopathic since no associated condition was found. Urinary cytology, rectal examination, prostate sonography and prostate-specific antigen were normal in all cases. The rarity of hemospermia in congenital bleeding disorders remains unexplained, although the strong perineal and sphincter muscles may exercise a compressive hemostatic effect which could prevent or reduce bleeding.

Thrombin activatable fibrinolysis inhibitor (TAFI)--how does thrombin regulate fibrinolysis?

The thrombin-catalysed conversion of plasma fibrinogen into fibrin and the development of an insoluble fibrin clot are the final steps of the coagulation cascade during haemostasis. A delicate balance between coagulation and fibrinolysis determines the stability of the fibrin clot. Thrombin plays a central role in this process, it not only forms the clot but it is also involved in stabilizing the clot by activating thrombin activatable fibrinolysis inhibitor (TAFI). Activated TAFI protects the fibrin clot against lysis. Here we will discuss the mechanisms for regulation of fibrinolysis by thrombin. The role of the coagulation system for the generation of thrombin and for the activation of TAFI implies that defects in thrombin generation will directly affect the protection of clots against lysis. Thus, defects in activation of TAFI might contribute to the severity of bleeding disorders. Vice versa an increased activation of TAFI due to an increased rate of thrombin generation might lead to thrombotic disorders. Specific inhibitors of activated TAFI or inhibitors that interfere with the generation of thrombin might provide novel therapeutic strategies for thrombolytic therapy. Besides having a role in the regulation of fibrinolysis, TAFI may also have an important function in the regulation of inflammation, wound healing and blood pressure.

A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome.

Scott syndrome (SS) is a bleeding disorder characterized by a failure to expose phosphatidylserine (PS) to the outer leaflet of the platelet plasma membrane. Because the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) is implicated in the exofacial translocation of PS, we assessed its role in the pathophysiology of a patient with SS. Substantially reduced levels of ABCA1 mRNA were found in the patient's leukocytes, compared with controls. The SS patient was heterozygous for a novel missense mutation c.6064G>A (ABCA1 R1925Q), absent from unaffected family members and controls. Both mutant and wild-type alleles were reduced in mRNA expression, and no causative mutation for this phenomenon was identified in the ABCA1 gene or its proximal promoter, suggesting a putative second mutation in a trans-acting regulatory gene may also be involved in the disorder in this patient. In vitro expression studies showed impaired trafficking of ABCA1 R1925Q to the plasma membrane. Overexpression of wild-type ABCA1 in SS lymphocytes complemented the Ca2+-dependent PS exposure at the cell surface. These data identify a mutation in ABCA1 that contributes to the defective PS translocation phenotype in our patient with SS.

Inherited defects of platelet function.

Inherited platelet defects bleeding syndromes underlie of varying severity. The Bernard-Soulier syndrome and Glanzmann thrombasthenia are disorders of membrane glycoproteins. In the former, a deficiency of the GPIb-IX-V complex leads to defective platelet adhesion, while in thrombasthenia, platelet aggregation does not occur in the absence of the integrin alphaIIbbeta3. Defects of primary receptors for stimuli are increasingly being described, and include a defect of a newly cloned Gi-protein-linked, seven transmembrane domain, ADP receptor. These lead to agonist-specific deficiencies in the platelet function response, as do abnormalities in the many intracellular signaling pathways of platelets. Defects affecting secretion from dense bodies and alpha-granules, of ATP production and generation of procoagulant activity, are also encountered. Some disorders are exclusive to megakaryocytes and platelets, while in others, such as the Chediak-Higashi, Hermansky-Pudlak and Wiskott-Aldrich syndromes; the molecular lesion extends to other cell types. Disorders affecting platelet morphology, the so-called "giant platelet" syndromes should also be considered. In familial thrombocytopenias, platelets are produced in insufficient quantities to assure hemostasis. Platelet disorders are examples of rare diseases; nevertheless they have provided essential information in the elucidation of the molecular basis of platelet function.