Thrombomodulin (TM) is essential in maintaining vascular homeostasis. Its anticoagulant function is mainly mediated through the formation of thrombomodulin-thrombin complex, which could accelerate the conversion of protein C (PC) to activated protein C (APC). We identified 3 patients who carried the mutation c.1288G > A, p.G430S in THBD and suffered from recurrent thrombosis. The objective of this study is to elucidate the molecular basis of thrombosis underlying the TM Gly430Ser mutation. We expressed the wild-type and Gly430Ser mutant TM in both full-length and soluble fragment forms in mammalian cells. The cofactor capacity of TM was evaluated by measuring the thrombin-dependent generation of APC and activated TAFI in a time- and concentration-dependent manner. The binding affinity between TM and thrombin was determined by surface plasmon resonance (SPR). Furthermore, the overall impact of TM was evaluated through thrombin generation test (TGT) and APTT prolongation test. The cofactor function of TM-G430S in promoting thrombin-mediated activation of PC and TAFI was substantially compromised. SPR analysis revealed that the binding affinity of TM-G430S for thrombin was significantly impaired, with only about 10

BackgroundPredictors for the successful eradication of neutralizing anti-FVIII alloantibodies (inhibitors) in hemophilia A patients receiving immune tolerance induction (ITI) therapy remain limited. Maternal microchimerism (MMc) showed potential to protect hemophilia A patients from inhibitor development.ObjectivesTo investigated the role of MMc in ITI therapy.Patients/MethodsThis observational study enrolled 121 pediatric with hemophilia A and inhibitors. MMc was determined using droplet digital PCR. Low-dose ITI (FVIII ~50IU/kg every other day) was administered, with adjunctive rituximab given to patients with higher risk clinical features.ResultsOf the 101 patients evaluable for MMc, 88 completed ITI, 18 were MMc positive (MMc+) and 70 were MMc negative (MMc-). Success was achieved in 75 (85.2%) patients, including 16 of 18 MMc+ patients (88.9%) and 59 of 70 MMc- patients (84.3%). Compared with MMc- patients, MMc+ patients had a lower peak inhibitor during ITI (median, 5.3 vs. 37.8 BU/ml, p = 0.029), less frequent rituximab use (27.8% vs. 61.4%; p = 0.011), and a shorter time to ITI success (median, 3.0 vs 9.9 months; p = 0.009). Multivariate Cox regression identified MMc+ (HR = 2.770), pre-ITI inhibitor <10BU/ml (HR = 2.663), peak inhibitor during ITI<200BU/ml (HR = 4.954) and non-large deletion/duplication F8 mutations (HR = 2.344) as independent predictors of shorter time to ITI success.ConclusionMMc was associated with more rapid ITI success in children with hemophilia A and inhibitors receiving low-dose ITI regimen, suggesting the potential role of MMc in facilitating the eradication of FVIII inhibitors.

Abstract Complement‐mediated thrombotic microangiopathy (CM‐TMA) and thrombotic thrombocytopenic purpura (TTP) arise from distinct triggers, yet converge on a final common pathway of von Willebrand factor (VWF)‐mediated platelet adhesion within the microvasculature. Current complement component 5 (C5) inhibition therapies effectively halt complement amplification but leave a therapeutic gap regarding the acute resolution of existing endothelial activation and VWF strings. To determine whether the type 2A von Willebrand disease (VWD) phenotype, characterized chiefly by loss of high‐molecular‐weight (HMW) VWF multimers, limits microvascular thrombosis, complement accumulation, and organ injury in a lipopolysaccharide (LPS)‐induced TMA‐like model. Using a type 2A VWD murine model with constitutively reduced prothrombotic HMW VWF multimers, we induced TMA‐like injury with LPS. Histologic injury, fibrin deposition, complement activation, shear‐dependent thrombosis, and inflammatory responses were evaluated in comparison with wild‐type (WT) mice. Compared with WT controls, type 2A VWD mice showed substantially reduced microvascular thrombosis and tissue injury after LPS challenge, accompanied by decreased fibrin deposition and complement accumulation in affected organs. Microfluidic assays confirmed reduced platelet adhesion and thrombus formation under high shear stress in type 2A VWD mice. Alternative‐pathway hemolytic activity was lower in VWD mice at 24 h, whereas circulating inflammatory mediators did not differ between genotypes. In this LPS‐induced TMA‐like model, the type 2A VWD phenotype was accompanied by reduced microvascular thrombosis, complement accumulation, and organ injury, implicating VWF multimer composition and function in the propagation of thrombo‐complement microvascular injury.

Background Activated protein C is an important physiological anticoagulant protein. It can downregulate thrombin generation by inactivating coagulation factors Va and VIIIa, thereby exerting an anticoagulant effect. We identified that several families presenting with thrombosis harbored heterozygous mutations at the identical Trp83 residue of PROC, c.247T>C, p.Trp83Arg (W83R) and c.248G>C, and p.Trp83Ser (W83S). While the mutations were previously reported in a large-scale screening of the Chinese population, functional studies to elucidate the pathogenic mechanisms remain uninvestigated. Objective This study aims to evaluate the contribution of PC-W83R and PC-W83S mutations to thrombosis risk and to elucidate the specific pathogenic mechanisms. Method We expressed the recombinant PC-W83R and PC-W83S in mammalian cells and characterized their properties in established coagulation and anti-inflammatory assay systems. Results Both PC-W83R and PC-W83S were expressed at levels comparable to that of PC-WT. The activation rate of PC-W83R by thrombin was higher than that of WT, whereas the activation rate of PC-W83S was comparable to WT. However, when activated by the thrombin and thrombomodulin complex, both PC-W83R and PC-W83S variants exhibited reduced activation rates when compared with PC-WT. The catalytic efficiency toward the chromogenic substrate was comparable to that of the wild type. Both mutants exhibited reduced interaction with phospholipids and led to a dramatically attenuated capacity for FVa inhibition. Additionally, the anti-inflammatory activity of activated protein C-W83R and activated protein C-W83S was also impaired. Conclusion The W83R and W83S mutations impair the binding of PC to phospholipids, consequently leading to a substantial reduction in its anticoagulant function, which increases the thrombosis risk in patients.

Background Coagulation factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. While elevated FVIII levels are an established risk factor for venous thromboembolism (VTE), genetic variants in the F8 gene directly causing such elevations remain scarce. Here, we report a novel complete F8 tandem duplication identified in a female patient with splanchnic venous thrombosis (SVT). Methods We performed genetic testing using a thrombophilia panel targeting 35 genes involved in thrombosis and haemostasis to detect both point variants and copy number variations (CNVs). Family co-segregation analysis and phenotypic assays for FVIII and von Willebrand factor (VWF) were conducted. The structural basis of the identified F8 copy number gain was elucidated using optical genome mapping (OGM). Full-length F8 mRNA amplification, quantitative PCR, plasma FVIII Western blotting, and X-chromosome inactivation analysis were performed to assess the functional consequences of the duplication. Thrombin generation test (TGT) was employed to assess the hypercoagulable state. Results Genetic testing identified three copies of all 26 exons of the F8 gene in the proband, which was also detected in her mother (CNVs = 3) and son (CNVs = 2). One-stage clotting and chromogenic assays confirmed persistently elevated FVIII activity in the proband and her mother, accompanied by increased FVIII antigen levels. The OGM analysis confirmed a 229 kb tandem duplication including the F8 gene on one of the proband's X chromosomes. The junction regions exhibited high sequence homology and were rich in repetitive sequences, which precluded precise breakpoint mapping. Full-length F8 mRNA amplification revealed no aberrant transcripts, whereas quantitative PCR showed increased F8 mRNA expression in all carriers. Plasma FVIII Western blotting indicated FVIII heavy and light chains of expected molecular weights with increased band intensity in carriers. X-chromosome inactivation analysis in female carriers showed no significant skewing. TGT in two available carriers showed increased thrombin generation compared with a normal control at both low (1 pM) and high (5 pM) tissue factor concentrations. Conclusion We identified a novel complete F8 tandem duplication associated with increased FVIII expression and a hypercoagulable phenotype in a female patient with SVT. These findings support F8 gene dosage gain as a rare gain-of-function mechanism contributing to elevated FVIII levels and thrombophilia, while variation in VWF levels and acquired risk factors may modify thrombotic penetrance.

Protein C (PC) is an important physiological anticoagulant factor in humans. Activated protein C (APC) is generated from the PC zymogen through proteolytic activation by thrombin. APC inhibits thrombin generation by inactivating activated factors V and VIII via limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent cytoprotective and anti-inflammatory properties. We have identified a young male with multiple-site thrombosis, who carries a heterozygous mutation c.1151A > G,p.Asn384Ser(N384S) in PC. Although this mutation has been previously documented, limited functional research has been conducted to elucidate its pathogenesis. To elucidate the functional alternations of the N384S mutant protein C and delineate the molecular mechanism underlying thrombosis in the patient carrying this mutation. We expressed the recombinant PC-N384S in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems. The expression level of the PC-N384S was reduced to approximately 7% of that observed for PC-WT. The activation of PC-N384S by thrombin or thrombin–thrombomodulin (TM) complex was significantly impaired, although the addition of TM exhibited a slight enhancement in the activation process. In terms of cleaving a chromogenic substrate, the catalytic efficiency reduced to approximately 50% of that observed in the wild type. In addition, in comparison with APC-WT, APC-N384S demonstrated a pronounced decline in amidolytic activity following an extended incubation period at 37°C. APC-N384S exhibited slightly impaired anticoagulant activity in either FVa inhibition assay or plasma-based assay systems. Furthermore, anti-inflammatory activity of APC-N384S was dramatically impaired as determined by evaluating the barrier-protective effect. The Asn384Ser mutation impairs both the anticoagulant and barrier-protective activities of protein C, thereby increasing the thrombosis risk in the heterozygous young male.

Thrombomodulin (TM) is an important regulator in various physiological processes, including coagulation, fibrinolysis and inflammation. TM exerts its cofactor activities for protein C (PC) activation and thrombin-activatable fibrinolysis inhibitor (TAFI) activation through its binding with thrombin. Numerous studies have reported that mutations of TM are associated with thromboembolic diseases. Here we report a heterozygous substitution of arginine to lysine at amino acid residue 403 (Arg403Lys, R403K) within the fourth epidermal growth factor (EGF)-like domain of TM, which was identified in five unrelated patients with thrombophilia. To elucidate the role of the R403K mutation in TM, we constructed expression plasmids for both the full-length (residues Met1 to Leu575) and extracellular fragment (residues Met1 to Ser515) of TM to study its functions on surface of endothelial cells and in vitro system as well. We observed a reduced cofactor activity of PC activation by the TM-R403K variant across several assays and demonstrated a weakened interaction between the mutant and PC as well. These results indicate that the R403K mutation in TM induces detrimental alterations in the anticoagulant function of TM, which might elucidate the thrombotic predisposition observed in patients. This study provides further compelling evidence suggesting that mutation at the terminus of the EGF4 domain in TM may be associated with thrombophilia, thereby enhancing our comprehension of the physiological role of TM.

Background: Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC).Material and Methods: We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation.Results: We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site ((NTT254)-N-252) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC.Conclusion: The new N-glycosylation site ((NTT254)-N-252) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established.Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared.Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34+ CD61+ MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34+ CD61+ MKs (≧20.00%) had lower platelet counts (36.00 × 109/L vs. 88.50 × 109/L, p = 0.015) and more profound emperipolesis (p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34+CD61+MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay.Conclusion High proportion of CD34+ CD61+ MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

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Background: Mutations of ABO gene may cause the dysfunction of ABO glycosyltransferase (GT) that can result in weak ABO phenotypes. Here, we identified two novel weak ABO subgroup alleles and explored the mechanism that caused Ax phenotype. Materials and methods: The ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the mutations was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. Results: The results of serological showed the subject RJ23 and RJ52 both were Ax phenotypes. The novel A alleles, Avar-1 and Avar-2 were identified according to the gene analysis. Both Avar-1 and Avar-2 harbored recombinant heterozygous alleles, specifically A2.05 and O.01.02. These alleles showcased substitutions at positions c.106G > T, c.189C > T, c.220C > T, and c.1009A > G in their respective exons. It is worth noting that the crossing-over regions of these two alleles differed from each other. In vitro expression study showed that GTA mutant impaired H to A antigen conversion, and the mutant did not affect the production of GTA though the Western bolt. In silico analysis showed that GTA mutant may change the local conformation and the stability of GT. Conclusion: The Avar-1 and Avar-2 alleles were identified, which could cause the Ax phenotype through changing the local conformation and reducing stability of the GTA.

BACKGROUND:Venous thromboembolism (VTE) is predisposed by thrombotic mutations in patients with hereditary thrombophilia. Although prothrombin deficiencies caused by homozygous or compound heterozygous mutations are associated with bleeding diathesis, rare cases have shown a correlation between heterozygous prothrombin mutations and thrombosis. MATERIALS AND METHODS:We surveyed genetic variants involved in thrombosis and hemostasis in 347 patients with unprovoked VTE or having a positive family history of thrombosis. For patients identified with heterozygous prothrombin mutations, we conducted family investigations and performed a thrombin generation test (TGT) to elucidate the thrombotic risk. Novel mutants were expressed and subjected to functional assays to clarify the underlying thrombotic mechanisms. RESULTS:Heterozygous prothrombin mutations were identified in 3.5% of patients (12/347), including three novel mutations Phe382Ser, Phe382Leu, and Asp597Tyr found in one patient each, as well as previously reported Arg541Trp mutation in four patients and Arg596Gln mutation in five patients. A total of 42 mutation carriers were identified within the 12 pedigrees, among whom 64.3% (27/42) had experienced thrombotic events. TGT results demonstrated hypercoagulability for carriers of the five mutations, with Arg596Gln showing the highest thrombin generation potential followed by Arg541Trp. The Phe382-associated mutations severely impaired thrombomodulin-binding ability of thrombin, resulting in obviously reduced protein C (PC) activation. The Asp597Tyr mutation exhibited a mild reduction in both inactivation by antithrombin and PC activation reactions. CONCLUSION:The presence of heterozygous prothrombin mutations represents a potential genetic predisposition for VTE. All thrombosis-associated mutations potentiate coagulation activity by either conferring antithrombin resistance and/or impairing PC pathway activity.

Introduction Hereditary Hemorrhagic Telangiectasia (HHT) is charactered by telangiectasia and arteriovenous malformations (AVMs). Recurrent visceral and mucocutaneous bleeding is frequently reported among HHT patients, while data on the prevalence of thrombosis remains limited. This study aims to describe the clinical manifestations and molecular biological characteristics of HHT patients. Methods We conducted a retrospective study at Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. A total of 24 HHT patients, observed between January 2019 and December 2023, were included. We recorded the biological, clinical, and therapeutic events, with particular attention to bleeding and thrombotic events. Gene mutation analysis and blood constituent measurements were performed. Results The prevalence of bleeding among all HHT patients was 100 %, while thrombotic events were noted in 41.70 % of cases. Hepatic arteriovenous malformations (HAVMs) were identified in six patients, pulmonary arteriovenous malformations (PAVMs) in five patients, and cerebral arteriovenous malformations (CAVMs) in one patient. For patients with thrombosis, the discontinuation rates were 23.08 % for antiplatelet therapy and 33.33 % for anticoagulant therapy due to the increased risk of bleeding. Genetic mutations related to HHT were present in 16 patients, with ACVRL1 (activin A receptor-like type 1) mutations being the most frequent at 41.67 %, followed by ENG (endoglin) mutations at 20.83 %, and GDF2 (growth differentiation factor 2) mutations at 4.17 %. The incidence of PAVMs was 75.00 % in HHT1 patients with ENG mutations and 20 % in HHT2 patients with ACVRL1 mutations, while HAVMs occurred in 0 % and 40.00 % of these groups, respectively. Patients were divided into non-AVMs and AVMs groups. Compared to normal controls, von Willebrand factor (vWF) activity was significantly increased in all HHT patients (149.10 % vs. 90.65 %, P < 0.001). In the non-AVMs group, the median level of stromal cell-derived factor-1 (SDF-1) was significantly elevated (124.31 pg/mL vs. 2413.57 pg/mL, P < 0.05), while vWF antigen levels were markedly higher in the AVMs group (165.30 % vs. 130.60 %, P = 0.021). Further grouping of HHT patients based on bleeding and thrombosis phenotypes revealed that those with thrombosis had significantly higher median percentages of schistocytes (3.50 % vs. 0 %, P = 0.002), ferritin concentrations (318.50 μg/L vs. 115.50 μg/L, P = 0.001), and lactate dehydrogenase (LDH) levels (437 U/L vs. 105 U/L, P < 0.001). There were no significant differences in the activity of vWF, protein C (PC), protein S (PS), and factor VIII (FVIII) between the two groups. Conclusion This study highlighted the complex relationship between arteriovenous malformations and genetic mutations in HHT patients. A comprehensive assessment of bleeding and thrombosis risks should be conducted for each HHT patient, additionally, further clinical studies are needed to explore the risk factors for thrombosis and anticoagulant-related bleeding in HHT.

Background: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported. Objectives: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC. Methods: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays. Results: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC. Conclusion: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.

Most thrombophilia patients are caused by insufficient suppression of coagulation reactions, which could be either mediated by deficiencies of protein C, protein S,antithrombin, or inhibition-resistant coagulation factor mutants, such as Factor V Leiden and prothrombin Yukuhashi. The thrombosis caused by coagulation factor mutants with increased procoagulant activity is very rare and so far only two Factor IX (FIX) variants involving residue Arg384, FIX Padua (p.Arg384Leu) and Shanghai (p.Arg384Gln), which increased FIX activity by up to 8 folds, had been reported to be associated with the incidence of thrombosis. In a young male venous thrombosis patient we had identified a novel F9 gene missense mutation c.1018G>A, resulting in p. Glu340Lys substitution (designated as FIX Shanghai II). The patient's APTT time was 28.9 seconds (normal reference 22.3~38.7s), and his PT was also within the normal reference range (11.5 seconds, normal reference 10.0~16.0s). Although the patient had the FIX: Ag comparable to the normal reference, 128.20±6.07%, the his FIX:C was around 201.69±10.38% of normal control (normal reference 50~150%). Thrombin generation assay (TGA) showed that compared with normal control, the patient had a shorter Lag time (2.67 vs 3.33 min), elevated ETP (1649.43 vs 1311.62 nM*min), and higher Peak value (387.34 vs 200.86 nM), indicating increased global coagulation activity of the patient. The activity of protein C, protein S, and antithrombin were all within the normal reference ranges and the patient was negative for antiphospholipid antibodies. Consistent with the patient's coagulation laboratory manifestations, the purified recombinant FIX Glu340Lys mutant showed 1.82 folds higher procoagulant activity than wild type FIX as determined by FIX:C/FIX:Ag ratio. The recombinant FIX Shanghai II mutant can be converted into FIXa by activated factor XI (FXIa) in a similar fashion as FIX WT. The kinetic study of FIXa-catalyzed FX activation showed that p. Glu340Lys mutation led to accelerated FX activation by FIXa. The p.Glu340Lys mutation also enhanced the procoagulant activity of previously reported thrombosis-causing FIX mutants, FIX Shanghai and Padua, in a similar fashion as it did on FIX WT. The FIX:C/FIX:Ag ratio of double mutants containing Glu340Lys and Arg384Gln (FIX Shanghai) or Arg384Leu (FIX Padua) was about twice of FIX Shanghai or Padua alone, 11.48- and 22.53-folds vs 6.16- and 8.13- folds higher than FIX WT respectively.

We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved similar to 5-20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling.

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebrand disease.

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 (52.6seconds) and prothrombin time (19.4seconds) 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.