In mammalian coagulation, the complex network of integrated biochemical events is composed primarily of fi ve proteases (factor II or prothrombin, factor VII, factor IX, factor X, and protein C), which inter- act with fi ve cofactors (tissue factor, factor VIII, factor V, thrombomodulin, and membrane pro- teins) to generate fi brin [2]. Data from protein struc- ture and gene and sequence analysis suggest that coagulation regulatory proteins probably emerged earlier than 400 million years ago from duplication and diversifi cation of two gene structures: a vitamin K-dependent serine protease with an epidermal growth factor (EGF)-like domain (common to factors VII, IX, and X, and protein C) and a second domain structure common to factors V and VIII.
Prothrombin, also a vitamin K-dependent serine protease, contains kringle domains rather than EGF domains, which suggests a replacement during gene duplication and exon shuffl ing. Thrombin has
active-site amino acid residues that distinguish it from other serine proteases, supporting its position as the ancestral blood enzyme [3]. Furthermore, there is evidence that local duplication and/or trans- location may have contributed to the evolution of multigene families residing on disparate chromo- somal regions [4]. The mammalian coagulation genome itself probably evolved from invertebrate or early vertebrate species, where the corresponding genes (i.e., orthologs) for the primary coagulant and fi brinolytic proteins exhibit homology to mamma- lian sequences [5].
In evolutionary terms, however, hemostasis is a more recent phenomenon [6], arising fi rst in verte- brates along with a closed, high-pressure circulatory system [7]. Concomitantly, there was a need for a hemostatic system that could respond rapidly, since even a minor injury could cause lethal bleeding, but that would be tightly regulated, to avoid a poten- tially lethal excessive response [6]. Evolutionary forces fostered a highly integrated hemostatic system that is characterized by many points of interface with an underlying architecture of numerous func- tional genes. In fact, genetic mutations have been identifi ed in nearly every component of the human hemostatic system [6].
Cellular model of arterial thrombosis
The complexity and interdependence of coagulation proteins, platelets, hemodynamic forces, and the vessel wall in acute thrombosis are best understood by employing a “biological systems” approach – highlighted by distinct individual components within a dynamic yet highly orchestrated network [8]. Landmark studies employing a cell-based model of coagulation demonstrated a pivotal role for tissue factor-bearing cells as the primary site of initiation, and platelets as the primed template for thrombus growth or propagation [8]. Coagulation protein lower extremities. Deep tendon refl exes were
diminished on the right with a right plantar extensor response.
A complete blood count showed
thrombocytopenia with a platelet count of 110 × 109 per liter. The other cell lines were within normal
limits, as were the red blood cell indices. Renal, hepatic, and electrolyte laboratory studies were unremarkable. A 12-lead ECG revealed normal sinus rhythm with normal intervals and nonspecifi c T- wave abnormalities. A brain CT scan was also unremarkable.
complexes are assembled on these cellular surfaces, providing an effective means for localizing proco- agulant substrates to the site of vascular injury [8].
Coagulation is highly regulated by stoichiometric and dynamic reactions that operate on these sur- faces, and the proteome of blood coagulation can now be studied in biochemically quantifi able terms [9,10] (Figure 12.1).
Paradigm of atherothrombosis
The vast majority of acute occlusive diseases in the arterial circulatory system require the participation of two conditions – atherosclerosis and thrombosis.
It has become increasingly clear that each process affects the other substantially, and the pathobiologi- cal interface of atheroma formation and thrombosis, beginning with endothelial cell injury, occurs long before the phenotypic expression of atherothrom- bosis is clinically manifested [11]. The vascular endothelium is vital for thromboresistance and infl ammoresistance, producing nitric oxide, throm- bomodulin, tissue factor pathway inhibitor, and heparan sulfate proteoglycans that augment the
inhibition of serine proteases by antithrombin [12].
In some cases, however, endothelial injury may be less dependent on traditional atherogenic risk factors, and a heightened state of coagulation may be suffi cient for thrombosis to occur [13]. Seem- ingly “normal” endothelium has been shown to produce substantial amounts of a biologically active, alternatively spliced soluble tissue factor isoform upon stimulation with infl ammatory cytokines [14].
The potential for undisrupted endothelium to par- ticipate actively in arterial thrombosis is further sup- ported by observations that cytokine-stimulated endothelium from human internal mammary arter- ies is suffi cient to initiate thrombin generation [15].
Vascular bed specifi city of thrombotic disorders Despite the assumption that inherited or acquired thrombophilias should affect all vascular beds equally, this is often not the case [16]. For instance, congenital defi ciencies of antithrombin III, protein C, and protein S are associated with a heightened IX
TF
TF-bearing cell
Activated platelet
Platelet
TF Va
VIIIa
Va VIIa
VIIa X
XaXa
VIIa VIIa IX
IXa IXa IXa
IXa
XIa XIa II
X
IIa IIa
II
V Va XI XIaXIa
VIII/vWF
VIIIa
Propagation
Amplification Initiation
Xa
Xa IIaIIa
Integrated pathways of thromboresistance, fibrinolytic potential, and cellular repair on the
endothelial surface
Fig. 12.1 Cell-based model of coagulation. Schematic showing the three phases of coagulation: initiation, amplifi cation, and propagation.
TF, tissue factor; vWF, von Willebrand factor.
risk of deep vein thrombosis of the lower extremi- ties, whereas a clear and consistent link with arterial thrombosis is uncertain [17]. This may be the result of several distinctions between venous and arterial thrombogenesis, creating a permissive environment within the venous but not the arterial circulation. In particular, high shear stress within the arteries governs the expression of profi brinolytic and plate- let-regulating endothelial cell-derived genes, includ- ing tissue-type plasminogen activator (tPA) and nitric oxide synthase [18]. In contrast, thrombosis in the lower shear stress environment of the venous system predominantly involves tissue factor/tissue factor pathway inhibitor and thrombin-regulating (thrombomodulin, activated protein C [APC], protein S) local systems [19].
Vascular-bed specifi city for thrombosis may apply not only to differences between the arterial and venous circulation but also to organ-specifi c vessels [16]. Inherited and acquired thrombophilias are discussed in greater detail in subsequent sec- tions. The genetics of stroke is highlighted in Chapter 13.
Inherited thrombophilias and stroke risk Coagulation proteins
The pathobiology of inherited thrombophilias with potential, of varying degrees, to cause arterial throm-
bosis falls into three major categories: factors governing thrombotic potential (coagulation pro- teins and platelets), thromboresistance (fi brinolytic proteins and intrinsic regulatory pathways), and response to vascular injury (endothelial progenitor cells and associated mechanisms of vascular repair).
The focus of our chapter will be on the fi rst two categories and the important infl uence of environ- mental factors on the phenotypic expression of disease (Figure 12.2).
Fibrinogen
Fibrinogen is the precursor of fi brin and also serves as a linker protein in platelet aggregation. Elevated fi brinogen levels have been associated with both stroke and myocardial infarction (MI) [20]. Genetic factors contribute to nearly 50% of variability in fi brinogen levels [21]. Polymorphisms in the genes encoding the three pairs of fi brinogen polypeptide chains (α, β, and γ) have been studied in relation to arterial thrombotic risk, with most studies focusing on variants within the gene encoding β-fi brinogen.
The –455G/A and BclI variants are in linkage dis- equilibrium with one another. The AA genotype of the –455G/A single nucleotide polymorphism (SNP) has been associated with 10% higher fi brinogen levels when compared with the GG genotype [21].
Studies have also shown that the A allele binds less
Genetic factors Phenotype/biological effects
Gene–environment interactions Atherothrombosis (clinical disease) 1 Acute coronary syndrome 2 Ischemic stroke 3 Acute limb, visceral ischemia
DM/metabolic syndrome Diet (folate, vitamin B12 deficiency, western diet)
Smoking Sedentary lifestyle Oral contraceptives, hormone replacement
Pregnancy and puerperium Air pollution
Coagulation proteins 1 Factor V Leiden (G1691A) 2 G20210A prothrombin variant 3 Fibrinogen β-chain -455 G/A.
854 G/A and Bc/1, α-chain Thr312Ala Fibrinolytic system
1 PAI-1 -6754G/5G
2 TAFI Ala147Thr, TAFI 1542C/G
Platelet receptors
1 GPIIIa Leu33Pro, GP1BA -5T/C, GP6 13254T/C
Metabolic
1 MTHFR 677C/T (homocysteine)
Gene-gene interactions
Activated protein C resistance (factor V Leiden)
Fibrinogen, thrombin, factor VII, factor VIII PAI-1, vWF (coagulation and fibrinolytic protein polymorphisms)
Platelet activation/aggregation (GP polymorphisms)
Hyperhomocysteinemia (MTHFR 677C/T)
Environmental factors
Fig. 12.2 Pathophysiology of arterial thrombophilias.
DM, diabetes mellitus; GP, glycoprotein; MTHFR, methyltetrahydrofolate reductase; PAI, plasminogen activator inhibitor; TAFI, thrombin- activatable fi brinolysis inhibitor; vWF, von Willebrand factor.
well to a putative repressor protein complex, result- ing in increased fi brinogen-β chain transcription [22]. However, the relationship between this par- ticular fi brinogen gene variant and the risk of arte- rial thrombosis, including ischemic stroke, remains unclear, with some studies showing an association and others showing none [23–29]. In one study, the –455 allele was associated with a 2.5-fold increase in risk of multiple cerebral lacunar infarcts but not with large-artery strokes [30]. A variant in the α- fi brinogen gene Thr312Ala produces extensive α- chain cross-linking [31] and has been shown to be associated with increased stroke-related mortality [32].
Factor V Leiden (FVL) and prothrombin G20210A The FVL and prothrombin G20210A SNPs have been investigated for their potential association with arterial thrombotic disorders (reviewed by Kim and Becker) [33,34]. The prevalence of the FVL mutation among both ischemic stroke patients and controls is approximately 7% [33,34]. Most large case-control and cohort studies have failed to establish FVL as an independent risk factor for arterial ischemic stroke in the general population and suggest that FVL may be seen as relevant in the same settings as proteins S, C, and AT. A few studies reported a positive association of FVL with ischemic stroke, especially in women [35] and in children [36] and one of prothrombin 20210 with stroke in children [36]. In a meta-analysis of greater than 17,000 patients, the association between these inherited gene mutations and arterial ischemic events was modest (FVL odds ratio [OR]
1.21 [95% confi dence interval {CI} 0.99–1.49]) – prothrombin G20210A OR 1.32 (95% CI 1.03–
1.69), with subgroup analyses of patients less than 55 years old and of women showing slightly stronger association overall (see gain-of-function gene variants) [33,37]. Additionally, cigarette smoking may induce arterial thrombosis in patients with FVL [17].
Prothrombin 20210A, in individual studies, has not been independently associated with ischemic stroke specifi cally, although a meta-analysis by Bushnell and Goldstein has suggested a modest increased risk conferred by the presence of the mutation – an OR of 1.4 with a 95% CI of 1.03–1.9 [38].
Antithrombin III, protein C, and protein S defi ciencies
The association between intrinsic thromboresis- tence proteins, antithrombin III, protein C, and protein S defi ciencies and venous thromboembo- lism is incontrovertible; however, there is no clear and consistent association with ischemic stroke [39].
The only exception to this generally accepted prin- ciple is in homozygous mutations characterized by early onset and recurrent venous, and still less commonly, arterial thrombotic events to include stroke.
Factor VII
Given the role of factor VII in the initiation of coag- ulation, the factor VII gene and the alterations of plasma levels have been a topic of interest in arterial thrombogenesis. Factor VII levels have been incon- sistently associated with coronary artery disease (CAD) and polymorphisms in the factor VII gene account for less than 30% of the variability in factor VII plasma levels [21]. The relationship, if any, with ischemic stroke is poorly defi ned.
Factor XIII
Factor XIII stabilizes fi brin clot through covalent linkage. Studies have reported an association between a polymorphism in the A subunit of factor XIII (Val34Leu) and arterial thrombotic risk, with the 34Leu allele being protective against stroke (reviewed by Ariens and colleagues [40]). This SNP infl uences the transglutaminase activity of FXIII, and the homozygosity for the mutation is associated with increased enzyme activity [41].
Thrombomodulin
Plasma thrombomodulin levels have been associ- ated with increased risk of MI [42]. Two polymor- phisms in the thrombomodulin gene are associated with MI (Ala455Val [43] and Ala25Thr [44]). The association between thrombomodulin mutations and ischemic stroke remains poorly defi ned.
Tissue factor
Tissue factor, the major initiator of the blood coagu- lation cascade, has been investigated in relation to the genetic risk of thrombotic disease. Sequencing of the promoter region of the TF gene revealed six novel polymorphisms, and the 1208D allele was
found to be associated with lower levels of circulat- ing tissue factor and, concomitantly, was [45] pro- tective against VTE, though it did not infl uence the arterial thrombotic risk [46].
von Willebrand Factor (vWF)
vWF, a large mutimeric protein synthesized pre- dominantly by vascular endothelial cells, partici- pates directly in platelet adhesion to sites of vessel injury and platelet aggregation under high shear conditions. Multimers of vWF can be as large as 20,000 kDa and are highly functional.
Elevated vWF levels have been associated with the occurrence of acute ischemic stroke [1]. In addition, plasma vWF levels may provide an incremental value to clinical risk stratifi cation schemes for stroke and other vascular events among individuals with nonvalvular atrial fi brillation [2,3].