INTRODUCTION:Aging and renal impairment may prolong the half-life and lead to accumulation of low molecular weight heparins. Correct dosing is critical to prevent bleeding or thrombosis.MATERIALS AND METHODS:Open, parallel study. Healthy adult [n=13] and elderly (>65yrs) [n=12] volunteers; and subjects with mild (ClCr≥50 to ≤80mL/min, n=8), moderate (ClCr≥30 to <50mL/min, n=7), and severe (ClCr<30mL/min, n=8) renal impairment received four prophylactic doses (3,500IU/24h) and a single therapeutic dose (115IU/kg) of bemiparin with an interim washout period. Anti-FXa activity and the potential need for dose adjustment were evaluated.RESULTS:There were statistically significant differences in the severe renal impairment group vs. adult volunteers in all anti-FXa related parameters, but no significant differences in any of the anti-FXa related parameters between the adult and the elderly. Anti-FXa simulations after 10 prophylactic doses predicted mean Amax=0.59IU/mL in subjects with severe renal impairment and 0.33-0.39IU/mL in the rest. Simulations in the severe renal impairment group with dose adjustment (2,500IU/24h) predicted all individual Amax<0.60IU/mL (mean Amax=0.42IU/ml). Simulations after 10 therapeutic doses predicted mean Amax=1.22IU/mL in severe renal impairment group and 0.89-0.98IU/mL in the rest. Simulations in the severe renal impairment group with 75% dose adjustment predicted individual Amax≤1.60IU/mL (mean Amax=0.91IU/mL).CONCLUSIONS:No dose adjustments are required in elderly with preserved renal function. A dose adjustment of bemiparin is only advisable in patients with severe renal impairment when using prophylactic or therapeutic doses.

Objective— Elevated plasma levels of coagulation factor XI (FXI) are implicated in the pathogenesis of venous thromboembolism and ischemic stroke, and polymorphisms in the F11 gene are associated both with risk of venous thromboembolism and an elevated plasma FXI level. Methods and Results— Here, we report the first hypothesis-free genome-wide genetic analysis of plasma FXI levels. Two genome-wide significant loci were detected in the family-based Genetic Analysis of Idiopathic Thrombophilia 1 cohort: one located in the kininogen 1 gene ( KNG1 ) (rs710446; P =7.98×10 −10 ) and one located in the structural F11 gene (rs4241824; P =1.16×10 −8 ). Both associations were replicated in a second population-based Swedish cohort. A significant effect on KNG1 mRNA expression was also seen for the 2 most robustly FXI-associated single nucleotide polymorphisms located in KNG1 . Furthermore, both KNG1 single nucleotide polymorphisms were associated with activated partial thromboplastin time, suggesting that FXI may be the main mechanistic pathway by which KNG1 and F11 influence activated partial thromboplastin time and risk of thrombosis. Conclusion— These findings contribute to the emerging molecular basis of venous thromboembolism and, more importantly, help in understanding the biological regulation of a phenotype that has proved to have promising therapeutic properties in relation to thrombosis.

The Protein C anticoagulant pathway regulates blood coagulation by preventing the inadequate formation of thrombi. It has two main plasma components: protein C and protein S. Individuals with protein C or protein S deficiency present a dramatically increased incidence of thromboembolic disorders. Here, we present the results of a genome-wide association study (GWAS) for protein C and protein S plasma levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. A total number of 397 individuals from 21 families were typed for 307,984 SNPs using the Infinium® 317 k Beadchip (Illumina). Protein C and protein S (free, functional and total) plasma levels were determined with biochemical assays for all participants. Association with phenotypes was investigated through variance component analysis. After correcting for multiple testing, two SNPs for protein C plasma levels (rs867186 and rs8119351) and another two for free protein S plasma levels (rs1413885 and rs1570868) remained significant on a genome-wide level, located in and around the PROCR and the DNAJC6 genomic regions respectively. No SNPs were significantly associated with functional or total protein S plasma levels, although rs1413885 from DNAJC6 showed suggestive association with the functional protein S phenotype, possibly indicating that this locus plays an important role in protein S metabolism. Our results provide evidence that PROCR and DNAJC6 might play a role in protein C and free protein S plasma levels in the population studied, warranting further investigation on the role of these loci in the etiology of venous thromboembolism and other thrombotic diseases.

INTRODUCTION:RO-14 is a novel ultra low molecular heparin. The purpose of this study was to evaluate the safety and pharmacodynamic profile of RO-14 in healthy males.MATERIALS AND METHODS:We conducted a two-stage, single-center, open-label, randomized study. Two cohorts of 6 volunteers were randomly assigned to 12 single, ascending subcutaneous doses (1750-19950IU of anti-FXa activity) in an alternating crossover fashion. Safety was assessed by spontaneous/elicited adverse events, medical examination and laboratory tests. Anti-FXa activity and anti-FIIa activity were assessed throughout the 24hours after dosing. Dose proportionality and linearity of the anti-FXa activity were evaluated.RESULTS:All doses were well tolerated and there were no bleeding events. At the lowest dose, anti-FXa activity A(max) was 0.16 (±0.02) IU/mL and AUC(0-24) was 1.11 (±0.24) IU*h/mL, At the highest dose anti-FXa activity A(max) was 1.67 (±0.15) IU/mL; AUC(0-24) was 21.48 (±4.46) IU*h/mL and t½ was 8.05h. Mean T(max) (all doses) was 2.86 (±0.39) h. RO-14 showed proportional and linear pharmacodynamics [normalized A(max) among doses (p=0.594) and normalized AUC(0-24) (p=0.092), correlations between A(max-)dose (R(2)=0.89, p<0.001) and AUC(0-24)-dose (R(2)=0.86, p<0.001)]. Anti-FIIa activity was below the detection limit (0.1IU/ml) at all dose levels. No clinically significant changes were observed in the platelet count, APTT, PT, TT, fibrinogen and antithrombin.CONCLUSIONS:In this phase I study, RO-14 exhibited a good safety profile, anti-FXa activity for either prophylaxis or treatment of venous thromboembolism, linear pharmacodynamics, a longer elimination half-life than currently marketed low molecular weight heparin and no anti-FIIa activity.

Background and Purpose-We tested the hypothesis that proteins of hemostasia could be associated with hematoma growth (HG) in patients with acute intracerebral hemorrhage.Methods-We prospectively studied patients with spontaneous supratentorial intracerebral hemorrhage within the first 6 hours after the onset of symptoms. HG was defined as an increase >33% in the volume of hematoma on CT obtained 24 to 72 hours after the onset of symptoms in comparison with the CT obtained at admission. We collected admission and follow-up blood samples. We measured fibrinogen, factor XIII, thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor, plasminogen, alpha(2)-antiplasmin, tissue plasminogen activator, D-dimer, thrombomodulin, thrombin-antithrombin complex, and plasmin-antiplasmin complex.Results-We included 90 patients with a mean age of 71 +/- 10.8 years; 61% were men. HG was observed in 35 (39%) of the patients. Mean baseline and follow-up protein measurements showed no difference between the groups with and without HG. The analysis of variance showed that factor XIII activity decreased in the non-HG group in the 24 to 72 hours sample, whereas it increased in the HG group (P=0.001).Conclusion-Factor XIII was the only measured protein related to HG. The levels at the follow-up sample decreased in the non-HG group and increased in the HG group. Further studies are needed to confirm this association. (Stroke. 2010;41:2976-2978.)

The remission rate with plasma exchange (PE) in thrombotic thrombocytopenic purpura (TTP) exceeds 80%, but the disease relapses in up to 20–30% of the cases. Clinical characteristics and response to treatment of relapsed TTP are not well defined. The objective of the present study was to compare the clinical and biological characteristics at presentation and the response to treatment between de novo and relapsed TTP. For such purpose, a total of 102 episodes of idiopathic TTP (70 de novo and 32 relapses) included in a recent multicentric prospective cohort study were analysed. All patients were homogeneously treated with daily PE and costicosteroids. In comparison with de novo TTP, episodes of relapsed TTP showed a higher Hb level (median, 122 g/l versus 91 g/l, p < 0.001) and lower serum lactate dehydrogenase (2.2- versus 4.5-fold above the upper limit of normality, p < 0.001). Neurological symptoms and fever were less frequently observed in patients with relapsed TTP than in patients with de novo TTP. Patients with relapsed TTP needed fewer PE sessions (five versus ten, p = 0.02) and a smaller volume of plasma (221 ml/kg versus 468 ml/kg, p = 0.004) to achieve remission than those with de novo TTP. There was no significant difference in the rate of recrudescence under treatment, the need of complementary treatments or the frequency of refractoriness to PE therapy. In conclusion, relapsed TTP has a milder clinical profile and responds more easily to PE than de novo TTP.

Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is a protein that potently attenuates fibrinolysis. A considerable proportion of its variability levels is genetically determined. It has been associated with arterial and venous thrombosis. We conducted a Genome Wide Scan for genes affecting variation in plasma TAFI levels in 398 subjects from 21 extended Spanish families. The data were analyzed by a variance-component linkage method. A strong linkage signal was found on the long arm of Chromosome 13, near the DNA marker D13S156, where the structural gene encoding for TAFI is located. In addition, other new linkage signals were detected on chromosome regions 5p and 7q. More importantly, we performed another multipoint linkage analysis of functional TAFI conditioned on TAFI antigen levels. We detected a strong linkage signal on Chromosome 19 (LOD = 3.0, P = 0.0001) suggesting a novel QTL in this region involved in the specific functional activity of TAFI, regardless of the TAFI antigen levels. One notable aspect of this study is the identification of new QTLs that reveal a clearer picture of the genetic determinants responsible for variation in TAFI levels. Another is the replication of the linkage signal of the CPB2 gene, which confirms an important genetic determinant for TAFI antigen levels. These results strongly suggest an oligogenic mode of inheritance for TAFI, in which CPB2 gene accounts for a proportion of the variation of the phenotype together with other unknown genes that may represent potential risk factors for thrombotic disease.

Background and Purpose— We evaluated the association between recombinant tissue-type plasminogen activator recanalization and change in hemostatic markers. Methods— We studied 40 patients. Recanalization was measured with transcranial Doppler. We evaluated the change in markers of coagulation (fibrinogen) and fibrinolysis (thrombin activatable fibrinolysis inhibitor and α 2 -antiplasmin) in patients with ischemic stroke treated with recombinant tissue-type plasminogen activator. Samples were obtained before and 90 minutes after recombinant tissue-type plasminogen activator infusion. Results— The analyses (2-way analysis of variance) showed that the change in the value of each marker did not depend on the vascular patency status. Conclusion— From a practical point of view, the measurement of these hemostatic markers is probably not useful for predicting recanalization.

Lupus anticoagulant (LA) and antiphospholipid antibodies (APA) are immunoglobulins directed at phospholipid-protein complexes that have been associated frequently with thrombophilia. Protein S (PS) is a protein with high affinity for phospholipids and can be a target for APA. It is a cofactor in the

Patent foramen ovale (PFO) is more frequent in cryptogenic stroke patients than in the general population. The aim of this study was to determine prothrombotic markers regarding PFO in young cryptogenic stroke patients. We prospectively included consecutive cryptogenic stroke patients younger than 55 years. PFO was diagnosed with simultaneous transcranial Doppler and transesophageal echocardiography. We analyzed the following prothrombotic markers: antiphospholipid antibodies (APS), protein C and protein S deficiencies, factor V Leiden FVG1691A, prothrombin gene mutation PTG20210A and coagulation factor XII mutation FXIIC46T. From June 2005 to July 2006 we studied 39 patients, mean age 44.7 +/- 8.6 years, 48.7% men. PFO was detected in 17 patients (43.6%). We found no differences between PFO and non-PFO patients regarding prothrombotic markers: APS (P = 0.851), protein S deficiency (P = 0.851), protein C deficiency (P = 0.249), FVG1691A (P = 0.202), PTG20210A (P = 0.401) or FXIIC46T (P = 0.966). Female gender was the only variable related to prothrombotic markers, independent of PFO (P = 0.001). The only prothrombotic marker related to PFO size (large PFO) was APS (P = 0.043). Large PFO were also related to deep venous thrombosis (P = 0.040) and atrial septal aneurysm (P = 0.010). PFO patients do not present more prothrombotic markers than non-PFO patients, but APS are more frequent in large PFO.

The aim of this study was to assess postprandial changes in thrombin activatable fibrinolysis inhibitor (TAFI) antigen, a thrombin-dependent fibrinolysis inhibitor with anti-inflammatory properties, and soluble markers of endothelial dysfunction in normotriglyceridemic type 2 diabetic patients. Fasting and postprandial TAFI antigen, thrombomodulin, tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibitor 1 were assessed in 12 normotriglyceridemic type 2 diabetic patients treated with diet (hemoglobin A1c, 6.80% ± 0.67%) and 14 controls. Fasting low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, free fatty acids and apolipoprotein B, and fasting and postprandial triglyceride, glucose, and insulin were also measured. Fasting TAFI was higher in the control group (102% ± 16.9% vs 72.9% ± 15.9%; P < .0005) and was inversely correlated with glycemic control. It decreased 4 hours after the meal (31.8% reduction [P < .005] for controls and 12.6% [P < .05] for diabetic patients) and returned to fasting levels after 8 hours. This decrement was correlated with fasting TAFI, glucose and hemoglobin A1c, and the area under the curve of glucose. Thrombomodulin, TFPI, and plasminogen activator inhibitor 1 were similar in both groups, with thrombomodulin and TFPI showing a transient postprandial increase. A fat-rich meal produces a transient increase in markers of endothelial dysfunction and a temporary reduction in TAFI, an anti-inflammatory molecule whose concentration is low in type 2 diabetes mellitus.

In a family-based study called GAIT (Genetic Analysis of Idiopathic Thrombophilia) that included a genome-wide scan we demonstrated that a polymorphism (46C-->T) in the F12 locus jointly influences variability of plasma (Factor XII) FXII levels and susceptibility to thrombotic disease. It then became germane to determine the prevalence of the 46C-->T polymorphism and its relative risk of thrombotic disease. We followed up evidence for genetic linkage with a case-control study, including 250 unrelated consecutive Spanish patients suffering from venous thrombotic disease and 250 Spanish subjects matched for sex and age as a controls. We measured FXII levels and genotyped the 46C-->T polymorphism, as well as a number of classical risk factors for thrombotic disease. We confirmed that individuals with different genotypes for this polymorphism showed significant differences in their FXII levels. Most importantly, the mutated T allele in the homozygous state (genotype T/T) was associated with an increased risk of thrombosis (adjusted OR of 4.82; 95% CI 1.5-15.6), suggesting that the polymorphism itself is an independent risk factor for venous thromboembolism. This study confirms that the 46C-->T polymorphism is a genetic risk factor for venous thrombosis in the Spanish population. In addition, our results confirm that a genome-wide scan coupled with a classical case-control association study is an extremely valuable approach to identify DNA variants that affect complex diseases.

To our knowledge, there is little information about functional thrombin activatable fibrinolysis inhibitor (TAFI) levels and the risk of acute coronary artery disease (CAD). We investigated the risk of acute CAD related to plasma levels of functional TAFI. We found that functional TAFI levels in plasma (above 126%), increased the risk of acute CAD almost 4-fold.

Our aim was to determine the associations of functional thrombin‐activatable fibrinolysis inhibitor (TAFI) levels in plasma with conventional cardiovascular risk factors, sex and age, and possible correlations with other hemostatic factors in a Spanish population. We included 303 individuals from a Spanish population. Hemostatic factors such as von Willebrand Factor, VII ag, VIIIc, XIc, XIIc, APCR, protein S, protein C, antithrombin, fibrinogen, and t‐PA antigen were assayed. The functional TAFI assay was based on the activation of plasma TAFI with thrombin–thrombomodulin, and the measure of TAFIa activity on the hippuryl‐Arg substrate. There were no statistical differences in mean values of functional TAFI among the various female age groups or among the different male age groups, with or without cardiovascular risk factors. Only women younger than 30 years of age showed lower levels of functional TAFI compared to older women. No differences were found among men of different ages. Adjusted for sex and age, hemostatic factors did not show a correlation with functional TAFI levels in plasma. Women with hypercholesterolemia showed higher levels of TAFI; other conventional cardiovascular risk factors did not modify functional TAFI levels either in men or in women. We also found no correlation of functional TAFI levels related to any other hemostatic factors. Am. J. Hematol. 76:348–352, 2004. © 2004 Wiley‐Liss, Inc.

Objective— Protein S (PS) is a component of the protein C anticoagulant system. PS deficiency is associated with myocardial infarction and venous thromboembolism, two highly prevalent causes of death in industrialized nations. As part of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) project, we conducted a genome-wide linkage screen to localize genes influencing variation in free PS (fPS) plasma levels. Methods and Results— fPS levels were measured in 397 individuals in 21 Spanish families. A total of 363 highly informative microsatellite markers were genotyped to provide a 10-cM genetic map, and variance component linkage methods were used. A region on chromosome 1q32, flanked by markers D1S425 and D1S213, showed strong evidence of linkage with fPS levels (LOD score, 4.07; nominal P =7.5×10 −6 ; genome-wide P =0.0024). This region contains two positional candidate genes, the complement component 4-binding protein α and β chains, which encode the principal binding protein for PS. Suggestive evidence for linkage was also observed on chromosomes 11p and 19p. Conclusions— These results represent one of the first genomic screens for quantitative variation in a component of the hemostatic pathway and provide strong evidence for a locus on chromosome 1q influencing fPS levels.

SummaryDeficiency of the anticoagulant vitamin K-dependent protein S (PS) is associated with increased risk of venous thrombosis. In human plasma, PS circulates in two forms: as free protein (free PS) and PS bound to C4b-binding protein (C4BP), a regulator of the complement system. Assays for free PS have higher sensitivity and specificity for protein S deficiency than assays for total protein S. We have extensively evaluated the analytical performance of a novel assay for free PS, the IL Test™ Free Protein S, which takes advantage of the affinity of C4BP for free PS, and compared its performance to existing methods. IL Test™ Free Protein S is a rapid, fully automated turbidimetric assay consisting of two reagents: a C4BP coated latex and an anti-PS monoclonal antibody coated latex. The test range, precision and linearity were adequate and the assay tolerated high concentrations of interfering substances of clinical significance. The reference range agreed with previously published studies. The analysis of 903 patient samples belonging to 20 different clinical categories with the new assay yielded free PS results that agreed well with those obtained using the assays established in the participating laboratories. The study demonstrated the IL Test™ Free Protein S to be rapid, reliable and easy to perform.

Objective: To evaluate the plasma levels of coagulation and fibrinolysis parameters in the third trimester of gestation and on the third day after delivery.Methods: Normal pregnant women (n = 65), pregnant women with essential hypertension (n = 10), pregnant women with gestational hypertension without proteinuria (n = 13), and pregnant women with preeclampsia (n = 12) formed the study population. Coagulation and fibrinolysis parameters were estimated using commercial tests. Antithrombin III, thrombin-antithrombin III complexes, heparin cofactor II(HCII), protein C, protein S, tissue plasminogen activator, D-dimer, and plasminogen activator inhibitors levels in uncomplicated pregnancies and in hypertensive pregnancies were determined.Results: No differences were found in coagulation inhibitor levels between normal, chronic essential hypertension, and gestational hypertension groups. However, preeclamptic women showed lower levels of antithrombin III and heparin cofactor II. The concentration of thrombin-antithrombin III was higher in hypertensive disorders, mainly in preeclampsia, than in normal pregnancy. No differences were found in the levels of tissue plasminogen activator and plasminogen activator inhibitors type 1 and type 2 in the normal pregnancy, chronic essential hypertension, and gestational hypertension groups. However, preeclamptic women had higher tissue plasminogen activator and lower plasminogen activator inhibitor type 2 levels.Conclusion: The finding that HCII levels were decreased only in the preeclamptic group shows its potential utility in the diagnosis of preeclampsia.