Robert A. S. Ariëns

The genetics of haemostasis: a twin study

BACKGROUND The concentrations of fibrinogen, factor VII and VIII, von Willebrand factor, plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator have been associated with coronary-heart disease. In addition, polymorphisms in the genes coding for fibrinogen, factor VII, PAI-1, and factor XIII have been reported to affect both protein concentrations and cardiovascular disease risk. METHODS We did a classic twin study to assess heritabilities of these haemostatic factors. We enrolled 1002 female twins; 149 pairs of monozygotic and 352 pairs of dizygotic twins. 89 monozygotic and 196 dizygotic twin pairs were analysed for factor VII. FINDINGS Quantitative genetic model fitting showed that genetic factors contributed to about 41-75% of the variation in concentrations of fibrinogen, factor VII, factor VIII, PAI-1, tissue plasminogen activator, factor XIII A-subunit and B-subunit, and von Willebrand factor. Factor XIII activity showed higher (82%) and factor XIIa lower (38%) heritability. INTERPRETATION We have shown that genetic factors have a major effect on plasma concentrations of haemostatic proteins. Our results stress the importance of research into the genetic regulation of proteins involved in haemostasis and atherothrombotic disorders, including myocardial infarction and stroke.

Fibrin Clot Structure and Function: A Role in the Pathophysiology of Arterial and Venous Thromboembolic Diseases

The formation of fibrin clots that are relatively resistant to lysis represents the final step in blood coagulation. We discuss the genetic and environmental regulators of fibrin structure in relation to thrombotic disease. In addition, we discuss the implications of fibrin structure for treatment of thrombosis. Fibrin clots composed of compact, highly branched networks with thin fibers are resistant to lysis. Altered fibrin structure has consistently been reported in patients with several diseases complicated by thromboembolic events, including patients with acute or prior myocardial infarction, ischemic stroke, and venous thromboembolism. Relatives of patients with myocardial infarction or venous thromboembolism display similar fibrin abnormalities. Low-dose aspirin, statins, lowering of homocysteine, better diabetes control, smoking cessation, and suppression of inflammatory response increase clot permeability and susceptibility to lysis. Growing evidence indicates that abnormal fibrin properties represent a novel risk factor for arterial and venous thrombotic events, particularly of unknown etiology in young and middle-aged patients.

Tissue-factor antigen and activity in human coronary atherosclerotic plaques.

BACKGROUND Coronary atherosclerotic-plaque thrombosis is a key event in the pathogenesis of unstable angina and myocardial infarction. Although plaque rupture or fissuring frequently occurs in atherosclerosis, only a small proportion of ruptured plaques develop thromboses. METHODS Tissue-factor antigen and activity were measured in atherectomy samples from 50 consecutive patients with coronary artery disease (stable angina n = 19, unstable angina n = 24, and myocardial infarction n = 7). FINDINGS Median tissue-factor antigen and activity concentrations were significantly higher in plaques from patients with unstable angina and myocardial infarction than in those from patients with stable angina (antigen: 66.1 pg/mg [interquartile range 43.8-82.5] vs 32.4 pg/mg [9.8-43.4], p = 0.0001; activity: 0.22 mU/mg [0.17-0.41] vs 0.13 mU/mg [0.05-0.16], p = 0.0004). INTERPRETATION Tissue-factor, an initiator of the coagulation cascade, may account for the different thrombotic responses to the rupture of human coronary atherosclerotic plaques.

Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk

BACKGROUND Polymorphisms in the fibrinogen and factor XIII genes are associated with atherothrombotic risk, but clinical studies have produced inconsistent results and laboratory studies have not explained these findings. We aimed to investigate interactions between polymorphisms in the factor XIII and fibrinogen genes, fibrinogen concentrations, and other cardiovascular risk factors in relation to fibrin structure and function. METHODS We used permeation analysis and electron microscopy to investigate interactions between fibrin structure, factor XIII Val34Leu, fibrinogen Aalpha Thr312Ala, fibrinogen Bbeta Arg448Lys, and fibrinogen concentrations in plasma and purified systems. FINDINGS Increased fibrinogen concentrations were associated with decreases in permeability, with tighter clot structures in the presence of factor XIII 34Val alleles compared with those in the presence of 34Leu alleles. Findings were confirmed by scanning electron microscopy of fibrin. Similar changes in permeability were noted for Aalpha fibrinogen 312Ala compared with that for 312Thr. INTERPRETATION Our results show interactions between coding polymorphisms in fibrinogen and factor XIII and fibrinogen concentrations that modify fibrin and explain the apparent paradox between epidemiological studies of factor XIII 34Leu and reported in-vitro effects on fibrin structure and function. We suggest a potential complexity of gene-gene and gene-environment interactions in determining cardiovascular risk.

Altered Fibrin Clot Structure in the Healthy Relatives of Patients With Premature Coronary Artery Disease

Background—A family history of premature coronary artery disease (CAD) is an independent cardiovascular risk factor. Fibrin clots composed of dense fiber networks are found in young CAD patients and may occur in the relatives of such individuals. Methods and Results—The ex vivo fibrin structure of 100 healthy male relatives of patients with premature CAD and 100 age-matched control subjects was assessed by measurement of permeability (Ks), fiber mass-length ratio (&mgr;), and turbidity (lag phase and maximum absorbency [max &Dgr;Abs]). Scanning electron microscopy was performed on selected samples. Relatives and controls shared similar levels of conventional cardiovascular risk factors. Ks was lower in relatives than in controls, 12.2 (11.1 to 13.3) versus 15.2 (14.0 to 16.5) ×10−9 cm2 (P <0.001), associated with a smaller decrease in &mgr;, 8.5 (7.7 to 9.2) versus 9.7 (8.9 to 10.5) × 1013 Da/cm (P <0.05), respectively. Lag phase was shorter in relatives than in controls, 39 (37 to 41) versus 47 (44 to 50) seconds (P <0.001), and max &Dgr;Abs was higher in relatives, 0.78 (0.74 to 0.82) versus 0.71 (0.67 to 0.74) in controls (P =0.02), which indicates the presence of thicker fibers in relatives. After adjustment for fibrinogen levels, lag phase and Ks remained significantly different between relatives and control subjects. Scanning electron microscopy images confirmed increased fiber diameter in relatives, possibly of reduced density. Factor XIII Val34Leu and fibrinogen A&agr; Thr312Ala and B&bgr; -455 G/A showed no association with clot structure. Conclusions—The male relatives of patients with premature CAD form fibrin clots that begin polymerization more quickly, have thicker fibers, and are less permeable than those of control subjects.

Genetic and Environmental Determinants of Fibrin Structure and Function: Relevance to Clinical Disease

The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. The structure of the fibrin clot and the genetic and environmental factors that modify it have effects on its biological function. Alterations in fibrin structure and function have implications for the clinical presentation of vascular disease. This review briefly describes the key features involved in the formation of a fibrin clot, its typical structure, and function. This is followed by a review of the current literature on genetic and environmental influences on fibrin structure/function and the relationship to clinical disease.

Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin

Activated platelets secrete a negatively charged polymer, polyphosphate (polyP). Here, we explore the interactions of polyP with fibrin(ogen) and its effect on fibrin structure and fibrinolysis. Electrophoretic mobility and binding assays indicate that polyP interacts with fibrinogen and soluble fibrin. Clots formed in the presence of polyP exhibited reduced turbidity and permeability indicative of a tighter fibrin network, but these changes were not related to cross-linking or fibrinopeptide release. Microscopy showed a change in fibrin distribution in clots formed with polyP; with formation of tight aggregates of fibrin fibers interspaced with large pores in contrast to homogenous fiber distribution in control clots. Lysis by tissue plasminogen activator (tPA) and plasminogen or plasmin was delayed in clots formed with polyP and depended on both the activator and polyP concentration. Adding polyP to the clot after fibrin formation or to repolymerizing soluble fibrin did not affect lysis, indicating changes induced by polyP occur at the level of conversion of fibrinogen to fibrin. Surface plasmon resonance showed that the presence of polyP reduced the binding of both plasminogen and tPA to partially lysed fibrin surfaces. These data show that polyP directly influences fibrin architecture and attenuates fibrinolysis through reduced binding of fibrinolytic proteins.

Subunit Antigen and Activity Levels of Blood Coagulation Factor XIII in Healthy Individuals: Relation to Sex, Age, Smoking, and Hypertension

Factor (F) XIII covalently cross-links and stabilizes the fibrin-clot. Recent evidence suggests a role for FXIII in atherothrombotic diseases, but no information is available regarding the association of FXIII with common risk factors. The aim of this study was to investigate the relationship of FXIII with age, sex, smoking, and hypertension. Plasma levels of FXIII A-subunit antigen, FXIII B-subunit antigen, and FXIII cross-linking activity were measured in 612 healthy individuals (250 men and 362 women). FXIII A- and B-subunit levels were correlated significantly with age in both men (r=0.21, P=0.001, and r=0.17, P=0.008, respectively) and women (r=0.20, P<0.0005, and r=0.13, P=0.011, respectively). FXIII B-subunit levels and activity were correlated significantly with FXIII A-subunit levels (r=0.60, P<0.0005, and r=0.14, P<0.0005, respectively) and fibrinogen (r=0.26, P<0.0005, and r=0.14, P=0.001, respectively). Women had higher levels of FXIII A-subunit (111.8% versus 105.2%, P<0.01) and B-subunit (109.5% versus 103.8%, P<0.01) than did men. FXIII A-subunit was significantly increased in smokers (117.0% versus 104.6%, P<0.0005) and in subjects with hypertension (114.9% versus 107.8%, P<0.05). In a multiple regression model, FXIII A-subunit was significantly increased by female sex (+6.4%, P<0.007), smoking (+12.3%, P<0.0005), and increasing age (+3.7% per 10 years, P<0.0005). FXIII B-subunit was significantly related to female sex and fibrinogen, and FXIII activity was significantly related to fibrinogen levels. In conclusion, the FXIII A-subunit level increases significantly with female sex, age, and smoking, whereas FXIII B-subunit and FXIII activity are associated with FXIII A-subunit level and fibrinogen. Although evidence for a causal relationship between FXIII A-subunit and vascular disease is not available, these results might suggest a role for elevated FXIII A-subunit levels in the pathogenesis of vascular disease.

Diagnosis and classification of factor XIII deficiencies

H. P . K OHLER ,* A. IC H I NO SE , R . SE ITZ ,§ R . A . S . AR I ENS– and L . MUSZBEK ,** ON BEHALF OF THE FACTOR X I I I AND F IBR INOGEN SSC SUBCOMMITTEE OF THE ISTH *Laboratory for Hemostasis Research, Department of Hematology, University Hospital of Bern, Bern; Department of Internal Medicine, Spital Netz Bern Hospitals, Bern, Switzerland; Department of Molecular Patho-Biochemistry, Yamagata University School of Medicine, Yamagata, Japan; §Paul-Ehrlich-Institut, Langen, Germany; –Section on Mechanisms of Thrombosis, Leeds Institute for Health, Genetics and Therapeutics, University of Leeds, Leeds, UK; and **Clinical Research Center and Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary

International Registry on Factor XIII Deficiency: A basis formed mostly on European data

FXIII deficiency is known as one of the rarest blood coagulation disorders. In this study, the phenotypic and in part genotypic data of 104 FXIII-deficient patients recorded from 1993 - 2005 are presented. The most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency. The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G>A). This mutation was found in eight (17%) of 46 analyzed families. The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. The international registry (http://www.f13-database.de) will provide clinicians and scientists working on FXIII deficiency with a helpful tool to improve patient care and direct future studies towards better understanding and treatment of the disease.