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Biomedical subjects

P Gaussem

Publications and source records attributed to P Gaussem.

At least 37 records · Page 2Linked to original sources
PubMed1998

Venous thrombosis in older people: prevalence of the factor V gene mutation Q506.

OBJECTIVES: Old age is usually considered to be a risk factor for venous thromboembolism, in conjunction with other factors such as heart failure, major surgery, cancer, long-term immobilization, and antiphospholipid antibodies. Genetic risk factors, especially inherited deficiencies in coagulation inhibitors, also play a role in the pathogenesis of thrombosis, but these are usually diagnosed in thrombophilic patients before the age of 50. The factor V Q506 mutation, responsible for activated protein C resistance, was recently linked to thromboembolic disease. We therefore investigated the prevalence of biological risk factors in older hospital patients with venous thromboembolism. DESIGN: A 2-year study period. SETTING: Ivry sur Seine (Paris), France. PARTICIPANTS: Seventy-nine geriatric patients (60 women and 19 men, mean age 83+/-6.8 years, range 70-102 years) who had had at least one proven episode of venous thromboembolism were enrolled over a 2-year period. MEASUREMENTS: Lupus anticoagulant and antithrombin (AT), protein C (PC), and protein S (PS) levels were determined in plasma. The factor V Q506 mutation was detected on genomic DNA. RESULTS: Lupus anticoagulant was detected in two women, one of whom also had a high level of anticardiolipin IgG, leading to the diagnosis of an antiphospholipid syndrome. No hereditary deficiency in AT, PC, or PS was found, but one patient had an acquired AT deficiency. Interestingly, nine of the 79 patients (11.4%, six women and three men) were heterozygous for the factor V Q506 mutation, although none were homozygous. The only major risk factor for thrombosis identified in these patients was prolonged immobilization in four cases. Four of the nine patients who were heterozygous for the factor V Q506 mutation had recurrent thromboembolism, and two of these patients had been immobilized for long periods. CONCLUSIONS: This study confirms that hereditary deficiencies in coagulation inhibitors, and the lupus anticoagulant, are rarely involved in the pathogenesis of venous thromboembolism in older subjects. In contrast, the factor V Q506 mutation was frequently associated with thrombosis (11.4% of our patients) and should, therefore, be considered an important risk factor in the older people.

Activated Protein C Resistance↗
PubMed1997

Protein C and protein S deficiencies.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.

Chromosome Mapping↗
PubMed1996

[Exploration of hemostasis in current practice].

The exploration of coagulation in biological practice implies simple routine tests. A normal bleeding time associated with a normal platelet count allows to exclude any primary haemostasis abnormality. A prolonged bleeding time is most frequently the result of a thrombocytopenia, the etiology of which remains to be determined. A prolonged bleeding time is also observed in von Willebrand disease and in thrombopathia. Such diagnosis requires appropriate investigation. The exploration of the coagulation cascade includes prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen level. A congenital or acquired deficiency in one or more coagulation factors should be distinguished from a lupus anticoagulant or from the occurrence of an antibody against a coagulation factor. Indeed, a prolonged APTT is not always related to a bleeding risk. Only the identification of the coagulation abnormality can predict the bleeding or thrombotic risk during surgery. In each case, an appropriate preventive therapy should be prescribed.

Blood Coagulation Disorders↗
PubMed1995

Amino acids 225-235** of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex.

Protein C (PC) is a vitamin K-dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin-thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium-dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin-thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225-235 in an Elisa assay. The peptide inhibited PC activation by the thrombin-thrombomodulin complex. These data confirm that the calcium-binding loop of the serine-protease domain is involved in the interaction of PC with the thrombin-thrombomodulin complex.

Amino Acid Sequence↗
PubMed1995

Human thrombin variable region 1, including E39, is involved in interactions with alpha 1-antitrypsin M358R and protein C.

We used an antithrombin autoantibody (IgG D), the epitope of which encompasses ABE1 and amino acids located within variable region 1, to study thrombin interactions with R358 alpha 1-AT and protein C. IgG D inhibited the thrombin interaction with R358 alpha 1-AT, while hirugen had no effect, indicating that the interaction of R358 alpha 1-AT with thrombin may involve the VR1 subsite. We also obtained evidence that VR1 may be involved in the activation of protein C by thrombin in the absence of thrombomodulin. Moreover, IgG D attenuated the inhibitory effect of calcium ions during protein C activation by thrombin, probably by masking E39 within the VR1 site.

Amino Acid Sequence↗
PubMed1995

Identification of 15 different candidate causal point mutations and three polymorphisms in 19 patients with protein S deficiency using a scanning method for the analysis of the protein S active gene.

To screen for point mutations causing protein S deficiency, we used a sequence of techniques specifically for the study of the protein S active gene, PS alpha. This strategy comprises amplification of exons and intron/exon junctions by means of the polymerase chain reaction (PCR) and electrophoresis of the amplified fragments in polyacrylamide gel containing a gradient of denaturing agents (denaturing gradient gel electrophoresis). Only fragments with altered melting behavior are sequenced after asymmetric PCR. Beside the frequent polymorphism already described on Pro 626, we detected 18 different sequence variations by studying exons II, IV, V, VIII, X, and XV in 19 of 100 consecutive patients with protein S deficiency. Fifteen were candidate causal mutations, 4 of which were associated with a qualitative deficiency (type IIa or IIb). The remaining three sequence variations were probably polymorphisms.

Base Sequence↗
PubMed1995

Serum antibodies to distinct epitopes of the tissue-type plasminogen activator (t-PA) in patients with systemic lupus erythematosus.

Defective fibrinolysis due to decreased tissue-type plasminogen activator (t-PA) activity is a well-established finding in patients with systemic lupus erythematosus (SLE). The possibility that this decrease in t-PA activity may be related to the presence of autoantibodies directed against t-PA, and the possible role of these autoantibodies in the pathophysiology of fibrinolysis in SLE, were investigated. Serum samples from 115 SLE patients and 63 normal volunteers were analyzed for the presence of such antibodies. The search for antibodies to t-PA was performed by means of several systems, allowing for the identification of epitopes presented in different conformational physical states of t-PA: free or associated to its inhibitor (PAI-1) in plasma, specifically bound to fibrin surface, or passively adsorbed to solid supports. Antibodies in variable amounts were detected by all systems used; however, t-PA activity was not inhibited by the IgG fraction of the positive sera in a fibrin-agar fibrinolysis system. Moreover, the demonstration of serum anti-t-PA antibodies was not associated with clinical or laboratory abnormalities related to vasoocclusive episodes. These results indicate that, as in the case of other autoantibodies, their detection in serum does not imply their direct participation in the pathophysiology of thrombosis in SLE.

Adolescent↗
PubMed1994

An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation.

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion-binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration-dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma-thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion-binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

Anions↗
PubMed1994

Influence of six mutations of the protein C gene on the Gla domain conformation and calcium affinity.

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1994

A new assay based on thrombin generation inhibition to detect both protein C and protein S deficiencies in plasma.

Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.

Amino Acid Sequence↗
PubMed1993

Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity.

We describe five families presenting with type II hereditary protein C deficiency characterized by normal antigen and amidolytic activity levels but low anticoagulant activity. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining amplification by the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis of the amplified fragments, and direct sequencing of fragments displaying altered melting behavior. We detected five novel mutations. Three were located in the C-terminal part of the propeptide encoded by exon III: Arginine (Arg)-5 to tryptophan (Trp), Arg-1 to histidine (His), and Arg-1 to cysteine (Cys) mutations. The two others, located in exon IX, affected Arg 229 and serine (Ser) 252, which were respectively replaced by glutamine (Gln) and asparagine (Asn). DNA studies of the other exons from affected individuals showed no other abnormalities. These novel mutations provide further insight into the importance of the affected amino acids located close to the active site, near Asp 257, one of the three amino acids of the catalytic triad. The low anticoagulant activity of the abnormal protein C indicated that Arg 229 and Ser 252 play a key role during the interaction between protein C and its cofactor protein S, phospholipids, or factors Va and VIIIa. The Arg-1 to Cys mutation led to the dimerization of protein C with another plasmatic component, as evidenced by the presence in the plasma of a high molecular weight form of protein C that disappeared after reduction. No molecular mass abnormalities were observed in heavy and light chains of all other protein C mutants. In the five families explored, 9 (64%) of the 14 subjects bearing the mutations reported thrombotic events. This suggests that the protein C amino acids affected by the mutations are very important for the in vivo expression of the antithrombotic properties of protein C.

Amino Acid Sequence↗
PubMed1993

Sodium dodecyl sulfate-induced dissociation of complexes between human tissue plasminogen activator and its specific inhibitor.

The stability of complexes between serine proteinases and their inhibitors after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis has been claimed to indicate covalent bond formation. In this work we have investigated the effects of SDS on the stability of complexes between single-chain or two-chain tissue plasminogen activator (t-PA) and its inhibitor (PAI-1). Complexes formed by incubation of t-PA with PAI-1 for 15 min at 22 degrees C were further incubated with various amounts of SDS before being subjected to SDS-polyacrylamide gel electrophoresis. The molecular species in the gels were identified both by zymography or by autoradiography after immunoblotting with antibodies directed against either t-PA or PAI-1. It was demonstrated that the interaction of SDS with t-PA.PAI-1 complexes before electrophoresis resulted in a transition from the complexed state to the free forms of t-PA and PAI-1 in a time- and dose-dependent manner. The first-order dissociation rate constant in the presence of 35 mM SDS at 22 degrees C had a koff value of 1.4 x 10(-2) min-1, which corresponds to a half-life of 49.5 min. The t-PA released from the complexes was fibrinolytically active, whereas the released PAI-1 inhibited activator-dependent fibrinolysis. In a similar fashion, the well characterized nonacylated pair alpha 1-proteinase inhibitor-elastase was dissociated by SDS treatment, confirming the validity of our experimental approach to demonstrate the reversibility of t-PA.PAI-1 complexes. These results demonstrate that SDS-polyacrylamide gel electrophoresis traps the molecular species in the state in which the proteins existed prior to the analysis, and they suggest that under the conditions used, the interaction of t-PA with PAI-1 results in the formation of nonacylated reversible complexes. This phenomenon may be relevant to the pathophysiology of fibrinolysis and to the general mechanism of serine proteinase-inhibitor complex formation.

Humans↗
PubMed1991

The formation of complexes between human plasminogen activator inhibitor-1 (PAI-1) and sodium dodecyl sulfate: possible implication in the functional properties of PAI-1.

The effect of the anionic detergent sodium dodecyl sulfate (SDS) on human PAI-1 present in plasma, platelet extracts and endothelial cell cultures, was examined. Using the dye partitional extraction method of Mukerjee [1956) Anal. Chem. 28, 870-873) to quantitate ionic surfactants, and a discontinuous spectrophotometric assay for the titration of PAI-1 based on the measurement of residual active t-PA, we found (i) that SDS remains tightly bound to PAI-1 after equilibrium dialysis and (ii) that the activity of the latter was closely related to the amount of SDS carried over by the PAI-1 solution. The highest concentrations of SDS (ratio of SDS to protein greater than 0.1) were detected in the platelet-derived sources of PAI-1 which also showed the lowest residual t-PA activity. Moreover, it is demonstrated by SDS-PAGE and autoradiography that the tight binding of SDS to PAI-1 decreases its ability to form complexes with t-PA. Similar results were obtained with PAI-1 previously inactivated at 37 degrees C: the inability of PAI-1 to form complexes with t-PA was unchanged after SDS treatment. These observations suggest that the decrease in the residual activity of t-PA observed with the SDS-treated PAI-1 preparations is not related to an increase in the inhibitory activity of PAI-1. In fact, SDS was able to produce a decrease in both the binding of t-PA to fibrin and the activation of plasminogen by fibrin-bound t-PA. Bovine PAI-1 has been shown to exist in a latent SDS-activatable form. Our data indicate that such a form might not be present in the human sources of PAI-1 we have tested.

Blood Platelets↗
PubMed1990

A plasma clot lysis assay based on the release of fibrin degradation products: application to the diagnosis of hypofibrinolytic states.

Using a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (less than 2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 micrograms of fibrin degradation products per mg of fibrinogen (delta T 200). Two among the 27 patients with effective VO were bad responders with a delta T 200 less than 3 h (whereas all the others had delta T 200 greater than 10 h). These patients had respectively a deficient tPA release (delta tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal delta T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.

Adult↗
PubMed1988

Functional identification of t-PA in crude and purified systems.

In the present study the activation of glu-plasminogen by fibrin-bound t-PA was determined by integrating spectrophotometric and computer analysis of the reaction. Our aim was to determine the parameters of the activation and to characterize the functional activity of t-PA. Our results indicate that the t-PA present in purified or crude sources such as plasma or culture supernatants, can be specifically identified using the methodology described in this report.

Fibrin↗