Congenital Protein C Deficiency Research Articles

Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

Perioperative management for fracture in a child with homozygous congenital protein C deficiency

Congenital protein C (PC) deficiency is one type of hereditary thrombosis. Patients with hereditary thrombosis are at high risk for thrombosis in the perioperative period, but a standard management strategy has not been established. Here we report a case of perioperative management of a fracture in a child with homozygous congenital PC deficiency. The patient was a 3-year-old boy who was diagnosed with congenital PC deficiency at birth. He sustained a traumatic supracondylar fracture of the right humerus and underwent emergency surgery. To prepare for open surgery for fixation of the fracture, warfarin was discontinued, and an activated PC (APC) concentrate was used in combination with vitamin K antagonism. However, warfarin was administered during the scheduled nail extraction because the operation was minimally invasive. No thrombotic or bleeding complications occurred in either operation. In emergency surgery in patients with congenital PC deficiency, the combination of vitamin K and APC concentrate is considered a maintenance option for PC deficiency. Postoperative PT-INR control was difficult in our patient due to the administration of vitamin K and withdrawal of warfarin, and this issue must be addressed in the future. Further case experience is desirable to standardize perioperative management.

Term Infant with Bilateral Parenchymal Brain Hemorrhages.

Term Infant with Bilateral Parenchymal Brain Hemorrhages.

Clinical and genetic features of Chinese pediatric patients with severe congenital protein C deficiency who first presented with purpura fulminans: A case series study and literature review

IntroductionPurpura fulminans (PF) is a hematological emergency that can be caused by severe congenital protein C (PC) deficiency. It has been rarely reported in the Chinese population. We aimed to characterize the clinical and genetic features of Chinese pediatric patients with severe congenital PC deficiency who first presented with PF. Materials and methodsTwelve pediatric patients were diagnosed with severe congenital PC deficiency with PF, which was diagnosed based on our hospital records and previous reports from 1988 to July 2021 in China. We evaluated the clinical and genetic features of these patients. ResultsNine patients (9/12, 75%) had onsets that were observed within the first 48 h after birth. Six patients had a family history of thromboembolism. There was no consanguinity. Other symptoms were intracranial thrombosis or hemorrhage (4, 33.3%), ocular lesions (2, 16.7%), gastrointestinal hemorrhage (2, 16.7%) and kidney infarction before birth (1, 8.3%). All but one of the patients (one case not detected) had a plasma PC activity of <10%. The genetic study indicated that in the eight patients with inherited PC deficiency, two were homozygous, five were compound heterozygous and one was heterozygous for PC deficiency. ConclusionThis is the first and largest case series of Chinese pediatric patients with severe congenital PC deficiency who first presented with PF. It has been shown that treatment with both fresh frozen plasma and anticoagulants is recommended when PC concentrate is not easily available, especially in developing countries.

A case of perioperative management in a patient with congenital protein C deficiency and repeating purpura fulminans since the neonatal period

Severe congenital protein C (PC) deficiency, one of the autosomal dominant diseases, causes purpura fulminans (PF) during the neonatal period. These patients usually receive long-term anticoagulant therapy to prevent thrombosis, therefore we should pay attention to both thrombotic tendency and hemorrhagic diathesis when performing surgical treatment. We report a case of perioperative management of a tooth extraction in a patient with severe congenital PC deficiency who has repeatedly developed PF since the neonatal period. A 20-year-old woman, previously diagnosed with severe congenital PC deficiency, was referred to our department for extraction of the left mandibular first and second molars with severe caries. We planned to carry out the tooth extraction without suspending anticoagulant therapy, with the attending physician in charge of hematology. The patient underwent the tooth extraction with sufficient local hemostasis. She left hospital 4 days after surgery without any complications. However, she was readmitted two days after discharge because of post-extraction bleeding. The bleeding was easily stopped with local hemostatic treatment, however purpura developed on her right leg. It was treated with antithrombotic therapy and gradually disappeared without necrosis.

Congenital protein C deficiency with renal vein thrombosis and central retinal venous occlusion in a term neonate due to novel mutation in the PROC gene

Severe protein C (PC) deficiency is a rare disorder caused by homozygous or compound heterozygous genetic anomalies. PC deficiency is inherited as autosomal dominant, and most patients are heterozygous for a genetic defect. Homozygous or compound heterozygous for a mutation or other genetic defect affecting PC is typically due to inheritance of an abnormal allele from both parents. In the neonatal period, homozygous PC deficiency is usually associated with the development of severe, and often fatal, purpura fulminans and disseminated intravascular coagulation and if untreated is incompatible with life. Here, we report a case of congenital PC deficiency due to novel homozygous mutation in the protein coding gene (PROC) in a term neonate which presented with severe purpura fulminans, perinatal right renal vein thrombosis (RVT), bilateral adrenal hemorrhage, and early neonatal central retinal venous occlusion with retinal detachment. Perinatal RVT and early neonatal central retinal vein occlusion have not been reported previously in the literature.

Simultaneous Replacement of Endothelial Thrombomodulin and Plasma Protein C: A Novel Therapeutic Strategy for Sepsis-Induced Disseminated Intravascular Coagulation

Sepsis induces a procoagulant state, which in its most extreme form can result in disseminated intravascular coagulation (DIC), a syndrome of widespread microvascular thrombosis, ischemia, and organ dysfunction. Suppression of the protein C (PC) pathway has long been thought to be an important part of the pathophysiology, leading to development of a number of candidate therapeutics, including soluble human thrombomodulin (shTM, or ART123), currently being evaluated in a phase III clinical trial. While conferring less risk of bleeding toxicity than previous pharmacologic interventions into the protein C pathway, shTM fails to reproduce the spatial localization of its endogenous counterpart, preventing optimal interaction with PC bound to its endothelial receptor. To test the importance of localization to the endothelial membrane, we compared the antithrombotic activity of shTM to that of hTM/R6.5, a fusion protein therapeutic that anchors recombinant human TM to endothelial ICAM-1. In modified thrombin generation assays, in which platelet-poor plasma was exposed to cytokine-activated human endothelial cells, cell bound hTM/R6.5 was more potent than shTM mixed into the plasma. A similar result was found in a human whole blood microfluidic assay of sepsis-induced microvascular thrombosis, in which endothelial bound hTM/R6.5 more effectively inhibited fibrin deposition than its soluble counterpart. To investigate its mechanism of action, the fusion protein was tested in the setting of moderate PC deficiency, a common occurrence in patients with DIC. While PC depletion alone had no effect on fibrin deposition in this model, it significantly reduced the efficacy of hTM/R6.5, which was restored by supplementation using plasma-derived PC concentrate. These results indicate that endothelial bound TM acts not only as a direct inhibitor of thrombin, but derives antithrombotic activity from the activation of PC, such that it may be insufficient to reverse the coagulopathy of patients with significant deficiency in plasma PC. Likewise, the data match the clinical experience with PC supplementation, which appears to be of nominal benefit in sepsis or DIC, despite repeated demonstration of efficacy in patients with severe congenital PC deficiency, who have normal levels of endothelial TM and EPCR. The synergistic effect observed with simultaneous replacement of plasma PC and endothelial TM, however, suggest that this combination may represent a novel, translational therapeutic strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High prevalence of congenital thrombophilia in patients with pregnancy-related or idiopathic venous thromboembolism/pulmonary embolism.

Congenital thrombophilia which is characterized by deficiencies in proteins such as antithrombin (AT), protein C (PC) and protein S (PS), is a major cause of venous thromboembolism (VTE). A total of 130 patients with VTE were evaluated for congenital thrombophilia based on the activity of AT, PC, or PS. Fifteen VTE patients with congenital AT deficiency (11.5%), 16 with congenital PC deficiency (12.3%) and eight with congenital PS deficiency (6.2%) were diagnosed using DNA analysis. The frequency of congenital AT deficiency was significantly higher in subjects with pregnancy-related and idiopathic VTE than in those with VTE due to other causes, and congenital PC and PS deficiency were frequently associated with idiopathic VTE. Among the groups examined, the plasma levels of AT were the lowest in subjects with pregnancy-related VTE. Although our findings may have been influenced by some unintentional bias, congenital thrombophilia is nevertheless a major cause of VTE in pregnant patients as well as in young or middle-aged patients without any underlying diseases.

Skin Necrosis and Purpura Fulminans in Children With and Without Thrombophilia—A Tertiary Center's Experience

Purpura fulminans (PF) is a very rare clinicopathologic skin disorder comprising dermal microvascular thrombosis associated with perivascular hemorrhage of multiple origins. It may occur as the presenting symptom of severe congenital deficiency of protein C (PC) or protein S (PS) during the newborn period, or later in life following oral anticoagulant therapy with vitamin K antagonists, or of sepsis that may be associated with disseminated intravascular coagulation. Treatment consists of anticoagulants and PC concentrates during acute episodes. We report our experience in the diagnosis and management of pediatric PF. The medical records of the 6 children aged 2–16 years (median: 5 years) who presented with PF to our tertiary care center between 1996 and 2013 were studied. The thrombophilia workup revealed either the presence of congenital homozygous PC deficiency, prothrombotic polymorphisms (factor V Leiden and FIIG20210A heterozygosity), acquired PC/PS deficiency, or no discernible thrombophilia. The skin necrosis resolved following conservative fresh-frozen plasma/anticoagulant therapy in 2 cases, whereas 3 children required interventional plastic surgery. The sixth case, a 10-year-old child with severe PC deficiency, heterozygous factor V Leiden, and FIIG20210A, received recombinant activated PC. PF in childhood is rare and has multiple etiologies. Understanding of the variable pathogenesis and risk factors will facilitate diagnosis and appropriate clinical management.

Protein-C deficiency presenting as pulmonary embolism and myocardial infarction in the same patient

Plasma protein-C exerts anticoagulatory effects by inactivating factors V and VIII. Hereditary protein C deficiency is transmitted as an autosomal dominant disorder. Homozygous individuals usually develop purpura fulminans as newborns; heterozygous protein C-deficient individuals are at increased risk for venous thrombosis and pulmonary embolism. However, arterial thrombosis occurring as a result of congenital protein-C deficiency is still controversial. We describe a young patient with heterozygous protein-C deficiency who experienced both pulmonary embolism as well as myocardial infarction due to thrombotic occlusion without underlying major risk factors. Acute myocardial infarction in young without underlying major risk factors may be evaluated for protein c deficiency.

Frequent association of thrombophilia in cerebral venous sinus thrombosis

Thrombophilia is frequently associated with venous thromboembolism (VTE) including cerebral venous sinus thrombosis (CVST). The possibility of thrombophilia was examined in 12 patients with CSVT diagnosed in the past 9 years. Thrombophilia due to abnormalities in antithrombin (AT), protein C (PC), or protein S (PS) or antiphospholipid syndrome (APS) was evaluated. Nine patients with abnormally decreased AT, PC or PS and one patient with APS were examined. Of the nine patients examined by a gene analysis of AT, PC, or PS, one had a congenital AT deficiency, one had a congenital PC deficiency, and two had congenital PS deficiencies including a novel mutant (Gly189Ala). AT, PC and PS levels were all decreased in one patient, PS level was decreased in three patients, and AT level was decreased in one patient at the onset of CVST, but these concentrations improved after treatment. CVST is frequently associated with thrombophilia and a transient decrease in AT, PC or PS may be a causal factor.

Utilisation des modulateurs de la voie de la thrombine et de la protéine C dans les CIVD : résultats des essais cliniques

A correlation between acquired antithrombin (AT) and protein C (PC) deficiencies, multiple organ dysfunction and mortality has been demonstrated in various clinical conditions. In addition, the evolution of these deficiencies over time seems to have a more pronounced predictive value. Thrombin and PC pathways modulation in DIC has been evaluated in various clinical trials. This chapter aims at reviewing the clinical situations associated with DIC and in which the effects of these therapies have been evaluated. Congenital PC deficiency and heparin resistance are not considered here. AT and PC therapies are reviewed with regard to their influence on DIC, multiple organ dysfunction and mortality. AT supplementation improves DIC biomarkers and in some situations, organ dysfunction scores. Its effect on mortality remains to be demonstrated. PC concentrates seem to favourably improve coagulation disorders observed in DIC. However, no controlled study has evaluated its effect on mortality. Activated protein C has been demonstrated to improve survival in patients with severe sepsis at increased risk of death and DIC biomarkers but is associated with an increased risk of bleeding. Finally, heparin improves coagulation parameters observed in DIC but has not been demonstrated to improve survival. Its influence on the effects of other DIC therapies has to be evaluated.

Laboratory Diagnosis of Hereditary Thrombophilia

Genetic defects of antithrombin (AT) or one of the components of the protein C pathway are associated with hereditary thrombophilia. Laboratory assays are currently available to diagnose and type hereditary thrombophilia due to deficiency or dysfunction of one of the anticoagulant factors antithrombin (AT), protein C (PC) and protein S (PS), and APC resistance without the need of DNA analysis. There are no functional tests for the prothrombin mutant G20210A and thrombomodulin mutations, which can be diagnosed by a PCR-based test or by gene analysis, respectively. Hereditary AT deficiency is classified in a quantitative type I and three functional type II deficiencies affecting the reactive site (RS), heparin binding site (HBS), or pleiomorphic site of the AT protein. All four types of hereditary AT deficiencies can be diagnosed by a heparin cofactor assay and one immune assay in combination with crossed immunoelectrophoresis of the AT protein. The combination of an enzyme-linked immunoadsorbent assay (ELISA) and a functional Protac-APTT-based assay for PC will detect quantitative type I and dysfunctional type II PC deficiencies. There is a significant overlap in PC antigen and functional levels between heterozygotes of PC deficiency and normals leaving a gray zone of uncertainty in differentiating congenital PC deficiency and normal individuals. Accurate diagnosis of hereditary PS deficiency should be a combination of tests aimed to measure free PS activity and antigen and total PS antigen levels. APTT-, Xa-, and RVVT-based APC-resistance tests, when test plasmas are diluted in factor V deficient plasma, have increased in sensitivity and specificity to 100% for the discrimination of normal individuals from heterozygotes and homozygotes for factor V Leiden. The RVVT-based APC-resistance test provides better separation of factor V Leiden and normals in the various clinical settings, lupus anticoagulant in particular. The modified APC-resistance tests also claim a separation between heterozygotes and homozygotes for factor V Leiden in the normal population, asymptomatic subjects, and thrombosis patients. Below a certain cut-off level, a minor overlap of normalized APC ratios between heterozygotes and homozygotes for factor V Leiden of thrombosis patients has been shown in one study, which still points to the need to perform the more time consuming and expensive DNA test to identify heterozygotes from the more clinically significant homozygotes. The prothrombin-based APC-resistance test, which measures thrombin activated factor Va in highly diluted test plasma, appears to be the most sensitive and specific of all APC-resistance tests and separates normal individuals from heterozygotes and heterozygotes from homozygotes for factor V Leiden without the need of confirmation by a DNA test.

Gene Analysis of an Abnormal Protein C with a Mutation of GAG for Glu26 to AAG for Lys

We report a missense mutation of protein C (PC) gene in a family with type II congenital. PC deficiency. The proband was a 17-year-old high school student with deep venous thrombosis and pulmonary embolism. Hemostatic examinations revealed normal amidolytic activity and PC antigen level measured by a chromogenic snake venom assay and an EIA utilizing a polyclonal antibody, respectively. However, anticoagulant activity measured by the activated partial thromboplastin time method and immunological antigen level estimated by a monoclonal antibody recognizing the Ca2+-dependent conformational change were reduced. Two other members. of this family (father and paternal grand-mother) showed the identical defect, but they had been asymptomatic. The functional abnormalities were most likely to be due to alternation in the Gla domain. Subsequently, a polymerase chain reaction (PCR) strategy and an allele-specific oligonucleotide (ASO) hybridization were used to evaluate exon 3 of the PC gene. Sequence analysis following amplification of exon 3 and its flanking regions showed a single G to A transition, resulting in the conversion of Glu26 codon (GAG) to Lys26 codon (AAG). Since this missense mutation generated no new restriction site, the PCR-amplified exon 3 fragments from this family member were hybridized with ASOs corresponding to the normal and mutated sequences. An identical mutation was detected in the affected family members of the proband by the ASO hybridization. These data indicate that conversion of the Glu26 codon (GAG) to Lys26 codon (AAG) was responsible for the type II congenital PC deficiency inherited in this Japanese family.

Intracardiac thrombosis diagnosed by echocardiography in childhood: predisposing and etiological factors

Eleven cases of intracardiac thrombi caused by different factors including protein-C deficiency are presented for discussion of the etiology and predisposing factors of intracardiac thrombi during infancy and childhood, and to stress the importance of protein-C deficiency as an etiological factor. Thrombi were localised in the left heart in five patients and right heart in five patients. One patient had both-sided thrombi. Four of our patients had dilated cardiomyopathy, one had mitral valve hypoplasia, and one had pulmonary valvar stenosis as the predisposing factors for thrombus formation. In three patients whose cardiac anatomies were completely normal, we determined protein-C deficiency as an etiological factor of thrombus formation. One of these had congenital protein-C deficiency and the other two had acquired temporary protein-C deficiency due to sepsis. In conclusion we recommend that protein-C deficiency should be investigated as an etiological factor in all cases of intracardiac thrombi irrespective of whether or not another predisposing factor is identified.

Protein C: Comparison of different assays in normal and abnormal plasma samples

Two assay methods for Protein C (PC) are described. Both tests are based on the activation of PC by a fraction of the venom of the Copperhead snake. Activated PC can be measured either by cleaving of a chromogenic substrate or by an APTT test. Both tests were compared with ELISA tests for PC. In 27 healthy persons the results were around 100% by all test systems when compared with normal pooled plasma. In 13 patients under stable anticoagulant therapy the average results were 54% for the ELISA and 52% for the chromogenic substrate test whilst the corresponding figures were 22% for the APTT test and 25% for the Quick test. In 4 cases of liver cirrhosis PC was diminished and there was no difference between the three tests. In two patients with congenital PC deficiency all results were close to 50% and in three patients the ELISA and the substrate tests were normal but the APTT test showed results of 44, 28 and 54% respectively. These results give rise to the conclusion that the snake venom activator is also capable of activation of decarboxylated PC molecules which can thereby split a chromogenic substrate but remain inactive in an APTT test.

Multicenter Comparison of Five Functional and Two Immunological Assays for Protein C

Five functional assays and two immunoassays for protein C (PC) were evaluated in parallel for the same plasma samples collected from healthy subjects, patients with congenital and acquired PC deficiencies or patients with conditions associated with high PC levels. For 7 patients starting warfarin therapy and for 15 patients during stabilized warfarin therapy, there were significant between-assay differences. For these groups immunoassays gave higher values than most functional assays and the latter also gave varied results, probably depending on their respective capacity for recognizing a carboxylated PC. On the other hand, there were no significant between-assay differences nor discrepancies between PC activity and antigen levels for healthy subjects (n = 39), patients with congenital PC deficiency (n = 10), myocardial infarction (n = 25), chronic liver disease (n = 19), disseminated intravascular coagulation (n = 35), in the post-operative period (n = 20) or in women taking oral contraceptives (n = 20). This comparison of PC assays indicates that PC levels measured by different functional or immunological assays are very close in the majority of clinical conditions, but not for patients on oral anticoagulants.

A Functional Assay of Protein C in Human Plasma

A three-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: (1) adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; (2) activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; (3) addition of antithrombin III and heparin to the system to inhibit thrombin and other coagulation enzymes generated during incubation and measurement of the activated PC with a synthetic (chromogenic) substrate. The assay appears to be specific for PC because: (a) PC-depleted plasma (by immunoadsorption) is inactive; (b) addition of purified PC to PC-depleted plasma reconstitutes its activity; and (c) no enzymatic activity is generated in the absence of the thrombin-thrombomodulin complex. Mixtures of a normal plasma pool with PC-depleted plasma yielded an amount of enzymatic activity proportional to the fraction of normal plasma. Using this as a standard curve, the amount of PC in the plasma of 23 normal subjects was 97% +/- 15%. The within-assay coefficient of variation was 3.5% and the between-assay coefficient 6.5%. A linear correlation (r = 0.86) was found between PC as measured with the functional assay and with a radioimmunoassay. In 3 patients with congenital PC deficiency, the functional PC level was 37% +/- 9% and the antigen level 64% +/- 11%. It is concluded that the present assay may be used for reliable and accurate estimation of activatable PC in human plasma.

A functional assay of protein C in human plasma

A three-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: (1) adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; (2) activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; (3) addition of antithrombin III and heparin to the system to inhibit thrombin and other coagulation enzymes generated during incubation and measurement of the activated PC with a synthetic (chromogenic) substrate. The assay appears to be specific for PC because: (a) PC- depleted plasma (by immunoadsorption) is inactive; (b) addition of purified PC to PC-depleted plasma reconstitutes its activity; and (c) no enzymatic activity is generated in the absence of the thrombin- thrombomodulin complex. Mixtures of a normal plasma pool with PC- depleted plasma yielded an amount of enzymatic activity proportional to the fraction of normal plasma. Using this as a standard curve, the amount of PC in the plasma of 23 normal subjects was 97% +/- 15%. The within-assay coefficient of variation was 3.5% and the between-assay coefficient 6.5%. A linear correlation (r = 0.86) was found between PC as measured with the functional assay and with a radioimmunoassay. In 3 patients with congenital PC deficiency, the functional PC level was 37% +/- 9% and the antigen level 64% +/- 11%. It is concluded that the present assay may be used for reliable and accurate estimation of activatable PC in human plasma.