Liver transplantation for congenital protein C deficiency with initial poor graft function: a case report with literature review.

Congenital deficiency of protein C (PC) is a rare disease that causes thrombophilia during the neonatal and infantile periods. Despite anticoagulative treatments, purpura fulminans and major vessel thrombosis often occur. We report a 7-year-old girl with congenital PC deficiency who underwent deceased donor liver transplantation (LT) and experienced complications accompanied by initial poor graft function (IPGF). Before LT, she had cerebral and ophthalmic hemorrhage, and seven episodes of purpura fulminans. The operation was successfully performed; however, the liver graft developed IPGF. Hyperammonemia and coagulopathy required continuous hemodiafiltration and infusion of fresh frozen plasma. It took 22 days for PC activity to reach reference levels. The changes in clotting and anticlotting activities in the patient's plasma were revealed using clot waveform analysis and the HemosIL ThromboPath® assay. PC activity remained normal for 5 years after LT. Even when IPGF occurs, liver function including PC activity can remain normal for a long time after recovery from IPGF. LT can be a curative treatment for congenital PC deficiency.

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.

An Embolic Stroke in a Patient With PROC p.Lys193del.

We report a 58-year-old woman who suddenly developed brain infarction with weakness of the left lower extremity and left perioral dysesthesia during postoperative tamoxifen therapy for breast cancer and prednisolone therapy for rheumatoid arthritis. Diffusion-weighted images detected multiple areas of hyperintensity in the posterior circulation system of the brain. Despite extensive examinations, we could not identify any embolic sources except hypoplasia of the right vertebral artery. We found decreased activity of protein C against its antigen level (activity: 59% versus antigen: 122%) with enhanced activity of coagulation factor VIII (178%) and von Willebrand factor (285%). DNA sequencing identified trinucleotide deletion of the PROC gene leading to 1 amino acid deletion at Lys-193 (p.Lys193del). We speculate that the PROC gene polymorphism may have participated in tamoxifen- and prednisolone- associated hypercoagulable state, leading to development of an embolic stroke in this patient.

Protein C and protein S deficiencies may be related to survival among hemodialysis patients.

BACKGROUND: Thrombophilia due to protein C (PC) and protein S (PS) deficiencies is highly prevalent among patients with stage 5 chronic kidney disease and is reported to arise due to extracorporeal circulation during hemodialysis (HD). This study aimed to evaluate the relationship between HD treatment and thrombophilia. METHODS: A total of 114 Japanese patients on maintenance HD (62 men, 52 women) were followed during 2008-2011. Their survival rates were compared against the duration of HD. Prior to each HD, coagulation/fibrinolysis parameters and PC and PS activities were measured using standard techniques. The patients were divided into two groups: Group 1, with PC and/or PS deficiencies (n = 32), and Group 2, without PC and PS deficiencies (n = 82). The influence of such deficiencies and duration of dialysis on survival was examined. Time-to-event analysis was applied using Kaplan-Meier estimates, and the log-rank test was proposed to test the equivalence of relative survival data. Hazard ratios and 95% confidence intervals (CI) were calculated. RESULTS: Of the 114 patients, 37 died (Group 1, 22; Group 2, 15). The hazard ratio (95% CI) was higher (p = 0.004) in Group 1 than Group 2. Gene analyses of PC and PS were performed in 14 patients from Group 1. No mutations in either protein were observed. We analyzed the causes of death in both groups; however, the estimated thrombophilia-related incidence of death could not be determined due to small sample size of HD patients. CONCLUSIONS: Our results suggest that PC and PS deficiencies may be related to survival in HD patients. However, this finding warrants additional research.

Hemostatic assessment of combined anticoagulant therapy using warfarin and prothrombin complex concentrates in a case of severe protein C deficiency.

Patients with severe congenital protein (P)C deficiency require long-term anticoagulant management. Recombinant PC concentrates for prophylactic use are not available in Japan; prothrombin complex concentrates (PCC), containing factors (F)II, VII, IX, X, and PC (PPSB-HT®), have been used 'off-label' in a few patients. We investigated the combined use of prophylactic PCC and Warfarin (VKA; PT-INR 2.0-2.5) in a severely PC-deficient patient in whom VKA alone did not prevent recurrent purpura. Plasma VKA-dependent factor levels and global PC function (Thrombopath®) were assessed. Plasma activity levels of FII/FVII/FIX/FX post-infusion of PCC (6.3 unit/kg) increased 35/27/27/35 (initial level) to 59/60/38/83 IU/dl, respectively. FVII:C and FIX:C rapidly returned to baseline levels 12-24 h post-infusion, but FII:C and FX:C returned more slowly. PC antigen (< 5%) increased to ~ 15%, followed by return to baseline levels 24 h post-infusion. Global PC function was very low (%PiCi 24%), but improved post-PCC infusion. This potential was slightly detectable even at an undetectable PC level. At day 3, high levels of D-dimer and FDP were observed without thrombotic event, but these improved post-infusion. Although PCC restored VKA-dependent coagulation factors, PC contained in PCC significantly improved global anticoagulation, and was clinically beneficial in this severely deficient patient.

Diagnostic challenge of the newborn patients with heritable protein C deficiency.

ABSTARCT: OBJECTIVE: The diagnosis of neonatal-onset protein C (PC) deficiency is challenging. This study aimed to establish the neonatal screening of heritable PC deficiency in Japan. STUDY DESIGN: We determined the changes in plasma activity levels of PC and protein S (PS) in healthy neonates, and studied newborn patients with PROC mutation in the Japanese registry. RESULT: Physiological PC and PS levels increased with wide range. The PC/PS-activity ratios converged after birth. The PC/PS-activity ratios of 19 patients with biallelic mutations, but not, 9 with monoallelic mutation, were lower than those of 13 without mutation. The logistic regression analyses established a formula including two significant variables of PC activity (cut-off < 10%, odds ratio = 30.0) and PC/PS-activity ratio (cut-off < 0.35, odds ratio = 22.7), with 93% sensitivity and 44% specificity for determining patients with mutation(s). CONCLUSION: The PC/PS-activity ratio is an effective parameter for the genetic screening of neonatal-onset PC-deficiency in Japanese population.

Pulmonary thromboendarterectomy in a combined thrombophilia patient.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a potentially correctable cause of secondary pulmonary hypertension. Surgical treatment remains the primary treatment for patients with CTEPH. Pulmonary thromboendarterectomy (PEA) with deep hypothermic circulatory arrest is the standard and recommended surgical technique for the treatment of these patients. The prevalence of CTEPH after an acute pulmonary thromboembolism (PTE) has been found in various studies to be between 0.6 and 8.8%. Mortality rates in elective PEA cases with CTEPH are reported to be between 1.9 and 4.5%. We report on a 50-year-old female patient with combined inherited thrombophilia, including protein C and protein S deficiencies, who was diagnosed with CTEPH and was successfully treated with pulmonary thromboendarterectomy.

[Thrombophilia in systemic lupus erythematosus: A case-control study]. / Les marqueurs de thrombophilie au cours du lupus érythémateux systémique : une étude cas-témoin.

INTRODUCTION: To investigate the thrombotic tendency in patients with systemic lupus erythematosus (SLE) by evaluating congenital and acquired abnormalities with an increased risk of thrombosis. PATIENTS AND METHODS: A total of 53 patients with SLE were included in the study. Fifty-three healthy controls paired by age and sex were assessed. Anticardiolipin antibodies (aCL), anti ß2 glycoprotein (aß2GP), lupus anticoagulant (LAC), protein C (PC), protein S (PS), antithrombin (AT), acquired activated protein C, and homocysteinemia were evaluated. Comparisons for categorical variables were analyzed by Chi2 and student tests. RESULTS: The patients were all female and had a mean age of 30.6 years (16/58). The healthy controls were all female and their mean age was 30.8 years (17/56). Five patients (9.4%) developed venous thrombosis during the 24 months of follow-up. The antiphospholipid antibodies were positive in 17 patients (32.1%) and negative in all healthy controls (P=0.01). PS deficiency was noted in 17 patients (32.1%) and in 5 controls (P=0.004). Hyperhomocysteinemia was noted in 16 patients (30.2%) versus 3 controls (5.6%) (P=0.002). Test for PC deficiency and acquired activated protein C showed no significant difference between the two groups. No AT deficiency was found in the patients. The study of clinical and biological correlations based on the presence and absence of thrombophilic parameters concluded to a significant association between Protein C deficit and thrombosis (P=0.02) and acquired activated protein C resistance and thrombosis (P=0.04). There was no significant association between the APL and thrombosis. CONCLUSION: Thrombophilic abnormalities were significantly more frequent in lupus patients than in healthy controls. Thrombotic events were significantly associated with PC deficit and acquired protein C resistance. There was no correlation between antiphospholipid antibodies and thrombosis.

The relationship between the levels and function of endothelial progenitor cells and factor V Leiden and protein C deficiency in patients with primary Budd-Chiari syndrome.

OBJECTIVE: Budd-Chiari syndrome (BCS) is a life-threatening hepatic disease characterized by hepatic venous obstruction at the level of hepatic vein, hepatic venules, or inferior vena cava. No evidence reported the relationship between the endothelial progenitor cells and the deficiency of factor V Leiden and protein C in patients with primary Budd-Chiari syndrome. PATIENTS AND METHODS: We recruited participants between June 2014 and July 2015. For primary BCS group, 28 patients were collected. 20 patients were included in the NAFLD group. Another 73 healthy participants were recruited into the control group. None of the patients and participants had received interventional therapy or had undergone surgery prior to being recruited. Levels and functions of endothelial progenitor cells (EPCs) were examined. The factor V Leiden mutation, protein C deficiency and protein S deficiency were evaluated. Finally, the relationship between the levels and function of endothelial progenitor cells and factor V Leiden and protein C deficiency in patients with primary Budd-Chiari syndrome was analyzed. RESULTS: The results showed that no significant differences were found between the BCS (and NAFLD) and control group considering age, sex, BMI, smoking (p>0.05 for variables). However, significant differences were observed in TG, TC, HDL-C, white blood cells, hemoglobin, ALT, AST, ALP, γ-GT, total bilirubin, and albumin (p<0.05 for variables). Compared with the healthy participants, significant downregulation was found in BCS and NAFLD patients regarding CD34+/CD45-, late outgrowth endothelial cells (OECs) colonies, OECs proliferation, and OECs tubulogenesis (p<0.001 for variables). Among the 28 BCS patients, factor V Leiden mutation (n=10, 35.71%, OR 12.67, 95% CI 5.24-27.93) and hereditary protein C deficiency (n=4, 14.29%, OR 7.48, 95% CI 2.02-21.43) were more prevalent than those in the control group. These results suggested that factor V Leiden mutation and protein C deficiency were major risk factors for BCS. Finally, we demonstrated that factor V Leiden and protein C deficiency may negatively regulate the OECs levels and functions in BCS patients. CONCLUSIONS: It's important to improve the OECs levels and functions, and to prevent the deficiency of factor V Leiden and protein C in the treatment of BCS.

Acquired Versus Congenital Neonatal Purpura Fulminans: A Case Report and Literature Review.

Neonatal purpura fulminans (PF) is a life-threatening disorder caused by congenital or acquired deficiencies of protein C (PC) or S. PF presents as a cutaneous manifestation of disseminated intravascular coagulation. We describe a case of PF in a newborn with left leg ischemia and undetectable PC levels soon after birth. Despite anticoagulation therapy and PC concentrate, left foot amputation was required. Genetic testing of PROC for congenital PC deficiency was normal. This case highlights the course of PF due to acquired PC deficiency in a newborn treated with PC concentrate which is rarely described in the literature.

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.

Purpura Fulminans: Mechanism and Management of Dysregulated Hemostasis.

Purpura fulminans (PF) is a highly thrombotic subtype of disseminated intravascular coagulation that can accompany severe bacterial, and more rarely, viral infections. PF is associated with an extremely high mortality rate, and patients often die of overwhelming multisystemic thrombosis rather than septic shock. Survivors typically experience amputation of involved extremities and significant scarring in affected areas. Despite the devastating clinical course associated with this hemostatic complication of infection, the mechanism of PF remains poorly understood. Severe acquired deficiency of protein C and dysfunction of the protein C-thrombomodulin pathway as well as other systems that exert a negative regulatory effect on coagulation have been implicated. Management of PF involves treatment of the underlying infection, aggressive anticoagulation, and robust transfusion support aimed at correcting acquired deficiencies in natural anticoagulant proteins. In this review, we address the diagnosis and management of PF with a focus on a rational approach to this condition informed by the available data. Proposed mechanisms underlying the dysregulation of coagulation seen in PF are also covered, and implications for therapy are discussed.

Characterization of thrombosis in patients with Proteus syndrome.

Patients with overgrowth and complex vascular malformation syndromes, including Proteus syndrome have an increased risk of thromboembolism. Proteus syndrome is a mosaic, progressive overgrowth disorder involving vasculature, skin, and skeleton, and caused by a somatic activating mutation in AKT1. We conducted a comprehensive review of the medical histories and hematologic evaluations of 57 patients with Proteus syndrome to identify potential risk factors for thrombosis. We found that six of ten patients, who were deceased, died secondary to deep venous thrombosis and/or pulmonary embolism. Of the remaining 47 living patients, six had thromboembolic events that all occurred postoperatively and in an affected limb. Eleven of 21 patients had an abnormal hypercoagulable panel including Factor V Leiden heterozygotes, antithrombin III deficiency, positive lupus anticoagulant, or Protein C or S deficiencies. We observed that eight of 17 patients had an abnormal D-dimer level >0.5 mcg/dl, but deep venous thromboses occurred in only four of those with D-dimer >1.0 mcg/dl. We conclude that the predisposition to thrombosis is likely to be multifaceted with risk factors including vascular malformations, immobility, surgery, additional prothrombotic factors, and possible pathophysiologic effects of the somatic AKT1 mutation on platelet function or the vascular endothelium. The D-dimer test is useful as a screen for thromboembolism, although the screening threshold may need to be adjusted for patients with this disorder. We propose developing a registry to collect D-dimer and outcome data to facilitate adjustment of the D-dimer threshold for Proteus syndrome and related disorders, including PIK3CA-Related Overgrowth Spectrum.

Gly74Ser mutation in protein C causes thrombosis due to a defect in protein S-dependent anticoagulant function.

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

Rational Design of Protein C Activators.

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

[Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency].

OBJECTIVE: To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detection and model analysis. METHODS: Chromogenic substrate method and enzyme linked immunosorbent assay (ELISA) were used to determine the plasma protein C activity (PC: A) and protein C antigen (PC: Ag) in the two probands and their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCR and analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members were also amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutations and Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutations were analyzed by Swiss-PdbViewer software. RESULTS: The PC: A and PC: Ag of proband 1 was 30% and 35%, while PC:A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missense mutations were found in exons 7 and 5 of the PROC gene, namely c.565 C>T (p.Arg147Trp) and c.383 G>A (p.Gly86Asp). His father and aunt were carriers for c.565 C>T, while his mother had carried c.383 G>A. The PC: A of proband 2 and his son were 50% and 64%, respectively. And they were both positive for p.Arg147Trp. Analysis of PolyPhen-2 indicated that p.Arg147Trp was benign, while p.Gly86Asp was damaging. Clustal X analysis indicated that the p.Arg147Trp was non-conservative, while the p.Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trp147 in p.Arg147Trp destroyed the two hydrogen bonds between Arg147-Lys146 and Arg147-Lys151, and steric hindranted with Arg178. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion. CONCLUSION: Both probands had hereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Arg147Trp and p.Gly86Asp were the main cause for PC: A activity decrease. Among these, p.Gly86Asp was discovered for the first time.

Changing Concepts of Cirrhotic Coagulopathy.

The state of clinical art of the coagulopathy of cirrhosis changed considerably over the last decade. Until 2005, cirrhosis was considered as the epitome of the hemorrhagic coagulopathies and the abnormal hemostasis tests associated with the disease were corrected with infusion of fresh frozen plasma or platelets to minimize the risk of bleeding. Since that time, a great deal of work has been done and there is now a change of paradigm. The prothrombin time once considered as an isolated measure of bleeding risk was rejected, and cirrhosis shifted from a purely hemorrhagic construct to a mixed and thrombosis-prone paradigm. In this article we examine the interesting history of how these conceptual changes came about.

Warfarin-Induced Skin Necrosis in Patients With Low Protein C Levels.

Warfarin-induced skin necrosis (WISN) is a rare complication of anticoagulant therapy associated with a high incidence of  morbidity and mortality requiring immediate drug cessation. At particular risk are those with various thrombophilic abnormalities, especially when warfarinisation is undertaken rapidly with large loading doses of warfarin. Cutaneous findings include petechiae that progress to ecchymosis and hemorrhagic bullae. With the increasing number of patients anticoagulated as out-patients for thromboprophylaxis, we are concerned that the incidence of skin necrosis may increase. We present a case of WISN with low protein C level. He was a 50-year-old male who came to our department because of acute infarction in irrigation area of the superior cerebellar artery. He had intermittent atrial fibrillation and was started on anticoagulant therapy.  After few day of therapy, he developed skin necrosis, and his level of protein C was low. Warfarin-induced skin necrosis is a rare but serious complication that can be prevented by routine screening for protein C, protein S or antithrombin deficiencies or for the presence of antiphospholipid antibodies before beginning warfarin therapy.