Genetic polymorphism and risk of thromboses

We have attempted to establish a systematic pathogenetic analysis of thrombophilia by including assays of antithrombin III(AT III), protein C(PC), protein S(PS), fibrinogen, plasminogen and heparin cofactor II by both functional and immunological methods as well as detecting lupus anticoagulants. Such a comprehensive scheme was instrumental in systematically identifying and confirming the pathogenesis of 164 cases which otherwise would have escaped detection since 1994 in our laboratory (Kyushu University Hospital). The analysis was conducted on 485 consecutive patients with venous thrombosis, arterial thrombosis and disorders in which small vessel thrombosis were implicated. Hundred and sixty four patients, (40% of the examined patients), were found to have low activities of PS, PC, ATIII etc. Among them, seventy five patients(46%) had low PS activity, and twenty nine(18%) had low PC activity. Genetic analyses performed on specimens with low PS/PC activities resulted in the confirmation of 24 genetic abnormalities. Such genetic abnormalities, however, does not solely lead to the pathogenesis of thromboses. We have found that some genetic polymorphisms, such as PS Tokushima, factor XII 46C allele, were also additional risk factors for thromboses.     

Venous thrombosis: prevalence of prothrombotic defects in north Indian population

431 patients with thrombosis of different venous system were evaluated for underlying acquired and inherited prothrombotic states. Associated acquired risk factors were observed to be present in 28.7% patients and possible inherited in 32.3%, in the rest, no cause could be identified. Major acquired risk factors included coexistence of liver disease (12.2%), oral contraceptives (4.1%), puerperium (2.5%), malignancy (2.3%) and lupus anticoagulant (2%). Low levels of protein C were detected in 21.1% and of which 11.3% were attributed to acquired factors. Protein S deficiency was found in 19.0% and of these 10.4% cases were associated with acquired risk factors. Antithrombin III (AT III) deficiency was detected in 6.4% of patients, of which 4.8% were secondary to acquired factors. In the rest, deficiency of protein C, protein S and AT III were attributed to inherited factors as no associated acquired risk factor was present. Activated protein C resistance (APC-R) was present in 12.5% cases.     

D-D和抗凝蛋白检测在下肢深静脉血栓形成患者诊治中的意义

目的 研究下肢深静脉血栓形成（LDVT）的主要发病机制.方法 应用日本Sysmex CA7000型全自动血液凝固仪检测55例LDVT患者（38例初发,17例复发）和60例健康人的血浆D-二聚体（D-dimer,D-D）、抗凝血酶（AT）、蛋白S（PS）,蛋白C（PC）的活性水平.结果 LDVT组与正常对照组相比、LDVT复发组与初发组相比,AT、PS、PC活性明显降低,D-D水平明显升高,均有极显著差异（P〈0.01）.结论 D-D水平升高和先天性或获得性抗凝蛋白缺陷是LDVT发病和复发的重要原因.因此有必要对LDVT患者进行D-D水平和抗凝蛋白水平的筛选.     

PROS1 analysis in 87 pedigrees with hereditary protein S deficiency demonstrates striking genotype-phenotype associations

Hereditary protein S (PS) deficiency predisposes to venous thrombosis. Previously, we demonstrated a difference in risk of venous thrombosis between PS deficiency type I and type III. We used direct sequencing, multiplex ligation-dependent probe amplification (MLPA), and linkage analysis to study whether this difference could be explained by molecular heterogeneity. The study contained two sets of families with PS deficiency type I (cohort 1; 35 probands, 155 relatives) or type III (cohort 2; 52 probands, 241 relatives). In cohort 1, a mixed type I/type III PS-deficient phenotype was observed in 66% of the pedigrees. A total of 34 probands carried a mutant PROS1 allele, compared to one proband in cohort 2 (P<10(-10)). The proband's mutation was identified in all type I, but only in 57% of type III PS deficient relatives. MLPA-analysis in the mutation negative families did not reveal PROS1 deletions or insertions. Linkage analysis in 16 families showed cosegregation of PROS1 markers in the family with type I deficiency, but not in the 15 families with type III deficiency. The genotype-phenotype associations point to differences in genetic architecture. Whereas PS deficiency type I is a monogenic disease due to PROS1 allelic heterozygosity, PS deficiency type III is most likely a more complex or heterogeneous disorder.     

Low prevalence of activated protein C resistance and coagulation factor V Arg506 to Gln mutation among Korean patients with deep vein thrombosis.

Activated protein C (APC) is a naturally occurring anticoagulant that interacts with factor V and VIII to inhibit the clotting cascade. The prevalence of APC resistance among Korean patients with deep vein thrombosis is ill defined. The aim of the present study was to investigate the prevalence of APC resistance and factor V Leiden mutation in Korean patients with deep vein thrombosis. The presence of factor V Leiden mutation was determined in 49 patients who visited Asan Medical Center. APC ratio was performed in 33 individuals from the above 49 patients. Three patients were excluded from the analysis because their baseline aPTT was prolonged. Resistance to APC was diagnosed when the APC ratio was below 2.55. APC resistance was documented in 8 individuals, representing 27% (8/30) of the patients on whom APC resistance test was performed. The 2 patients, who showed APC resistance, were positive for lupus anticoagulant. None of the 49 patients demonstrated factor V Leiden mutation. These findings indicate that factor V Leiden mutation is rare and APC resistance is less prevalent in Korean patients with deep vein thrombosis than in Caucasians. APC resistance not caused by factor V Leiden mutation may be a risk factor for deep vein thrombosis in this population.     

Hemodialysis with defibrotide: effects on coagulation parameters.

In a crossover study conducted with eight uremic patients maintained on hemodialysis, the Authors compared the effects of heparin (100 IU/kg at the start of dialysis) and defibrotide (400 mg at the start, repeated at 2 hours of ongoing dialysis) on the parameters of blood coagulation (VIII:C, AT III, TAT, PC antigen and activity, PS, and FPA), each being assessed before dialysis and at 2, 3 and 4 hours of the ongoing procedure. Heparin-assisted dialysis resulted in a significant rise of VIII:C and AT III; with defibrotide, instead, there was evidence of thrombin activation (increased FPA and TAT). PC levels were raised with both dialysis modalities; however, PC activity and PS levels were increased only in defibrotide-assisted dialysis. There were no adverse reactions or evidence of fibrin formation. These results confirm the antithrombotic activity of defibrotide in the course of dialysis and indicate that this action is independent of thrombin neutralization.     

Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion.

The objective of this study was to establish whether or not patients with unexplained recurrent abortion have an increased incidence of haemostatic or metabolic abnormalities. Fifty-two patients with a history of unexplained habitual abortion (two or more spontaneous abortions before 16 weeks' gestation) were tested for protein S, protein C and antithrombin (AT) III deficiency, activated protein C (aPC) resistance, hyperhomocysteinaemia and anticardiolipin antibodies (ACA). The control group consisted of 67 healthy women with a history of only uncomplicated pregnancies. Blood samples were taken for measuring protein S, protein C, AT III, ACA and activated protein C resistance and a methionine loading test was performed. Of the 46 patients tested for protein S deficiency, 8 (17.4%) were positive. Of the 43 patients tested, two (4.7%) were protein C deficient and none was AT III deficient. Of the 42 patients tested for ACA, eight (19.1%) had detectable antibodies. Of the 44 patients tested for aPC resistance, two (4.6%) were positive. Finally, 35 patients were tested for hyperhomocysteinaemia and six (17.1%) were positive. It was concluded that parous women with a history of unexplained recurrent abortion have an increased incidence of hyperhomocysteinaemia and a trend of increased incidence of ACA can be found.     

A heparin binding site Arg79Cys missense mutation in the SERPINC1 gene in a Korean patient with hereditary antithrombin deficiency

We describe a case of heparin binding site Arg79Cys mutation in the gene encoding antithrombin, SERPINC1, in a Korean patient with hereditary antithrombin (AT) deficiency. The patient was a 34-year-old Korean man who presented with deep vein thrombosis (DVT) in his right leg without precipitating factors. On outpatient evaluation, coagulation tests without anticoagulation revealed a decreased AT III activity level at 48%, but normal AT III antigen level at 103%, indicating type II AT deficiency. Family studies revealed that his father (62 years of age) had decreased AT activity (48%) but had normal AT antigen levels (116%), indicating that the proband had a paternally inherited type II AT deficiency. Direct sequencing of the SERPINC1 gene in the patient and his father revealed a heterozygotic missense mutation, a cytosine to thymine substitution at nucleotide position 235 in exon 2 of the SERPINC1 gene (p.Arg79Cys). To our knowledge, this is the first report of Arg79Cys heterozygote mutation in family members with venous thromboembolism.     

Alteration of some natural anticoagulants in dogs with chronic renal failure

The diagnosis of hypercoagulation is essential for the identification of individuals at high risk for thrombosis and for early treatment of thrombotic disorder. The objective of the study was to evaluate some parameters for assessing the prothrombotic state in dogs with chronic renal failure (CRF). Some natural anticoagulants, protein C (PC), protein S (PS), and antithrombin III (AT III), as well as fibrinogen concentration and clinical chemistries, were concentrated. The study groups consisted of 42 dogs with CRF and 34 age- and sex-matched clinically healthy control dogs. The level of AT III in the CRF group was significantly lower (P < 0.05), but the fibrinogen concentration was significantly higher (P < 0.05) than in the control group. Additionally, the cholesterol level in the CRF group was significantly higher than in the control group (P < 0.05) and was positively correlated to creatinine (R = 0.5, P < 0.05). Elevated levels of PC and PS were exhibited in eight dogs with subcutaneous edema. The increased levels of PC and PS may counterbalance the reduction of AT III and may be related to the magnitude of hypoalbuminemia and proteinuria. These seem to be preventive mechanisms against thromboembolic phenomena. Simple correlations among parameters were determined for the CRF group. The fibrinogen concentration was correlated inversely with the AT III level (R = −0.63, P < 0.05). A negative correlation between AT III and azotemic parameters (creatinine: R = −0.68, P < 0.05; blood urea nitrogen (BUN): R = −0.65, P < 0.05) was observed also. In contrast, the fibrinogen concentration was positively correlated to creatinine (R = 0.66, P < 0.05) and BUN (R = 0.67, P < 0.05). The study concluded that there was a significant reduction in AT III and hyperfibrinogenemia, which were predictable parameters for thrombotic tendency in the dogs with CRF. Hypercholesterolemia was the other risk factor.     

Inherited abnormalities in the protein C activation pathway

The protein C (PC) anticoagulant pathway plays a crucial role in the regulation of fibrin formation via proteolytic degradation of the procoagulant cofactors factor Va and VIIIa by activated PC (APC). PC circulates in plasma as a zymogen, which is activated, on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another endothelial cell-specific protein, the endothelial cell PC/APC receptor (EPCR), binds PC on the endothelial cell surface and further enhances the rate of PC activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of at least four proteins: thrombin, thrombomodulin (TM), PC and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced APC generation and an increase in the risk of thrombosis. In the last years, several reports have suggested the association between mutations in TM and EPCR genes and venous and arterial thrombosis. Surprisingly, no studies have been reported linking mutations with levels of circulating APC, the final product of the interaction between thrombin, TM, PC and EPCR. Here, we describe the previously reported mutations in the TM and EPCR genes, and present the design and evaluation of a new strategy to investigate TM, EPCR, PC and prothrombin gene mutations in arterial and venous thrombosis.     

Blood coagulation and fibrinolysis in patients with Beh?et's disease

In patients with Beh?et's disease, venous thrombosis has often been described as a complication. The pathogenesis of this complication, however, has not been fully understood. In this work, various parameters of blood coagulation and fibrinolysis were studied in 20 patients with Beh?et's disease and 13 sex-matched healthy volunteers. Patients were classified into three subgroups according to the number of clinical signs involved; group I (no sign): 4 patients; group II (one or two signs): 11 patients; group III (more than three signs): 5 patients. Patients with Beh?et's disease, showed an activation of blood coagulation, such as the shortening of prothrombin time (p less than 0.001), decreases in concentrations and activities of plasma antithrombin III (AT-III) (p less than 0.01) and elevated levels of plasma thrombin-antithrombin-III complex (TAT) (p less than 0.01), compared to the control group. Plasma levels (p less than 0.01) and activities (p less than 0.01) of protein C (PC) and total protein S (PS) levels (p less than 0.05) were increased in the patients. Decreased levels of alpha 2-plasmin inhibitor (p less than 0.001) also indicated an activation of fibrinolysis in the patients. When analyzed among the subgroups, patients belong to group II and III showed higher levels of plasma FDP D-dimer (p less than 0.05) and lower levels of plasminogen (p less than 0.05), as compared with patients in group I or control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mesenteric venous thrombosis and antithrombin III deficiency.

Of the 123 patients with acute mesenteric infarction treated over the past 12 years, 16 (13%) had mesenteric venous thrombosis. Eight of the patients with mesenteric venous thrombosis survived the initial episode; two have since died. The remaining six patients were studied for evidence of haemostatic deficiencies or abnormalities. Antithrombin III deficiency, which is known to be associated with recurrent venous thrombosis, was found in three patients. It is recommended that all patients with mesenteric venous thrombosis should be screened for antithrombin III deficiency as treatment with coumarin anticoagulants may be indicated, providing effective prophylaxis against further thrombotic episodes.     

Homozygous antithrombin type HBS deficiency; a family study

Congenital antithrombin (AT) deficiency is the most thrombotic genetic abnormality of haemostasis. Total quantitative deficits are lethal as early as life intra-uterine. Only homozygous mutations concerning the heparin-binding site are compatible with life. We report here the case of an 18 years old patient with recurrent deep venous thrombosis of the inferior members. Haemostasis exploration shows a decreased AT activity (11%) in the presence of heparin while AT progressive activity and AT antigen are normal. Two other homozygous sisters are identified in this family study. Molecular study of AT gene show Arg47-Cys substitution, already reported in the literature with patients of different geographic origins. Treatment of patients with homozygous AT type HBS deficiency is similar that for patients with heterozygous AT deficiency; a continuous prophylactic anticoagulant treatment is always necessary and AT concentrates infusions are required in all situations needing curative heparin treatment.     

Normal functional protein S activity does not exclude protein S deficiency

Protein S (PS) deficiency appears to increase the risk of venous thrombosis. PS deficiency is classified into three phenotypes using antigenic levels and functional activity. By definition, all three phenotypes of PS deficiency should result in low activated protein C cofactor activity. We compared the results of functional PS activity testing to free antigenic PS testing in order to determine if a normal functional PS activity assay result could eliminate the need for free antigenic PS testing. The sensitivity of the functional assay is 45.5% (95% confidence interval, CI, 36-55%), specificity 95.3% (95% CI 93-97%), negative predictive value 88.6% (95% CI 86-91%) with a positive predictive value of 68.5% (95% CI 57-79%). In conclusion, a normal functional PS activity result does not exclude free antigenic PS deficiency. Functional PS activity testing should not be used as a screening test to eliminate free antigenic PS testing for the laboratory diagnosis of PS deficiency.     

Screening for identified and unidentified protein C pathway defects by the Agkistrodon contortrix venom test in consecutive patients.

To determine the real performance of the Agkistrodon contortrix venom (ACV) screening test for protein C (PC) pathway defects, we studied 400 consecutive patients referred for the study of personal venous thrombosis or for family study. All 82 patients with factor V Arg 506 Gln (FV R506Q) (n = 82), 6 patients with activated PC resistance without FV R506Q, 17 patients with PC deficiencies, and 9 patients with combined defects were identified by an abnormal ACV result. Three of 6 protein S deficiencies overlapped the normal range. Among the 280 patients without a PC pathway defect, 63 of 193 with thrombosis and 18 of 87 asymptomatic subjects (relatives of patients with thrombosis) had an abnormal ACV result. A significant linear inverse relationship was observed between the ACV results and factor VIII. However, 31 of 63 patients (49%) with thrombosis and 15 of 18 (83%) asymptomatic subjects with an abnormal ACV had a normal factor VIII level. This test, with a 100% negative predictive value, is reliable for screening for PC deficiencies and for FV R506Q and can be used safely as an exclusion test. Moreover, the test may be useful to indicate, in the PC pathway, unidentified risk factors for venous thrombosis.     

Acquired activated protein C resistance in postmenopausal women is dependent on factor VIII:c levels.

Activated protein C (APC) resistance is an established risk factor for venous thromboembolism. In 5% to 10% of patients with venous thromboembolism, the APC resistance phenotype is observed in the absence of factor V Leiden mutation. Moreover, some physiologic and pathologic conditions are associated with an "acquired" APC resistance, not caused by the Leiden mutation, such as inflammatory diseases, pregnancy, or oral contraceptive therapy. Several studies have demonstrated the effect of menopause on the hemostatic system, but no data are available about APC resistance. We found a high prevalence of APC resistance in postmenopausal women, not associated with factor V Leiden mutation. The mechanism that underlies this acquired APC resistance may be related to the higher levels of factor VIII, which showed a strong inverse correlation with APC resistance, whereas no correlation was found between the normalized APC ratio, factor V levels, and protein S values. Higher levels of factor VIII correlated with a marker of coagulation activation, such as prothrombin fragments 1 plus 2. Therefore, to identify women receiving hormone replacement therapy who have a greater risk for deep venous thrombosis, the APC resistance coagulation test should be used instead of the genetic study.     

Frequent use of fresh frozen plasma is a risk factor for venous thrombosis in extremely low birth weight infants: a matched case-control study

Percutaneously inserted central catheters (PICCs) are often used in neonatal medicine. Venous thrombosis (VT) is one of the complications associated with PICC use. According to some reports, fresh frozen plasma (FFP) may be a risk factor for VT. The purpose of this study was to determine whether FFP use is associated with VT in extremely low birth weight infants (ELBWIs). We performed a matched case-control study on risk factors for VT in ELBWIs born over a period of 5 years in the neonatal intensive care unit of a tertiary hospital. Controls were infants from the unit matched for gestational age and birth weight. We performed univariate analyses and created receiver operating characteristic (ROC) curves for the cut-off values of continuous parameters such as FFP. We also conducted multivariate conditional logistic regression analysis and calculated adjusted odds ratios and their 95% confidence intervals. Thirteen VT cases and 34 matched controls were examined. Using an ROC curve, FFP by day 5 > 50 mL/kg was selected as the cut-off value. In multivariate conditional logistic regression analysis, FFP by day 5 > 50 mL/kg exhibited an adjusted odds ratio of 5.88 (95% confidence interval: 1.12-41.81, p = 0.036). FFP by day 5 > 50 mL/kg may be a risk factor for VT in ELBWIs.     

Novel mutation (E113X) of antithrombin III gene (AT3) in a woman with gestational recurrent thrombosis

A 35-year-old Japanese woman with a low level (42-54%) of blood antithrombin (AT) III, experienced two induced abortions due to deep venous thrombosis at 8 weeks of gestation (GW) and cerebral thrombosis at 10 GW. The present pregnancy was successfully managed with intravenous administration of AT III (6,000-8,000 U/wk). Analysis of polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) for exons 3A and 4 of the AT III gene (AT3) using her DNA revealed extra expansion bands with altered migration. The DNA sequencing demonstrated novel mutations in exon 3A of AT3: a G to T substitution at nucleotide position 5333 in codon GAG for Glu 113, causing a stop codon (E113X), and an A to T substitution at position 5338 in codon AAA for Lys 114, forming Asn (K114N). These novel mutations, especially E113X, in AT3 may be related to recurrent thrombosis in the pregnancy.     

The role of d-dimer as first marker of thrombophilia in women affected by sterility: implications in pathophysiology and diagnosis of thrombophilia induced sterility

Background  

D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by thrombosis. Yet, several studies showed that also infertility, in particular secondary infertility due to recurrent fetal losses, has been often related to thrombotic events, in particular in women carrying thrombotic risk factors such as inherited thrombophilia (MTHFR_C677T, PTHR_A20210G, Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia (primary antiphospholipid syndrome, acquired protein C, protein S, AT III deficiency, drugs induced thrombophilia). However, because its high predictive negative value in case of suspected thrombosis, increased d-dimer has been often associated to subclinical thrombophilia. The aim of this study is to investigate the role of d-dimer as first marker of thrombophilia in women affected by unexplained infertility and subsequently to search the cause of increased d-dimer, such as inherited and/or acquired thrombophilia.     

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C->G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C->T, 182G->C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.