421
Review Article
Zinc: An important cofactor in haemostasis and thrombosis
Trang T. Vu1,2; James C. Fredenburgh1,3; Jeffrey I. Weitz1,2,3,4
1Thrombosis
and Atherosclerosis Research Institute, Hamilton, Ontario, Canada; 2Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada; 3Department of
Medicine, McMaster University, Hamilton, Ontario, Canada; 4Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
Summary
There is mounting evidence that zinc, the second most abundant transition metal in blood, is an important mediator of haemostasis and
thrombosis. Prompted by the observation that zinc deficiency is associated with bleeding and clotting abnormalities, there now is evidence that zinc serves as an effector of coagulation, anticoagulation
and fibrinolysis. Zinc binds numerous plasma proteins and modulates
their structure and function. Because activated platelets secrete zinc
into the local microenvironment, the concentration of zinc increases in
the vicinity of a thrombus. Consequently, the role of zinc varies de-
Correspondence to:
Dr. Jeffrey Weitz, MD
Thrombosis and Atherosclerosis Research Institute
237 Barton St. E., Hamilton, Ontario L8L 2X2, Canada
Tel.: +1 905 574 8550, Fax: +1 905 575 2646
E-mail: [email protected]
pending on the microenvironment; a feature that endows zinc with
the capacity to spatially and temporally regulate haemostasis and
thrombosis. This paper reviews the mechanisms by which zinc regulates coagulation, platelet aggregation, anticoagulation and fibrinolysis and outlines how zinc serves as a ubiquitous modulator of haemostasis and thrombosis.
Keywords
Zinc, coagulation, contact system, fibrinolysis, platelets
Financial support:
This work was supported in part by the Canadian Institutes of Health Research (MOP
3992, MOP 102735, and CTP 79846) and the Heart and Stroke Foundation of Ontario
(T4792 and T4730). T.T.V was supported by an Ontario Graduate Student of Ontario
Award. J.I.W. holds the Heart and Stroke Foundation of Ontario/J. Fraser Mustard
Endowed Chair in Cardiovascular Research and the Canada Research Chair (Tier 1) in
Thrombosis.
Received: July 5, 2012
Accepted after major revision: November 27, 2012
Prepublished online: January 10, 2013
doi:10.1160/TH12-07-0465
Thromb Haemost 2013; 109: 421–430
Introduction
Next to iron, zinc is the most abundant transition metal in the
body and is critical for maintaining normal physiology (1). Zinc’s
biological importance results from its ability to bind proteins and
modulate protein-protein interactions and enzymatic activity (2,
3). Accordingly, the levels of zinc are tightly maintained (4, 5). The
plasma concentration of zinc ranges from 10 to 20 μM, and virtually all of the zinc is bound to proteins. Although it can bind to
numerous haemostatic proteins to modulate their activity, the majority of zinc forms a low affinity interaction with albumin, which
serves as a zinc repository (2, 6, 7). Because most of the zinc is protein-bound, the concentration of unbound or free ion in plasma is
only 0.5-1 μM (2, 5, 6). However, the plasma concentration of zinc
is not static, and can change under certain conditions. For
example, because of competition with zinc for albumin binding,
zinc levels rise when the plasma concentration of free fatty acids
increases (7). A second mechanism for modulating zinc levels involves platelets. Platelets accumulate zinc in their cytoplasm and
α-granules, such that the concentration of zinc in platelets is 30- to
60-fold higher than that in plasma (8). In the milieu of activated
platelets, the free zinc concentrations can reach 7-10 µM (9). Furthermore, erythrocytes, lymphocytes and neutrophils can also release zinc into the blood (10). Third, the affinity of zinc for plasma
proteins is altered when the pH falls because of local ischaemia
and subsequent tissue hypoxaemia. A low pH alters the capacity of
proteins to bind zinc, thereby modifying its effect on protein structure and function (11-15). The potential for modulation of the
local zinc concentration and activity supports the contention that
zinc is a dynamic regulator of haemostasis and thrombosis.
The importance of zinc in haemostasis first came to attention in
1982 when bleeding and clotting abnormalities were described in
men on a low zinc diet (16). Zinc deficiency is associated with impaired platelet aggregation, and cutaneous bleeding and platelet
dysfunction are noted in some zinc-deficient cancer patients;
problems that are reversed with zinc supplementation (16-19).
Subsequent studies have shown that zinc regulates the coagulation,
anticoagulation and fibrinolytic pathways. The interaction of zinc
with factor (F)XII, the initiator of the contact pathway and the kallikrein/kinin system, was the first described role for this ion in coagulation (20). Zinc also modulates platelet aggregation and fibrin
formation. Release of zinc from activated platelets results in a local
surge in the zinc concentration that facilitates propagation of coagulation. Zinc also regulates ancillary steps in anticoagulant and
fibrinolytic pathways. Therefore, zinc plays an essential part in
regulating multiple reactions in haemostasis and thrombosis. An
overview of these roles is summarised in ▶ Figure 1.
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Vu et al. Zinc regulation of haemostasis and thrombosis
Figure 1: Zinc as a
dynamic regulator of
haemostasis and
thrombosis. Zinc modulates the activity of proteins in the coagulation
(intrinsic and extrinsic
pathways), anticoagulation and fibrinolytic
pathways. Local concentrations of zinc can increase in the vicinity of a
thrombus because activated platelets secrete
zinc.
If zinc is such an important cofactor in these processes, why has
it not received greater recognition? Zinc has long been overlooked
for several reasons. First, calcium, which was identified as coagulation FIV, is perceived as the predominant divalent cation because
it is more abundant in plasma than zinc and has an obligate role in
numerous haemostatic reactions (21). Second, citrate is commonly
used as an anticoagulant when preparing plasma for coagulation
or platelet function testing or for studies of other haemostatic reac-
tions. Citrate chelates all divalent cations including zinc, but only
calcium is routinely reconstituted prior to in vitro assays (21). Because the affinity of citrate for zinc is higher than that for calcium,
it is likely that many zinc-dependent reactions in plasma have
been overlooked. Recognising the importance of zinc in haemostasis and thrombosis, this review describes the mechanisms by
which zinc modulates coagulation, platelet aggregation, anticoagulation and fibrinolysis.
Role of zinc in the regulation of coagulation
Contact system
Figure 2: Effects of zinc on contact-mediated activation of coagulation on polyanionic surfaces. Zinc binding to FXII induces a conformational change in the zymogen rendering it more susceptible to cleavage by
FXIIa and kallikrein (K). Zinc binding to high-molecular-weight kininogen
(HK) localises the contact system proteins to polyanionic surfaces, thereby
promoting contact-mediated activation of coagulation. As a dynamic switch,
zinc has the capacity to inhibit FXIIa activation of prekallikrein (PK), although
its effects on FXIa generation are unknown. Upward and downward pointing
triangles represent stimulatory and inhibitory actions of zinc, respectively.
Coagulation is initiated by tissue factor (TF) or via the contact system and intrinsic pathway. Although the TF pathway triggers
thrombus formation at sites of vascular injury, current thinking is
that the intrinsic pathway is important in the propagation phase
(22). Thus, emerging evidence indicates that the contact system
plays an important role in thrombosis, even though it is not essential for haemostasis (23, 24). This section outlines the role of zinc
as a cofactor for initiating contact-mediated activation of coagulation on polyanionic surfaces and for the assembly of contact system proteins on endothelial cells (ECs) and platelets.
FXIIa generation on polyanionic surfaces
The contact system is initiated when FXII comes into contact with
a polyanionic surface. One way in which FXIIa is generated is
through FXII autoactivation, whereby FXIIa cleaves its own zymogen to amplify activation (▶ Figure 2). Zinc binds to FXII and
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Vu et al. Zinc regulation of haemostasis and thrombosis
Figure 3: Effects of zinc on the assembly and activation of the contact system on endothelial cells. Zinc is a cofactor that promotes the interaction of HK and FXII with endothelial cell binding sites, cytokeratin 1
(CK1), urokinase plasminogen activator receptor (uPAR) and complement
component C1q receptor (gC1qR). HK localises PK and FXI onto the endothelium. Subsequent activation of PK by prolylcarboxypeptidase (PRCP) generates kallikrein (K), which then activates FXII to generate FXIIa and cleaves HK
to release bradykinin (BK). FXIIa cleaves FXI to generate FXIa.
induces a conformational change that potentiates surface-independent and surface-dependent autoactivation by at least 10-fold
(11, 12, 25, 26). During the process of FXII autoactivation, the zymogen undergoes two distinct conformational changes, which
prime it for optimal proteolysis (25, 27). The first conformational
change occurs when FXII binds to polyanionic surfaces, whereas
the second coincides with zinc binding to histidine residues on
FXII (11, 28, 29). Studies of FXIIa generation in prekallikrein
(PK)-deficient plasma reveal that polyanionic surface alone is insufficient to induce FXII autoactivation. Instead, optimal FXII
autoactivation requires both the surface and zinc (27). These findings suggest that zinc is an essential cofactor for activation of the
contact system on artificial surfaces. However, the extent to which
zinc is required for contact-mediated activation of coagulation on
physiological surfaces, such as nucleic acids and polyphosphates, is
as yet unstudied (30, 31).
There is ample evidence that zinc binds FXII and induces conformational changes. These changes could augment the interaction of FXII with surface or with its activators, or enhance FXII
cleavage (25-27, 32). There are four zinc binding sites on FXII (26,
33, 34). One binding site is lost when FXII is converted to α-FXIIa,
and further conversion to β-FXIIa results in the complete loss of
zinc binding; findings that suggest that zinc interacts predominately with the heavy chain of FXII (26). Zinc promotes the binding of FXII and α-FXIIa, but not β-FXIIa, to dextran sulfate. These
observations suggest that by binding to the heavy chain, zinc enhances the capacity of FXII to bind to surfaces (29). There also is
evidence that zinc induces a conformational change in FXII that
renders it a better substrate for its activators, FXIIa and kallikrein
(12). Although zinc does not bind directly to the catalytic domain
of FXIIa, zinc inhibits FXIIa-mediated activation of PK in a dosedependent manner, thereby providing further evidence of a zincinduced conformational change (12, 25). The capacity of zinc to
serve as both a cofactor for FXII activation and an inhibitor of
FXIIa may provide a regulatory switch for control of the contact
system.
In addition to its effect on FXII auto-activation, zinc also potentiates kallikrein-mediated activation of FXII, which provides a
second positive feedback loop that further amplifies FXII activation. This pathway is dependent on high-molecular-weight kininogen (HK), a cofactor that bridges PK and kallikrein onto
negatively charged surfaces and cells (9, 20, 35, 36). Although PK
and kallikrein binds to surfaces independently of HK, activation of
FXII by kallikrein is less efficient in the absence of HK. The mechanism by which zinc augments kallikrein-mediated generation of
FXIIa may reflect the capacity of zinc to bind HK and to promote
its interaction with anionic surfaces (37). Alternatively, the zincmediated conformation change in FXII may render it more susceptible to kallikrein cleavage (11). Therefore, there are multiple
avenues by which zinc potentiates FXIIa generation on anionic
surfaces.
Contact system on endothelial cells
Zinc also facilitates the assembly of the contact system on quiescent ECs (▶ Figure 3). In the presence of zinc, both HK and FXII
form high-affinity interactions (Kd values of 7-54 nM) with EC
binding sites, such as urokinase-type plasminogen activator recep-
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Vu et al. Zinc regulation of haemostasis and thrombosis
tor (uPAR), complement component C1q receptor (gC1qR), and
cytokeratin 1 (CK1) (9, 38-42). The majority of circulating PK and
FXI is bound to HK, and HK can bridge PK and FXI to ECs. Because zinc facilitates HK binding to ECs, zinc serves to localise
these zymogens onto the endothelial surface (38, 43). The amount
of zinc required to facilitate FXII binding is 33- to 50-fold higher
than that needed for HK binding (0.3 μM), suggesting that HK is
the preferred ligand for the EC binding sites (9, 44, 45). Nonetheless, the local increase in zinc levels that occurs upon platelet activation raises the zinc concentration to levels sufficient for FXII to
compete with HK for binding to ECs, which may provide a regulatory switch that modulates the activity of these proteins (9, 46). On
the EC surface, prolylcarboxypeptidase converts PK to kallikrein
more efficiently than FXIIa; a finding that suggests that prolylcarboxypeptidase is the physiological activator of PK on ECs (47, 48).
Once generated, kallikrein cleaves HK to release bradykinin, a potent vasodilator (49). Unlike kallikrein, generation of FXIa on ECs
is dependent on FXIIa and this process is regulated by zinc because the ion localises both factors onto the endothelium (48, 50).
Therefore, by mediating the interaction between the proteins in
the contact system and the endothelium, zinc regulates thrombosis. Assembly of the contact system on the endothelium is also important for regulating vascular tone, inflammation and angiogenesis; the effects of zinc on these processes are discussed in detail
elsewhere (49-53).
FXIa activity on the platelet surface
Propagation of coagulation occurs on the platelet surface and the
first step is mediated by FXIa (reviewed in [54]). There are two
mechanisms through which FXI binding to platelets can be modulated. The first involves calcium and prothrombin, while the second requires zinc and HK (55, 56). Zinc facilitates the binding of
FXI to glycocalicin, a glycoprotein present on the external portion
of glycoprotein lb (GPlb). However, it is unclear whether zinc acts
alone or in concert with HK to localise FXI onto activated platelets
(55, 56). On the platelet surface, FXIIa, FXIa or thrombin activates
FXI to FXIa, which in turn activates FIX to FIXa. The intrinsic tenase complex, which consists of FIXa and its activated cofactor,
FVIIIa, assembles on the activated platelet surface and propagates
the initial procoagulant response (55, 56). Because zinc contributes
to the regulation of FXIa binding to platelets, it is an important
mediator of coagulation.
Protease nexin-2 (PN-2), which is secreted by activated platelets, inhibits FXIa. Zinc reduces the Ki for FXIa inhibition by
PN-2 by at least 3-fold (57). Because platelet-bound FXIa is protected from PN-2 inhibition, whereas free FXIa is not, the release
of zinc from platelets may localise FXIa activity to the platelet surface (57, 58).
Role of zinc in platelet aggregation and
fibrin formation
Activation of the coagulation system results in thrombin generation. Thrombin is a potent platelet agonist and induces release of
zinc from the platelet cytoplasm and α-granules. The surge in zinc
concentrations in the local microenvironment of an expanding
thrombus promotes fibrin formation and triggers key signalling
events that lead to platelet aggregation. Therefore, zinc release
from platelets can be viewed as an essential transition phase that
promotes the propagation of coagulation. This section outlines the
role of zinc in platelet aggregation and fibrin formation; two processes that are critical for thrombus formation.
Platelet aggregation
Evidence from animal models and humans suggests that zinc is an
important effector of platelet aggregation. Tail bleeding times are
prolonged in rats with acute zinc deficiency and their platelets exhibit impaired aggregation in response to agonists such as adenosine diphosphate (ADP) and thrombin (16-18). Zinc enhances collagen- and ADP-induced aggregation of washed platelets (59, 60)
and, even in the absence of agonists, zinc augments platelet aggregation by promoting fibrinogen binding to αIIbβ3, its cognate receptor on the platelet surface (59, 61). Thus, zinc not only augments agonist-induced platelet aggregation, but also promotes the
binding of adhesive proteins to platelets.
The intracellular calcium concentration, which is an important
determinant of platelet activation, depends on the dynamic balance between calcium uptake and release (62, 63). Zinc regulates
the capacity of platelets to take up extracellular calcium, but does
not affect the flux of calcium from intracellular pools (59, 64).
Therefore, by enhancing calcium influx, zinc modulates processes
important in platelet aggregation. This phenomenon may explain
why platelets from zinc deficient animals and humans exhibit impaired aggregation, and why addition of zinc promotes agonist-induced platelet aggregation (16-18).
It is unclear how zinc modulates extracellular calcium uptake.
Calcium channels are dependent on sulfhydryl (SH) groups for
normal function (65). When rats are fed a low zinc diet, the concentration of SH groups on the membrane proteins of their platelets is reduced by about 15% and this is associated with impaired
uptake of extracellular calcium and reduced ADP-induced platelet
aggregation (66). Therefore, current thinking is that zinc regulates
the redox state of SH moieties in the calcium channels. Consequently, in the absence of zinc, SH groups are oxidised and form a
disulfide bond, which inactivates the channel (64). Rearrangement
of disulfide bonds on the platelet membrane, which is crucial for
platelet aggregation, secretion and post-aggregation events, is primarily mediated by protein disulfide isomerase (PDI) (67, 68).
Zinc induces oligomerisation of PDI, thereby attenuating its activity (69); an observation that raises the possibility that zinc may
modulate the redox state of calcium channels in an indirect
fashion. Furthermore, the activation of protein kinase C, a family
of calcium-dependent kinases that modulate platelet signalling
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Vu et al. Zinc regulation of haemostasis and thrombosis
and aggregation, is also regulated via a zinc redox mechanism (70,
71). Taken together, these studies demonstrate the importance of
zinc in modulating key signalling events that result in platelet aggregation.
Fibrin formation
Once generated, thrombin converts fibrinogen to fibrin, which
then stabilises the platelet plug that forms at sites of injury. Zinc
has multiple effects on the fibrin clotting process. Zinc inhibits the
amidolytic activity of thrombin in a dose-dependent manner (72,
73). This finding is consistent with the observation that zinc attenuates thrombin-mediated fibrinopeptide A release (74). However, the same group also reported that zinc accelerates the rate of
thrombin-induced fibrin clot formation (72, 74). In contrast,
neither magnesium nor manganese had an effect on thrombin clot
times, suggesting that modulation of thrombin-induced conversion of fibrinogen to fibrin is unique to zinc (72).
It is unclear how zinc attenuates the activity of thrombin, yet
promotes fibrin clot formation. One hypothesis is that zinc interacts with fibrinogen/fibrin and potentiates the assembly of fibrin
into a three-dimensional network. In support of this concept, zinc
binds to fibrinogen and fibrin with Kd values that ranges from 8 to
18 µM and with a stoichiometry of six zinc atoms per molecule of
fibrin(ogen) (75). The zinc binding sites on fibrin(ogen) are likely
localised to the D and/or the αC-domains because zinc binds to fibrinogen fragments D and X, but not to fragment E (76). Additionally, analysis of fibrin clot structure reveals that zinc, but not
manganese, increases the fiber thickness of thrombin-generated fibrin clots. Zinc-containing fibrin fibrils are thicker and have fiber
diameters that are ~10-fold greater than those formed in its absence (75, 77). Furthermore, the zinc binding sites on fibrin(ogen)
are distinct from those of calcium, a known effector of fibrin polymerisation (75, 77-79). Although zinc enhances the lateral association of protofibrils to a greater extent than calcium, fibrin clots
formed in the presence of zinc have reduced visco-elastic strength
compared with those formed in the presence of calcium alone.
Therefore, zinc modulates fibrin formation in a manner distinct
from that of calcium (75, 77, 80). Together, these observations suggest that the ability of zinc to promote fibrin formation is not
necessarily the result of altered thrombin proteolysis of fibrinogen,
but more likely reflects modulation of fibrin polymerisation (74,
79).
limiting the propagation of the extrinsic pathway. In addition, zinc
also modulates the activity of the protein C (PC) anticoagulant
pathway and heparin-mediated inhibition of coagulation. This
section provides an overview of the mechanisms by which zinc attenuates coagulation and highlights the contention that zinc serves
as a dynamic, regulatory switch in haemostasis and thrombosis.
Contact system on platelets
In addition to its effects on platelet aggregation, zinc also modulates platelet activation. Zinc promotes the binding of HK and
FXII to GPlb on the platelet surface (81-83). The light chain of HK
binds to anionic surfaces, whereas the heavy chain binds to GPlb
(81, 84, 85). HK binds to both resting and activated platelets with a
Kd value of 1-20 nM. This interaction is dependent on zinc, but not
calcium, suggesting that zinc modulates the binding of HK to platelets (20, 36). Current thinking is that cleavage of protease activated receptors 1 and 4 by thrombin triggers signalling events that
lead to platelet activation. However, thrombin may also induce a
platelet procoagulant response via activation of GPlb (86). Because
HK and FXIIa can compete with thrombin for GPlb binding,
thrombin-mediated platelet activation is attenuated (81-83, 87).
The capacity of zinc to serve as both an activator and an inhibitor
of platelet activation may provide a regulatory switch for control of
haemostasis.
Inhibition of FVIIa
Extrinsic tenase, the complex of FVIIa with its cofactor TF, activates FX and FIX. The activity of FVIIa is allosterically regulated
by TF and divalent cations. In addition to calcium, the crystal
structure of p-aminobenzamidine-inhibited FVIIa in complex
with soluble TF reveals two zinc atoms in the protease domain
(88). Functional studies indicate that zinc attenuates the amidolytic activity of FVIIa regardless of whether it is in complex with
TF, although calcium negates this effect (89, 90). It is likely that
zinc targets the protease domain of FVIIa because the amidolytic
activity of mutant FVIIa lacking gamma-carboxyglutamic acid
(Gla) residues is attenuated by zinc to the same extent as the native
protein (89, 90). However, zinc does not significantly alter the affinity of FVIIa for TF (89). Because zinc has the capacity to
dampen FXa generation, the ion may attenuate thrombosis in the
vicinity of a platelet rich thrombus, where the concentration of
zinc is elevated.
Role of zinc in the regulation of
anticoagulant pathways
Modulation of activated protein C generation and
activity
The increase in the local zinc concentration that occurs upon platelet activation provides a positive feedback mechanism that ensures propagation of coagulation. However, zinc is also involved in
several ancillary reactions that counteract coagulation. First, by
regulating the assembly of the contact pathway proteins on platelets, zinc attenuates platelet activation. Second, zinc binds to
FVIIa and protein S (PS) and attenuates FXa generation, thereby
The PC pathway plays a key role in anticoagulation. PC is converted into activated PC (APC) by the thrombin-thrombomodulin
(TM) complex on ECs in a calcium-dependent manner (91, 92).
By positioning PC in proximity to the thrombin-TM complex, the
endothelial cell protein C receptor (EPCR) augments APC generation by 20-fold compared with thrombin-TM alone (reviewed in
[93]). Zinc binds to PC and induces a conformational change that
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Vu et al. Zinc regulation of haemostasis and thrombosis
increases the affinity of PC for EPCR by 5- to 10-fold compared
with calcium alone (92). In addition to facilitating phospholipid
interactions, the Gla-domains of PC and APC also mediate their
interaction with EPCR (94). PC and APC contain 8-10 zinc binding sites, 6-8 of which are localised to the Gla-domain. Therefore,
zinc binding to the Gla-domain regulates PC and APC binding to
EPCR. The ability of zinc to potentiate APC generation is only observed in the presence of calcium, suggesting that the two ions
work in concert to promote APC generation (92). Collectively,
these studies suggest that zinc augments APC generation by enhancing the interaction of PC with EPCR.
APC serves as an anticoagulant by inactivating the activated
cofactors, FVa and FVIIIa (95). In addition to binding to the Gla
domain of APC, zinc also binds to its protease domain and induces
a conformational change. In the absence of calcium, zinc inhibits
the amidolytic activity of APC by ~7-fold, but the impact of this
phenomenon on the anticoagulant activity of APC is unclear (92,
96). Zinc attenuates the capacity of APC to inactivate FVa in the
presence of phospholipid vesicles (96), but not on the EC surface
(92); differences that suggest that APC activity varies depending
on the membrane surface (97). Together, these studies suggest that
zinc acts as a regulatory switch for APC by augmenting its generation and attenuating its activity.
Protein S anticoagulant activity
PS is a cofactor that enhances the anticoagulant activity of APC
(98). However, PS also has PC-independent anticoagulant functions that are regulated by zinc. PS binds one zinc atom; an interaction that is lost with some PS purification methods (99, 100). In
the absence of zinc, PS augments the capacity of tissue factor pathway inhibitor (TFPI) to inhibit FXa generation by extrinsic tenase
(101, 102). However, in the presence of zinc, PS dampens the generation of FXa by extrinsic tenase in a TFPI-independent manner.
Thus, in the presence of zinc, PS binds to FX, FXa and TF and attenuates assembly of the extrinsic tenase complex (103). An antibody directed against PS residues 621-635 only recognises zinccontaining PS, suggesting that zinc modulates PS conformation
and activity (99). Moreover, zinc increases the affinity of PS for
FXa by 16-fold, thereby enhancing PS-mediated inhibition of FXa
activity (99, 100). Collectively, these observations suggest that zinc
binds PS and modulates its anticoagulant function.
Anticoagulant activity of heparin
Heparin and its derivatives are commonly administered anticoagulants. Heparin exerts its anticoagulant activity by binding to
antithrombin (AT) and enhancing its activity (104). There are
multiple mechanisms by which zinc alters the anticoagulant activity of heparin. Zinc increases the affinity of heparin for fibrin(ogen) by ~4-fold (105). By promoting heparin-fibrin(ogen)
interactions, zinc renders less heparin available to bind to AT,
thereby attenuating its anticoagulant activity (105). A consequence
of the interaction of heparin with fibrin is the formation of the ternary heparin-thrombin-fibrin complex, which protects fibrin-
bound thrombin from inhibition by heparin-catalysed serpins,
such as AT and heparin cofactor II (106).
Approximately 10-15% of circulating fibrinogen molecules are
heterodimers containing a variant γ-chain (γA/γ') and their abundance in plasma is associated with cardiovascular disease (107,
108). Because thrombin forms a higher affinity interaction with
γA/γ'-fibrin than with γA/γA-fibrin, thrombin bound to γA/γ'-fibrin is more protected from inhibition by AT and heparin cofactor
II (106). However, in the presence of heparin, zinc augments the
affinity of thrombin for γA/γA-fibrin by 4-fold (105). Consequently, thrombin bound to γA/γA-fibrin in the presence of zinc
and heparin is protected from AT to a similar extent as thrombin
bound to γA/γ'-fibrin (105).
Pro- and anticoagulant functions of histidine-rich
glycoprotein
The capacity of zinc to serve as a pro- and anticoagulant effector is
highlighted by its interaction with the abundant plasma protein,
histidine-rich glycoprotein (HRG) (15, 109). HRG is composed of
multiple domains, the most notable being a histidine-rich region.
Zinc binds to this region and induces a conformational change in
the molecule, which facilitates HRG binding to various haemostastic factors, including heparin, fibrinogen and FXIIa (15,
110-113).
Binding of HRG to heparin abrogates its anticoagulant activity
in both purified and plasma systems (114, 115). This effect is zincspecific because neither copper nor magnesium influences the
reaction (115). These studies suggest that zinc serves as a cofactor
for heparin neutralisation by HRG, which dampens the anticoagulant activity of AT, thereby promoting thrombosis.
Conversely, HRG also exhibits zinc-dependent anticoagulant
activity. Zinc potentiates the binding of HRG to FXIIa by
1,000-fold (112). By binding FXIIa, HRG dampens FXII autoactivation and FXIIa-mediated activation of FXI; and zinc is a cofactor
in these processes (112). Moreover, in the presence of zinc, HRG
binds to the carboxy-terminus of the γ'-chain of γA/γ'-fibrinogen
with a Kd of ~0.8 nM. Because the carboxy-terminus of the
γ'-chain also serves as a high affinity thrombin binding site, in the
presence of zinc, HRG competes with thrombin for binding to this
region (113). Fibrin-bound thrombin remains active and is protected from inhibition by fluid phase inhibitors (106, 116). By displacing thrombin from fibrin, zinc-containing HRG may, therefore, have antithrombotic properties.
Role of zinc in modulation of the fibrinolytic
pathway
Compared with coagulation and anticoagulation, little is known
about the role of zinc in fibrinolysis. Fibrinolysis is initiated when
tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) converts plasminogen (Pg) to plasmin (Pn).
Pn then degrades fibrin to generate soluble fibrin degradation
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Vu et al. Zinc regulation of haemostasis and thrombosis
Table 1: Overview of
the zinc-binding proteins and the effects
of zinc on haemostasis
and thrombosis.
Target
Binding region
Consequence
Effect on haemostasis
Factor XII/XIIa
Heavy chain
↑ factor XII autoactivation
Coagulation
High-molecular-weight
kininogen (HK)
Light chain
↑ HK binding to polyanionic surfaces
Coagulation
Platelet calcium channels
Sulfhydryl-groups
↑ platelet aggregation
Coagulation
Fibrin(ogen)
D and/or
αC-domains
↑ lateral association of fibrin
protofibrils
↑ formation of ternary thrombinfibrin-heparin complexes
Coagulation
Factor VIIa
Protease domain
↓ amidolytic activity
Anticoagulation
Protein C/activated protein C
(APC)
Gla and protease
domains
↓ APC generation on endothelial cell protein C receptor
↓ APC amidolytic activity
Anticoagulation
Protein S
Unknown
↑ factor Xa generation by extrinsic tenase
↓ factor Xa activity
Anticoagulation
Histidine-rich glycoprotein
Histidine-rich region ↑ interaction with heparin
↑ interaction with fibrin(ogen)
Plasmin(ogen)
Unknown
↓ plasminogen activation by tPA
↓ plasmin proteolytic activity
Anti-fibrinolytic
Tissue plasminogen
activator (tPA)
Unknown
↓ proteolytic activity
Anti-fibrinolytic
Contact system proteins
Numerous
↑ release of tPA from endothelial cells
↑ activation of uPA by kallikrein
↑ activation of plasminogen
Pro-fibrinolytic
Single-chain urokinase
plasminogen activator (uPA)
Unknown
↑ interaction with fibrin
Pro-fibrinolytic
Fibrin
D and/or
αC-domains
↑ fibrin fiber thickness
Pro-fibrinolytic
products (117). In vitro studies suggest that zinc influences the activity of key components of the fibrinolytic pathway.
Plasmin generation and fibrin degradation
Zinc may be an effector of fibrinolysis because it modulates the activity of tPA, uPA and Pn. Zinc inhibits the catalytic activity of tPA
and Pn in a dose-dependent manner and as a result, Pn generation
by tPA is also dampened by zinc (118, 119). Although zinc binds to
tPA, it is currently unknown whether it also binds to Pg and/or Pn
(118, 119). The effects of zinc on fibrinolysis are unclear because
none of the studies performed to-date has included fibrin. It is
well-established that tPA binds to fibrin and uPA does not (117,
120). However, zinc, but not calcium or magnesium, promotes
binding of single-chain (sc-) uPA to fibrin in a reversible and saturable manner with a Kd value of 300 nM and a stoichiometry of
three sc-uPA molecules per fibrin monomer. The binding of scuPA to fibrin is specific because neither two-chain (tc-) uPA nor
its degradation product, low-molecular-weight tc-uPA, binds fibrin in the presence of zinc (120). Pn and kallikrein convert scuPA to tc-uPA, and there is evidence that zinc accelerates this process on the fibrin surface (117). These results are interesting and
Coagulation and
anticoagulation
posit a novel role for zinc in regulating the activity of fibrinolytic
proteins.
Despite substantial evidence that zinc regulates the activity of
proteins important in fibrinolysis, how this affects fibrin degradation remains unclear. There is some evidence that zinc potentiates
lysis by altering fibrin clot structure. On scanning electron images,
zinc-containing clots are coarser and composed of thicker fibrils
than clots formed in the absence of zinc (75, 77-79). This may explain how zinc affects fibrinolysis because thicker fibers undergo
more rapid lysis than thinner fibers (121, 122). Therefore, zinc has
the capacity to impact fibrinolysis by modulating processes important in fibrin clot formation, Pg activation and lysis.
Contact system and fibrinolysis
Components of the contact system are also involved in fibrinolysis
and these processes are regulated by zinc. Zinc mediates the localisation of HK and kallikrein onto the EC surface (40-42) and, in
turn, kallikrein cleaves HK to generate bradykinin, a potent inducer of tPA release from ECs (123). Moreover, zinc potentiates
FXIIa-mediated Pg activation in the presence of kaolin (124). In
addition, in the presence of zinc and sulfatides, FXIIa is as potent
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427
428
Vu et al. Zinc regulation of haemostasis and thrombosis
an activator of Pg as uPA (125). Therefore, these studies suggest
that the contact pathway is also important in fibrinolysis and that
zinc modulates these processes.
Conclusion
Zinc is important in platelet aggregation, coagulation, anticoagulation and fibrinolysis (▶ Table 1). Zinc promotes initiation of the
contact system in both soluble and cellular compartments. Once
activated, platelets secrete zinc, which provides a positive feedback
mechanism that is essential for the propagation of coagulation.
Zinc further stimulates coagulation by potentiating platelet aggregation and fibrin clot formation. Furthermore, zinc has secondary
effects on anticoagulant pathways and fibrinolysis, which dampen
the procoagulant response. Consequently, zinc not only serves as a
regulatory switch that ensures entry into the propagation phase of
coagulation, it also limits these processes by activating compensatory inhibitory pathways. The seemingly paradoxical role played
by zinc is, in fact, a trait shared with calcium, which also simultaneously participates in numerous pathways in haemostasis.
Zinc binds to proteins and alters their enzymatic activity or
promotes protein interactions (2, 113). Several mechanisms exist
to regulate local concentrations of zinc and its activity; processes
that enable zinc to serve as a dynamic regulator of haemostatic
reactions. The concentration of free zinc increases in the vicinity
of activated platelets, and its capacity to bind to proteins is modulated by the pH in the local microenvironment. Consequently, the
ability of zinc to act as an effector molecule is attenuated under ischaemic, hypoxic or acidic conditions (11, 13-15). Since the processes that maintain haemostasis and thrombosis are spatially and
temporally regulated, the effects of zinc on these reactions may
also vary depending on the location and sequence of events. Such
regulation endows zinc with the capacity to act as a dynamic regulator of haemostasis and thrombosis.
The procoagulant activity of zinc is particularly relevant to contact-initiated coagulation. Because there is increasing evidence
that the contact system contributes to thrombosis (23, 24), zinc
may be an important modulator of thrombosis. Collectively, these
observations suggest that a better understanding of the mechanisms of zinc regulation in haemostasis and thrombosis may lead to
novel strategies to mitigate and treat thrombotic disorders.
Conflicts of interest
None declared.
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