Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall.

BACKGROUND: Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS: Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS: An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION: SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Familial risks of unusual forms of venous thrombosis: a nationwide epidemiological study in Sweden.

OBJECTIVE: This is the first nationwide study to determine familial risks of unusual forms of venous thrombosis amongst offspring of affected parents and amongst siblings. DESIGN AND SETTINGS: The Swedish Multigeneration Register of 0- to 75-year-old subjects was linked to the Hospital Discharge Register for the period 1987-2007. Standardized incidence ratios (SIRs) were calculated for individuals whose relatives were hospitalized for venous thromboembolism (VTE), as determined by the International Classification of Diseases, compared to those whose relatives were not affected by VTE. RESULTS: The total number of hospitalized patients with VTE was 45 362, of which 1824 (4.0%) were affected by a rare thrombotic condition. The familial SIRs in cases with a history of VTE in parents or siblings were significantly increased for migrating thrombophlebitis (1.81; 95% confidence interval (CI) 1.40-2.31), portal vein thrombosis (2.35; 95% CI 1.77-3.06), vena cava thrombosis (1.96; 95% CI 1.42-2.64) and cerebral venous thrombosis (1.74; 95% CI 1.30-2.28). Budd-Chiari syndrome (SIR, 0.92; 95% CI 0.24-2.38) and renal vein thrombosis (SIR, 1.72; 95% CI 0.62-3.77) were not significantly associated with parental or sibling history of VTE; however, these two conditions were very rare, and therefore, we cannot draw any definite conclusions from this finding. CONCLUSIONS: Family history is an important risk factor for most unusual forms of VTE. Moreover, even the paraneoplastic phenomenon, migrating thrombophlebitis (Trousseau's syndrome), is associated with a family history of VTE. Thus, our data suggest that most rare forms of VTE have a familial background.

COVID-19 susceptibility variants associate with blood clots, thrombophlebitis and circulatory diseases.

Epidemiological studies suggest that individuals with comorbid conditions including diabetes, chronic lung, inflammatory and vascular disease, are at higher risk of adverse COVID-19 outcomes. Genome-wide association studies have identified several loci associated with increased susceptibility and severity for COVID-19. However, it is not clear whether these associations are genetically determined or not. We used a Phenome-Wide Association (PheWAS) approach to investigate the role of genetically determined COVID-19 susceptibility on disease related outcomes. PheWAS analyses were performed in order to identify traits and diseases related to COVID-19 susceptibility and severity, evaluated through a predictive COVID-19 risk score. We utilised phenotypic data in up to 400,000 individuals from the UK Biobank, including Hospital Episode Statistics and General Practice data. We identified a spectrum of associations between both genetically determined COVID-19 susceptibility and severity with a number of traits. COVID-19 risk was associated with increased risk for phlebitis and thrombophlebitis (OR = 1.11, p = 5.36e-08). We also identified significant signals between COVID-19 susceptibility with blood clots in the leg (OR = 1.1, p = 1.66e-16) and with increased risk for blood clots in the lung (OR = 1.12, p = 1.45 e-10). Our study identifies significant association of genetically determined COVID-19 with increased blood clot events in leg and lungs. The reported associations between both COVID-19 susceptibility and severity and other diseases adds to the identification and stratification of individuals at increased risk, adverse outcomes and long-term effects.

Recurrent superficial venous thrombophlebitis because of mutations in the protein C and fibrinogen genes in a young Argentinian female.

: Hypodysfibrinogenemia and protein C deficiency are coagulopathies and in this report, we describe a young patient with both defects confirmed by molecular genetic tests. The patient was a 24-year-old woman referred for recurrent thrombophlebitis and finally deep venous thrombosis. Routine coagulation studies revealed mild decrease of protein C (0.49 IU, reference values 0.7-1.40 IU) and hypodysfibrinogenemia (0.88 g/l and 1.83 g/l for activity and antigen, respectively, reference values 2.0-4.0 g/l). Direct sequencing analyses were performed on FGA, FGB, and FGG genes to confirm hypodysfibrinogenemia and on the protein C gene to confirm protein C deficiency. As a result, the patient was shown to be heterozygous p.Ala82Gly in the FGG gene (Fibrinogen Dunedin) and for compound heterozygous missense mutation in protein C gene. To our knowledge, this is the first report on a case of combined dysfibrinogenemia and protein C deficiency confirmed by molecular genetic tests.

A case of hereditary protein S deficiency presenting with cerebral sinus venous thrombosis and deep vein thrombosis at high altitude.

A 35-year-old healthy male with no history of any past medical illness developed severe headache, vomiting and drowsiness while at high altitude (4,572 m) in the eastern Himalayan ranges. He was evacuated to a tertiary-care hospital where he was diagnosed to have cerebral sinus venous thrombosis (CSVT) on magnetic resonance imaging, with deep vein thrombosis (DVT) of his right popliteo-femoral vein on color Doppler study. Investigation for thrombophilia revealed protein S (PS) deficiency in this patient. Family screening revealed low levels of PS in two elder brothers. One brother had a history of 'stroke in young' at the age of 20 years with the other being asymptomatic. This established the hereditary nature of PS deficiency. We are not aware of any previously published report on hereditary PS deficiency combined with CSVT and DVT occurring at high altitude. However, 1 case of protein C deficiency with CSVT has been reported previously.

Integrating genomic based information into clinical warfarin (Coumadin) management: an illustrative case report.

Warfarin is a well established oral anticoagulant for the treatment of thromboembolic disorders. Warfarin therapy is complicated by a narrow therapeutic index and marked inter-individual dose variability with therapeutic doses ranging from 1 mg to 10 mg/day. Recently genetic variation and resultant drug metabolizing polymorphisms have been found to contribute to warfarin dose variability with resultant hemorrhagic or thromboembolic complications. Cytochrome P4502C9 alters the rate of warfarin metabolism and clearance. A second enzyme, Vitamin K Epoxide Reductase Complex (VKORC) binds and reduces Vitamin K which is necessary for activation of clotting Factors II, VII, IX and X. The VKORC1 gene encodes for Vitamin K Epoxide Reductase Complex subunit 1, a key component of VKORC. The combination of physiologic factors (30%), CYP2C9 variations (20%) and VKORC1 variants (25%) accounts for approximately 75% of warfarin dose variability. This illustrative case report demonstrates the clinical importance of this new information. Clinicians need to incorporate these new genomic findings into appropriate management of warfarin dose anticoagulation.

Deficiency of protein C in congenital thrombotic disease.

A family with a history of recurring thrombosis was studied to determine if a plasma protein deficiency could account for the observed disease. Protein C levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his father, and his paternal uncle, who are severely affected, had 38-49% of normal levels of protein C antigen, whereas unaffected family members had normal levels. There was no familial deficiency of antithrombin III and plasminogen. Because activated protein C is a potent in vitro anticoagulant enzyme and an in vivo profibrinolytic agent, it is suggested that the recurrent thrombotic disease in this family is due to an inherited deficiency in protein C.

Abnormal plasminogen. A hereditary molecular abnormality found in a patient with recurrent thrombosis.

A patient who suffered a recurring thrombosis over the last 15 yr has been investigated. The only abnormality found in this patient was a significantly depressed level of plasminogen activity in plasma. In spite of the depressed plasminogen activity, the patient was found to have a normal level of plasminogen antigen concentration. It was calculated that the activity per milligram of plasminogen of the patient was approximately one-half the values of normal subjects. The same discrepancy between biological activity and antigen concentration was found in the other members of the kindred. A niece was found to have practically no plasminogen activity but possessed a normal concentration of plasminogen antigen. Both her parents were found to have approximately half the normal plasminogen activity and normal antigen levels. These studies suggested that the molecular abnormality was inherited as an autosomal characteristic, and the family members who had half the normal levels of activity with normal plasminogen antigen were heterozygotes whereas the one with practically no plasminogen activity was homozygote. Subsequent studies showed that the pattern of gel electrofocusing of purified plasminogen of the heterozygotes consisted of 10 normal bands and 10 additional abnormal bands, each of which had a slightly higher isoelectric point than each corresponding normal component. This indicates that plasminogen of the heterozygote is a mixture of normal and abnormal molecules in an approximately equal amount, which was substantiated by active site titration of purified plasminogen preparations obtained from the propositus and a normal individual. The gel electrofocusing pattern of the homozygote consisted of abnormal bands only. The defect is a hereditary abnormality of plasminogen.

Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)

A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4 degrees C and rapidly at 41 degrees C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.

Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C.

Resistance to activated protein C (APC) is the most prevalent inherited cause of venous thrombosis. The APC resistance phenotype is associated with a single point mutation in the factor V gene, changing Arg506 in the APC cleavage site to a Gln. We have investigated 50 Swedish families with inherited APC resistance for this mutation and found it to be present in 47 of them. Perfect cosegregation between a low APC ratio and the presence of mutation was seen in 40 families. In seven families, the co-segregation was not perfect as 12 out of 57 APC-resistant family members were found to lack the mutation. Moreover, in three families with APC resistance, the factor V gene mutation was not found, suggesting another still unidentified cause of inherited APC resistance. Of 308 investigated families members, 146 were normal, 144 heterozygotes, and 18 homozygotes for the factor V gene mutation and there were significant differences in thrombosis-free survival curves between these groups. By age 33 yr, 8% of normals, 20% of heterozygotes, and 40% of homozygotes had had manifestation of venous thrombosis.

Lemierre's syndrome and genetic polymorphisms: a case report.

BACKGROUND: Lemierre's syndrome presents a classic clinical picture, the pathophysiology of which remains obscure. Attempts have been made to trace genetic predispositions that modify the host detection of pathogen or the resultant systemic reaction. CASE PRESENTATION: A 17-year old female, with no previous medical history, was admitted to the intensive care unit for septic shock, acute respiratory distress syndrome and Lemierre's syndrome. Her DNA was assayed for single nucleotide polymorphisms previously incriminated in the detection of the pathogen, the inflammatory response and the coagulation cascade. We observed functional variations in her Toll like 5 receptor (TLR 5) gene and two coagulation variations (Tissue Factor (TF) 603 and Plasminogen-Activator-Inhibitor-1 (PAI-1) 4G-4G homozygosity) associated with thrombotic events. CONCLUSION: The innate immune response and the prothrombogenic mutations could explain, at least in part, the symptoms of Lemierre's syndrome. Genomic study of several patients with Lemierre's syndrome may reveal its pathophysiology.

Does use of hormonal contraceptives among women with thrombogenic mutations increase their risk of venous thromboembolism? A systematic review.

Because use of combined oral contraceptives (COCs) confers some risk of venous thromboembolism (VTE), there is concern that this effect may be greater among women with thrombogenic mutations. We searched the MEDLINE and EMBASE databases for all articles published from January 1966 through September 2004 for evidence relevant to hormonal contraception and thrombogenic mutations. Of 301 articles identified by the search strategy, 16 evaluated COCs, and no studies were found for other hormonal methods. We used standard abstract forms and grading systems to summarize and assess the quality of the evidence. A total of 10 studies together provided "good" evidence of a greater risk of VTE (risk ratios of 1.3-25.1) and cerebral vein or cerebral sinus thrombosis among COC users with factor V Leiden mutation when compared with nonusers who have the mutation. The evidence for prothrombin and other thrombogenic mutations was not as strong as for factor V Leiden mutation. It is unclear whether the type of COC or duration of use modifies the risk of VTE among women with thrombogenic mutations.

The molecular genetics of familial venous thrombosis.

Inherited defects of antithrombin III, protein C, protein S, heparin cofactor II, plasminogen and the fibrinogens are thought to be responsible for between 10 and 15% of all patients presenting with recurrent venous thrombosis. The structure, function and expression of these genes and the nature of the gene lesions underlying the deficiency states are reviewed in detail.

Risk factors for venous thrombosis: prevalence, risk, and interaction.

Annually, 1 in 1,000 individuals is affected by venous thrombosis. Risk factors that are known to increase the risk of thrombosis may be either genetic or acquired, or have a combined origin. Many of these risk factors are very frequent, among which several have been recently identified, such as resistance to activated protein C by factor V Leiden, hyperhomocysteinemia, high levels of factors VIII, as well as the classical acquired risk factors, such as surgery and malignancies. When the prevalence of risk factors is high, it becomes likely that in some individuals two or more risk factors will be present simultaneously. The question "What happens to the risk in these circumstances?" is one involving interaction, also known as effect modification or synergy. In this article we review the prevalence and risk estimates for the various genetic and acquired risk factors for venous thrombosis, discuss the concept of interaction, and give an overview of the evidence for interaction of these risk factors.

Regulation of thrombin formation by activated protein C: effect of the factor V Leiden mutation.

Activated Protein C (APC) resistance, one of the most common genetic risk factors for venous thrombosis, is caused by a single base mutation (G1691-->A) in the factor V (FV) gene resulting in the replacement of Arg506 by Gln at a predominant cleavage site for APC. Great progress in understanding the mechanism of downregulation of FVa activity via the protein C pathway has been achieved by studying APC-mediated inactivation of FVa purified from homozygous APC-resistant individuals. This review briefly summarizes the role of FVa in prothrombin activation and the structure-function relationship of FV and FVa. Subsequently, APC-dependent inactivation of FVa and FVa Leiden and its modulation by protein S and factor Xa in model systems containing purified proteins is discussed. FV also has a function in increasing the inactivation of FVIII/VIIIa by APC. This cofactor activity appears diminished in FV Leiden. Thus, an intricate mechanism of regulation of thrombin formation via the protein C pathway is starting to emerge. Extensive studies in plasma milieu will be needed to gain more insight into the relation between the presence of FV Leiden and impaired downregulation of thrombin formation in APC-resistant individuals.

Familial thrombophilia: a complex genetic disorder.

Familial thrombosis has long been considered as an autosomal dominant trait, caused by a dominant gene defect with a reduced penetrance for the disease. Recently, this view has changed and today familial thrombophilia is considered as a complex genetic disorder caused by the segregation of two or more gene defects (known and unknown) in a family. Here we briefly discuss the known genetic defects (protein C, protein S, and antithrombin deficiency and activated protein C resistance associated with factor V Leiden) with special focus on the relation between gene mutation and plasma abnormality and on the association between abnormality and thrombosis in affected families and in the population. Finally, the evidence is reviewed that indicates familial thrombosis as an oligogenetic disorder and on the basis of these data strategies are discussed for the identification of new genetic risk factors for thrombosis via a genetic approach.

Antithrombin Cambridge II, 384 Ala to Ser. Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins.

Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only function abnormality was a minor but consistent decrease in the heparin-induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G----T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re-entry into the A-sheet of the molecule is necessary for optimal inhibitory activity.

Congenital antithrombin III deficiency. Incidence and clinical features.

Antithrombin III (ATIII) deficiency is inherited as an autosomal dominant trait. Three types of ATIII deficiency are recognized clinically. The prevalence of ATIII deficiency is uncertain; it has been estimated to occur in between one in 2,000 and one in 20,000 subjects. ATIII deficiency is found in between 4 and 6 percent of young patients with venous thrombosis, similar to but slightly lower than the prevalence of protein C and protein S deficiency in young subjects with thrombosis. The chances of finding a deficiency is increased if there is a history of familial or recurrent venous thrombosis. Cross-sectional reports in the literature are that between 30 and 80 percent of carriers have thrombosis. Thrombosis is uncommon in the first decade, but the risk rises sharply between the ages of 15 and 30. The major clinical manifestations of ATIII deficiency are young age at onset, idiopathic thrombosis, family history, and recurrent venous thromboembolism. Pregnancy and surgery are predisposing factors. Approaches to prophylaxis and treatment are discussed.

Congenital antithrombin III deficiency: insights into the pathogenesis of the hypercoagulable state and its management using markers of hemostatic system activation.

Hereditary antithrombin III (ATIII) deficiency predisposes patients to venous thrombosis. The prothrombin fragment F1+2 radioimmunoassay demonstrates that many asymptomatic patients with this disorder not receiving antithrombotic therapy have elevated plasma factor Xa activity. The hemostatic system hyperactivity as measured by this assay could be specifically corrected by rising plasma ATIII levels of several persons into the normal range. This indicates that the prethrombotic state can be defined as an imbalance between the production and inhibition of factor Xa enzymatic activity. The effects of warfarin on factor Xa enzymatic activity in persons with congenital ATIII deficiency have also been evaluated. At equivalent intensities of oral anticoagulation, the mean plasma F1+2 level in patients with ATIII deficiency was significantly elevated as compared with anticoagulated persons without this inherited thrombotic disorder. It is concluded that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-ATIII mechanism. This suggests that the F1+2 radioimmunoassay can be employed to improve the understanding of the hypercoagulable state associated with antithrombin III deficiency as well as to develop more effective treatment strategies to prevent thromboembolic events in patients with this disorder.

Clinical significance of the FV:Q506 mutation in unselected oncology patients.

PURPOSE: A common germline mutation in the factor V gene (FV:Q506) has been associated with hypercoagulability in families with heritable predisposition to thrombosis. We examined the prevalence and clinical significance of the FV:Q506 mutation in cancer patients. PATIENTS AND METHODS: We performed a retrospective cohort study by examining 353 consecutive, unselected patients in a general hematology/oncology clinic. We ascertained risk factors, obtained the clinical clotting history, and determined the heterozygous or homozygous presence of the FV:Q506 allele for each patient. RESULTS: We detected a germline mutation in 5.4% (19 of 353) of patients, of whom 18 were heterozygous and 1 was homozygous for the FV:Q506 mutant allele. In 17 of 18 heterozygous patients, there was no history of venous thrombosis or catheter-associated thrombosis. These asymptomatic patients included 13 patients who had been diagnosed with cancer or leukemia for a mean of 66.2 months (median 69) and had received a variety of local and systemic treatments. In contrast, 1 of 18 heterozygous and 1 of 1 homozygous patients had developed deep vein thrombosis that was associated, respectively, with either recurrent thrombotic events or a strong family history for pulmonary embolus. CONCLUSIONS: Routine screening for the FV:Q506 mutation in cancer patients without a personal or family history for venous thrombosis is not helpful in guiding management. In contrast, an episode of venous thrombosis in a patient with a mutant germline FV:Q506 allele was associated with recurrent thrombotic events. These findings suggest that patients heterozygous for the FV:Q506 allele may require an independent "susceptibility" element to manifest a venous hypercoagulable state. In addition, only 2 of 25 clinic patients with a venous clot carried the FV:Q506 allele suggesting this genetic defect plays a minor role in the hypercoagulable state of cancer.