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A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient's plasma clotting time indicated that both activated partial thromboplastin time (52.6 seconds) and prothrombin time (19.4 seconds) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired approximately fivefold. Thrombin generation assay using patient's plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased; however, the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with approximately 12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased by approximately 10-fold; however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated approximately 20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient's normal hemostatic phenotype.

Key Points Deleterious F8 mutations do not necessarily lead to the incidence of inhibitors in hemophilia A patients receiving replacement therapy. Maternal chimeric cells migrated into a fetus with hemophilia A during pregnancy could induce tolerance toward FVIII.

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC. We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.

Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.

Protein C belongs to vitamin K-dependent family of serine protease zymogens in plasma which upon activation by thrombin in complex with thrombomodulin (TM) functions as an anticoagulant to degrade factors Va and VIIIa (FVa and FVIIIa) by limited proteolysis [1–3]. Activated protein C (APC) has a multi-domain structure composed of an N-terminal non-catalytic light chain linked by a disulfide bond to the C-terminal catalytic heavy chain with a trypsin-like specificity [4,5]. APC functions as an anticoagulant by proteolytic cleavage of at least two peptide bonds on heavy chains of factors FVa and FVIIIa bound to negatively charged membrane surfaces in extrinsic and intrinsic pathways of the clotting cascade [6–9]. The APC cleavage of cofactors culminates in disassembly of procoagulant activation complexes and inhibition of thrombin generation. The anticoagulant function of APC in degradation of FVa and FVIIIa requires the cofactor function of protein S, bound to the same negatively charged membrane surfaces in the presence of calcium [10,11]. In addition to its key anticoagulant role, APC also exhibits potent cytoprotective and antiinflammatory activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 [12–14]. Thus, protein C deficiency is associated with dysregulation of both coagulation and inflammatory pathways. Protein C deficiency has an autosomal dominant pattern of inheritance with heterozygous deficiency increasing the risk of venous thromboembolism and homozygous deficiency causing purpura fulminans, which is fatal if not treated by protein C replacement therapy [15,16]. Complete deficiency of protein C is not compatible with life in knockout mice [17]. Protein C deficiency is divided into type-I and type-II. Type-I deficiency is characterized by equally low antigen (PC:Ag) and activity (PC:A) levels, whereas type-II is defined by only a lower activity level [18]. Type-I deficiency is characterized by specific ELISA assays and type-II deficiency is measured by analysis of either amidolytic activity of APC after activating plasma protein C by the snake venom Protac or by anticoagulant activity in plasma-based assays using aPTT reagents [19]. If both amidolytic and anticoagulant activities are decreased, the deficiency is called type-IIa and if only anticoagulant activity is reduced the deficiency is classified as type-IIb [18,19]. Most type-IIa deficiencies occur on the catalytic doamin since mutations on the non-catalytic light-chain rarely influence APC amidolytic activity, whereas mutations on both heavy and light chains can adversely affect the APC anticoagulant activity. In this study, we identified novel and not previously reported protein C-deficient patients who experience venous thrombosis. Initial analysis of subjects’ plasma PC:Ag levels by a commercial ELISA and PC:A levels by both chromogenic and clotting assays showed these values are ~50% of normal, suggesting patients have type-I deficiency. However, results from a second commercial ELISA indicated PC:Ag levels of 84% and 102% for patients. Genetic analysis indicated subjects carry a heterozygous mutation (c.715G>A) in PROC, leading to Gly197 to Arg substitution (p.Gly197Arg) on the catalytic domain (G43R in chymotrypsin numbering) [20]. We expressed this protein C variant and discovered the variant is activated by thrombin with ~10-fold improved rate, however, TM has no cofactor activity in promoting its activation as tested in purified and plasma-based assay systems. Moreover, the APC mutant exhibited several hundred-fold impaired activity in both amidolytic and plasma-based coagulation assays. Structural-based analysis by molecular modeling indicated an Arg side-chain in APC can cause major distortions of the local structure, and adversely affect the folding of catalytic pocket and/or the calcium-binding-loop of the mutant. In support of this hypothesis, we discovered the initial commercial ELISA used to determine the PC:Ag level in subjects’ plasma, is incapable of detecting this mutant, explaining the 50% antigen level in the plasma. Thus the first commercial ELISA mischaracterized this type-IIa protein C deficiency as type-I.

Objective: Integrin β3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether β3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with β3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The β3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that β3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine) 720 –722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/β3/LPL complex that is required for β3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in β3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH 720– 722 -mutated β3 genes. Conclusions: This study reveals a hypertriglyceridemia in both β3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of β3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with β3-deficient Glanzmann thrombasthenia.

SummaryProtein C is a vitamin K–dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin (TM) down-regulates the clotting cascade by a feedback loop inhibition mechanism. Activated protein C (APC) exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa. We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal, but its anticoagulant activity is only 34% of the normal level. Genetic analysis revealed that the proband and her younger brother carry a novel heterozygous mutation c.346G>A, p.Gly74Ser (G74S) in PROC. Thrombin generation assay indicated that the TM-dependent anticoagulant activity of the proband’s plasma has been significantly impaired. We expressed protein C-G74S in mammalian cells and characterised its properties in established coagulation assays. We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa. However, it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired. Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired. The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected. These results provide insight into the mechanism through which G74S substitution in APC causes thrombosis in the proband carrying this mutation.

SummaryWe have characterised the pathogenic basis of dysprothrombinaemia in a patient exhibiting paradoxical bleeding and thrombotic defects during pregnancy and postpartum. Genetic analysis revealed that the proband is homozygous for the prothrombin Arg382His mutation, possessing only ~1 % clotting activity. The proband experienced severe bleeding episodes during her pregnancy, which required treatment with prothrombin complex concentrates, and then pulmonary embolism and deep-vein thrombosis at 28 days postpartum, which required treatment with LMWH and fresh frozen plasma. Analysis of haemostatic parameters revealed that the subject had elevated FDP and DD and decreased fibrinogen levels, indicating the presence of hyperfibrinolysis. Thrombin generation and clotting assays with the proband’s plasma in the presence of soluble thrombomodulin and tissue-type plasminogen activator indicated a defect in activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Unlike normal plasma, no TAFI activation could be detected in the patient’s plasma. The expression and characterisation of recombinant prothrombin Arg382His indicated that zymogen activation by prothrombinase was markedly impaired and the activation of protein C and TAFI by thrombin-Arg382His was impaired 600-fold and 2500-fold, respectively. The recombinant thrombin mutant exhibited impaired catalytic activity toward both fibrinogen and PAR1 as determined by clotting and signalling assays. However, the mutant activated factor XI normally in both the absence and presence of polyphosphates. Arg382 is a key residue on (pro)exosite-1 of prothrombin and kinetic analysis of substrate activation suggested that the poor zymogenic activity of the mutant is due to its inability to bind factor Va in the prothrombinase complex.Supplementary Material to this article is available online at www.thrombosis-online.com.

Objective: Defective PC (protein C) pathway predisposes patients to venous thromboembolism (VTE) and is mostly, but not exclusively, attributed to hereditary PC or PS (protein S) deficiencies and activated PC resistance caused by factor V Leiden mutation. Approach and Results: In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the 3 pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. Conclusions: In summary, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin’s procoagulant activity over anticoagulant function.

Background Inherited Factor XIII deficiency (FXIIID) is one of the most severe and under-diagnosed rare bleeding disorders. Only 5 large deletions involving one or more exons in F13A1 have been reported, and lacking of multiplex ligation-dependent probe amplification (MLPA) assay might underestimate the copy number variations (CNVs) in F13A1 and F13B. We had characterized the clinical presentation of two unrelated severe FXIIID probands and explored the pathogenic mechanisms. Results Both probands experienced several episodes of fatal bleeding and delayed wound healings prior to diagnosis. FXIII activity was measured by the ammonia release assay, and FXIII-A and FXIII-B antigens were determined by ELISA. All the exons including exon-intron boundaries and promoter regions of F13A1 and F13B were amplified and directly sequenced. Copy number variations (CNVs) of F13A1 and F13B were detected by the CNVplex® method. Breakpoints of the F13A1 large deletion were identified by quantitative primer walking combined long-range PCR (LR-PCR) strategies. Proband 1 was found to have compound heterozygous mutations of a novel small deletion (c.1147del) and a missense mutation p.Arg383Ser. Proband 2 was compound heterozygous for a novel large deletion (g.[77815_112815del;112837_116628del]) and a missense mutation p.Arg716Gly in F13A1. Bioinformatics analysis of the large deletion breakpoints predicted that two fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) events with two homologies of TCT and C might be responsible for the complex rearrangement. Prophylactic replacement therapy was immediately administered for the two probands upon establishment of the diagnosis. Conclusions We detected two type I FXIIID pedigrees and adopted CNVplex® method to detect CNVs of F13A1 and F13B for the first time. A large heterozygous deletion of g.[77815_112815del;112837_116628del] in F13A1, mediated by two FoSTeS/MMBIR events, was identified. Electronic supplementary material The online version of this article (10.1186/s13023-019-1144-z) contains supplementary material, which is available to authorized users.

Table S1. The target sequences for CNV detection of F13A1 and F13B in probands 1 and 2. Table S2. Primer sequences for quantitative primer walking detection and LR-PCR amplification. Table S3. Quantitative PCR results of 10 fragments in intron 6 and intron 8 of F13A1 for the primer walking detection in proband 2. Table S4. Primer sequences for mRNA analysis of the large deletion of F13A1. Figure S1. Effect of the F13A1 large deletion on transcription analyzed by RT-PCR in proband 2. Figure S2. Instability analysis of the DNA sequences around breakpoints and insertion in proband 2. (DOCX 4664 kb)

The changes in the coagulation, fibrinolytic, and endothelial functions are correlated with the pathophysiology of the thromboembolic diseases during acute illness. However, these changes in patients with hereditary thrombophilia who were not in the acute stage of venous thromboembolism (VTE) are unclear. A panel of 4 biomarkers, including thrombin–antithrombin complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC), tissue-type plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAIC), and soluble thrombomodulin (sTM), were assayed in 100 healthy controls and 100 patients with thrombophilia. Although significantly higher concentrations of TAT, PIC, t-PAIC, and sTM were observed in patients with thrombophilia than in healthy controls, 70 patients showed absolutely normal levels of the above 4 biomarkers. Among the other 30 patients who had at least 1 biomarker out of the corresponding reference interval, 26 of them presented elevated PIC with or without increased TAT. Except for sTM, other 3 biomarkers did not show significant differences in patients with previous VTE compared to those without. Patients with single episode of VTE had obviously lower t-PAIC than those with multiple episodes of VTE, whereas the levels of TAT, PIC, and sTM were unassociated with the number of thrombosis episodes. Most thrombophilia patients who were not in the acute stage of VTE showed normal coagulation, fibrinolytic, and endothelial functions. Thus, we were unable to show that the one-time response of this panel was clinically helpful in determining thrombosis risk in thrombophilia individuals. Future studies should focus on the dynamic monitoring during the chronic phase of VTE to offer special advantages for patients with thrombophilia.

Combined factor V (FV) and FVIII deficiency (F5F8D) is a rare autosomal-recessive bleeding disorder caused by mutations in lectin mannose binding-1 (LMAN1) and multiple coagulation factor deficiency-2 (MCFD2). Six causative homozygous mutations (5 in LMAN1 and 1 in MCFD2) were identified in 6 patients with F5F8D. A thrombin-generation assay, triggered with tissue factor (1 pM) in F5F8D plasma, paradoxically exhibited enhanced thrombin generation compared with normal plasma. Significantly lower free tissue factor pathway inhibitor (fTFPI) was found in F5F8D patients compared with healthy controls (P < .01). Normalizing tissue factor pathway inhibitor α (TFPIα) in F5F8D plasma greatly delayed and reduced thrombin generation. Increasing FV concentrations by adding plasma FV to F5F8D plasma only caused a gradual decrease in thrombin generation, suggesting that low levels of TFPIα and FV cocontributed to the elevated thrombin generation by reducing anticoagulant effects. On the contrary, thrombin generation in F5F8D platelet-rich plasma (PRP) was significantly lower than in normal controls (P < .05); however, it was fully corrected by normalizing FVIII or after 1-deamino-8-d-arginine vasopressin (DDAVP) infusion, indicating that the hypocoagulable state of F5F8D patients is associated with low FVIII levels. In addition, plasma and platelet FV in F5F8D PRP were sufficient to support normal thrombin generation, and low TFPIα may have no effect on thrombin generation. DDAVP infusion induced a complete response in 5 F5F8D patients and a partial response in the remaining patient. Based on our findings, we suggest that DDAVP may be considered a potential substitute for FVIII concentrates, and fresh-frozen plasma (FFP) infusion may not be necessary for F5F8D patients with minor bleeding challenges.

Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree.

Disseminated intravascular coagulation (DIC) is a common life-threatening complication in critically ill patients. The diagnostic scoring systems of DIC enable a more prompt and accurate diagnosis of DIC, such as the International Society on Thrombosis and Haemostasis (ISTH), the Japanese Association for Acute Medicine (JAAM) and the Japanese Ministry of Health and Welfare (JMHW). This study prospectively evaluated the newly proposed Chinese DIC Scoring System (CDSS), which was conducted at 18 centers in China during a one-year period. Receiver operating characteristic (ROC) curves showed that, for diagnosis of DIC and for prediction of the 28-day all-cause mortality, the CDSS had larger areas under the ROC curve (AUCs) than the ISTH and the JAAM in different groups. The CDSS also had larger AUC than the JMHW for the ISTH DIC in non-infectious diseases. All of the AUCs of the CDSS were greater than 0.8, accompanied with both high sensitivity and high specificity. Furthermore, the CDSS score was an independent predictor of mortality (odds ratio, 1.882; p

Defective protein C (PC) pathway predisposes patients to venous thromboembolism (VTE)and is mostly, but not exclusively, attributed tohereditary PC or protein S (PS) deficiencies and activated PC resistance caused by factor V Leiden mutation. In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the three pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. In conclusion, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function. Figure Disclosures Ding: Shanghai General Hospital affiliated to Shanghai Jiao Tong University, Shanghai: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work.. Wang:Ruijin Hospital, Shanghai Jiao Tong University School of Medicine: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work.. WU:Ruijin Hospital, Shanghai Jiao Tong University School of Medicine: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work..

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1 × 1011 vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This “hybrid vector” retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.

SummaryFactor X (FX) is a vitamin K-dependent coagulation zymogen, which upon activation to factor Xa assembles into the prothrombinase complex to activate prothrombin to thrombin. FX can be activated by either factor VIIa-tissue factor or factor IXa-factor VIIIa in extrinsic and intrinsic pathways, respectively. In this study, we identified a bleeding patient with moderate FX deficiency who exhibits a clotting defect only in the intrinsic pathway. Exome sequencing revealed that the patient carries a novel homozygous missense mutation that results in substitution of Thr211 with Pro in the activation peptide of FX. Thr211 is the site of an O-linked glycosylation in the activation peptide of FX. We postulated that the lack of this post-translational modification specifically impacts the activation of FX by intrinsic Xase, thereby impairing thrombin generation in the subject. To test this hypothesis, we expressed both wild-type FX and FX containing this mutation in mammalian cells and following the purification of the zymogens to homogeneity characterized their properties in both purified and plasma-based assay systems. Analysis of the results suggests that Thr211 to Pro substitution renders the FX mutant a poor substrate for both physiological activators, however, at physiological concentration of the substrate, the clotting defect manifest itself only in the intrinsic pathway, thus explaining the bleeding phenotype for the patient carrying this mutation.

SummaryProtein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the pro-coagulant cofactors Va and VIIIa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits ∼3 times lower affinity for binding to EPCR and the K150del variant has ∼2–3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.

Summary Background Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized. Objectives To identify large deletions in the F8 gene and to characterize the molecular mechanisms leading to these deletions. Patients and methods We used AccuCopy technology, a copy number variation (CNV) genotyping method based on multiplex competitive amplification, to confirm deletions in index patients and to screen potential female carriers in 10 HA families. Also, breakpoints of these large deletions were characterized by a primer walking strategy and genome walking technique. Results Ten large deletions and four female carriers were identified by AccuCopy. The extents of deleted regions ranged from 1.3 to 68.5 kb. Exact breakpoints of these deletions were successfully characterized. Eight of them presented microhomologies at breakpoint junctions and several recombination-associated elements (repetitive elements, non-B conformation forming motifs and sequence motifs) were also observed in close proximity to the junctions. Conclusions AccuCopy technology is a reliable and efficient tool for detecting large deletions in the F8 gene and identifying HA female carriers. The genome walking technique is a highly specific, efficient and versatile method for characterizing the deletion breakpoints. Molecular characterization of deletion breakpoints revealed that non-homologous end joining and microhomology-mediated replication-dependent recombination were the major causative mechanisms of the 10 large deletions in the F8 gene.

Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P

Factor X (FX) deficiency is a rare autosomal-recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were1% in both intrinsic and extrinsic pathway assays and 1.71% in chromogenic assay, the level of FX antigen (FX:Ag) was 53.36% in the proband. Two novel heterozygous mutations (IVS5+1GA and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1GA) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense-mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185-189 loop and impaired binding of the loop to sodium ions (Na(+) ), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.

High-molecular weight heparins promote the protein Z-dependent protease inhibitor (ZPI) inhibition of factors Xa (FXa) and XIa (FXIa) by a template mechanism. To map the heparin-binding site of ZPI, the role of basic residues of the D-helix (residues Lys-113, Lys-116, and Lys-125) in the interaction with heparin was evaluated by either substituting these residues with Ala (ZPI-3A) or replacing the D-helix with the corresponding loop of the non-heparin-binding serpin α(1)-proteinase inhibitor (ZPI-D-helix(α1-PI)). Furthermore, both the C-helix (contains two basic residues, Lys-104 and Arg-105) and the D-helix of ZPI were substituted with the corresponding loops of α(1)-proteinase inhibitor (ZPI-CD-helix(α1-PI)). All mutants exhibited near normal reactivity with FXa and FXIa in the absence of cofactors and in the presence of protein Z and membrane cofactors. By contrast, the mutants interacted with heparin with a lower affinity and the ~48-fold heparin-mediated enhancement in the rate of FXa inhibition by ZPI was reduced to ~30-fold for ZPI-3A, ~15-fold for ZPI-D-helix(α1-PI), and ~8-fold for ZPI-CD-helix(α1-PI). Consistent with a template mechanism for heparin cofactor action, ZPI-CD-helix(α1-PI) inhibition of a FXa mutant containing a mutation in the heparin-binding site (FXa-R240A) was minimally affected by heparin. A significant decrease (~2-5-fold) in the heparin template effect was also observed for the inhibition of FXIa by ZPI mutants. Interestingly, ZPI derivatives exhibited a markedly elevated stoichiometry of inhibition with FXIa in the absence of heparin. These results suggest that basic residues of both helices C and D of ZPI interact with heparin to modulate the inhibitory function of the serpin.

To study the molecular pathogenesis of protein C (PC) deficiency in a patient with pulmonary embolism and in his family members.Anticoagulated blood samples were collected from the proband and his family members to detect PC, PS and AT activities. PC antigen level was measured using ELISA. The genomic DNA was extracted to amplify all the 9 exons and their flanking sequences of PC gene using PCR, and the PCR products were sequenced. The mutated exons identified were amplified and sequenced for the other family members.The proband and his parents and sister were identified as carriers of PC gene mutation, which led to type II PC deficiency. Sequencing of the proband's PC gene showed two heterozygous point mutations in exon 3 (G5540A) and exon 7 (C10230T) to cause compound heterozygous mutations of PC E29K and PC R147W, which were inherited from his father and mother, respectively. His sister was a heterozygote of PC R147W.The proband is a compourd heterozygous mutations carrier of PC E29K and PC147W. PC E29K is a novel PC mutation, and PC R147W is a reported PC gene mutation seen in patients with type II hereditary PC deficiency and recurrent thrombosis.

Background Inherited deficiency of antithrombin, protein C and protein S, three important, naturally occurring coagulation inhibitors, might play a major role in the occurrence of venous thromboembolism in Chinese. The establishment of age- and gender-related normal ranges of these inhibitors is crucial for an accurate diagnosis of these deficiencies.Design and Methods We designed a prospective cross-sectional study recruiting healthy adults from four university–affiliated hospitals in China. Antithrombin, protein C and protein S were studied by measuring their activity. Gene analysis was performed when natural anticoagulant deficiency was suspected. Polymorphisms of the factor V gene were searched for among subjects who were positive for activated protein C resistance.Results In 3493 healthy Chinese adults (1734 men, 1759 women; age 17–83 years), we found higher age-adjusted activities for protein C and protein S in men than in women but no sex difference for antithrombin. In women, mean protein C and protein S activities increased with age. In men, mean protein C levels increased with age up to the age of 49 but decreased after 50 years old; mean protein S levels decreased after 50 years of age. Antithrombin levels remained stable over time in women but decreased significantly after 50 years of age in men. Reference values according to age and sex allowed the identification of 15 genetic variants (protein C: 10, antithrombin: 3, protein S: 2) in subjects with protein activity below the 1st percentile.Conclusions This is the largest survey ever conducted in the healthy general Chinese population. These normal ranges provide the essential basis for the diagnosis and treatment of thrombosis in Chinese.

Molecular imaging of thrombus formation at initial stage requires a robust thrombus-specific contrast agent with high sensitivity. In this study, we report a novel P-selectin-targeted paramagnetic molecular imaging agent and the agent’s potential to sensitively detect occult microthrombi on the intimal surface of endothelium. Platelet clots and blood clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that was improved with increasing gadolinium level. In vivo contrast enhancement under part of circulation conditions was assessed in dogs. The micro-thrombi around the femoral vein of dog demonstrated higher signal intensities than the control clots and the adjacent muscle. Histology was performed on regions likely to contain thrombus as indicated by MRI. These results suggest that molecular imaging of P-selectin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of P-selectin and may allow for early, direct identification of microthrombi, leading to early diagnosis.

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

Summary. Background: Hemophilia A (HA) is an X-chromosome-linked recessive disorder. Aim: We report the case of a female HA patient with a moderate decrease of factor (F) VIII activity and antigen (FVIII:C 3.4%, FVIII:Ag 4.2%) and severe bleeding symptoms. Methods: The patient's father had mild FVIII deficiency (FVIII:C 6.9%, FVIII:Ag 7.4%), and her mother had normal FVIII activity. The von Willebrand disease antigen and von Willebrand factor ristocetin cofactor activity were normal in all family members. The genomic DNA was extracted from the peripheral blood lymphocytes of the patient and her family members. Long-distance polymerase chain reaction (PCR) was employed to screen for the intron 22 inversion of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. Results: Two heterozygous mutations were identified in the patient: one a substitution of nucleotide 5981T by C that leads to a missense mutation Leu1975Pro, and the other an insertion of an ‘A’ between nucleotides 3637 and 3638 (3637_3638insA) that shifts the reading frame and predicts a premature stop codon downward. The mutation Leu1975Pro was identified in the father's F8; however, 3637_3638insA was a de novo mutation that occurred in the patient's maternal-derived F8. Real-time PCR was applied to analyze the level of ectopically F8 gene transcripts in the peripheral lymphocytes of family members. The ectopic transcripts of F8 of the patient were less abundant than the normal control (patient:normal control ratio 0.67), whereas her parents showed no significant difference from the normal control. Conclusion: The FVIII deficiency of the HA patient resulted from a de novo occurrence of a frameshift 3637_3638insA in her maternal-derived F8 and a novel missense mutation Leu1975Pro inherited from her father.

To evaluate levels of coagulation /anticoagulation factors and distribution of their gene polymorphisms in patients with venous thromboembolism(VTE) and acute myocardial infarction(AMI). Of 95 VTE, 95 AMI patients and 95 normal controls were be studied. The results showed that the levels of fibrinogen and D-Dimer, the activies of FII, FV and FVIII were remarkably higher in patient with VTE than those in healthy group, and the activities of FIX, PC and AT were of no significant difference between VTE group and healthy group. The mean levels of fibrinogen, D-Dimer, FV:C and FVIII:C were significantly higher in AMI group than that in controls; while, no significant difference of FII:C, FIX:C, PC:A and AT:A between AMI patients and the controls. In controls, -148CT + -148TT was associated with higher fibrinogen concentrations, the genotypes of other gene polymorphisms were not associated with levels of coagulation /anticoagulation factors. Clear difference were observed for the T allele frequencies of -148C/T and distribution frequencies of FII A19911G were found between VTE group and normal group. there was significant difference of -1476TT genotype distributions and T allele frequencies in -1476A/T between VTE and control subjects. There was significant difference of AC+CC genotype distribution in 1298 A/C polymorphisms between AMI and control, and it was so with the GA genotype in 1793 G/A polymorphisms. But neither genotype frequencies nor allele frequencies of other polymorphisms were found to be no of significant difference between VTE /AMI group and control group. This study proved the importance of combined functions of several coagulant factors in causing VTE and AMI. The carriage of the C148T mutation, FII 20210G/A and -1476A/T polymorphism might be associated with an increased risk of VTE, while the MTHFR 1298A/C and 1793G/A polymorphisms may be related to the development of AMI. Only some of joint occurrence of above polymorphisms might increase the occurrence rate of VTE or AMI.

Introduction: The inherited risk factors for venous thromboembolism differ between Caucasians and Chinese, antithrombin (AT), protein (PC) and protein (PS) deficiencies being the most common in Chinese patients. However, the age- and gender-related normal activity ranges of AT, PC, PS and the prevalence of their deficiencies in the general Chinese population remained to be determined. Methods: 3493 healthy volunteers, 1759 women and 1734 men, blood donors or individuals taking routine health checkup, were recruited in four Chinese medical centers, Beijing, Tianjin, Shanghai and Suzhou, between February and May 2006. The inclusion criteria were: informed consent, normal renal and liver functions, no anticoagulant, contraceptive or hormonal substitutive therapy, no previous thrombosis. The plasma levels of AT, PC, PS activities and APC-R were measured with the same batches of reagents and the same type of analyzer (Stago). For the individuals with low activities, mutations of PROC gene, PROS1 gene or SERPINC1 gene were determined by direct sequencing analysis. Restriction fragment length polymorphism analysis was performed in those APC-R positive samples to detect 5 single nucleotide polymorphisms (SNPs) in FV gene, including R306T, R306G, R506Q, H1299R and I359T. Results: The plasma activities (mean±SD) were: AT 109.4%±10.1%, PC 110.2%±20.4%, PS 95.4%±21.6% and APC-R 155sec±20sec. The normal ranges (mean±2SD) were: AT 89–130%, PC 69–151%, PS 52–139% and APC-R 114–195sec. Using multiple linear regression models, we identified significant (p Conclusions: This is the largest population study ever conducted in China to determine the normal activity levels of the three main natural anticoagulants in the healthy population and their genetic deficiencies. The prevalence of the genetic deficiency of the three proteins was 4.3 per 1000. Contrary to previous studies in China and other Asian areas, PC deficiency was most frequent, followed by PS deficiency and AT deficiency. Six novel mutations were found and there was no recurrent mutation. It has been confirmed again that the common SNPs in FV gene responsible for APC-R in the Caucasians were not detectable in Chinese population. The normal plasmatic ranges according to age and gender should be considered when evaluating the thrombotic risk or events in the Chinese population.