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We evaluated free plasma levels of protein S, a natural anticoagulant factor, the prevalence of anti-protein S antibodies, a possible cause of protein S deficiency, and their correlation with anti-phospholipid antibodies in 53 patients with inflammatory bowel disease (IBD) and 53 age- and sex-matched controls. Mean free plasma protein S levels (+/- SD) were significantly lower in IBD patients (0.98+/-0.32 IU/ml) than in controls (1.06+/-0.28 IU/ml) (P < 0.05); only one patient showed protein S deficiency. Specific antibodies to protein S were found in four IBD patients (7.5%) and in one control (1.9%) (P = NS). Five IBD patients (9.4%) and none of the controls showed anti-phospholipid antibodies (P < 0.06). No correlation was found between free protein S levels and anti-protein S antibodies or between anti-protein S and anti-phospholipid antibodies. In conclusion, free plasma protein S levels are slightly but significantly decreased in IBD patients. The prevalence of anti-protein S and antiphospholipid antibodies is increased in IBD patients. Anti-protein S antibodies do not appear to determine low protein S levels or to overlap with or belong to anti-phospholipid antibodies. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.

The composition of atherosclerotic plaques is a crucial factor in determining rupture, thrombosis and clinical events. In this study, we analyzed gene expression in coronary plaques from patients with stable or unstable angina using gene arrays. Total RNA was extracted from eight plaques collected by therapeutic directional coronary atherectomy. cDNA probes, generated by amplification, were hybridized to nylon arrays containing 482 genes. Here we report the results for the inflammation, adhesion and hemostasis subsets. Many genes not previously associated with atherosclerosis, such as the lymphocyte adhesion molecule MadCAM, were expressed in the plaques. anova analysis showed higher tissue factor (TF) expression in unstable angina samples. Five genes were expressed at lower levels in unstable angina samples: anticoagulant protein S, cyclooxygenase (COX)-1, interleukin (IL)-7 and chemokines monocyte chemotactic protein (MCP)-1 and -2. Gene arrays provide a new approach to study plaque composition and identify candidate markers of plaque instability.

Background: Coagulation Factor XIII is implicated in fibrin stabilization and wound healing. Plasma levels of Factor XIII are reduced in inflammatory bowel disease patients; recently, a valine 34 to leucine polymorphism of the Factor XIII-A subunit gene with a defined protective effect against thrombosis and as yet undetermined effect on wound healing has been described., Aim: To evaluate Val34Leu Factor XIII polymorphism distribution and to find possible correlations with clinical features in Italian inflammatory bowel disease patients., Study Population: A total of 152 inflammatory bowel disease patients, 90 with ulcerative colitis and 62 with Crohn's disease and 130 healthy volunteers were studied., Methods: Val34Leu polymorphism was detected by RFLP with BsaH I. Statistical analysis was performed by means of Fisher exact test., Results: In inflammatory bowel disease, 57.2% of patients showed the wild type status, 37.5% were heterozygous and 5.3% were homozygous for the 34Leu allele; the frequency of the mutated allele was 24.0%. In controls, 66.1% of subjects showed the wild type status, 28.5% were heterozygous and 5.4% were homozygous for the 34Leu allele; the frequency of the mutated allele was 19.7%. There was no difference in genotype distribution and prevalence of the mutated allele between inflammatory bowel disease patients and controls., Conclusions: The present data do not show any differences in Val34Leu Factor XIII polymorphism distribution between inflammatory bowel disease patients and controls. The prothrombotic state described in inflammatory bowel disease patients does not depend on an altered distribution of Val34Leu Factor XIII polymorphism.

EPCR is a type I transmembrane protein, highly expressed on the endothelium of large vessels, that binds protein C and augments its activation. In this study, a 23bp insertion in the EPCR gene was found in 4/198 survivors of myocardial infarction and 3/194 patients with deep vein thrombosis. The EPCR gene with the insertion predicts a protein that lacks part of the extracellular domain, the transmembrane domain and the cytoplasmic tail. Expression studies showed that the truncated protein is not localized on the cell surface, cannot be secreted in the culture medium, and does not bind activated protein C. Since protein C activation depends on the concentration of EPCR, patients with the EPCR insertion could have a diminished protein C activation capacity. Further clinical studies of adequate samples size are necessary to establish whether or not the EPCR insertion predisposes to the development of thrombotic events.

Late fetal loss can be associated with placental insufficiency and coagulation defects. Thrombomodulin (TM) and the endothelial protein C receptor (EPCR) are glycoprotein receptors expressed mainly on the endothelial surface of blood vessels and also in the placenta; they both play a key physiological role in the protein C anticoagulant pathway. Defects in these proteins might play an important role in the pathogenesis of late fetal loss. We performed a case-control study in 95 women with unexplained late fetal loss (> 20 weeks), to elucidate whether TM or EPCR gene mutations were associated with an increased risk for this complication of pregnancy. The control group comprised 236 women who gave birth to at least one healthy baby and had no history of late fetal death or obstetrical complications. The entire TM and EPCR genes, including the promoter region, were screened. In total, five mutations were identified in the TM gene in 95 patients and three in 236 control subjects, and two mutations were identified in the EPCR gene in 95 patients and one in 236 control subjects. The relative risk for late fetal loss when having a mutation in the TM or EPCR gene was estimated by an odds ratio of 4.0 (95% CI 1.1-14.9). In conclusion, identified mutations in the TM and EPCR genes of women with unexplained fetal loss are more prevalent compared with women with no obstetrical complications.

Two mutations in exons 3 and 9 of the protein C gene were identified by amplification and sequencing from symptomatic probands referred for venous thromboembolism and thrombophilia screening. The phenotype associated with the mutations is a type II protein C deficiency with normal amidolytic activity. In one family, the mutation in exon 3 (G3545-->A), which predicts an R9 to H substitution in the Gla domain, was identified. A mutation in exon 9 (G10899-->A), which predicts an R352 to W substitution in the catalytic site, was identified in the second family and has been reported previously in association with type II deficiency with low amidolytic activity. Western blotting of the purified proteins from the probands' plasma did not show any abnormal migratory pattern. Molecular modelling suggested a possible impairment in the recently described Na+ binding pocket for the R352-->W mutant. No conclusions could be drawn relative to the R9-->H mutant.

By single strand conformational polymorphism, nucleotide sequencing and enzyme restriction, we analyzed the protein S alpha gene in 17 protein S-deficient probands and in their available family members. The relationship between genotype and phenotype was also evaluated. Twelve different sequence variations were identified in 17 probands. Ten were putative causal mutations distributed in 16 probands: 4 were nonsense, 5 missense and one a splice site mutation. In most families in which a mutation was identified, more than one phenotype of PS deficiency was present. The same splice site mutation (intron j G-A, exon 10+5) was associated with type I deficiency in one family and with type I/III in another unrelated family. A phenotypic discrepancy was also observed for the Arg474Pro, Gly597Asp and Arg410stop mutations. Glu26Ala, previously reported in kindreds with type I deficiencies, was found in association with I, II and III phenotypes in four unrelated kindreds. Phenotypic analysis of protein S deficiency is poorly related to the underlying genetic defect.

With the aim of establishing whether the HR2 haplotype in factor V affects the risk of venous thromboembolism, a retrospective multicenter cohort study was performed in 810 family members identified through 174 probands who suffered from at least 1 episode of deep vein thrombosis and/or pulmonary embolism and had an inherited defect associated with thrombophilia (antithrombin, protein C, or protein S deficiency; factor V R506Q or prothrombin G20210A). Fifty-eight percent (468/810) of the family members had an inherited defect and 10% (47/468) were symptomatic. The HR2 haplotype was found in association with factor V R506Q more frequently in family members with venous thromboembolism (18%) than in those without (8%). Double heterozygosity for factor V R506Q and HR2 conferred a 3- to 4-fold increase in the relative risk of venous thromboembolism compared with factor V R506Q alone. The median age at first event was lower when the 2 defects were associated (46 v 52 years). No increase in risk of venous thromboembolism could be demonstrated when the HR2 haplotype was associated with inherited thrombophilic defects other than factor V R506Q. Because both factor V R506Q and the HR2 haplotype are very frequent, the effect of their coinheritance on the risk of venous thromboembolism might represent a clinically relevant issue, and screening for HR2 in carriers of factor V R506Q should be considered.

Deficiency of the naturally occurring anticoagulant proteins, such as antithrombin, protein C and protein S, and activated protein C resistance due to the factor V Leiden gene mutation is associated with inherited thrombophilia. So far, no direct comparison of the thrombotic risk associated with these genetic defects is available. In this study, we wish to compare the lifetime probability of developing thrombosis, the type of thrombotic symptoms, and the role of circumstantial triggering factors in 723 first- and second-degree relatives of 150 index patients with different thrombophilic defects. We found higher risks for thrombosis for subjects with antithrombin (risk ratio 8.1, 95% confidence interval [CI], 3.4 to 19.6), protein C (7.3, 95% CI, 2.9 to 18.4) or protein S deficiency (8.5, 95% CI, 3. 5 to 20.8), and factor V Leiden (2.2, 95% CI, 1.1 to 4.7) than for individuals with normal coagulation. The risk of thrombosis for subjects with factor V Leiden was lower than that for those with all three other coagulation defects (0.3, 95% CI, 0.1 to 1.6), even when arterial and superficial vein thromboses were excluded and the analysis was restricted to deep vein thrombosis (0.3, 95% CI, 0.2 to 0.5). No association between coagulation defects and arterial thrombosis was found. The most frequent venous thrombotic manifestation was deep vein thrombosis with or without pulmonary embolism (90% in antithrombin, 88% in protein C, 100% in protein S deficiency, and 57% in factor V Leiden), but a relatively mild manifestation such as superficial vein thrombosis was common in factor V Leiden (43%). There was a predisposing factor at the time of venous thromboembolism in approximately 50% of cases for each of the four defects. In conclusion, factor V Leiden is associated with a relatively small risk of thrombosis, lower than that for antithrombin, protein C, or protein S deficiency. In addition, individuals with factor V Leiden develop less severe thrombotic manifestations, such as superficial vein thrombosis.

Levels of free activated protein C are a measure of the activation of the protein C pathway in vivo. The aim of this study was to establish if the protein C pathway is triggered in familial thrombophilia and if activated protein C levels correlate with type of defect or symptoms. We measured activated protein C in 133 patients with a deficiency of antithrombin (n = 31), protein C (n = 24) or protein S (n = 27) or with resistance to activated protein C (n = 51). Levels of activated protein C were evaluated also in 97 healthy individuals. Results indicate that the levels of activated protein C are higher in patients who have experienced a thrombotic event than in patients who have not and that 71% of patients with levels of activated protein C above the normal reference range had had a venous thromboembolic event. We conclude that the protein C pathway is triggered in patients with thrombophilia and that in symptomatic patients, activated protein C levels are increased and may reflect heightened coagulation activation and scavenging through the protein C pathway.

A recent study suggests that protein S deficiency is not a risk factor for venous thrombosis. Since this unexpected finding would have important clinical implications if confirmed, we performed a case-control study with the aim to determine the prevalence of protein S deficiency in patients with thrombosis and in healthy individuals taken from the general population and the relative risk of thrombosis in protein S-deficient patients. Free protein S concentration was measured in 327 consecutive patients with at least one venous thrombotic episode and in 317 age- and sex-matched control individuals. Different normal reference ranges were obtained and adopted for men and women. Protein S deficiency was found in 3.1% (95% CI: 1.5-5.2) of patients and in 1.3% of controls (95% CI: 0.3-2.8). Ten patients and 4 control subjects had protein S deficiency, which determined a relative risk of thrombosis (sex- and age-adjusted odds ratio) of 2.4 (95% CI: 0.8-7.9). When men and women were analyzed separately, the risk was 5.0 (95% CI: 0.6-43.6) and 1.6 (95% CI: 0.4-6.7) respectively. PS-deficient men had more thrombotic episodes than women and later in life. Multivariate analysis established that sex was an independent determinant of the number of episodes, as was age, while PS deficiency was not. However sex and PS deficiency status were both determinants of age at first thrombotic episode.

Factor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.

Four hundred and ninety-three consecutive patients referred for arterial or venous thrombosis were screened for congenital and acquired abnormalities of blood coagulation predisposing to thrombosis, and were compared to 341 age- and sex-matched controls. The aim of the study was to determine the prevalence and clinical characteristics of resistance to activated protein C (APC), a defect shown to have different prevalences in different ethnic groups and to be associated with an increased risk of thrombosis. Seventy-three (15%) patients had both APC resistance and the 1691 G to A factor V gene mutation, compared to 6/341 (2%) controls. Seven patients had antithrombin deficiency (1.4%), 11 had protein C deficiency (2.2%), and 4 had protein S deficiency (0.8%). The relative risk of thrombosis in APC-resistant patients was 9.4. Resistance to APC was associated mainly with venous thrombosis, the most frequent being deep-vein thrombosis of the lower limbs. Fifty-eight percent of APC-resistant patients had an associated risk factor at the first thrombotic event: pregnancy and oral contraceptive intake were associated with the first thrombotic episode in 35% and 30% of women, respectively. APC resistance is the most frequent defect of blood coagulation in the general population and in the unselected thrombotic population studied by us.

Venocclusive disease (VOD) of the liver is the major dose-limiting complication of pretransplant regimens for bone marrow transplantation. Recent reports from different groups point to the involvement of the hemostatic mechanism in the development of VOD. We measured the naturally occurring anticoagulants protein C, antithrombin III, and protein S in 45 patients undergoing bone marrow transplantation for hematologic malignancies before cytoreductive therapy and after transplant. The aim of this prospective study was both to evaluate the status of the naturally occurring anticoagulant pathway in patients who develop VOD compared with patients who do not, and to find a predictive marker of VOD. In transplant patients, protein C decreased from before cytoreductive therapy to posttransplant, whereas protein S and antithrombin III did not. In a multivariate analysis, protein C was the only variable that could independently discriminate between VOD and non-VOD patients at all times. Discriminant function analysis established that low protein C levels before cytoreductive therapy predicted the occurrence of VOD with good sensitivity and specificity.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.

Twenty-nine of 54 uremic patients had low levels of protein C measured as anticoagulant activity, contrasting with normal levels measured as amidolytic activity or antigenic concentration. We demonstrate that this discrepancy is due to the presence of a soluble plasma inhibitor that interferes specifically with the anticoagulant activity of activated protein C. The inhibitor does not interfere with other coagulation assays. It is resistant to diisopropylfluorophosphate, high temperatures and repeated freezing and thawing. It can be dissociated from protein C by anti-protein C antibodies or by dialysis in vitro and in vivo. It binds to positively charged resins and can be eluted with high salt concentrations without losing its inhibitory capacity. The inhibitory effect is correlated with plasma creatinine levels and fluctuates with time.

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin-thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.