Protein C Deficiency Research Articles

Factor V G1691A and Factor II G20210A Mutations and Clinical Expression of Severe Hemophilia A (< 2%) in Children: Impact on Bleeding Episodes and Joint Damage.

It has been recently shown that the first bleeding onset in children with severe hemophilia A (HA) carrying prothrombotic risk factors is significantly later in life than in non-carriers.1 The present multicenter study was performed to determine whether the factor (F) V G1691A or the F II G20210A are associated with decreased annual bleeding episodes (ABE) in 106 pediatric PUP patients with severe HA (Intron 22 58.6%) consecutively admitted to German pediatric hemophilia treatment centers. Treatment was initiated according to the frequency of bleedings, and most patients received on demand therapy with a switch over to prophylactic therapy 3x/week (40–60 IU/kgKG factor VIIII concentrate) when more than three bleedings (range 2–6) had occurred into the same joint (n=49). Prospective median(range) patient follow-up was 14(4–35) years. Heterozygosity of the FV mutation was found in 8 subjects, homozygosity in one, and 5 children carried the FII mutation once combined with protein C-deficiency. Carriers of the FV and FII mutations had significantly fewer ABE than non-carriers (p=0.004). 66 of 106 PUP patients developed at least one target joint with a median(range) Pettersson score of 1(0–12) available in 57 patients clearly dependent on age (p=0.039) as well as ABE (p=0.037). The “Nuss” joint score available in 33 subjects highly correlated with the Pettersson score (p=0.007). Data presented here give evidence that the clinical expression of severe HA in children is influenced by the co-expression of the FV and FII mutation.

Prothrombotic Risk Factors in Infants of Diabetic Mothers

Infants of diabetic mothers (IDMs) are at an increased risk for thromboembolic disease. The mechanism(s) to explain this association is unclear. We hypothesized that the pathophysiology of thrombosis in IDMs is multifactorial and likely involves interactions among genetic and acquired factors affecting the procoagulant, anticoagulant and fibrinolytic pathways. To compare the prevalence of common prothrombotic risk factors in a cohort of IDMs to a matched control group. Full-term infants born to mothers with diet controlled (A1-IDM) (N=17), insulin requiring diabetes (ID-IDM) (N=20) and healthy term infants (controls) (N=20) matched for mode of delivery had cord blood collected at delivery. Samples were analyzed for the following: factor V Leiden (FVL), prothrombin 20210A (P20210A), methylenetetrahydrofolate reductase C677 T (MTHFR), Factor VIII (FVIII), Protein C (PC), Lipoprotein(a) (Lp(a)) and plasminogen activator inhibitor-1 (PAI-1). None of the infants had a clinically apparent thrombotic event. IDM mothers and their infants were clinically similar to controls except for a higher prevalence of hypoglycemia (30 vs 0%; p=0.005). There was no significant difference in the prevalence of the common genetic risk factors (FVL, P20210A, MTHFR) FVIII, or PAI-1 levels. Elevated Lp(a) levels were seen more frequently in IDMs than Controls (40 vs 20%) but this difference was not statistically significant. The PC activity (%) was significantly decreased in the IDM group compared to controls, 35+/-12 vs 44+/-9 (p<0.005). A1-IDM had lower PC activity compared to ID-IDM (p=0.05) and controls (p=0.001). PC deficiency is likely one mechanism to explain thrombosis in IDMs.

Theoretical and experimental analysis of analyte transport in a fiber‐optic, protein C immuno‐biosensor

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.

A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

During the systemic inflammatory state induced by sepsis, the potential for coagulopathy exists because of up-regulation of natural procoagulants and anti-fibrinolytics, and down-regulation of natural anti-coagulants, with protein C (PC) being a critical example of the latter case. PC functions as an anti-coagulant, profibrinolytic, and anti-inflammatory agent, and, thus, its administration or deficiency may affect the course and outcome of sepsis in patients. In this study, a cecal ligation and puncture model of septic peritonitis was applied to wild-type mice and littermates with a targeted heterozygous deficiency of PC (PC(+/-)) to characterize the importance of a PC-deficiency on polymicrobial sepsis. An enhanced mortality rate was found to accompany a PC deficiency. Plasma cytokines, as well as organ-specific expression of cytokine transcripts, were elevated in PC(+/-) mice. No signs of severe disseminated intravascular coagulation (DIC) were observed in wild-type or PC(+/-) mice, as indicated by an increase in fibrinogen levels and the invariability of platelet counts after cecal ligation and puncture. Consumption of coagulation factors was similar in both genotypes and a decrease in the PC mRNA and protein levels was more prominent in PC(+/-) mice. Renal and organ muscle damage was enhanced in PC(+/-) mice, as shown by increases in plasma blood urea nitrogen, creatinine, and creatinine kinase. Hypotension and bradycardia were more enhanced in PC(+/-) mice than in wild-type mice, thus provoking a more severe septic shock response. Thus, the hemodynamic role of PC during sepsis is of critical importance to the outcome of the disease.

Protein C system defects in Indian children with thrombosis.

As ethnic variations are known to exist in inherited genetic defects, the clinico-haematological profile of Indian children with thrombophilia may be different from that of Caucasians. The aim of the study was to analyse the phenotypic and genotypic causes of thrombophilia in Indian children. Forty patients with arterial (21 patients) and venous (19 patients) thrombosis were the subjects of the study. Their age ranged from 6 days to 15 years. All of the patients were initially screened by Pro C Global assay. Activated protein C resistance (APCR) was measured. In cases with low Pro C Global values, protein C (PC), protein S (PS) and factor V G1691A, prothrombin G20210A and MTHFR C677T polymorphism were tested in all 40 cases. Of the 21 patients with arterial thrombosis, 4 (19%) had PC deficiency, 7 (33.3%) had PS deficiency and 1 (4.8%) had combined deficiency of PC and PS. Of the 19 patients with venous thrombosis, 5 (26.3%) each had PC and PS deficiency and 4 (21%) had combined PC and PS deficiency. Heterozygous factor V G1691A defect was seen in one (4.8%) patient with arterial thrombosis and three (15.8%) patients with venous thrombosis. Heterozygous MTHFR C677T polymorphism was seen in five (23.8%) patients with arterial thrombosis and in four (21%) patients with venous thrombosis. Prothrombin G20210A polymorphism was absent in all patients and controls. Protein C system defect is common in Indian children with thrombosis.

Portal vein thrombosis in children and adolescents: The low prevalence of hereditary thrombophilic disorders

Purpose The aim of this study was to determine the frequency of thrombophilic disorders in children and adolescents with portal vein thrombosis (PVT) as well as assessing the hereditary character of this disorder. Methods A 2-year prospective study was carried out in pediatric PVT patients (n = 14), their parents (n = 25), and an age-matched control group free of liver disease (n = 28). The presence of PVT was assessed by means of Doppler ultrasound scan or angiography. None of the PVT patients presented biochemical or histologic signs of liver disease. Results The frequency in PVT patients of protein C (PC), protein S (PS) and antithrombin (AT) deficiency was 42.9% ( P < .05 v controls), 21.4% ( P > .05) and 7.1% ( P > .05), respectively. None of the controls or parents of PVT patients presented hereditary PC, PS, or AT deficiency. One PVT patient and one control ( P = .999) presented prothrombin G20210A mutation. Homozygous methylenetetrahydrofolate reductase C677T genotype was observed in 3 of 14 (21.4%) PVT patients and in 5 of 28 (17.9%; P = .356) controls. None of these patients presented factor V G1691A mutation. Conclusions PC deficiency was frequent in pediatric PVT patients and does not seem to be an inherited condition. The hereditary prothrombotic disorders do not seem to play a vital role in thrombosis in children and adolescents with PVT.

P0351 DEFICIENCY OF COAGULATION-INHIBITING PROTEINS IN CHILDREN AND ADOLESCENTS WITH CIRRHOSIS

Introduction: A deficiency of coagulation-inhibiting proteins may be acquired or hereditary. Cirrhosis is considered one of the acquired causes of protein C (PC), S (PC) and antithrombin (AT) deficiency because these proteins are synthesized principally in the liver. Very few studies on the prevalence of coagulation-inhibiting proteins deficiency in patients with cirrhosis are available in the literature. The object of this study is to determine the frequency of such deficiencies. Methods: A prospective study was carried out, including 24 children and adolescents with cirrhosis (n = 24) and an age-matched control group without liver disease (n = 28). The presence of cirrhosis was diagnosed by hepatic biopsies. The activity of the proteins C and S was determined by the coagulometric method and antithrombin activity was measured with chromogenic substrate. The Chi2 test and Fischer exact test was used for statistical analysis. Results: The mean and standard deviation for age of the patients with cirrhosis (Group 1) and of the control group (Group 2) was 8,2 years ± 5,5 years and 8,10 years ± 4,5 years, respectively. In Group 1, 14/24 patients (58,3%) were female and 23/24 (95,8%) were Caucasians. The etiological factor of the patients of group 1 was extrahepatic biliary atresia in 10/24 (41,7%), auto-immune hepatitis in 3/24 (12,5%), Virus B hepatitis in 1/24 (4,2%) and Vírus C hepatitis associated with thalassemia em 1/24 (4,2%). No etiologic factor was identified in the remaining patients. According to the Child-Pugh classification, 12/24 (50%) of the patients fall into category A, 8/24 (33,3%), B e 2/24 (8,3%), C. The majority of the children with cirrhosis (16/24, 66,7%) presented a reduction in the activity level of one or more coagulation-inhibiting proteins. PC deficiency was observed in 14/24 (58,3%), of PS em 11/24 (45,8%) and of AT in 11/24 (45,8%). The average activity of PC, PS e AT in Group 1 was 49,23 ± 33,10; 69,08 ± 29,63 and 78,38 ± 38,59 (p < 0,05 vs. controls), respectively. In the Child-Pugh Group A, the average of PC, PS e AT was 70,92 ± 32,66; 86,75 ± 27,65 e 102,08 ± 35,11 (p < 0,05 vs controls), respectively, and in the Child-Pugh B and C categories 27,92 ± 14,69; 51,42 ± 19,75 e 54,67 ± 25,58 (p < 0,05 vs controls), respectively. Conclusion: The coagulation-inhibiting proteins activity decreases in accordance with the gravity of the hepatic disease. The assessment of this hemostatic parameter could be of assistance in the evaluation of the progression of liver impairment.

Protein C levels are regulated by a quantitative trait locus on chromosome 16: results from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project.

Protein C (PC) is a component of the protein C anticoagulant pathway. PC deficiency is a risk factor associated with venous thromboembolism. As part of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project, we conducted a genome-wide linkage scan to localize genes that influence variation in PC plasma levels. PC levels were measured in 398 individuals belonging to 21 Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1-cM genetic map. Variance component linkage methods were used to evaluate linkage and to detect quantitative trait loci (QTL). A region on chromosome 16 (16q23), flanked by markers D16S3106 and D16S516, showed strong evidence of linkage with PC levels (LOD=3.69). This region contains 1 positional candidate gene, the NAD(P)H:dehydrogenase quinone 1 (NQO1), involved in vitamin K metabolism. The association of 1 SNP of this gene with PC levels (P=0.005) strongly supports the implication of NQO1 gene in the variability of PC levels. These results illustrate the application of genomic scans to identify the genetic determinants of quantitative variation in a component of the hemostatic pathways. They provide strong evidence for a locus (QTL) on chromosome 16 that influences PC levels.

Pregnancy-associated thrombosis.

Venous thromboembolism (VTE) occurs infrequently but is a leading cause of illness and death during pregnancy and the puerperium. In the general population the incidence of pregnancy associated VTE is approximately 1 in 1500 deliveries The risk of VTE is five times higher in a pregnant than in a non-pregnant woman. Postpartum the VTE-risk is even higher. Women with congenital abnormalities or persistent presence of antiphospholipid antibodies have an increased risk of VTE during pregnancy and the puerperium. In individuals with well defined hereditary thrombosis risk factors, such as the factor V:R506Q mutation, the factor II:G20210A variation, antithrombin-deficiency or protein C-deficiency, a relative risk of pregnancy associated VTE between 3.4 and 15.2 has been found. Women with previous VTE have an approximately 3.5 fold increased risk of recurrent VTE during pregnancy compared to non-pregnant periods. Our ability to diagnose pregnancy-associated VTE clinically is generally poor, since dyspnea, tachypnea, swelling and discomfort in the legs are common. Objective diagnosis is essential for treatment decisions. Exposure to radiation of less than 50,000 microGy (5 rad) has not been associated with a significant risk of fetal injury Therefore, besides sonography, routine diagnostic procedures should be performed, if clinically necessary. Heparin does not cross the placenta and is therefore the anticoagulant of choice. In case of acute thrombosis during pregnancy, treatment is performed like in nonpregnant patients. There is ongoing debate, whether or not pregnant women with previous venous thrombosis should routinely receive prophylactic anticoagulation. In patients who have hereditary antithrombin deficiency, antiphospholipid antibodies, a combined abnormality or a history of a severe thrombotic event (pulmonary embolism, extended deep vein thrombosis) should be advised to use prophylactic heparin during pregnancy, starting during the first trimester. Post partum prophylaxis should be given in all women with an increased risk for VTE.

The prevalence of protein C, protein S, and antithrombin III deficiency in non-APS/SLE Chinese adults with noncardiac cerebral ischemia.

Protein C (PC), protein S (PS), and antithrombin III (AT-III) are vital thrombin antagonists in circulation. However, the prevalence of these natural inhibitors for cerebral ischemia is barely mentioned in the Chinese population. The prevalence of PC, PS, and AT-III deficiency in Chinese adults with cerebral ischemia is reported. The study subjects were free of antiphospholipid antibody syndrome or systemic lupus erythematosus. Cardiac, liver, and renal function were normal. An overall rate of thrombophilia was 27%. PS deficiency was the most common disorder, followed by PC with PS and PC deficiency. There was only one patient with AT-III deficiency. No gender was specific for thrombophilia. However, PS deficiency was predilected in young adults. A positive correlation between PC and AT-III was achieved in patients with a normal PC activity but not PC deficiency. There was no correlation between AT-III or PS. The odds ratios of PC and PS were 5.29 and 2.86, respectively. Accordingly, an inability for thrombin antagonization by the PC/PS axis may relate to the occurrence of cerebral ischemia in the Chinese population. AT-III seems to display a minor role only.

Defective Sorting to Secretory Vesicles in Trans-Golgi Network Is Partly Responsible for Protein C Deficiency

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Incidence of thrombophilia detected in southern Taiwanese patients with venous thrombosis.

In order to analyze the incidence of thrombophilia in southern Taiwan, we studied the prevalence of antithrombin (AT), protein C (PC), and protein S (PS) deficiencies, the prevalence of factor V Leiden mutation, and the presence of acquired lupus anticoagulant (LA) and anticardiolipin antibody (ACA) in 56 patients < or =65 years old with deep venous thrombosis (DVT). Of 56 patients, 30 were male, 26 female, and the mean age of the patients was 43 years (18-65 years). None had factor V mutation or activated PC resistance; 21 patients (37.5%) showed abnormal results: 4 (7.1%) had AT deficiencies, 6 (10.7%) PC deficiencies, 6 (10.7%) PS deficiencies, 2 (3.6%) a combined PC and PS deficiency, and 3 (5.4%) LA and ACA. Only PC and PS deficiencies were significantly associated with increased risk for the development of thrombosis with an odds ratio of 4.2 (95% confidence interval: 1.2-15.0, P=0.018) and 8.1 (95% confidence interval: 1.6-40.6, P=0.003), respectively. We concluded that the prevalence of heritable thrombophilia (34.0%) in Taiwan is higher than that in Western countries, but that it is lower than previously reported in Hong Kong and Taiwan. We attribute this to selection bias.

Sensing improvement of protein C biosensor by sample circulation.

Protein C (PC) is one of the most important proteins involved in the blood hemostasis. It is a potent anticoagulant, antithrombotic, and anti-inflammatory agent. Individuals with heterozygous PC deficiency have, by the age of 40, an 8–10 times higher risk of the thrombotic complications1. Accordingly, PC deficiency may lead to problems of oxygen and nutrient transport to tissue. Therefore, early diagnosis of PC deficiency is crucial to prevent thrombo-embolic episodes, such as, lung embolism, stroke, and/or heart attack.

Development of an economic miniantibody production process for the purification of Protein C (anti-coagulant/anti-thrombotic).

Protein C (PC) is a vital anticoagulant and antithrombotic in the blood coagulation cascade. Because PC is activated only when it is needed, it does not cause internal bleeding complications as does heparin or coumadin. PC deficiency can lead to blood clot formation in blood vessels, which can eventually lead to heart attack, stroke, skin necrosis, or even death. About 1,400 people worldwide currently die each day of septic shock and recently, PC has also been proven highly effective for septic shock treatment. Therefore, an inexpensive supply of PC is important for PC deficient patients, sepsis patients, and those undergoing major surgeries.

Preliminary study for the protein C purification using mini-antibodies produced from recombinant E. coli.

Protein C (PC) is an anticoagulant, antithrombotic, and anti-inflammatory protein in the blood. PC deficiency can lead to blood clots and these clots can prevent the nutrient and oxygen from being transported to tissues. In addition, blood clots detached from the origin can lead to pulmonary embolism, heart attack, and stroke. Purified PC has valuable therapeutic functions for PC-deficient patients, sepsis patients1, and patients undergoing major surgeries2,3. The current purification method for PC is expensive due to the high cost of monoclonal antibodies (by animal cell culture) used as a ligand in immunoaffinity chromatography separation3. In an attempt to lower the PC purification cost, a cheaper ligand, the single chain variable fragment (ScFv; mini-Ab) produced by recombinant E. coli is currently being studied.

Inherited thrombophilia, pregnancy, and oral contraceptive use: clinical implications.

Inherited thrombophilia has been reported to be associated with an increased risk for complications of pregnancy, including venous thromboembolism (VTE) as well as preeclampsia (PEC), fetal loss (FL), fetal growth retardation (FGR), and abruptio placentae (AP), the latter probably due to inadequate placental perfusion. The estimate of risk largely depends on the kind of thrombophilia and on the criteria applied for the selection of the patients, producing in some cases contradictory results. Convincing evidence is available that deficiency of antithrombin III (AT), protein C (PC), and protein S (PS) is a risk factor for VTE and late FL. Factor V (Leiden) is associated with an increased risk for VTE, unexplained recurrent FL, late FL, and perhaps PEC; prothrombin G20210A is a weak risk factor for VTE. So far, the data available for FGR and AP are scarce. However, the absolute risk for VTE during pregnancy and puerperium is between 1 and 3%, in comparison with the baseline risk of 0.08%. Antithrombotic prophylaxis with subcutaneous heparin is warranted during puerperium in all women with thrombophilia and throughout all pregnancy in women at higher risk (AT deficiency, homozygosity for factor VLeiden, and perhaps PC and PS deficiencies); treatment with subcutaneous heparin for prevention of FL among women with thombophilia is under investigation. Presence of inherited thrombophilia increases the risk for VTE due to oral contraceptives up to an absolute risk of 3 per 1000 person-years, in comparison with the baseline risk of 3 to 6 per 10000 person-years; the risk is further increased by first usage, the use of preparations containing third-generation progestins, and thrombophilia due to AT, PC, and PS deficiency as well as homozygous factor V (Leiden) and combined defects.

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.

Molecular biological basis and diagnosis of hereditary defect of antithrombin III, protein c and protein S

Recent progress in molecular biology enabled the elucidation of the nucleotide sequences of the genes for antithrombin III (AT III), protein C (PROC) and protein S (PROS). Furthermore numerous mutations were identified causing genetic defects of the important inhibitors of blood coagulation. As the genes for AT III (13.8 kb) and PROC (11.2 kb) are small and easy to analyze a great number of molecular defects already are described in extensive databases (50, 73): 79 different mutations for AT III and 160 for PROC are included. The identification of mutations leading to AT III and PROC deficiency has given important information on the structure-function relationships of the proteins. In case of protein C deficiency the clinical relevance of DNA analyses is most important because the diagnosis at the protein level is often uncertain. The gene for PROS is not so easy to analyze like the other two genes. The PROS gene is large and also a PROS pseudogene exists. Although a number of mutations have been identified, there has not been published a database until now. The clinical relevance to identify gene defects in PROS deficiency is as important as for PROC deficiency. Presumably the elucidation of PROS gene defects will advance in the near future.

Sensing performance of protein C immuno-biosensor for biological samples and sensor minimization

Protein C (PC) is an important anticoagulant and antithrombotic agent in human blood plasma. PC deficiency can result in clotting complications that interfere with oxygen and nutrient transport. A fiber-optic biosensor is being developed to provide real time diagnosis of PC deficiency. The PC sensor was tested to quantify PC level in human plasma. The signal intensity obtained from the plasma sample was 30% of the buffered sample, possibly due to the increased viscosity. The feasibility of monitoring PC level in animal cell culture broth and animal milk was tested. For the cell culture broth, 80% of signal was observed. However, the decrease was consistent over the sensing range. For whole and 1:100 diluted bovine milk, the signals were 60 and 78% of buffered sample, respectively. The biosensor length was reduced from 12.5 to 6 cm with sufficient sensitivity. To increase the sensor reusability, various elution buffers were applied after each sensing. Triethylamine elution buffer provided the best sensor regeneration capability and increased the number of assays from 2.5 to 7 times for 6 cm fibers.

Chelator, metal ion and buffer studies for protein C separation

Protein C (PC) is the pivotal anticoagulant and antithrombotic in the human coagulation cascade. PC deficiency can result in major medical problems such as deep vein thrombosis (DVT), leading to tissue oxygen deprivation. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. One source of PC is transgenic animal milk. The major components in the milk, such as α-casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin, are proteins that must be separated from PC. Immobilized metal affinity chromatography (IMAC) is an inexpensive separation technology with relatively high specificity, and it has great potential for difficult protein separations. After systematic studies of different chelator, metal ion and buffers, the combination of iminodiacetic acid (IDA) and Fe was found to be effective to separate PC from major milk components. α-Lactalbumin and β-lactoglobulin fell through the column in the starting buffer. PC was eluted. α-Casein, β-casein, κ-casein remained bound in the column after PC elution. This technology might be applied for PC separation from transgenic animal milk. It is very important for PC production in large quantities and at low cost to treat PC-deficient patients.