Accelerated phase development in a late-onset adolescent Chediak-Higashi syndrome patient caused by compound novel LYST mutations in the setting of SARS-CoV-2 infection.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive genetic disorder characterized by severe immunodeficiency, albinism and coagulation deficiency. Mostly diagnosed in early childhood, this devastating condition is associated with lysosomal abnormalities attributed to the absence or impaired function of lysosomal trafficking regulator caused by mutations in the CHS1/LYST gene. In current study, we report a case of late-onset CHS caused by two novel compound heterozygous CHS1/LYST mutations: c.8407C > T, leading to early termination of translation at residue Gln2803 (p. Gln2803Ter), and a small deletion c. 4020_4031del, resulting in an in-frame deletion of three amino acid residues (p. Asp1343_Val1346del). Both variants retain a large part of the CHS/LYST protein, particularly p. Asp1343_Val1346del, which preserves critical functional BEACH and WD40 domains in the C terminal, potentially maintaining residual activity and alleviating patient symptoms. The timeline of SARS-CoV-2 infection and rapid symptom progression suggests that the viral infection may have trigger the accelerated phase development leading to a poor prognosis.

Structural basis of von Willebrand factor multimerization and tubular storage.

The von Willebrand factor (VWF) propeptide (domains D1D2) is essential for the assembly of VWF multimers and its tubular storage in Weibel-Palade bodies. However, detailed molecular mechanism underlying this propeptide dependence is unclear. Here, we prepared Weibel-Palade body-like tubules using the N-terminal fragment of VWF and solved the cryo-electron microscopy structures of the tubule at atomic resolution. Detailed structural and biochemical analysis indicate that the propeptide forms a homodimer at acidic pH through the D2:D2 binding interface and then recruits 2 D'D3 domains, forming an intertwined D1D2D'D3 homodimer in essence. Stacking of these homodimers by the intermolecular D1:D2 interfaces brings 2 D3 domains face-to-face and facilitates their disulfide linkages and multimerization of VWF. Sequential stacking of these homodimers leads to a right-hand helical tubule for VWF storage. The clinically identified VWF mutations in the propeptide disrupted different steps of the assembling process, leading to diminished VWF multimers in von Willebrand diseases (VWD). Overall, these results indicate that the propeptide serves as a pH-sensing template for VWF multimerization and tubular storage. This sheds light on delivering normal propeptide as a template to rectify the defects in multimerization of VWD mutants.

Structural and functional characterization of novel F7 mutations identified in Chinese factor VII-deficient patients.

Hereditary factor VII (FVII) deficiency is a rare recessive bleeding disorder with an estimated prevalence of 1/500 000. We had investigated 50 unrelated Chinese patients with FVII deficiency and identified, in total, 25 mutations, including 18 missense mutations and 5 splicing mutations, on the F7 gene. The nucleotide transition c.1224T>G (p.His408Gln) in exon 9 constitutes a hotspot of mutation, with 19 patients harbouring this genetic variance. Few patients were homozygous or compound heterozygous for deleterious mutations, such as non-sense mutations, large insertion or deletions, indicating that complete deficiency of FVII may not be compatible with life. The eight novel mutations identified in the study, including one small deletion (p.Glu49GlyfsTer101), three type I missense mutations, p.Cys238Phe, p.Gly420Asp, p.Ala252Val and four type II missense mutations, p.Val336Met, p.Ser342Gly, p.Gly432Ser and p.Ile213Asn, were further analysed by in vitro expression and functional studies. The laboratory phenotype and structural analysis confirmed the functional consequence of p.Ile213Asn mutation involving cleavage and activation site. The molecular dynamic simulations and binding energy calculations along with functional probing of p.Gly432Ser mutation revealed the critical role of residue Gly432 in the binding between activated factor VII (factor VIIa) and tissue factor.

Noninvasive fetal genotyping of single nucleotide variants and linkage analysis for prenatal diagnosis of monogenic disorders.

BACKGROUND: High-cost, time-consuming and complex processes of several current approaches limit the use of noninvasive prenatal diagnosis (NIPD) for monogenic disorders in clinical application. Thus, a more cost-effective and easily implementable approach is required. METHODS: We established a low-cost and convenient test to noninvasively deduce fetal genotypes of the mutation and single nucleotide polymorphisms (SNPs) loci by means of targeted amplification combined with deep sequencing of maternal genomic and plasma DNA. The sequential probability ratio test was performed to detect the allelic imbalance in maternal plasma. This method can be employed to directly examine familial pathogenic mutations in the fetal genome, as well as infer the inheritance of parental haplotypes through a group of selected SNPs linked to the pathogenic mutation. RESULTS: The fetal mutations in 17 families with different types of monogenic disorders including hemophilia A, von Willebrand disease type 3, Duchenne muscular dystrophy, hyper-IgM type 1, glutaric acidemia type I, Nagashima-type palmoplantar keratosis, and familial exudative vitreoretinopathy were identified in the study. The mutations included various forms: point mutations, gene inversion, deletions/insertions and duplication. The results of 12 families were verified by sequencing of amniotic fluid samples, the accuracy of the approach in fetal genotyping at the mutation and SNPs loci was 98.85% (172/174 loci), and the no-call rate was 28.98% (71/245 loci). The overall accuracy was 12/12 (100%). Moreover, the approach was successfully applied in plasma samples with a fetal fraction as low as 2.3%. CONCLUSIONS: We have shown in this study that the approach is a cost-effective, less time consuming and accurate method for NIPD of monogenic disorders.

A case-report of the unprovoked thrombotic event in a patient with thymoma and severe FVII deficiency.

BACKGROUND: Factor VII deficiency is a rare bleeding disorder caused by a deficiency of clotting factor VII. However, there have been some case reports of venous thrombosis in patients with factor VII deficiency, especially underlying the prothrombotic risk factors exposure. Patients with factor VII deficiency require special considerations before undergoing surgery to minimize the risk of bleeding or thrombogenesis. CASE PRESENTATION: Here, we described a patient with early-stage thymoma and severe factor VII deficiency who experienced an unprovoked thrombotic episode before thymectomy and a fatal thrombotic event after surgery. By adopting gene screening, a reported homozygous F7 mutation (p.His408Gln) and a novel heterozygous PROS1 mutation (p.Pro147Ala) were identified. The former resulted in severe factor VII deficiency but did not protect against thrombosis, and the latter was correlated with normal expression and cofactor activities of protein S through the thrombin generation test. The perioperative infusion of recombinant factor VII concentrate and the absence of antithrombotic prophylaxis may collectively contribute to her fatal thrombotic event after surgery. CONCLUSIONS: For the patients with severe factor VII deficiency undergoing surgery, uniform replacement therapy may not be recommended, and antithrombotic prophylaxis should be used in the case with thrombotic history to minimize the risk of bleeding and thrombogenesis.

Evaluation of prothrombotic risk of two PROC hotspot mutations (Arg189Trp and Lys193del) in Chinese population: a retrospective study.

BACKGROUND: R189W and K193del of protein C (PC) were hotspot mutations in Chinese population with venous thromboembolism (VTE), but almost two-thirds of patients with above mutations coexisting with other genetically or aquiredly prothrombotic risk factors. The aim of this study is to clarify the independent contributions of R189W or K193del to VTE risk. METHODS: 490 unrelated patients with a personal history of VTE and 410 healthy participants were enrolled in this study. Data of their demographics, family history, genetic and acquired thrombosis risk factors were collected and statistically analyzed. RESULTS: PC R189W and K193del were identified in 3/410 (0.7%) and 7/410 (1.7%) healthy controls, and in 27/490 (5.5%) and 43/490 (8.8%) patients with VTE, respectively. Notably, about 70% of these mutant carriers combined with other genetic or acquired thrombophilic factors. After adjustment for age, gender, other inherited and acquired risk factors, we demonstrated that R189W and K193del were associated with 5.781-fold and 4.365-fold increased risk of VTE, respectively, which were significantly lower than the prothrombotic risk of anticoagulant deficiencies induced from rare mutations. Independent R189W or K193del mutation was not associated with earlier first-onset age as well as higher recurrent rate of VTE. However, combination of other genetic or acquired thrombophilic factors had supra-additive effects on those consequences. The more additional risk factors the patients had, the younger first-onset ages and higher risk of recurrence would be. CONCLUSIONS: As the most frequent mutations for PC deficiency in Chinese population, both R189W and K193del mutations had limited independent contributions to VTE development compared with other rare mutations in PROC gene, but may act in concert with other genetic defects or acquired thrombotic risk factors to produce the final severe phenotype.

Effects of 14 F9 synonymous codon variants on hemophilia B expression: Alteration of splicing along with protein expression.

There is growing evidence that synonymous codon variants (SCVs) can cause disease through the disruption of different processes of protein production. The aim of the study is to investigate whether the 14 SCVs reported in the F9 variant database were the pathogenic causes of hemophilia B. The impacts of SCVs on splicing and protein expression were detected using a combination of in silico prediction, in vitro minigene splicing assay and cell expression detection. The splicing transcripts were identified and quantified by co-amplification fluorescent PCR. The mechanism of splicing was verified by a modified pU1snRNA and pU7snRNA approach. Aberrant splicing patterns were found in eight SCVs. Five of the 8 SCVs produced almost all aberrant splicing isoforms, which were expected to truncate protein, three of them presented a partial defect on both splicing and protein secretion, the overall effects were consistent with the residual Factor IX activity of the affected cases. Neither the pre-messenger RNA (mRNA) splicing process nor the protein function was impaired in the rest six SCVs. In conclusion, our study firstly revealed the pathogenic mechanism of the 14 F9 SCVs and highlighted the importance of performing mRNA splicing analysis and protein expression studies of SCVs in inherited disorders.

Unraveling the molecular basis underlying nine putative splice site variants of von Willebrand factor.

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.

Inhibitors: Diagnostic challenges, unknowns of inhibitor development, treatment of bleeding and surgery, and insights into diagnosis and treatment in China.

Factor (F) VIII inhibitors develop in around 30% of previously untreated patients (PUPs) with severe haemophilia, to a lesser extend in moderate and mild haemophilia A and in up to 10% in severe haemophilia B. Diagnostic challenges and questions remain including access to high quality testing, the role for functional inhibitor testing and binding antibody testing, and the adaptations needed in the presence of non-factor replacement therapy. Despite significant gains in knowledge there are still many unanswered questions underlying the immunologic mechanisms of inhibitor development and tolerance. Therapeutic options include eradication of inhibitors using immune tolerance induction therapy (ITI), prophylaxis with bypassing agents (i.e., recombinant activated factor VII /rFVIIa or activated prothrombin complex concentrate/aPCC) or non-factor replacement therapies (e.g., emicizumab) and treatment of bleeds or coverage of surgeries/invasive procedure. Recently a haemophilia centre capacity building program was launched in China to further develop the infrastructure and support needed to improve the diagnosis of haemophilia, detection of inhibitors, and continue to improve the care of patients with haemophilia and inhibitors.

Low factor V level ameliorates bleeding diathesis in patients with combined deficiency of factor V and factor VIII.

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.

Prothrombin Arg541Trp Mutation Leads to Defective PC (Protein C) Pathway Activation and Constitutes a Novel Genetic Risk Factor for Venous Thrombosis.

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.

Characterization of two large duplications of F9 associated with mild and severe haemophilia B, respectively.

INTRODUCTION: Only two large duplications of F9 causing haemophilia B (HB) have been reported. AIM: To analyse the pathogenic mechanisms of large F9 duplications. METHODS: We have identified two large duplications of F9 (dup ex 1-6 and dup ex 4-6) associated with mild and severe HB in probands A and B, respectively. Here, we localized the breakpoints of the two duplications using long-range PCR and genome walking combined with quantitative primer walking strategies. We traced the origin of dup ex 4-6 by haplotype analysis then performed somatic mosaicism detection in sporadic pedigree B and detected the effect of chimeric intron derived from the duplication on transcription by minigene assay. RESULTS: Mechanisms of fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) might be responsible for the formation of two tandem direct duplications. The dup ex 4-6 was traced to maternal grandmother of proband B, who was both somatic mosaicism and germline mosaic and the duplication might be formed during mitosis of her early embryonic cells. Minigene assay demonstrated that chimeric intron generated three transcripts, one minor transcript produced an in-frame protein adding duplicated 143 amino acids into the normal FIX, explaining the small amount of larger FIX shown in Western blot. The inter-F9 dup ex 1-6 adjacent to the original F9 copy created two identical promoters, and promoter competition might be the pathogenic mechanism of the duplication causing mild HB. CONCLUSIONS: This study highlights that duplications can be associated with diseases by complicated pathogenic mechanisms.

The characteristics and spectrum of F9 mutations in Chinese sporadic haemophilia B pedigrees.

INTRODUCTION: Sporadic haemophilia B (HB) without obvious familial history poses challenges for genetic diagnosis and counselling. AIM: To identify the F9 variants in sporadic HB patients and probe the origin of these de novo mutations. METHOD: A total of 294 unrelated HB pedigrees sought genetic diagnosis were analysed in this single-centre study. The F9 gene was analysed by direct sequencing, and AccuCopy technique was adopted to screen for gene copy number variations. Six short tandem repeats approximal or within F9 gene were applied for linkage analysis. Mosaicism of sequence variant was determined by ddNTP Primer Extension method. RESULTS: Sporadic HB patients constituted 36% (61/294) of cases enrolled in current study. The sporadic and familial HB patients shared similar spectrum of F9 variants, with single nucleotide substitution as predominant form of disease-causing mutation and no mutation prone hotspot sites, including CpG dinucleotide sequences, had been identified. Majority of the mothers of sporadic HB patients were F9 mutation carriers (70%, 43/61), and most of them (95%, 41/43) had the inherited bleeding trait traced back to maternal grandfathers. Although most de novo mutations occur in germ cells, 2 maternal grandfathers, who had somatic mosaic mutations of F9, were also revealed to be the source of genetic variations identified in patients. In our cohort, FIX inhibitor incidence was 1%, developed only in patients carrying null mutations. CONCLUSION: The diversity of F9 genetic variants and possible mosaicism of de novo mutation demand extensive study and more cautious in genetic counselling of sporadic HB.

Evaluation of the activity levels of rat FVIII and human FVIII delivered by adeno-associated viral vectors both in vitro and in vivo.

The development of a novel coagulation factor VIII (FVIII) expression cassette with an enhanced activity for gene therapy of hemophilia A (HA) is essential. The biological properties of several non-human FVIII sequences, such as porcine and canine, have been evaluated. Here, we compared the activity level of rat FVIII (rFVIII) and human FVIII (hFVIII) by using single-chain and dual-chain strategies in 293 T cells and the HA mice. In both in vitro and hydrodynamic injection studies, the activity of rFVIII detected by the activated partial thromboplastin time assay was higher than that of hFVIII both by single-chain (~2.96-fold and ~1.72-fold, respectively) and dual-chain (~7.69-fold and ~2.35-fold, respectively). Moreover, the dual chain exerted a potentially higher delivery efficacy compared with the single chain (~4.96-fold and ~2.99-fold, respectively). The blood loss of HA mice administrated with rFVIII was less than those with hFVIII. AAV-delivered rFVIII and hFVIII also exerted long-term therapeutic effects on HA mice and caused a transient ALT elevation. These data might help to the development of novel, optimized FVIII expression cassettes based on the amino acid difference between rFVIII and hFVIII. These data indicate that the dual-chain strategy would likely enhance the delivery efficiency of the AAV-mediated FVIII gene therapy.

The prevalence of heterozygous F12 mutations in Chinese population and its relevance to incidents of thrombosis.

BACKGROUND: The contribution of moderate coagulation factor XII (FXII) deficiency to development of thromboembolism is still undetermined. We have tried to show the relevance of FXII deficiency to incidences of venous thrombosis by exploring the prevalence of F12 gene mutations in Chinese patients with thrombotic disorders. METHODS: One hundred and six patients with venous thromboembolism (VTE) and 220 healthy controls were enrolled in study. The coding region and flanking sequences of F12 gene were amplified and sequenced to identify genetic variances. Patients with F12 mutations were also screened for other thrombotic risk factors. RESULTS: Heterozygous F12 gene mutations were identified in 6 individuals with VTE and 10 healthy controls. Q336X and R66W were found in two healthy individuals; D291E was identified in a patient with DVT; and A343P was a recurrent mutation with a prevalence of 4.7% (5/106) in patient group and 3.6%(8/220) in healthy control. The prevalence of heterozygous mutations between the two groups had no significant difference. The association of A343P mutations with VTE was weak with an OR of 1.31 (95% CI 0.42-4.11). No other thrombophilia risk factors screened were positive in patients harboring heterozygous F12 mutations. CONCLUSIONS: There were conflicting theories about the relationship between FXII deficiency and thrombosis formation. Heterozygous F12 mutation decreases the plasma FXII activity approximately by half and cause moderate FXII deficiency. Although multiple mutations were identified in both groups, the link between F12 heterozygous mutation and development of thrombotic disorders is weak and further studies are warranted to clarify their relationship.

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

BACKGROUND: The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. STUDY DESIGN AND METHODS: Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-Bweak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. RESULTS: While plasma total GTB transfer capacity was undetectable in this B3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the Bweak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B310 cDNA and B cDNA. The mRNA expression level of B310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. CONCLUSION: ABO c.28G>A mutation may cause B3 -like subgroup by affecting RNA splicing of the ABO gene.

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated following U50,488H and etorphine respectively. Thus KOPR internalization requires receptor phosphorylation above a certain threshold, and higher-order KOPR phosphorylation may be disproportionally important.

The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis.

Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl(3)-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy.