Severe Protein C Deficiency due to Novel Biallelic Variants in PROC and Their Phenotype Correlation.

Severe protein C deficiency due to biallelic PROC mutations is an extremely rare thrombophilia, most commonly presenting during the neonatal period as purpura fulminans. Despite treatment, severe morbidity and mortality are frequent. The current study reports 3 unrelated patients harboring novel homozygous PROC mutations and their clinical phenotypes. We discuss how the cytoprotective activity of protein C and its role in the stabilization of endothelial barriers may account for the unique symptoms of this thrombophilia.

Thrombophilia testing in patients with portal vein thrombosis.

Portal vein thrombosis (PVT) is a rare but severe condition. Several risk factors predispose to PVT. However, it remains unclear to which degree thrombophilia contributes to the risk of PVT and whether PVT patients should be routinely referred for thrombophilia testing. The aim of the present study was to investigate the prevalence of thrombophilia in PVT patients to clarify the relevance of thrombophilia testing in PVT patients. Clinical data and results from thrombophilia investigations were systematically obtained from all PVT patients referred to Centre for Hemophilia and Thrombosis, Aarhus University Hospital, Denmark for thrombophilia testing between 1st of January 2010 and 31st of December 2018 (n = 93). The investigated thrombophilias included factor V Leiden and prothrombin G20210A mutations, deficiency of protein S, protein C and antithrombin, antiphospholipid syndrome, and increased levels of factor VIII. The prevalence of thrombophilia was compared to healthy controls obtained from previously published data on thrombophilia distribution in cohorts of the Western European adult general population. Comparing PVT patients with healthy controls, significantly increased odds of presence of lupus anticoagulant (crude odds ratio (OR) 6.2, 95% confidence interval (CI) 1.8-20.6) were found, whereas no significantly increased odds of inherited thrombophilia were demonstrated. In conclusion, routine testing for inherited thrombophilia in PVT patients does not seem indicated. However, PVT patients should still be tested for antiphospholipid antibodies because patients meeting the criteria for antiphospholipid syndrome preferentially should receive vitamin K antagonists as anticoagulant therapy.

Management of severe congenital protein C deficiency with a direct oral anticoagulant, edoxaban: A case report.

A male patient diagnosed with severe congenital protein C (PC) deficiency during the neonatal period was treated with long-term warfarin but frequently developed purpura fulminans and bleeding. At four years of age, edoxaban was initiated (direct oral anticoagulant [DOAC]). His d-dimer and fibrin/fibrinogen degradation product levels were closely monitored. His PC activity increased from below the sensitivity range to 17%; this increase was thought to be due to a reduction in PC consumption during edoxaban therapy. After edoxaban introduction, he experienced just one episode of purpura fulminans over two years without any adverse events. Thus, DOAC may be a promising alternative for the management of congenital PC deficiency.

Cerebral Sinus Venous Thrombosis and Prothrombotic Risk Factors in Children: A Single-Center Experience From Turkey.

BACKGROUND: Cerebral sinus venous thrombosis (CSVT) is a rare cerebrovascular disease that may be life-threatening, especially in children. OBJECTIVE: The purpose of this study was to assess the clinical presentation, radiologic imaging, underlying conditions, treatment, and outcomes of children with CSVT. PATIENTS AND METHODS: In total, 23 consecutive children aged between 1 month to 18 years with CSVT, who were followed-up in Erciyes University Children's Hospital, were retrospectively enrolled in the study from January 2000 to December 2016. RESULTS: The median age of the 23 children (13 female patients, 10 male patients) at initial diagnosis was 60 months (1 to 204 mo). The most common clinical manifestation was headache/irritability (n=9). The most common site of the CSVT was the transverse sinus (n=16). The most common prothrombotic risk factor was protein C deficiency (n=4). Underlying risk factors were detected in 15 patients. Genetic risk factors such as protein C deficiency, infections, trauma, malignancies, autoimmune hemolytic anemia, neurometabolic disorders, asphyxia, and cardiac malformations were common risk factors. Six children died. Multiple sinus involvement and parenchymal hemorrhages were seen in 4 and in 3 of the 6 children who died, respectively. CONCLUSIONS: Protein C deficiency seemed to be relatively high in the presented children. Multiple sinus involvement and additional parenchymal hemorrhages represent poor prognostic features.

Severe Congenital Protein C Deficiency: Practical Aspects of Management.

Subcutaneous (SC) protein C (PC) was used in a child with purpura fulminans secondary to severe congenital PC deficiency. For maintenance, PC 80-120 IU/kg, given over 60-90 min SC Q48hr, has been successful as a home therapy for more than 3 years. The treatment was monitored by measuring trough PC chromogenic activity (target ≥15%) and D-dimer levels. No change in clinical course was appreciated after discontinuing enoxaparin (and leaving the patient on prophylactic PC replacement alone). A significant discrepancy between clotting-based and chromogenic-based PC activity is shown.

Different impact of two mutations of a novel compound heterozygous protein C deficiency with late onset thrombosis.

We investigated the alteration of coagulation state in a protein C (PC) deficiency pedigree and the impact of the PC gene mutations. The pedigree of a proband with cerebral hemorrhagic infarction had sixteen members with four generations. The plasma levels of PC activity (PC:A), protein S activity (PS:A), factor V:C and factor VIII:C, and routine coagulation tests were measured. Nine exons of the PC gene (PROC) were sequenced. Plasma PC:A and PC antigen (PC:Ag) of the proband were 26 and 18%, respectively, which was significantly lower than normal ranges. Two heterozygous missense mutations of PC in the proband were identified, T>G at site 6128 (exon 7) and G>C at site 8478 (exon 9) resulting in F139V and D255H, respectively. The family members with F139V (N = 4) or D255H (N = 4) had lower levels of PC:A and PC:Ag than members with wild-type PROC (N = 6). D255H mutation caused a more significant decrease in the levels of PC:A, PC:Ag and factor V:C as compared to F139V mutation (P < 0.05). Two independent mutations, F139V and D255H, of PROC reduce PC function. Compound heterozygous condition of the two mutations can cause synergistic PC deficiency, but resulting in later onset of cerebral thrombosis.

Paediatric presentation and outcome of congenital protein C deficiency in Japan.

Severe heritable protein C (PC) deficiency is quite rare, although heterozygous PROC mutation is the second leading cause of genetic predisposition to thrombosis in Japanese adults. The aim of the study was to search the optimal management, the paediatric onset and outcomes of PC deficiency were characterized in Japan. The genetic study, postmarketing survey of activated PC(aPC) concentrate (Anact(®)C) and intensive review in Japan for 20 years enabled the analysis of the disease onset, genotype, treatment and prognosis. Symptomatic PC deficiency was determined in 27 Japanese children. All but two patients presented within 16 days after birth (three prenatal and six neonatal onsets). Postnatal-onset cases had normal growth at full-term delivery. Of the 27 patients, 19 suffered intracranial thrombosis or haemorrhage (ICTH) (three foetal hydrocephalies), 16 developed purpura fulminans (PF) and 10 had both at the first presentation. ICTH preceded PF in both affected cases. Low PC activities of 18 mothers and/or 12 fathers indicated 20 inherited PC deficiencies (2 homozygotes, 11 compound heterozygotes and 7 heterozygotes) and seven unidentified causes of PC deficiency. Nine of 11 patients studied had PROC mutations. Four unrelated patients (50%) carried PC nagoya (1362delG). No PC-deficient parents had experienced thromboembolism. Of the 18 patients with aPC therapy, two died and eight evaluable survivors had neurological sequelae. This first comprehensive study of paediatric PC deficiency suggested that perinatal ICTH was the major presentation, occurring earlier than neonatal PF. PC nagoya was prevalent in paediatric, but not adult, patients in Japan. Early maternal screening and optimal PC therapy are required for newborns at risk of PC deficiency.

Acroangiodermatitis of Mali in protein C deficiency due to a novel PROC gene mutation.

Acroangiodermatitis of Mali is a dermatologic condition of kaposiform skin lesions that has been associated with chronic venous insufficiency. Here we report a case of a 28-year-old Chinese man with acroangiodermatitis which co-existed with chronic lower limb deep vein thrombosis. Investigations revealed protein C deficiency and a frame shift mutation, c246_247dupCT, of the PROC gene. Our report lengthens the list of male acroangiodermatitis of Mali cases with a Chinese patient harboring a novel PROC mutation with manifest protein C deficiency.

[Skin necrosis during long-term fluindione treatment revealing protein C deficiency]. / Nécroses cutanées tardives sous fluindione révélant un déficit en protéine C.

BACKGROUND: Cutaneous necrosis is a rare complication of vitamin K antagonist therapy. It presents as cutaneous hemorrhagic necrosis and usually occurs at the start of treatment. We describe an atypical case of recurrent skin necrosis after two years of treatment with fluindione. CASE REPORT: A 70-year old woman with a history of venous thromboembolism and obesity presented with a large haemorrhagic necrosis of the abdominal wall. She had been treated with fluindione for two years. Genetic protein C deficiency was discovered. Resumption of vitamin K antagonist therapy was followed by recurrence of skin necrosis despite concomitant administration of heparin. Treatment with vitamin K antagonists could not be continued. DISCUSSION: This observation is unusual due to the late onset of skin necrosis. The condition usually begins shortly after initiation of vitamin K antagonist therapy, generally between the third and the sixth day of treatment. It is due to a transient hypercoagulable state in patients with protein C deficiency or, in rare cases, protein S deficiency. This late-onset skin necrosis, occurring many years after initiation of anticoagulant therapy, may be explained by a sudden worsening of pre-existing protein C deficiency due to infectious and iatrogenic factors.

MiR-124 and miR-506 are involved in the decline of protein C in children with extra-hepatic portal vein obstruction.

The deficiency of protein C (PROC) can be partly rescued by Rex shunt through restoring portal blood flow in children with extra-hepatic portal venous obstruction (EHPVO). However, the decline of PROC is still found in some patients with a normal portal blood flow after Rex shunt. The aim of this study was to identify the candidate miRNAs involving in the decline of PROC and their mechanism. The protein level of PROC was detected by the ELISA assay, and was compared between sick and healthy groups. The expressions of miRNAs and PROC mRNA were measured using qRT-PCR, and were compared between sick and healthy groups. The correlation between PROC and candidate miRNAs was analysed by a Pearson correlation analysis to identify the most significant miRNAs. The expression of PROC mRNA was detected by qRT-PCR in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors and negative control (NC) mimics, which was compared among the different groups. The rates of liver cells' proliferation and apoptosis were detected in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors or with overexpressing PROC and negative control mimics by CKK8 assay and flow cytometry, which were compared among the different groups. The expressions of COX-2 and VEGF were measured by qRT-PCR, and were compared between the miRNAs groups and NC group. Western blot was assayed for detecting the protein levels of PROC, COX-2, VEGF, Bcl-2 and Bax, which were compared between the miRNAs groups and NC group. The expression of PROC mRNA was lower, and the expressions of miR-506-3p and miR-124-3p were higher in children with EHPVO than healthy group. PROC mRNA was negatively correlated with the expression of miR-506-3p and miR-124-3p. Compared to the NC group, the transcription activity of PROC was lower after exposure of miR-506 and miR-124 mimics in HL-7702 and LX-2 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. The rate of cell proliferation was lower after exposure of miR-506 and miR-124 than the NC group, which was increased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The apoptotic rate was higher after exposure of miR-506 and miR-124 than the NC group, which was decreased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The mRNA levels of COX-2 and VEGF were significantly higher after exposure of miR-506 and miR-124 mimics than those in the NC group. The protein levels of PROC and Bcl-2 were down-regulated, and the levels of COX-2, Bax and VEGF were up-regulated after exposure of miR-506 and miR-124 in HL-7702 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. MiR-506-3p and miR-124-3p may involve in the decline of PROC in protein and transcriptional level, in which the anti-proliferation and pro-apoptosis role of miR-506-3p and miR-124-3p for liver cells may involve in this mechanism.

Testing for inherited thrombophilias in arterial stroke: can it cause more harm than good?

BACKGROUND AND PURPOSE: Despite a paucity of evidence supporting a true association of ischemic stroke and the inherited thrombophilias, it is common practice for many neurologists to order these tests as part of the work-up of ischemic stroke, especially in young patients. Treatment with oral anticoagulation is often used in patients with positive results for the inherited thrombophilias. METHODS: We reviewed the literature focusing on case-control studies of the 5 most commonly inherited disorders of coagulation: protein C deficiency, protein S deficiency, antithrombin deficiency, and the factor V Leiden and prothrombin gene mutations in patients with stroke. We also analyzed the available data on stroke patients with inherited thrombophilia and patent foramen ovale. RESULTS: Multiple case-control studies have not convincingly shown an association of the inherited thrombophilias with ischemic stroke, even in young patients and patients with patent foramen ovale. CONCLUSIONS: If there is an association between the inherited thrombophilias and arterial stroke, then it is a weak one, likely enhanced by other prothrombotic risk factors. The consequences of ordering these tests and attributing causality to an arterial event can result in significant costs to the health care system and pose a potential risk to patients, because this may lead to inappropriate use of long-term oral anticoagulants, exposing patients to harm without a clearly defined benefit.

Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities.

Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC-/- mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1-18% of normal PC levels. In contrast to PC-/- mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC-expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease.

Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice.

Matings of mice heterozygous for a protein C (PC) deficient allele, produced by targeted PC gene inactivation, yielded the expected Mendelian distribution of PC genotypes. Pups with a total deficiency of PC (PC-/-), obtained at embryonic day (E) 17.5 and at birth, appeared to develop normally macroscopically, but possessed obvious signs of bleeding and thrombosis and did not survive beyond 24 h after delivery. Microscopic examination of tissues and blood vessels of E17.5 PC-/- mice revealed their normal development, but scattered microvascular thrombosis in the brain combined with focal necrosis in the liver was observed. In addition, bleeding was noted in the brain near sites of fibrin deposition. The severity of these pathologies was exaggerated in PC-/- neonates. Plasma clottable fibrinogen was not detectable in coagulation assays in PC-/- neonatal mice, suggestive of fibrinogen depletion and secondary consumptive coagulopathy. Thus, while total PC deficiency did not affect the anatomic development of the embryo, severe perinatal consumptive coagulopathy occurred in the brain and liver of PC-/- mice, suggesting that a total PC deficiency is inconsistent with short-term survival.

Focal arterial inflammation is augmented in mice with a deficiency of the protein C gene.

Increased risk of thrombosis, with propitious conditions for fibrin deposition, along with upregulation of inflammation, are important factors that enhance plaque formation in atherosclerosis. Evidence supporting the role of anticoagulant protein C (PC) as an inflammatory agent has emerged, supplementing its well-known function as an anticoagulant. Thus, we sought to examine whether a PC deficiency would lead to an enhanced response to an acute arterial hyperplasic challenge. The presentation of early arterial inflammation was studied using a copper/silicone arterial cuff model of accelerated focal neointimal remodeling in mice with a heterozygous total deficiency of PC (PC+/-). Increased inflammation, cell proliferation, cell migration, fibrin elevation, and tissue necrosis were observed in the treated arteries of PC+/- mice, as compared to arteries of equally challenged age- and gender-matched WT mice. These results indicate that PC+/- mice subjected to this challenge displayed enhanced focal arterial inflammation and thrombosis, leading to larger neointimas and subsequent localized occlusion, as compared to their WT counterparts.

An adult case of moyamoya syndrome that developed dural sinus thrombosis associated with protein C deficiency: case report and literature review.

We describe a 54-year-old woman exhibiting MMS who developed delayed dural sinus thrombosis associated with PCD. Angiographic findings of the patient were so unusual that bilateral internal carotid arteries were occluded between their origin and the carotid fork with extensive development of collateral circulation via the external carotid arteries and the posterior cerebral arteries instead of moyamoya vessels at the base of the brain. Seven years after bilateral cerebral revascularization surgery, intracerebral hemorrhage occurred caused by dural sinus thrombosis. In the treatment for the patient with MMS associated with PCD, risk of sinus thrombosis should be taken into account.

Sagittal vein thrombosis caused by central vein catheter.

Cerebral venous thrombosis, including thrombosis of cerebral veins and major dural sinuses, is an uncommon disorder in the general population. However, it has a higher frequency among patients younger than 40 years of age, patients with thrombophilia, pregnant patients or those receiving hormonal contraceptive therapy or has foreign body such as catheter in their veins or arterial system. In this case report, we described clinical and radiological findings in a patient with protein C-S deficiency and malposition of central vein catheter.

[A pedigree analysis of pulmonary embolism caused by compound heterozygous mutations of protein C].

OBJECTIVE: To study the molecular pathogenesis of protein C (PC) deficiency in a patient with pulmonary embolism and in his family members. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation.

AIMS: To identify the mutation and study the molecular mechanism of inherited protein C (PC) deficiency in a Chinese pedigree. METHODS: The plasma levels of PC activity (PC:A) and antigen (PC:Ag) were measured by chromogenic assay and ELISA, respectively. The PROC gene was amplified and sequenced for mutational screening. Wild type and Pro275Ser mutant PC cDNA expression plasmids were constructed and transfected into HEK 293T cells and COS 7 cells, respectively. The expression and transcription of PC were investigated by ELISA, Western blot and real time RT-PCR. Immunofluorescence staining was utilised to analyse the intracellular distribution of PC, and pulse-chase experiments were used to detect the intracellular stability of the mutant PC. RESULTS: The proband's plasma PC:A and PC:Ag were 5% and 13.9%, respectively. A missense mutation (p.Pro275Ser) was identified in exon 9 of PROC gene. In vitro expression study showed that Pro275Ser variant was present at 22.6% and 78.9% of wild type levels in culture supernatants and cell lysates, respectively. No significant differences in the molecular weights, mRNA levels or intracellular stability were observed between the mutant and wild type PC. Immunofluorescence staining revealed that the mutant protein was mainly located in the endoplasmic reticulum. CONCLUSIONS: A homozygous Pro275Ser mutation was identified in a Chinese pedigree of PC deficiency. Impaired secretion of the mutant PC might be the molecular mechanism of PC deficiency caused by Pro275Ser mutation.

Thrombophilic disorders: a real threat to patients with end-stage renal disease on hemodialysis and at the time of renal transplantation.

Management of end-stage renal disease is the mainstay of prevention of renal vascular complications and kidney rejection. We sought to describe the association of some disorders such as diabetic nephropathy, polycystic renal disease, hypertension, and thrombophilia with renal failure and discuss possible mechanisms explaining the implication of the thrombophilic states in kidney allograft thrombosis and renal rejection. Five hundred and sixty-eight patients were included in this case-control study and multivariate analysis was applied. Cases and controls were tested for all major types of thrombophilia. Diabetic nephropathy, autosomal dominant polycystic kidney disease, hypertension, and smoking are the strongest causal agents of end-stage renal disease in Tunisia. It should also be noted that the prevalence of factor V Leiden (P = 0.05) and protein C deficiency (P = 0.005) were significantly higher in ESRD patients awaiting renal transplantation than controls. The present study has raised the possibility that thrombophilic factors may play a pathophysiological role in renal failure. These results will serve as a basis for anticoagulant prophylaxis aimed at preventing kidney rejection and renal allograft thrombosis.