Blood Clotting

http://ntri.tamuk.edu/homepage-ntri/lectures/clotting.html

The circulatory system must be self-sealing to prevent life threatening injury. Bleeding is
rapidly stopped by a process known as hemostasis that involves three processes.

1. First, platelets adhere to damaged blood vessels and then to each other forming a
plug that can stop minor bleeding. This process is mediated by von Willebrandt
factor, a large (104 kD) multimeric plasma glycoprotein of subunit mass 225 kD.
This protein binds to both a specific receptor on the platelet membrane and to the
collagen and possibly other components of the subendothelial membrane.
2. As the platelets aggregate, they release several physiologically active substances,
including serotonin (5-hydroxytryptamine) and thromboxane A2, which stimulates
vasoconstriction, thereby reducing the blood flow at the injury.
3. Finally, the aggregating platelets and damaged tissue initiate blood clotting, which
is the major defense against bleeding.

Platelet Plug

Until platelets are needed for hemostasis, circulating platelets are maintained in a non-
reactive state for the following reasons:

1. Partly by inhibitory compounds secreted by endothelial cells , such as nitric oxide

and prostacyclin  [a prostaglandin (any of various oxygenated unsaturated
cyclic fatty acids of animals that perform a variety of hormonelike actions (as in
controlling blood pressure or smooth muscle contraction)that is a metabolite of
arachidonic acid, inhibits platelet aggregation, and dilates blood vessels].
2. The negative surface charge limits platelet interaction with circulating
erythrocytes, leukocytes, or the vascular endothelium.
3. The intact endothelium also represents a barrier separating platelets form adhesive
substrates in the subendothelial connective tissue matrix such as collagen.

Disruption of the endothelium by trauma, or by a disease such as atherosclerosis, allows

platelets to come in contact with and adhere to sub-endothelial matrix. Adhesion is the
first stage of platelet function. Platelets tend to stick to the exposed ends of injured blood
vessels. Actually, they adhere to any rough surface, particularly to the collagen (the chief
fibrous protein found in skin, bone, cartilage, tendon, and other connective tissue that yields gelatin when
boiled in water) in connective tissue that underlies the endothelial lining of blood vessel. At
lower shear rates, the integrins α2 Β1, α5 Β1 , and α6 Β3 bind to exposed collagen,
fibronectin [any of a group of glycoproteins  (a complex protein in which the nonprotein part is a
carbohydrate ) of cell surfaces, blood plasma, and connective tissue that promote cellular adhesion and
migration], and laminin , respectively (Helmler et. al., 1988). At higher shear rates, platelet
adhesion requires sub-endothelial von Willebrand factor (vWf) (Turrito et al. 1985). VWf
binds to the GPIb-IX-V complex on un-activated platelets (Coller et al. 1983) and to the
αIIb Β3 complex on activated platelets (Ruggeri et al. 1983).
Collagen is not only an important substrate for platelet adhesion but also a binding site
for vWf in the sub-endothelium ( Savage et al 1998). A number of platelet proteins
interact with collagen besides α2 Β1. Other that interact include αIIb Β3, GPIV, GPVI and
two other proteins with a molecular weight of 65,00 and 85,000 molecular weight protein
(Clemetson et. Al 1999). Clinical reports suggest that α2 Β1 and BPVI are the
physiological platelet receptors for collagen (Moroi et al. 1989, Mieuwenhuis et al. 1985,
and Arai et al. 1995)).

When platelets contact collagen, their shapes change drastically, and numerous spiny
processes begin to protrude from their membranes. At the same time, they tend to stick to
each other with fibrinogen forming a platelet plug in the vascular break. This starts the
second phase of platelet function aggregation. In contrast to adhesion, platelet
aggregation is an active metabolic process in which platelet agonist binding to specific
membrane receptor initiates signaling pathways that enable the integrins αIIbΒ3 to bind
soluble macromolecular ligands, such as fibrinogen or vWf (Bennett 1996). The
fibrinogen or vWf bound to a αIIbΒ3 cross-links platelets into a hemostatic plug or
thrombus.
Platelets are important for blood clotting and provide a good example of how cell-cell
interactions are modulated by controlling integrin activity. The αIIb Β3 integrin normally is
present on the plasma membrane of platelets but is unable to bind the blood protein
fibrinogen or the other protein ligands. Only after binding collagen or thrombin in a
forming clot activates the platelet can αIIb Β3 integrin bind fibrinogen. This interaction
accelerates the formation of the clot. Platelet activation is accompanied by a
conformational change in the αIIb Β3 integrin. The nature of this change is unknown, but
as platelet activation also involves a major change in the platelet cytoskeleton. This
change probably involves binding of a cytoskeletal protein to the integrin cytosolic
domain. Patients with genetic defects in the β3 integrin subunit are prone to excessive
bleeding.

Unstimulated platelets do not aggregate in circulation since they do not bind fibrinogen.
Antagonists such as ADP, epinephrine, thrombin, or prostaglandin endoperoxides
stimulate platelets exposing fibrinogen receptors to associate with αIIb Β3 integrin, which
results in fibrinogen binding and subsequent platelet aggregation.

Platelets are the smallest corpuscular components of human blood (diameter 2-4µm) - the
physiological number varies from 150,000 to 300,000/mm3 blood. Despite their
appearance on the face of it platelets are not cells, as they are not provided with a
nucleus. The origin of platelets is the bone marrow. Megakaryocytes as the results of
mitotic proliferation of a committed progenitor cells liberate platelets as the end product
of protrusions of their membrane and cytoplasm. The typical shape of resting platelets is
discoid (Figure 2 & 3), upon activation they undergo a shape change to a globular form
with pseudopodia (up to 5µm long).

Figure 2: Scanning electromicrograph of activated platelets.

 Figure 3: Scanning electromicrograph of platelets.

The membrane consists of a typical phospholipid bilayer (Figure 4). Embedded in

membrane are different kinds of glycoproteins (GP), the receptors for activation and
interaction with other cells (figure 4). The integrin α1β1 mediates platelet adhesion to
subendothelial collagen via von Willebrand factor (vWf) and activate the αIIb Β3 integrin.
Figure 4: Capillary with platelet plug

and
receptors of platelet aggregation.

Collagen will induce formation of aggregates as well as secretion of granule contents.

Platelet responses that are well recognized include development of stickiness, adhesion,
change in shape, irreversible aggregation and secretion of granule contents.

Blood Clotting Cascade

A blood clot (thrombus) forms through the action of a cascade of proteolytic reactions
involving the participation of nearly 20 different substances, most of which are liver-
synthesized plasma glycoproteins. The cascade is diagrammed below. All but two of
these factors are designated by both a roman numeral and a common name. Seven of the
clotting factors are zymogens of serine proteases [any of numerous enzymes that
hydrolyze proteins and are classified according to the most prominent functional
group(i.e serine)] that are proteolytically activated by serine proteases further up the
cascade. Other clotting proteins, termed accessory factors, which are also activated by
these serine proteases, enhance the rate of activation of some of the zymogens. In both
cases the active forms of a factor is designated by subscript a.
The above figure is the blood clotting cascade in humans showing the division of its
primary stages into the so-called intrinsic and extrinsic pathways. The clotting factors are
named in black. The active clotting factors, with the exception of fibrin, are serine
proteases indicated in red. Red arrows represent their proteolytic activation of other
factors in the cascade. Accessory factors including Ca2+ and phospholipid membrane (PL)
are indicated in green. There are many feedback reactions that accelerate the clotting
process.
 The blood clotting system is in all vertebrates, contains a number of homologous serine
proteases and therefore appears to have developed through a series of gene duplications.
The C-terminal ~250 residues of these proteases, which comprise their catalytically
active domains, are also homologous to the pancreatic serine proteases trypsin,
chymotrypsin, and elastase. The blood clotting proteases and digestive proteases are
activated by proteolytic cleavages that precede their C-terminal segments. The clotting
proteases differ from the digestive enzymes in the presence of Ca 2+ on an appropriate
phospholipid membrane. The resulting N-terminal fragments are quite large (150-582)
residues and, with the exception of prothrombin are linked to their C-terminal segments
via disulfide bonds so that these segments do not separate upon activation. The N-
terminal segments are thought to be responsible, at least in part, for the exquisite
specificities of the proteolytic blood clotting factors.

Fibrinogen to Fibrin

Blood clots consist of arrays of cross-linked fibrin that forms an insoluble fibrous
network. See the scanning electron micrograph of a blood clot showing a red cell
enmeshed in a fibrin network. Fibrin is made from the soluble plasma protein fibrinogen
(factor I) through a proteolytic reaction catalyzed by the serine protease thrombin (factor
II). Fibrinogen comprises 2 to 3% of plasma protein. A molecule of fibrinogen consists of
three pairs of chains Aα(610 residues), Bβ (461 residues) and γ (411 residues) and two
pairs of N-linked oligosaccharides of ~2.5 kD each. Here A and B represent the 16- and
14-residue N-terminal fibrinopeptides that thrombin cleaves from fibrinogen, so a fibrin
monomer designated α2β2γ2.
Electron microscopic and low-resolution X-ray crystallographic studies indicate that
fibrinogen is a two fold symmetric elongated molecule ~450 A in length, that has two
nodules at each end and one in the middle. Its 6 polypeptide chains are joined by 17
disulfide bonds, 7 within each half of the dimer and 3 linking these two protomers. The
central region of each protomer consists mainly of a three-stranded coiled coil of a
helices in which the α, β, and γ chains each contribute a strand. The peripheral nodules
diagrammed are formed by the C-terminal domains of the β and γ chains. The C-terminal
segment of the α chain apparently lacks a defined conformation and is therefore not
represented.

Fibrin Polymerization

Thrombin specifically cleaves the Arg-X peptide bond (in most animals X is Gly) joining
each fibrinopeptide to fibrin. Fibrin then spontaneously aggregates to form fibers that
have a banded structure that repeats every 225 A . This is one half repeat length of fibrin
monomer 450 A, which suggests that fibrin monomers associate as a half-staggered array
as shown below. The main reason the fibrin aggregates is that the loss of the
fibrinopeptides exposes otherwise masked sites that mediate intermolecular association.
In addition, fibrinogen aggregation is inhibited by charge repulsions. The fibrinopeptides
are highly anionic, and the fibrinogen has a charge of -8 at fibrinopeptide site whereas
that of fibrin is +5.

The diameters of fibrin fibers, which are fairly uniform (~50 nm) are important
determinants of a clot's physical properties. Through electron microscope studies the
fibrin is uniformly twisted. Consequently, the in-register molecules near the periphery of
a twisted fiber must traverse a longer path than molecules near the fiber's center. The
degree to which a molecule can stretch therefore limits the diameter of the fiber.
Molecules add to the outside of a growing fiber until the energy to stretch an added
molecule exceeds its energy of binding.

Fibrin-Stabilizing Factor

A soft clot is rather fragile. It is rapidly converted to a more stable hard clot by the
covalent cross-linking of neighboring fibrin molecules in a reaction catalyzed by fibrin-
stabilizing factors (FSF or XIIa). This transamidase initially joins the C-terminal
segments of adjacent γ chains by forming isopeptide bonds between the side chains of a
Gln residue on on γ chain and a Lys residue on another. Two such symmetrically
equivalent bonds are rapidly formed between each neighboring pair of γ chains as shown
below. The α chains are similarly cross linked to one another, but at slower rate.
The transamidation reaction forming the isopeptide bonds cross-linking fibrin monomers
in "hard" clots as catalyzed by activate fibrin-stabilizing factor (FSF, XIIIa).
FSF is present in both platelets and plasma. Platelet FSF consists of two 75 kD α chains,
whereas plasma FSF additionally has two 88 kD β chains. Both species of FSF occur as
zymogens that undergo thrombin-catalyzed cleavage of a specific Arg-Gly bond near the
N-terminus of each α chain with the consequent release of a 37 residue propeptide.

Thrombin Activation

Thrombin is a serine protease that consists of two disulfide-linked polypeptide chains. In

humans there are 36 residues for A chain and a 256 residues for B chain. The thrombin B
chain is homologous to trypsin and has similar specificity but is far more selective. It
cleaves only certain Arg-S and less frequently cleaves Lys-X bonds with a clear
preference for a Pro preceding the Arg or Lys.

Human thrombin is synthesized as a 579 residue zymogen, prothrombin (II), which is

activated by two proteolytic cleavages by activated Stuart factor (Xa), the product of the
preceding steps of the clotting cascade. The cleavage of prothrombin's Arg 271-Thr 272
and Arg 320-Ile 321 bonds releases its N-terminal propeptide and separates the A and B
chains. The latter cleavage, which yields active enzyme, results in the formation of an ion
pair between Ile 321 and Asp 524, much like that formed between chymotrypsinogen's
homologous Ile 16 and Asp194 in the activation of this zymogen. Thrombin then
autolytically cleaves its Arg 285-Thr 286 bond, thereby trimming away the N-terminal 13
residues of the A chain to yield thrombin.

Prothrombin's propeptide consists of three domains. An N-terminal 40 residue Gla

domain followed by two 40% identical ~115 residue kringle domains. Kringles are cross-
linked by three characteristically located disulfide bonds that gives three triple looped
sequence motifs. Gla domains and kringles occur in several of the proteins involved in
the formation and the breakdown of blood clots.

Vitamin K is an Essential Cofactor in the Synthesis of γ-Carboxyglutamate

Prothrombin, as well as the Factors VII, IX and X, is synthesized in the liver in a process
that requires and adequate dietary intake of vitamin K. Lack of vitamin K or the presence
of a competitive inhibitor such as dicoumarol or warfarin causes the production of an
abnormal prothrombin that is activated by factor Xa (Stuart factor) at only 1 to 2% of the
normal rate. This was puzzling because normal and abnormal prothrombins seemed to
have identical amino acid compositions. NMR studies eventually established however,
that normal prothrombin contains γ-Carboxyglutamate (Gla) residue, 10 of which occur
between residues 6 and 32 in human prothrombin. Abnormal prothrombin, in contrast,
contains Glu in the place of Gla residues. Vitamin K must therefore be a cofactor in the
post-translational conversion of Glu to Gla. The reason why prothrombin's Gla residues
were not initially detected is because they decarboxylate to Glu under the condition of
acid hydrolysis normally used in amino acid composition determinations.

Prothrombin Activation
Factor Xa (Stuart factor) by itself, is an extremely sluggish prothrombin activator. In the
presence of activated proaccelerin (Factor Va), Ca2+, and phospholipid membrane, its
activity is enhanced 20,000-fold. The membrane surface in contact with the activation
complex must contain negatively charged phospholipids such as phosphatidylserine in
order to stimulate this rate enhancement. Such phospholipids occur almost exclusively on
the cytoplasmic side of cell membranes which of course are normally not in contact with
the blood plasma. In addition, 20% of factor V (proaccelerin) in the blood is stored in the
platelets and released only upon platelet activation. Consequently, physiological
prothrombin activation normally takes place at a significant rate only in the vicinity of an
injury. Ca2+ is required for either prothrombin or factor Xa to bind phospholipid
membranes. These proteins are anchored to the membrane via Ca2+ bridges. Prothrombin
(II) and factor Xa (Stuart factor) from vitamin K-deficient animals have greatly reduce
membrane binding affinities compared to the corresponding normal proteins. The Gla
side chains, which are much stronger Ca2+ binding sites. Prothrombin (II), proconvertin
(VII), Christmas (XI), and Stuart (X) have homologous N-terminal segments which
contain the 9 to 12 conserved Gla residues. The excision of prothrombin's N-terminal
propeptide releases the resulting thrombin from the phospholipid membrane so that it can
activate fibrinogen in plasma. The other vitamin K-dependent zymogen (proconvertin
(VII), Christmas (XI), and Stuart (X) remain bound to the phospholipid membrane after
their activation Activated thrombin specifically cleaves prothrombin's propeptide at it's
Arg 155- Ser 156 bond to yield its 155 residue N-terminal segment, the so called
fragment 1, which consists of prothrombin's Gla domain and its kringle 1.

Activated proaccelerin (VA), the accessory factor in prothrombin, is activated by a

thrombin-catalyzed proteolytic cleavage. Prothrombin activation, in this indirect way, is
thereby autocatalytic. Thrombin, in vitro can also directly activate prothrombin by
cleaving its Arg 283-Thr 284 bond, but this reaction has been shown to be
physiologically insignificant. VA is subject to further thrombin-catalyzed proteolysis,
which inactivates it. Thrombin can proteolytically inactivate thrombin molecules. Clot
formation is therefore self limiting, a safeguard that helps prevent blood clots from
propagating away from the site of an injury.

Thrombin structurally resembles trypsin. The structure of the B-chain closely resembles
that of pancreatic serine proteases as had been expected from the high degree of sequence
homology. The thrombin's substrate-binding cleft is much deeper than those of the
pancreatic serine proteases. Steric hindrance by loops greatly restrict access to the active
site and presumably contributes to thrombin's high specificity and its poor binding of
most natural serine protease inhibitors. The specificity of thrombin for fibrinogen is
largely attributable to its so called anion-binding site, and extension of thrombin's
substrate binding cleft which is lined with positively charged side chains and which bind
the highly anionic fibrinopeptides.

Intrinsic Pathway

Factor X (Stuart factor) may be activated by two different proteases. By factor IXa
(Christmas factor) a product of the intrinsic pathway and factor VIIa (proconvertin) the
product of the extrinsic pathway. It has long been known that bringing blood into contact
with negatively charged surfaces, such as those of glass, or kaolin (clay) initiates clotting.
In vivo, collagen and platelet membranes are though to have the same effect.

The contact system consist of four glycoproteins:

1. Hageman factor (XII),

2. Prekallikrein,
3. Plasma thromboplastin antecedent or PTA (factor XII)
4. High molecular weigh kininogen (HMK)

Adsorption to a suitable surface is thought to somehow activate the Hageman factor

which, in the presence of high molecular weight kininogen (HMK), hydrolyzes to the
active protease kallikrein. Kallikrein in turn proteolytically activates Hageman factor do
that these two proteins reciprocally activate each other.

The nature of contact-activated Hageman factor is enigmatic; it is by no means certain

that physical adsorption to a surface cleaves the same bond as does kallikrein or, for that
matter, cleaves any bond at all. Much of the experimental difficulty in resolving this issue
is a consequence of the contact-activation process's autocatalytic nature. Prekallikrein,
contact activated Hageman factor's substrate is the zymogen of the protease that activate
Hageman factor. Consequently, in any measurement of its activity, the nature of contact
activated Hageman factor is immediately obscured by large amounts of rapidly generated
kalikrein activated Hageman factor.

The final reaction mediated by the contact system is the proteolytic activation of factor
XI by activate Hageman factor ((XIIa) in the presence of HMK. Although the contact
system is clearly effective in initiating in vitro clot formation, its in vivo importance is in
doubt because individuals deficient in Hageman factor, prokallikrein, or HMK do not
suffer from bleeding problems.

Factor XIa (plasma thromboplastin antecedent PTA) catalyzes the proteolytic activation
of Christmas factor (IX), a Gla-containing glycoprotein, in a Ca2+ requiring reaction that
takes place on a phospholipid membrane surface. No accessory factor are known for this
reaction. Christmas factor may also be activated by activated proconvertin (VIIa), a
product of the extrinsic pathway.

Factor X is proteolytically cleaved by activated Christmas factor (IXa) on a phospholipid

membrane in a reaction requiring Ca2+ and the accessory factor activated antihemophilic
factor (VIIIa). Antihemophilic factor, as is proaccelerin (V), is proteolytically activated by
thrombin in a second autocatalytic process leading to prothrombin activation. Not
surprising, proaccelerin and antihemophilic factor are homologous proteins.
Antihemophilic factor circulates in the plasma in complex with von Willebrandt factor; in
fact, the activities of these two substances were initially attributed to a single protein.

Extrinsic Pathway
The extrinsic pathway, the alternative arm of the clotting cascade, is initiated by the
proteolysis of proconvertin (VII), a process that can be catalyzed by activated Hageman
factor (XIIa) as well as by thrombin. Activated proconvertin (VIIa) mediates the activation
of Stuart factor (X). In the presence of phospholipid membrane, Ca2+ and tissue factor
thromboplastin (III), the rate is enhanced 16,000-fold.

Tissue factor thromboplastin (III) is an integral membrane glycoprotein that occurs in

many tissues and is particularly abundant in brain, lung, blood vessel walls and placenta.
Consequently, an injury that exposes blood to tissue rapidly initiates the extrinsic
pathway. In fact, the addition of tissue factor thromboplastin to the extrinsic system
causes clot formation in 12 s, where as the intrinsic system requires several minute to do
so. These observations suggest that the intrinsic pathway is normally of little significance.
However, the severity of the hemophilias resulting from intrinsic pathway clotting factor
deficiencies clearly establishes the importance of the intrinsic pathway in blood clotting.
The two pathways are not really independent since they are coupled through a number of
reactions.

Control of Clotting

The multilevel cascade of the blood clotting system permits enormous amplification of it
triggering signals. Proconvertin (VII), Stuart Factor (X) prothrombin (II) and fibrinogen
(I) are present in plasma in concentration of 1, 8, 150, and 4000 µg/ml respectively.
Clotting must be regulated since even one clot can have fatal consequences. Indeed,
blood clots are the leading cause of strokes and heart attacks, which are the two major
causes of human death in developed countries.

A variety of factors limit clot growth. There are several interactions among the various
clotting factors that inhibit blood coagulation. The dilution of active clotting factors by
blood flow reduces the risk of clotting. Clots are selectively removed from the circulation
by the liver. Plasma contains several serine protease inhibitors whose presence prevents
clots from spreading beyond the vicinity of an injury. For example, antithrombin (58 kD)
inhibits all active proteases of the clotting system except Proconvertin (VIIa) by binding
to them in a1:1 complex. The presence of heparin, a sulfated glycoaminoglycan, enhances
the activity of antithrombin by several hundred folds. Heparin occurs almost exclusively
in the intercellular granules of the mast cells that line certain blood vessels.

Protein C is another plasma protein that limits clotting. This Gla residue containing 62
kD zymogen is activated by thrombin to proteolytically inactivate proaccelerin (V) and
antihemophilic factor (VII). Activated protein C attacks the active forms of these
accessory factors more readily than their non-active forms. The importance of protein C
is demonstrated by the observation that individuals who lack it often die in infancy of
massive thrombotic complications.

Thrombomodulin is a 74 kD glycoprotein that projects from the cell surface membranes

of the vascular endothelium. Thrombomodulin specifically binds thrombin so as to
convert it to a form with decreased ability to catalyze clot formation and 1000 fold
increased capacity to activate protein C.

Clot lysis

Blood clots are only temporary patches; they must be eliminated as wound repair
progresses. This is a particularly urgent need when a clot has inappropriately formed or
has broken free into the general circulation. Fibrin is easily dismantled in a process
termed fibrinolysis. The demolition agent is a plasma serine protease named plasmin.
This enzyme specifically cleaves fibrin that is a triple coiled protein. Plasmin cuts away a
covalently linked α chain which is not in the triple coiled region. The rather open mesh-
like structure of a blood clot gives plasmin relatively free access to polymerized fibrin
molecules thereby facilitating clot lysis.

Plasmin is formed through the proteolytic cleavage of 82 kD zymogen plasminogen, a

protein that is homologous to the zymogen of the blood-clotting cascade. There are
several serine proteases that activate plasminogen, most notably the 54 kD enzyme
urokinase, which is synthesized by the kidney and occurs as its name implies in the urine.
Tissue type plasminogen activator (t-PA), a 70 kD protein, occurs in vascular tissues and
activates plasminogen. In addition, activated Hageman factor, in the presence of
prekallikrein and HMK activates plasminogen. Fibrinolysis is not as simple as just a
zymogen and its activators. There are several inhibitors. The 70 kD glycoprotein α2-
antiplasmin forms an irreversible equal molar complex with plasmin and prevents it from
binding to fibrin. The α2-antiplasmin cross-links to fibrin α chains through the action of
activated FSF (XIIIa) thereby making hard clots less susceptible to fibrinolysis than soft
clots.

Plasminogen activators have received considerable medical attention aimed at rapidly

dissolving the blood clots responsible for heart attacks and strokes. Streptokinase, a 45
kD protein produced by certain streptococci, has shown considerable utility in this regard
particularly when administered together with aspirin. Despite its name streptokinase
exhibits no enzymatic activity. Rather, it acts by forming a tight 1:1 complex with
plasminogen that proteolytically activates other plasminogen molecules. The use of
streptokinase to dissolve clots has the apparent disadvantage that it activates plasmin to
degrade fibrinogen as well as fibrin thereby increasing the risk of bleeding problems,
particularly strokes.

The therapeutic use of t-PA, which has been synthesized by recombinant DNA
techniques, is thought to eliminate these problems because this enzyme activates
plasminogen only in the presence of a blood clot.

Clinical Laboratory Procedures Clinical laboratory procedure used in hematology.

View an animation of eurythrocyte clearance

View an animation of how the sonoclot works

View an animation of how the Chronolog works

Strokes