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L'injection de progeniteurs endotheliaux (PEC) cultives ex vivo ou provenant de cellules mononucleees medullaires a montre une efficacite dans les modeles precliniques et cliniques de neovascularisation. Cependant, l'origine et la definition exacte de la population de cellules responsable de ces benefices cliniques restent controversees. Etant donne l'utilite potentielle des PEC comme produit de therapie cellulaire, leur caracterisation moleculaire est primordiale. Nous decrivons ici les deux principaux types de PEC obtenus en culture cellulaire, et la mise en evidence par notre equipe des BMP2/4 comme marqueurs de PEC tardifs qui, de plus, jouent un role important dans la differenciation et l'expansion des PEC. Cette revue recapitule egalement les differentes methodes decrites pour l'expansion ex vivo des PEC qui, dans la plupart des cas, sont issus de sang de cordon et s'averent difficilement transposables aux PEC de sang adulte. Ainsi, une des possibilites therapeutiques serait de deriver des cellules endotheliales a partir de cellules souches embryonnaires (cellules ES) ou, mieux encore, de cellules « iPS » (induced pluripotent stem cells) produites a partir de cellules adultes autologues. Leur utilisation comme produit de therapie cellulaire des maladies cardiovasculaires pose encore actuellement des problemes ethiques, d'une part, en ce qui concerne les cellules ES, et d'effets indesirables d'autre part, comme le risque de formation de teratomes. Enfin, la meilleure comprehension des facteurs impliques dans les proprietes de pluripotence, d'autorenouvellement et de differenciation permettra de mieux maitriser la biologie des cellules souches adultes.

Recent data have implicated a haplotype of the purinergic receptor P2Y, G-protein coupled, 12 gene (P2RY12), as potential risk determinant for atherothrombosis. However, to date, no prospective, genetic-epidemiological data are available. Using DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we examined the possible association of P2RY12 genetic variants, in particular a haplotype H2 (constituted by dbSNP rs10935838, rs2046934, rs5853517, and rs6809699) amongst 708 white males who subsequently developed a thromboembolic event (incident myocardial infarction (MI), ischemic stroke, or deep venous thromboembolism/pulmonary embolism (DVT/PE)) and amongst an equal number of age- and smoking-matched white males who remained free of reported vascular disease during follow-up (controls). The P2RY12 gene variants tested were in linkage disequilibrium. The haplotype H2 distribution was significantly different between the DVT/PE cases (12%) and their matched controls (21%), p-permuted=0.02. In an adjusted conditional logistic regression analysis, the haplotype H2 was significantly associated with a lower risk of incident DVT/PE as compared to the reference haplotype H1 (odds ratio=0.50, 95% CI=0.27-0.93, p=0.028). However, we found no evidence for an association of the P2RY12 variants or the haplotype H2 with incident MI or ischemic stroke. The present investigation provides evidence for an association of the P2RY12 haplotype H2 with lower risk of DVT/PE; however these findings require replication in other well-designed studies.

Thrombomodulin is expressed at the surface of endothelial cells and controls thrombin generation and thrombin-induced platelets and vascular cell activation. Several thrombomodulin gene polymorphisms have been associated with coronary events and brain infarction. In a previous analysis from the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC) study, we found that soluble thrombomodulin (sTM) concentration modulated the risk of and prognosis for brain infarction.In 474 brain infarction cases and 483 controls from the GENIC study, we investigated the relationship between three thrombomodulin gene polymorphisms (-1748G/C, -1208/-1209delTT, +1418C/T) and sTM levels, brain infarction risk and 5-year mortality after stroke.The three polymorphisms were in linkage disequilibrium and defined three major haplotypes with no influence on sTM concentration (all P values0.16). Single locus and haplotype analyses found no significant association with brain infarction, even when the analysis was restricted to individuals without a vascular history. After 5 years of follow-up, we found no relationship with vascular or total mortality (all P values0.64).Our results suggest that these three thrombomodulin gene polymorphisms do not contribute to sTM level variations and are not associated with risk of brain infarction and mortality after stroke.

The coagulation system is governed by a subtle balance between clotting activators and inhibitors. Many genes can contribute to the overall phenotype, and polymorphisms may act to up regulate or down regulate the generation of thrombin, the coagulation-key enzyme. An increase in coagulation factor (gain function) or/and a decrease in coagulation inhibitors (loss of function) may favor venous thromboembolism (VTE). It has been observed since a long time that VTE may be a familial disease, but it was only in 1965 that Egeberg published the first case of inherited antithrombin (AT) deficiency. This was followed by similar reports of protein C (PC) and protein S (PS) deficiencies. Hereditary thrombophilia was thus initially considered as a rare monogenic disorder with incomplete penetrance. AT, PC and PS deficiencies are due to multiple and mostly private mutations of the corresponding genes. Most patients are heterozygous and experience VTE at adult age. Homozygosity associated with severe thrombosis at birth has been observed in newborns with undetectable PC or PS concentrations. The discovery of factor (F) V Leiden and F2 g.20210 G>A, two gain of function mutations, challenged the view of thrombophilia as a rare monogenic disorder. FV Leiden and F2 g.20210 G>A are due to a founder effect and affect populations of European descent with frequencies at 5% and 3% respectively. These two mutations are moderate of risk factor for thrombosis and paved the way for gene-gene and gene-environment interactions. Patients carrying more than one genetic risk factor are at higher risk to develop VTE. The exposition to acquired risk factors such as estrogen based oral contraception may also have a synergistic effect favoring thrombosis in patients with FV Leiden or other genetic risk factors.

We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin γ-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (GlaFII-PS) or in PS deleted of the thrombin-sensitive region (TSR) (GlaFII-ΔTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into GlaFII-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.

Heparin-induced thrombocytopenia (HIT) occurs in nearly 3% of patients treated with heparin after cardiopulmonary bypass (CPB). HIT carries a risk of severe thrombotic complications, and must be diagnosed rapidly. To identify simple criteria for estimating the probability of HIT after CPB, we retrospectively analyzed the files of 84 patients with suspected HIT after CPB and we analyzed the usefulness of several variables collected at the time of HIT suspicion to estimate HIT probability. HIT was confirmed in 35 cases and ruled out in 49 cases, on the basis of a platelet increment after heparin withdrawal, detection of heparin-dependent antibodies, and absence of other clear cause of thrombocytopenia. A biphasic platelet count from CPB to the first day of suspected HIT, an interval of >/= 5 days from CPB to the first day of suspected HIT, and a CPB duration of

Background and Purpose— Increased soluble thrombomodulin (sTM) concentration has been associated with recurrent coronary events, whereas in one prospective study it predicted fewer first-ever coronary events. One study found no relationship between brain infarction (BI) and sTM levels. Among all subjects of the Étude du Profil Génétique de l’Infarctus Cérébral (GENIC) cohort and those free of previous vascular history, we investigated the relationship between sTM level and BI risk, and among cases, its relationship with BI prognosis. Methods— Patients with BI (n=492) were consecutively recruited from 12 centers. Hospital controls without a history of stroke (n=492) were individually matched for age, sex, and center. Blood samples were collected after hospitalization. Determination of sTM levels was centralized in a single laboratory. Results— Soluble TM concentration significantly increased with age and hypertensive status, but was similar in cases and controls. With analyses restricted to 278 pairs of subjects with no previous vascular history, sTM concentration >59.6 μg/L (second and third tertiles compared with the first) was associated with fewer first-ever BI (adjusted odds ratio of 0.56 (95% CI, 0.35 to 0.89; P =0.014). Among the cases, increased sTM concentration was associated with a higher death rate after a median follow-up of 5.2 (1.4 to 6.4) years. The adjusted hazard ratio per 1 SD of sTM concentration increase (34.2 μg/L) was 1.19 (95% CI, 1.02 to 1.39; P =0.028). Conclusions— Increased sTM concentration may be protective against BI in subjects with no previous vascular disease, whereas it may predict a fatal outcome in patients who have already had a BI. Consequently, sTM levels should be interpreted according to vascular history.

There is increasing evidence that serpin conformational alteration caused by single point mutations can be responsible for protein deficiency associated with human diseases. A typical example is the alpha1-antitrypsin deficiency caused by the Z variant carrying a Glu342Lys substitution. Only a few cases of "conformational disease" involving other serpins have been described so far. We investigated a severe antithrombin deficiency in a 13-month-old child with fever and cerebral venous thrombosis. The infant was found to be homozygous for a new antithrombin gene mutation (7396TC, predicting a Phe229Leu antithrombin variant), and heterozygous for the factor V Leiden mutation. Mild atypical antithrombin deficiency was found in both parents, who were first cousins, asymptomatic, and heterozygous for the same antithrombin gene mutation. The Phe229Leu variant, which does not readily fit into the current classification of antithrombin deficiency, was shown to be a thermolabile antithrombin that spontaneously polymerized in the proband's circulation. This points to a key role for the conserved Phe at position 229, which is near the reactive site loop in a region critical for serpin function and stability. Molecular modeling suggested how the mutation might destabilize this region of the protein and thereby favor reactive site loop insertion and polymerization. This study provides the first direct evidence of antithrombin polymerization in vivo causing antithrombin deficiency and severe thrombotic disease.

Protease-activated receptor 1 (PAR-1), the main thrombin receptor on vascular cells, plays a key role in platelet activation. We examined the range of PAR-1 expression on platelets, obtained twice, 1 week apart, from 100 healthy subjects and found a 2-fold interindividual variation in receptor numbers (95% CI = 858-1700). Because PAR-1 density was stable with time (r2 = 76%,P

Protein S (PS) possesses a sex-hormone-binding globulin (SHBG)-like domain in place of the serine-protease domain found in other vitamin K-dependent plasma proteins. This SHBG-like domain is able to bind a complement fraction, C4b-binding protein (C4b-BP). To establish whether the PS SHBG-like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG-like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b-BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG-like region is an independent folding unit. We also obtained the first direct evidence that the SHBG-like domain alone is sufficient to support the interaction with C4b-BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b-BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG-like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG-like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.

Aims We conducted a phase I placebo-controlled trial with two i.v. doses (0.5 mg h−1 and 3 mg h−1) of S 18326, a selective thrombin inhibitor that interacts with the catalytic site of thrombin, with the aim to study the relationships between increasing plasma levels of S 18326 and changes in coagulation tests and thrombin generation markers. Methods Thirty-six healthy male volunteers were divided into three groups. In each group, 10 volunteers were randomly assigned to receive S 18326 and two to receive a placebo. Following a bolus of 4.5 mg, doses were 0.5 mg h−1 in the first group and 3 mg h−1 in the two other groups, administered as an i.v. infusion for 24 h. Blood was drawn repeatedly up to 36 h after the bolus, and tested for the activated clotting time (ACT) and activated partial thromboplastin time (APTT). The APTT reagent was chosen among five commercial reagents to yield a linear increase in the clotting time among possible therapeutic S 18326 concentrations in vitro. To accurately measure the thrombin-inhibiting effects of low doses of S 18326 (

Genetic risk factors became a frequent predisposing cause of venous thromboembolism (VTE) since the discovery of two mutations: factor V Leiden and G20210A mutation of prothrombin gene. One of these both mutations is associated with around 25% of VTE events. Interaction of genetic risk factors, such as interaction of FV Leiden or G20210A mutation of prothrombin with antithrombin, protein C or protein S deficiencies, as well as interaction with acquired risk factors, have demonstrated that venous thrombosis is a multifactorial disease. The search for thrombophilia must be done in VTE occurring before the age of 45, in case of recurrencies and in case of familial history of VTE.

SummaryWe have previously identified, in intron 7 of the TFPI gene, a T to C single-base polymorphism (−33T→C) which is strongly associated with total circulating TFPI antigen levels. Here we examined the influence of this polymorphism on the risk of venous thromboembolism. The polymorphism was identified in the PATHROS study population (330 cases with venous thromboembolism and 826 controls). The CC genotype was found in 6.4% of cases and 10.2% of controls (age-adjusted odds ratio 0.6; 95% CI 0.3–0.9; p = 0.03). This protective effect persisted after adjustment for oral contraception and the factor V Leiden and prothrombin gene polymorphisms. In 171 controls and 49 cases in whom blood was taken at least three months after the thrombotic event, the CC genotype was associated with significantly higher total TFPI levels than the TT genotype. These results suggest that the CC genotype of the TFPI intron 7 polymorphism is an independent protective factor for venous thromboembolism, an effect probably mediated by increased TFPI levels.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the γ-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the γ-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

In the past 3 decades, numerous biological abnormalities linked with deep venous thrombosis have been described. Among the different possibilities, it is crucial to order tests that can modify the therapeutic attitude towards the patient and/or his family. A combined clinical and laboratory approach taking into account the history of the patient and his family, the prevalence of the defects, and also the accuracy of the tests should allow tailoring a laboratory testing program to each patient. It is essential to keep in mind that the more difficult task is not to perform the tests but to consider who will benefit from testing both for prevention and therapy of venous thromboembolism. This article provides answers to some of these issues. These answers should, however, be considered as tentative and provisional because new findings and study results will certainly modify them in the near future.

SummaryWe have previously reported that the 3363 inserted (Ins) C mutation in exon 6 of the protein C gene was present in four unrelated French patients and in four French Canadian families with type I protein C deficiency as well as in a large Vermont protein C deficient kindred of French Canadian origin. The present study was designed to investigate the likelihood of the existence of a founder effect for this mutation in protein C deficient individuals of French origin living in France, Québec and Vermont. In order to demonstrate a possible founder effect for the 3363 InsC mutation, we have previously constructed a high-resolution genetic map to locate several highly polymorphic markers close to the protein C locus. Thereafter, the markers D2S347, D2S2339, D2S383, D2S2271 and D2S2215 were genotyped in 117 heterozygotes from France (n = 7), Québec (n = 36) or Vermont (n = 74). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Our data suggest that patients from Québec and Vermont carry a common haplotype at the protein C locus. Moreover, in order to study the evolutionary history of the 3363 InsC mutation, we traced back the ascending genealogy of one proband in each of the families with this mutation. These results showed that the 3363 InsC mutation was most probably introduced in North America by a couple of French settlers who established themselves in 1669 on Isle d‘Orleans located near Québec City. All heterozygotes for the 3363 InsC mutation living in North America are related to these founders within 10 generations. Thus, these families afford a unique opportunity to evaluate the role of the protein C system in thrombophilia due to the high degree of linkage disequilibrium at the protein C gene, which in essence holds that variable more constant than in a more heterogeneous population.

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three-dimensional structure were analysed. The last mutation in exon 4 was associated with a type II 'reactive site' deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.

Abstract —Tissue factor (TF) is a transmembrane protein considered to be responsible for the initiation of coagulation. TF gene expression may be induced in monocytes and endothelial cells and is present in atherosclerotic plaque to initiate thrombus formation. To investigate whether individual differences in TF gene expression could predispose subjects to thrombosis, we sequenced the 5′ domain of the gene up to nucleotide 2732 and found 6 different polymorphisms: 4 of them were completely concordant and defined 2 haplotypes with similar frequencies, designated as 1208 D and 1208 I. Genotyping of patients with myocardial infarction in a case-control study involving 2354 subjects showed no association between the polymorphisms and nonfatal coronary thrombosis. In another study involving 255 patients with venous thromboembolism and 1204 controls, allele D was less common in the cases ( P =0.022). The odds ratio associated with the presence of at least 1 D allele was 0.72 ( P =0.031). Comparison of subgroups of control subjects who were homozygous for the D or I allele demonstrated a lower plasma TF concentration in DD homozygotes. These results indicate that the TF gene promoter exists in 2 major forms differing at 4 sites. The 1208 D haplotype is not associated with coronary thrombosis but is associated with reduced plasma TF levels and a lower risk of venous thrombosis.

SummaryWe analyzed the protein C gene (PROC) with the denaturing gradient gel electrophoresis (DGGE) scanning strategy in a series of 129 patients with suspected protein C (PC) deficiency (93 with low plasma PC levels and 36 with borderline level). At least one sequence variation was found in 104 of the 129 patients. Thirty-nine sequence variations (found in 72 patients) were already reported detrimental mutations. Thirty-three were novel sequence variations, of which 19 (found in 25 patients) were probably detrimental. Five novel mutations (A1T, R9H, S11R, S12R and K193Q) were associated with qualitative plasma PC deficiency, suggesting or confirming the functional importance of amino acids at these positions. This strategy confirmed the diagnosis of inherited PC deficiency in 79/93 (84.9%) patients with low plasma PC levels and 14/36 (38.8%) patients with borderline values. In order to explain abnormal PC levels observed in patients who did not carry detrimental mutations, screening for the -1654C/T and -1641A/G PROC promoter polymorphisms known to influence plasma PC concentrations was performed. The frequency of the CG allele associated with lower PC concentrations was slightly but not significantly lower in 82 heterozygotes for detrimental PROC gene mutations than in 36 patients with no identified detrimental mutations.

SummaryA monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i. e. the carboxyterminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.

Endothelial progenitor cells are selectively mobilised immediately after coronary artery bypass grafting or valve surgery

Protein C is a plasmatic inhibitor which regulates the blood coagulation mechanism by modulating the anticoagulant response. The improvement of its bioavailability would be beneficial for the treatment of the disorders caused by its homozygous deficiency or by an other plasmatic inhibitor deficiency. In this context, the protein C encapsulation into biodegradable nanoparticles could be used to approach the problem. However, the method used to prepare the nanoparticles requires the use of ultrasonication and of an organic solvent such as methylene chloride which interferes with protein activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that neither ultrasonication nor methylene chloride, singly or in combination, led to protein C aggregation or cleavage. Thus, a binding study using an ELISA assay with four characterized monoclonal antibodies was carried out to identify the epitopes damaged by these experimental constraints. The correlation between the immunological assay and a functional one i.e. by the means of the assay of its anticoagulant activity (activated partial thromboplastin time) made it possible to show that protein C amino acids 166-169 of the activation peptide were probably altered after ultrasonication and methylene chloride treatment. Indeed, it is likely that these residues were no longer surface-exposed but had moved inside the protein core.

The factor V (FV) Arg 506 to Gln mutation is the most common abnormality observed in familial thrombophilia. Many studies have shown that its clinical expression differs among families and among carriers. Some thrombotic patients carry an additional genetic risk factor such as protein C, protein S or antithrombin deficiency. We sought to identify other genetic risk factors potentially favouring expression of the thrombotic phenotype in 370 members of 43 families with the FV Arg 506 to Gln mutation. We analysed three candidate polymorphisms in genes involved in the PC anticoagulant pathway, consisting of two polymorphic sites in the 5' non-transcribed region of the PC gene, -1654 C/T and -1641 A/G, with three known combinations (TA, CA and CG) that influence the protein C plasma level; one polymorphic site (4070 A/G) in exon 13 of the FV gene, which influences the plasma factor V concentration, and one polymorphic site (677 C/T) in the methylenetetrahydrofolate reductase gene, which is often associated with moderate hyperhomocysteinaemia. The distribution of these different polymorphisms was similar in patients with a history of thrombosis and those who remained asymptomatic, ruling out the possibility that each of these polymorphisms alone can play a role in the onset of thrombosis in carriers of the FV Arg 506 to Gln mutation.

Abstract —The allele and haplotype frequency of the –1654 C/T and –1641 A/G protein C (PC) gene promoter polymorphisms was determined and analyzed according to circulating PC concentrations in 394 healthy subjects aged 20 to 60 years. The CG haplotype was associated with a lower PC concentration in both homozygous and heterozygous subjects compared with noncarriers. The TA allele had the reverse effect, but only in homozygotes. The distribution of the CG and TA alleles was significantly different in 242 patients, aged 17 to 60 years, with venous thromboembolism. The CG allele increased the risk of thrombosis, with an OR of 1.39 (95% confidence interval (CI), 1.04 to 1.87). The TA allele was protective in subjects aged TA was also significantly associated with older age at the first thrombosis. This study confirms the link between the PC gene promoter and circulating PC levels, and suggests a complex effect on the risk of thrombosis.

Abstract —The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides −293 to −12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.

SummaryThe A4070G polymorphism in exon 13 of the factor V (FV) gene, which replaces His by Arg at position 1299 of the B domain, was recently shown to influence circulating FV levels and to contribute to the activated protein C (APC) resistance phenotype. We examined the impact of this polymorphism in a population of unselected patients with venous thromboembolic disease (VTE). The prevalence of the G4070 (R2) allele was determined in 205 patients and 394 healthy subjects of similar age and sex distribution. Thirty-seven patients (18%) were heterozygous for the R2 allele and 1 (0.5%) was homozygous. Forty-four controls (11.2%) were heterozygous for the R2 allele and 1 (0.2%) was homozygous. Thus, the allelic frequency was significantly higher in the patients with VTE than in the healthy controls, with respective values of 9.5% and 5.8%. The odds ratio was 1.8 (95% CI: 1.1-2.8, p = 0.02), pointing to an increased risk of VTE in carriers of the R2 allele. After excluding subjects with putative or confirmed gene defects (mainly the FV R506Q mutation), the R2 allele was still a risk factor for VTE in the remaining patients, with an odds ratio of 2.0 (95% CI: 1.2-3.5, p = 0.01), demonstrating that this polymorphism is itself a risk factor. This study also confirms that the R2 allele influences APC resistance (APCR) in the absence of the FV R506Q mutation.

The genomic analysis of a 70-year-old man with recurrent deep venous thrombosis having a protein S (PS)-deficient phenotype corresponding to both type III and type II evidenced two different mutations: a +5 g→a mutation in the donor splice site of intron e (ivs e) and a ser 460 to Pro mutation. The propositus' son, who had a type II PS deficiency phenotype, only bore the ivs e +5 g→a mutation. The study of platelet PS mRNA prepared from this subject showed that the ivs e, +5 g→a mutation led to the generation of two abnormal transcripts, one lacking exon 5 and the other lacking exons 5 and 6. The presence of an additional PS band with a decreased molecular mass on immunoblots performed in reducing conditions suggested the presence of truncated PS lacking EGF1 (encoded by exon 5). Two monoclonal antibodies (MoAbs) were used to further characterize the nonfunctional plasma PS. Comparison of PS levels measured with each of these MoAbs and PS levels in conventional assays was consistent with the presence of an abnormal inactive protein in the plasma of both patients bearing the ivs e, +5 g→a mutation, suggesting that variant PS lacking EGF1 is secreted but is devoid of activated protein C cofactor activity.

Type III protein S (PS) deficiency, characterized by low levels of free PS and normal total PS levels, is often associated with the Ser 460 to Pro substitution. However, some patients bearing this mutation have normal PS levels, suggesting that another gene defect may account for this phenotype. We postulated that this defect was located in the C4b-BP beta-chain gene (C4BPB) and searched for a mutation in the coding regions of this gene in 35 propositi with type III PS deficiency and bearing the Ser 460 to Pro mutation. No mutations explaining the phenotype of type III PS deficiency were identified. We did, however, find two frequent nucleotide changes, one being located in the donor splice site of intron d and the second in the codon corresponding to Asn 137. We used these two polymorphisms to establish C4BPB gene haplotype in five informative type III PS-deficient families and exclude a role of the C4BPB gene in this phenotype of three of them. Finally, increased C4b-BP beta-chain levels were not responsible for the phenotype of type III PS deficiency as the C4BPB haplotype did not correlate with C4b-BP beta-chain levels.

SummaryThrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 μM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

SummaryHeparin-induced thrombocytopenia (HIT) involves heparin-dependent antibodies which induce platelet activation. In the present study, we searched for a relationship between the polymorphism of the Fc receptor (FcγRIIa) and the development of HIT. In this purpose, all the donors were genotyped for their FcγRIIA and HIT patients were selected on the basis of at least one positive answer by 14C-serotonin release assay (SRA). The frequency distribution of the FcγRIIa polymorphism in the HIT patient group was similar to that observed in the healthy control group. Moreover, a statistical analysis taking into account our results and those of 3 previously published studies, suggested at most only a weak association between HIT and the FcγRIIa-131 polymorphism.Laboratory tests used to diagnose HIT rely on the activation of normal donor platelets but fail to detect every HIT positive patient. We determined the role of FcγRIIa-131 polymorphism on the reactivity of control platelets to HIT plasmas. When control platelet FcγRIIa-131 was of Arg/Arg form, only 47% of the HIT plasmas were positive by SRA, compared to 81% and 74% for His/His or His/Arg forms, respectively. We also compared the level of anti PF4/heparin antibodies in the HIT plasmas with the response obtained by SRA. The mean anti PF4/heparin antibodies level in HIT plasma was significantly lower in negative SRA than in positive tests when using control platelets from FcγRIIa-Arg/Arg131 and heterozygous donors. Thus, the variability of control platelets to respond to HIT plasmas in the SRA test is related to both the FcγRIIa-131 polymorphism, and to the amount of anti PF4/heparin antibodies.

Considering that platelet response to thrombin receptor activation might be critical for the development of arterial thrombosis, we measured the dense granule release under stimulation by the thrombin receptor activating peptide (TRAP) in a series of 102 healthy volunteers. The threshold TRAP concentration which initiated a secretion ranged from 3 to 20 microM. A good concordance (79%, k=0.677) between two tests performed at a 1 month interval indicated that platelet response to thrombin receptor activation was characteristic of each individual donor. Since the threshold concentration required to initiate secretion corresponded to the threshold concentration which induced a biphasic aggregation, all volunteers were genotyped for the PlA2 polymorphism, the Pro33 variant of GPIIIa. Platelets from subjects with the PlA2 polymorphism required higher TRAP concentrations to aggregate than those from subjects with no PlA2 allele (P=0.0012). However, they also required a higher ADP concentration to aggregate. In order to exclude any influence of GPIIIa polymorphism on TRAP-induced secretion, we studied the variability of platelet response to TRAP among the 77 individuals with no PlA2 allele, and found the same interdonor variability with the same distribution of threshold TRAP concentrations as for the 102 individuals. The results suggest that (i) platelet secretion in response to thrombin receptor activation could be a genetically controlled phenotype independent of the GPIIIa polymorphism; (ii) the PlA2 polymorphism is associated with platelet hypoaggregability.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Patients with phenotypic PS deficiency may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. The molecular basis for hereditary PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele.

SummaryPurpura fulminans is associated with homozygous protein C and homozygous protein S deficiency or may follow bacterial or viral infections. We present 2 children from 2 unrelated Arab families with purpura fulminans who were double heterozygotes for factor V Leiden inherited from their fathers and protein S deficiency inherited from their mothers. No previous thrombotic events have occured in either patient or their respective family members. In one patient sepsis accompanied by disseminated intravascular coagulation appeared to be the trigger of purpura fulminans. In the other patient varicella infection preceded purpura fulminans and was also associated with disseminated intravascular coagulation. This report emphasizes the need for evaluation of hereditary defects in the inhibitory mechanisms of blood coagulation in patients with purpura fulminans at any age.

SummaryWe analyzed the clinical features of 36 patients homozygous for the Arg 506 to Gin factor V mutation and found a circumstantial event at risk for thrombosis in 29 of the 31 patients with thrombosis. The most frequent predisposing factors were the post-partum period and the use of oral contraceptives in women, and surgery in both sexes. Venous thrombosis recurred in 48% of the patients. One patient had a myocardial infarction at age 33 years, and also had an antiphospholipid syndrome. Homozygous Gin 506 mutation leads to far less severe thrombotic complications than homozygous protein C and protein S deficiencies and does not seem to predispose patients to arterial thrombosis.

“Ce qui a peut-etre transforme le plus profondement l'etude desetres vivants, c'est l'accsa l'analyse d'objets nouveaux,” or in other words, “That which had the greatest impact on the study of living subjects is access to the analysis of new objects” (Francois Jacob, 1965 Nobel Prize of Medicine). In the 20th century, it is recombinant-DNA technology that has opened a new chapter in the history of medicine, genetic medicine, because it has rendered the human genome accessible to analysis. The cardiovascular field has been rather slow to integrate the concepts and the tools of molecular biology and molecular genetics; however, this barrier has now been overcome, and it is an exciting time for the field. This article focuses on studies that are conducted in France in the context of national multidisciplinary INSERM networks and that deal with two types of hereditary forms of cardiovascular diseases, cardiomyopathies, and hereditary thrombophilia. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:207–210).

SummaryTo further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturating gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus’ gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gin mutation.

The clinical presentation of hereditary protein C deficiency is highly variable. Homozygosity and compound heterozygosity have been linked to severe thrombotic complications early in the life. Heterozygous patients have a moderate form of the disease with deep venous thrombosis during adulthood. In the French population, we found 53 different mutations in 90 families. The amount of the protein C produced by the mutant allele as well as the genetic status partly account for the variable clinical expression. Other gene may also be involved: Arg 506 to Gln factor V mutation shows a frequency of 10 to 20% in symptomatic protein C deficient patients. Some protein C gene mutations are associated with a non functional circulating protein; most of them are located in the GLA domain and in the serine protease domain. The biochemical characterization of a few of theses variants has shown the important role of some amino acids on the activation and the mechanism of action of protein C.

SummaryIn a series of 16 propositi with symptomatic protein S deficiency and a protein S gene mutation, we identified a sporadic case of a novel mutation that probably affects gene expression. The mutation, a G to C transversion leading to the substitution of Ala 484 by Pro, was not found in the protein S gene of the patient’s parents. Transmission of the paternal and maternal protein S alleles was apparently normal, on the basis of the frequent polymorphism in exon XV. We also checked the transmission of chromosomal material by analysing protein C gene polymorphisms, Β-globin gene frameworks and four variable number of tandem repeats (VNTRs). By combining the results of these analyses, we were able to rule out nonpaternity and to confirm the de novo nature of the mutation.

Summary Activated protein C resistance is the most prevalent cause of thrombophilia: it is found in 20 to 30% of patients with a deep venous thrombosis history. Activated protein C resistance is due to an arginine 506 to glutamine mutation in factor V. This mutation prevents normal inactivation of activated factor V by activated protein C. The estimated increase in relative risk of venous thrombosis is 5- to 10-fold in heterozygotes, and 50- to 100-fold in homozygotes. Activated protein C resistance does not seem to play a role in arterial thrombosis and in the occurrence of myocardial infarction.

The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.

The mutations observed in patients with antithrombin and protein C deficiencies are mostly substitutions of one nucleotide, or deletions/insertions of fewer than 10 nucleotides in the exons and intron-exon junctions. These genomic abnormalities result in missense changes (involving aminoacids important for protein folding), aberrant polypeptide chains and/or premature termination codons, or abnormal splicing precluding DNA transcription. The number of mutations so far identified is such that it is difficult to use genomic DNA analysis for diagnostic purpose. However, identification of the gene defect can be useful in well-defined situations, such as the risk of homozygosity, and complex or ambiguous plasma phenotypes, which frequently occur in protein C deficiency. Protein S deficiency, the molecular bases of which have been less extensively studied, is due to micromodifications of the coding sequence in only half the cases investigated so far. The mechanisms involved in the remaining cases remain to be identified.

In a series of 40 patients with symptomatic protein C deficiency, we identified two sporadic cases with novel mutations that probably affect gene expression. The mutations, a 5-bp deletion of the donor splice site of intron f (nucleotides 3455 to 3459) and a mutation of nucleotide 8523 in exon IX leading to the substitution of Ser 270 by Pro, were not found in the protein C gene of the patients' parents. Transmission of the paternal and maternal protein C alleles was apparently normal on the basis of frequent polymorphisms in exons I, VI, and VIII. We also checked the transmission of the chromosomal material by analyzing the beta-globin gene frameworks and three variable number of tandem repeats (VNTRs). By combining the results of intragenic polymorphism, VNTR and beta-globin gene framework analyses, we were able to exclude nonpaternity and confirm the de novo origin of the mutation.

SummaryWe report three novel mutations accounting for cases of inherited type I antithrombin (AT) deficiency. Using the polymerase chain reaction (PCR) and direct sequencing of the coding sequences of the AT gene, we found one mutation in exon 4 and two in exon 6. A deletion of 105 bp causing an in-frame deletion of 35 amino acids between Tyr 240 and Gly 276 was found in exon 4. In a second kindred, deletion of two adenines in codon 412-413 introduced a frameshift and a stop codon at position 431. The last mutation was an insertion of ACCG in codon 387, generating a frameshift with a stop codon located at the normal position.The finding of a sequence repeat of nine residues located at the 5’and 3’ ends of the deleted fragment might explain the 105 bp deletion by slippage and mispairing at the replication fork during DNA synthesis. The second mutation is the fourth described within a region of six amino acids (between Phe 408 and Arg 413), which seems to be a cluster of mutations. In this case, the presence of a double repeat sequence - TTCCT and AACA - flanking this region could be particularly favorable for slipped mispairing.These results confirm that human gene mutations are not random events but are strongly influenced by DNA flanking sequences.

SummaryTreatment monitoring based on a laboratory parameter increases the efficacy and safety of standard heparin therapy, but it is not known if this also applies to low-molecular-weight heparin (LMWH) therapy of acute deep vein thrombosis (DVT). In a prospective randomized trial involving 122 consecutive patients, group A (58 patients) received a weight adjusted dose of Fragmin (100 IU/kg) subcutaneously twice a day throughout the treatment period (10 days ± 1), while in group B (64 patients) the dosage was based on the results of an anti factor Xa (anti Xa) amidolytic assay to obtain a target concentration from 0.5 to 1 IU/ml. AntiXa and antithrombin activities were also measured retrospectively on frozen plasma from all patients. The two regimens were comparable in terms of hemorrhagic complications (4 in group A and 3 in group B). Bilateral ascending phlebography was performed before inclusion and at the end of LMWH treatment. Treatment efficacy, based on Marder’s score, did not differ between the two groups (p = 0.3). Dosage adjustment to between 0.5 to 1IU anti-Xa/ml does not therefore appear to improve the efficacy or safety of LMWH tieatment. However, correlations between the change in Marder’s score and both anti-Xa (p

SummaryThe association of two missense mutations, a Leu 223 to Phe and an lie 403 to Met, is described in a family presenting with various protein C deficiency phenotypes. In this family, two subjects were compound heterozygotes with protein C levels of about 25%, the other members being heterozygous for only one of the mutations. The Leu 223 to Phe mutation was also found in 9 members of 3 other families and, in all cases but one, resulted in protein C levels below 60% associated with a high incidence of thrombotic complications. The other mutation, an lie 403 to Met, was identified in those of the family’ members who presented with borderline protein C concentrations. In such a family, the genomic DNA analysis represents the only way to differentiate between the genetic status of each family member. The results highlight the importance of the genotype determination and the poor discriminative power of the plasma assays currently used.

SummaryUse of oral contraceptives (OC) that combine a progestogen with synthetic ethinyl estradiol (EE) is associated with increased risk of venous thromboembolism. NOMAC/E2 is a new monophasic OC that combines nomegestrol acetate (NOMAC), a highly selective progestogen, with 17β-estradiol (E2). The study objective was to compare the effects on markers of haemostasis of NOMAC/E2 (2.5 mg/1.5 mg) versus the second-generation OC, levonorgestrel (LNG)/EE (100 μg/20 μg). Healthy women (age 18–38 years) received once-daily treatment for three consecutive 28-day cycles in a double-blind, randomised study: either NOMAC/E2 for 24 days with a four-day placebo interval (n=45) or LNG/ EE for 21 days with a seven-day placebo interval (n=45) per cycle. Mean changes from baseline to end-of-treatment in coagulation markers, including prothrombin fragment 1+2 (primary endpoint), fibrinolysis markers and platelet functions were assessed. Mean prothrombin fragment 1+2 levels (primary endpoint) did not increase with NOMAC/E2 compared with LNG/EE ( –0.02 vs. +0.08 nM, p

Hereditary protein C (PC) deficiency is usually associated with a high risk of thrombosis. We report the results of a study undertaken to screen for molecular defects in families with hereditary quantitative PC deficiency. Using a strategy combining polymerase chain reaction amplification of selected gene fragments, denaturing gradient gel electrophoresis of the amplification products, and direct sequencing of fragments with altered melting behavior, we studied the PC gene exons and exon/intron junctions of subjects with hereditary type I PC deficiency. Computer simulation of DNA melting was used to design several sets of primers, each containing a GC-clamp, permitting the complete analysis of each amplified exon sequence. Using this procedure, we identified two previously undescribed mutations located in exon VII: a C-to-T substitution generating a nonsense codon in place of Arg 157 in the mature PC and a G-to-A substitution converting Arg 178 to GIn. The two mutations were detected in, respectively, 3 and 2 apparently independent families. This strategy is therefore a valuable tool for screening patients, and the results emphasize its advantages over plasma assays in individuals with a family history of thrombosis. © 1992 Wiley-Liss, Inc.