A new method to quantify ß-antithrombin glycoform in plasma reveals increased levels during the acute stroke event.

INTRODUCTION: ß-antithrombin, the minor antithrombin glycoform in plasma, is probably the major thrombin inhibitor in vivo because of its high heparin affinity. The levels and variability of this glycoform in general population and its relevance in thromboembolic diseases is unknown since there is no specific method to measure this glycoform in clinical samples. METHODS: Plasma and recombinant α- and ß-antithrombins were purified by heparin affinity chromatography. An anti-FXa chromogenic method in presence of pentassacharide was used with two NaCl concentrations (15mM and 1.1M). This method was applied to plasma samples from 97 healthy subjects and 117 consecutive patients with ischemic cerebrovascular disease during the acute event and one year later. SERPINC1 sequencing was done in cases with antithrombin deficiency. RESULTS: High salt concentrations specifically restricted the pentassacharide-induced activation of antithrombin to the ß glycoform. ß-antithrombin displayed a normal distribution in the general population (89.5%-103.5%), with no significant variations according to age or sex. In patients, whole antithrombin values remained within the normal range. Only five cases had antithrombin deficiency during the thrombotic event, one carrying the L99F mutation in SERPINC1. Interestingly, both ß-antithrombin and the ß/whole antithrombin ratio were significantly higher in patients during the acute event but normalized after one year. CONCLUSIONS: We have developed a rapid, simple, sensitive and specific method to quantify ß-antithrombin activity using 1µL of plasma. ß-antithrombin significantly increases in patients with ischemic cerebrovascular disease during the acute event, probably by its release from the vasculature.

Genetic variants of the extra-large stimulatory Gs protein alpha-subunit and risk of thrombotic and haemorrhagic disorders.

A polymorphism of the gene encoding the extra-large stimulatory G-protein alpha-subunit (XLalphas), originally identified in three patients with a bleeding tendency, involved a 36-bp insertion and two missense changes. A paternally-inherited insertion displayed a moderate platelet Gsalpha over-expression, which lead to platelet hypo-reactivity. These data prompted us to investigate the genetic, functional and clinical relevance of this polymorphism in the Mediterranean population. We included 414 healthy subjects and three case/control studies: 263 consecutive patients with a first episode of primary intracerebral haemorrhage, 195 patients with deep venous thrombosis, and 104 patients with cerebrovascular disease. Controls were selected by approximating criteria to match selected risk factors to patients. Moreover, we performed studies of platelet function. We developed a simple method to determine the methylated allele, by digestion of genomic DNA with Sma I before polymerase chain reaction amplification. We identified two new rare variants, resulting from the loss of repeat units 7 and 5. The AB genotype was present in 3.6% of healthy population and the prevalence of the B allele was similar among cases and controls. Accordingly, the non-methylated B allele did not modify either the expression of platelet Gsalpha or the platelet response to Gs-agonists. Thus, our study suggests a minor functional role of XLalphas polymorphism in thrombotic or in haemorrhagic disorders.

Role of factor XIII Val 34 Leu polymorphism in patients with migraine.

At present, it is contradictory to determine if the combination of certain prothrombotic polymorphisms and migraine increases the risk to develop ischaemic cerebrovascular disease. Recently, the common Val34Leu polymorphism of the A-chain factor XIII gene, associated with variations in factor XIII activity, has been suggested to play a significant role in the development of arterial and venous thrombotic disorders. We analysed the prevalence of this polymorphism in 17 patients with coexisting ischaemic cerebrovascular disease and migraine (5 with aura, and 12 without aura), 89 patients with migraine (43 with aura, and 46 without aura), 116 patients with ischaemic cerebrovascular disease, and 467 healthy Caucasian controls from the South of Spain. Genomic PCR amplification, using a mutated oligonucleotide, and allele-specific restriction assays were used for genotyping. The factor XIII Leu 34 variant was present in 47.1; 40.5; 34.9; and 35.1% of patients with coexisting ischaemic cerebrovascular disease and migraine, ischaemic cerebrovascular disease, migraine, and control subjects, respectively. These data suggest that the factor XIII Leu 34 allele does not play a protective role against these disorders in our population.

Polymorphisms of clotting factors modify the risk for primary intracranial hemorrhage.

Intracranial hemorrhage is the third most frequent cause of cerebrovascular disease, but few genetic risk factors have been associated with its development. Recently, it has been reported that some polymorphisms that affect clotting factors increase the risk for thrombosis. However, reports have analyzed the effect of polymorphisms influencing the hemostatic state in bleeding disorders insufficiently. A case-control study was conducted of 201 patients with spontaneous intracranial hemorrhage and 201 control subjects matched for age, race, sex, and selected risk factors (hypertension, smoking, and alcohol consumption). Genomic polymerase chain reaction was used to analyze the prevalence of 4 polymorphisms: factor V Leiden, prothrombin 20210A, factor VII-323 Del/Ins of a decanucleotide, and factor XIII V34L. Subjects with factor V Leiden had decreased risk for spontaneous intracranial hemorrhage (odds ratio, 0.19; 95% confidence interval, 0.03-0.95). The frequency of the prothrombin 20210A/G genotype was also lower among patients than controls (1.5% vs 3%, respectively). Moreover, carriers of the -323 Ins allele of factor VII had a 1.54-fold risk for intracranial hemorrhage (95% CI, 1.03-2.72). Finally, no significant differences were observed in the prevalence of factor XIII V34L polymorphism between patients and controls. Therefore, new genetic factors affecting the risk for spontaneous intracranial hemorrhage were identified. These data, together with the relevance of these polymorphisms in thrombotic diseases, support the idea that a polymorphism may play opposite roles in thrombosis and hemorrhage, suggesting an explanation for the high frequency of these polymorphisms in the general population.

Polymorphisms of P-selectin glycoprotein ligand-1 are associated with neutrophil-platelet adhesion and with ischaemic cerebrovascular disease.

P-selectin glycoprotein ligand (PSGL-1) shares common features with platelet glycoprotein Ibalpha. A recently described polymorphism in this receptor that results in a variable number of tandem repeats (VNTR) sequence present either 16, 15 or 14 times (alleles A, B or C) could, similar to GPIbalpha, be functionally relevant. The allelic frequency of this polymorphism was investigated in 469 individuals from the south of Spain, and was similar to that previously described in other Caucasian populations: 85% A, 14% B and 1% C alleles. We identified two new polymorphisms genetically linked to the C isoform, resulting in the Ser273Phe and Met274Val changes. To assess the functional consequence of the polymorphisms in the receptor, we performed flow cytometric analysis of P-selectin dependent neutrophil-platelet interaction. Neutrophils carrying the shortest C allele and the amino acid variations in residues 273 and 274 exhibited a significantly lower capacity to bind activated platelets than A/B and A/A samples (mean fluorescence intensity of CD42b+ neutrophils 262 versus 303 and 319 respectively, P < 0.05). The distribution of the VNTR was analysed in three case-control studies including 104 cerebrovascular (CVD), 101 coronary heart disease (CHD) and 150 deep venous thrombosis (DVT) patients. The results showed that smaller (B and C) alleles seem to be associated with a lower risk of developing CVD (P = 0.008) but not to be related to CHD or DVT. In conclusion, polymorphisms of the PSGL-1 receptor may influence the neutrophil-platelet binding, and represent a risk factor for CVD.

The FXIII Val34Leu polymorphism in venous and arterial thromboembolism.

BACKGROUND AND OBJECTIVE: Several hereditary disorders affecting coagulation factors have been identified as prothrombotic risk factors. Recently, the common Val34Leu polymorphism of the A-chain factor XIII gene, associated with high factor XIII activity, has been identified as a protective genetic factor against occlusive arterial and venous diseases in British and Finnish populations. The aim of our study was to investigate the role of this polymorphism in arterial and venous thromboembolic disorders in a distinct population. DESIGN AND METHODS: We analyzed the prevalence of this polymorphism in three case/control studies of consecutive patients from the south of Spain diagnosed as having acute coronary syndromes (101), acute cerebrovascular events (104), and deep venous thrombosis (97). RESULTS: No significant differences were detected in the prevalence of genotypes or alleles between patients and controls. INTERPRETATION AND CONCLUSIONS: The Leu 34 allele does not play an important role in the development of thromboembolic episodes in the Spanish population.

A common polymorphism flanking the ATG initiator codon of GPIb alpha does not affect expression and is not a major risk factor for arterial thrombosis.

The platelet membrane glycoprotein (GP) Ib alpha plays a key role in the initial formation of thrombi. Polymorphisms (VNTR and HPA-2) in this receptor are associated with increased risk of coronary heart disease (CHD) and cerebral vascular disease (CVD). We investigated whether a recently described polymorphism (S/R), due to a single base change (T-->C) five nucleotides upstream the initiator codon of GPIb alpha, might influence the expression of the protein, and be implicated in the development of arterial thrombosis. One hundred and thirty nine healthy individuals provided blood samples for DNA analysis of platelet GPIb alpha polymorphisms, and for flow cytometric analysis of the surface expression of the receptor. A group of 20 S/R normal individuals and an identical number of S/S participants, age and sex matched, was investigated for the analysis of the density of various platelet receptors. The distribution of the S/R polymorphism was also analyzed in two case/control studies including 104 CVD patients, 101 CHD patients, and one control age, sex, and environmental risk factors matched for each case patient. Surface density of GPIb alpha showed no wide variations between individuals, was not influenced by the presence of S or R alleles, nor associated with the VNTR or HPA-2 polymorphisms. The prevalence of the S/R genotype among CVD and CHD patients was not distinct from that in the control groups. We conclude that the S/R polymorphism of GPIb alpha, flanking the initiator codon of the receptor, does not seem to be associated with surface levels of the protein, and is not an independent risk factor for arterial thrombosis.

Factor XIII Val34Leu polymorphism in primary intracerebral haemorrhage.

INTRODUCTION: Recently, the common Val34Leu polymorphism of the A-chain factor XIII gene, associated with high factor XIII activity, has been identified as a protective genetic factor against occlusive arterial and venous diseases. Moreover, this polymorphism has been suggested to be the first one to increase the risk of cerebral haemorrhage in a small number of Caucasian patients. The aim of our study was to investigate the role of this polymorphism in patients with primary intracerebral haemorrhage from a distinct population. MATERIAL AND METHODS: Patients with non-traumatic primary intracerebral haemorrhage (n=116), age-, race-, sex- and risk factor-matched controls (n=116), and individuals from the general population (n=465) were genotyped for the factor XIII Val34Leu polymorphism by polymerase chain reaction and allele specific restriction assay. The relationships of the Val/Leu genotype with distinct intracerebral haemorrhagic risk factors and with early mortality associated with the haemorrhagic episode were also analysed. RESULTS: No statistical difference in terms of prevalence was detected between patients (P=0.190) and controls (P=0.181). The frequency of the FXIII Leu34 allele was similar in the general population (P=0.191). CONCLUSION: The results suggest that the Leu 34 allele of the A-chain factor XIII gene has a minor role in the development of non-traumatic primary intracerebral haemorrhage. Moreover, the simultaneous presence of the Leu 34 allele with selected risk factors for this disease does not increase the risk of developing this disease.

Prothrombotic genetic risk factors in patients with coexisting migraine and ischemic cerebrovascular disease.

The role of hemostatic elements in stroke has been clearly defined but several prothrombotic polymorphisms of hemostatic factors, important for other thromboembolic disorders, seem not to be very significant in stroke. Recently, the high prevalence of factor V Leiden in patients with stroke and a history of migraine has suggested an association between migraine and prothrombotic genetic risk factors. Stroke being a multifactorial disease, the aim of this study was to test whether prothrombotic tendencies increase the risk of stroke in patients with migraine. We determined the prevalence of four prothrombotic genetic risk factors (factor V R/Q 506, factor II 20210 G/A, decanucleotide insertion/deletion in the factor VII promoter, and the platelet HPA-1 alloantigen system) in 17 patients with coexisting ischemic cerebrovascular disease and migraine, 107 patients with ischemic cerebrovascular disease, 106 patients with migraine, and 202 control subjects. Genotyping for all polymorphisms analyzed in our study were performed after specific genomic polymerase chain reaction, and confirmed by single-strain conformation polymorphism analysis. In the group of patients with coexisting ischemic cerebrovascular disease and migraine, the prevalence of prothrombotic genotypes (factor V Leiden, 5.8%; factor II 20210 A, 0%; factor VII A1, 70.6%; and HPA-1b, 35.3%) was similar to that obtained in all other groups. We can conclude that the studied polymorphisms do not seem to be associated with the development of ischemic cerebrovascular disease in those patients with migraine.

Polymorphisms of platelet membrane glycoprotein Ib associated with arterial thrombotic disease.

Platelet membrane glycoprotein Ibalpha (GPIbalpha) is a major receptor for von Willebrand factor and thrombin, which plays a key role in the initial development of thrombi. Two polymorphisms (HPA-2 and VNTR) that affect phenotype have been described in GPIbalpha. The relevance of these polymorphisms to thrombotic disease was investigated by genotypic identification in three case-control studies: 104 case patients with acute cerebrovascular disease (CVD), 101 case patients with acute coronary heart disease (CHD), 95 patients with deep venous thrombosis (DVT), and one control age-, sex-, and race-matched for each case patient. Results show that the C/B genotype of the VNTR and the HPA-2b polymorphisms of GPIbalpha are strongly associated with increased risk of coronary heart disease and cerebral vascular disease but not with deep vein thrombosis. These two polymorphisms of GPIbalpha may represent newly identified risk factors for arterial thrombotic disease, but not for venous thrombosis.

Migraine and prothrombotic genetic risk factors.

It has been suggested that during attacks of migraine both platelet activation and plasma coagulability are increased. We investigated the prevalence of several prothrombotic genetic risk factors in patients with migraine: factor V R/Q 506, factor II 20210 G/A, decanucleotide insertion/deletion in the factor VII promoter, and the platelet HPA-1 and HPA-2 alloantigenic systems, by genotypic identification in an age- and sex-matched case-control study including 106 patients with migraine (49 with aura, and 57 without aura). The prevalence of all genotypes was similar among case patients and controls. No association in relation to the type of migraine was detected in the factor II, factor VII, HPA-1, or HPA-2 polymorphisms. Our results showed a high prevalence of factor V Leiden in those patients with migraine with aura (6.1%), though that association was not statistically significant. The studied prothrombotic genetic factors do not seem to be associated with the development of migraine and, therefore, are not likely relevant in the previously reported hypercoagulability and platelet hyperaggregability in this disease.

HPA-1 genotype in arterial thrombosis--role of HPA-1b polymorphism in platelet function.

Recently, the HPA-1b (PlA2) polymorphism of the platelet glycoprotein IIIa has been suggested as a genetic risk factor for coronary artery disease. We conducted two case-control studies of 103 patients with ischaemic cerebrovascular disease (CVD) and 101 patients with ischaemic heart disease (IHD). The groups were matched for age, race and sex. No significant differences regarding selected risk factors (hypertension, diabetes mellitus, hypercholesterolaemia and smoking) were found between case patients and controls. Moreover, we investigated 286 normal individuals from the Mediterranean area. Genotyping of HPA-1 was performed by PCR-allelic specific restriction and single-strand conformation polymorphism analysis. The prevalence of HPA-1b was similar among case patients and controls (29.2% vs. 25.3% and 26.7% vs. 34.6% for CVD and IHD case-control studies, respectively). The HPA-1b allele was found in 36.4% of the normal population. Finally, the analysis of platelet function in nine controls with the three possible HPA-1 genotypes (three a/a, three a/b and three b/b) indicates that HPA-1b genotype does not modify either the in vitro platelet aggregation and activation profile, nor the GP IIb/IIIa interaction with fibrinogen or von Willebrand factor. Our results do not support the role of HPA-1b polymorphism as an inherited risk factor for arterial thrombotic disease.

Factor-V (Arg506 --> Gln) mutation in ischemic cerebrovascular disease.

Factor V Leiden is already well established to be involved in venous thrombosis but not in coronary heart disease. Results are conflicting and even antagonistic in cerebrovascular disease (CVD). In this study we examine the prevalence of factor V Leiden in 125 consecutive patients with ischemic CVD 12 additional selected CVD patients with the first CVD episode before the age of 45 years, and 102 controls from the same area. Identification of the mutation was achieved using two molecular methods. The prevalence of factor V Leiden in patients with CVD was similar to that in the normal population. The presence of the mutation was independent of the age and severity of the ischemic episode. Finally, no significant clinical differences were found between patients with and those without factor V Leiden. Therefore, factor V Leiden cannot be considered as a genetic risk factor for CVD.

Detection of factor V Leiden from a drop of blood by PCR-SSCP.

Recently, a point mutation in the coagulation Factor V gene (G to A in position 1691) has been identified which makes the mutant Factor V (called Factor V Leiden) resistant to activated protein C. This defect is a well established genetic risk factor in venous thrombosis. Because of the high prevalence of Factor V Leiden in normal population (2-7%), it would be reasonable to perform a rapid and simple method for screening the genetic abnormality in population at risk. We have developed a simple, reproducible, rapid and cheap procedure that, using PCR and SSCP, allows the identification of the mutation responsible for Factor V Leiden. Specificity of this method has been tested in 319 samples: 304 normal, 14 heterozygous and 1 homozygous for Factor V Leiden. This assay allows non-isotopic Factor V Leiden identification by using frozen whole blood in 3 h. All these features make this test adequate for routine screening of this mutation in a large number of samples.

[Testicular seminoma manifested by carcinomatous neuropathy: an unusual presentation. Review of the literature]. / Seminoma testicular manifestado por neuropatía carcinomatosa: una presentación poco común. Revisión de la literatura.

Contribution of one patient with tetraparesic picture originated by a major sensory-motor neuropathy, due to paraneoplastic syndrome produced by a testicular seminoma. The patient underwent inguinal orchiectomy and radiotherapy, with favourable evolution of his neurological symptomatology. Review of the literature and confirmation of the extraordinary rarity of such clinical picture.