1. HOW DOES BLOOD CLOT ROSHNI KULKARNI MD USHA M REDDY MD
Professor, Department of Pediatrics & Human Development, Michigan State University
USHA M REDDY MD
Visiting Scholar, MSU
Multimedia, Graphics, Webpage Consultant
BRUCE L EVATT MD
Director, Hematologic Disease Branch Center for Disease Control (CDC)

2. How does blood clot Platelet HEMOSTASIS Hemostasis overview
Platelet activation
Extrinsic pathway
Intrinsic pathway
Tissue factor pathway
Platelet
Platelet Production
Platelet Membrane
Platelet Body
Platelet Granules
Platelet role in Hemostasis

3. HOW DOES BLOOD CLOT - OVERVIEW
Overview of hemostasis    Hemostasis is the balance between bleeding and clotting (thrombosis). It is achieved by the following components in the blood:  1. Blood vessel wall  2. Platelets  3. Clotting proteins or factors  4. Fibrinolytic system  5. Naturally occurring anticoagulants.    Introduction    The purpose of the blood clotting or coagulation system is two-fold:    1. Keep blood in the fluid state such that it circulates.    2. Prevent leakage of blood whenever there is an injury to the blood vessel wall or from normal wear and tear, by sealing defects in the vessel wall or endothelium.    When a blood vessel is injured, it narrows (vasoconstriction) thereby diverting and decreasing the amount of blood flow. At the site of the injury, blood is exposed to the subendothelium (the layer below the endothelium); bleeding is then rapidly stopped by a process known as hemostasis. There are three components of hemostasis:    First the platelets, a disc shaped tiny blood cells, stick or adhere to the damaged blood vessel and then to each other (aggregation) forming a platelet clump that can plug and stop minor bleeding. A glue like protein called von Willebrand factor is produced by the endothelium as well as the platelets and binds the platelet to the site of injury.    As the platelet adhere and aggregate, they change shape (like starfish) and are called “activated”. Such platelets release chemicals such as serotonin that further potentiate vasoconstriction, thromboxaneA2 that causes platelet aggregation. Activated platelets also express on their surface certain proteins (receptors) that attach to von Willebrand factor, fibrinogen (glycoprotein IIb-IIIa), collagen (glycoprotein Ia) and a crucial clotting protein called thrombin. A “platelet plug” is sometimes sufficient to stop minor bleeding.    Finally the activated platelets and the damaged tissue initiate blood clotting by activating a number of clotting proteins. A clot often stops major bleeding. Excess clot formation is prevented or removed by proteins called anticoagulants (shown here as the revolving blade termed plasmin). Platelet release a factor called platelet derived growth factor (PDGF) that promotes wound repair and healing.  

4. PLATELET ACTIVATION

5. EXTRINSIC PATHWAY
The Extrinsic pathway The extrinsic pathway is so called because it is initiated by tissue factor/VIIa complex. Exposure to inflammatory cytokines or injury to cell membrane causes TF to be expressed on the surface of endothelium, activated platelets or monocytes. Tissue factor (TF) is normally not detected in circulation. The extrinsic pathway is assessed in the laboratory by the prothrombin time (PT). · Tissue factor activates FVII to VIIa, in the presence of calcium. · The TF/VIIa complex activates FX to Xa in the presence of calcium. · When Xa has been formed, activation can proceed down the common pathway of both the intrinsic and extrinsic system as follows: § Factor V binds factor Xa and factor II in the presence of a phospholipid surface so that factor Xa can cleave factor II to form thrombin. § The final step in this series of reactions is proteolytic conversion of fibrinogen to fibrin (measured clinically by the thrombin time). For this reaction, thrombin splits off fibrinopeptides A and B from fibrinogen. § After the removal of the fibrinopeptides, fibrin monomers undergo spontaneous polymerization by hydrogen bonding to form a network. § This network is then stabilized by factor XIII. The cross-linked fibrin is not only far more insoluble than the fibrin polymer; it is also more resistant to digestion by plasmin. Abnormalities of this system (i.e., absence or abnormalities of factors VII, X, V, II or fibrinogen, and inhibitors of these factors) produce an abnormal prothrombin time (PT). Similarly, abnormalities of fibrinogen and inhibitors of the conversion of fibrinogen to fibrin result in an abnormal thrombin time.  

6. INTRINSIC PATHWAY
THE INTRINSIC PATHWAY The primary platelet plug, by itself, is only a temporary seal, and the formation of the clot is necessary to secure the repair of the damaged vessel. The proteins involved in the coagulation process can be described as falling into 4 general categories: · Zymogens-enzyme precursors that circulate in an inactive state until activated. · Cofactors (factor VIII and factor V) that organize proteins, thereby increasing the rate of enzymatic reactions. · Fibrinogen (factor I)—a protein that once cleaved by thrombin, forms a gel. · Inhibitors of coagulation—that act to interfere with the coagulation process in a controlled way so that the thrombus (clot) remains at the site of injury. There are two pathways: historically termed the extrinsic system and the intrinsic pathways. These systems reflect how clotting occurs in the test tube during tests. Clotting in the body is initiated slightly differently. However, it is clinically useful to think about the clotting process in terms of these two systems, because the physician can direct investigation towards only a few tests using the concept of this coagulation scheme. The intrinsic Pathway THE INTRINSIC Pathway so called because the components are contained within the vascular system, is assessed in the laboratory by the APTT test. In the test tube, the initiation of clotting via the intrinsic system (measured by the activated partial thromboplastin time or APTT) begins with the activation of factor XII when it is exposed to the glass surface. This surface activation of factor XII depends upon two other molecules found in plasma high molecular weight kininogen (Fitzgerald factor) and prekallikrein (Fletcher factor). · Contact with surface (i.e., a glass test tube), alters conformation of FXII molecule. Factor XII, also called Hageman factor, is activated primarily by negatively charged surfaces such as kaolin, ellagic acid, glass etc., as well as phospholipids. · The bound FXIIa activates prekallikrien to kallikrein, which in turn causes proteolysis of FXIIa. · In addition, kallikrein converts low as well as high-molecular-weight kininogen (HMWK, also called Fitzgerald factor) into bradykinin. The latter is a potent vasodilator. · HMWK (Fitzgerald factor) facilitates activation of prekallikrein to kallikrein. · Assists in activation of both XII and XI by facilitating binding of proteins to surfaces in proximity to each · FXIIa converts plasminogen to plasmin. · The FXIIa/HMWK complex activates FXI to XIa. · The FXIa activates FIX to IXa. · Factor VIII probably serves as a regulatory or organizing protein and allows factor IXa and factor X to come in close proximity for proper proteolysis to occur. In vivo a phospholipid requirement is probably provided by platelets. The product of this reaction (IXa, VIII, and X) is Xa. · The FVIII circulates bound to von Willebrand Factor (vWF) that protects it from proteolysis. · Factor Xa forms the crossroads of the two clotting systems by participating in both the extrinsic and the intrinsic pathways. · Once factor Xa is formed, activation can proceed down the common pathway as described below. · Factor Xa binds to FVa and activates II (prothrombin) to IIa (thrombin). · Factor IIa or thrombin cleaves fibrinogen, liberates fibrinopeptides, allowing the fibrin monomers to link and form fibrin clot. DETAILS OF IMPORTANT PROTEIN INTERACTIONS WITHIN THE CASCADE. Mechanism of Factor XII (Hageman) activation. Activation of the coagulation via Factor XII is predominately an in vitro (in the test tube) event. It is however, important clinically, for it forms the basis of initiating the APTT and can give test results that may confuse some clinicians. When the glass surface combines with factor XII, a conformational change in the molecule occurs that allows factor XII to be more susceptible to the proteolytic action of prekallikrein. High molecular weight kininogen attaches factor XI to the surface in close proximity to factor XII. Although factor XII, prekallikrein (Fletcher factor), and high molecular weight kininogen (Fitzgerald factor) are all essential for normal clotting in the test tube, their biological purpose for clotting is uncertain. Persons who are deficient in these factors have no significant bleeding disorders. In fact Mr. John Hageman, the person who was first described with FXII deficiency died of a pulmonary embolism, following a hip fracture. · Deficiency of XII, prekallikrein and HMW kininogen all result in extremely long partial thromboplastin times (APTT), blood that is extremely slow clotting in a test tube, but are not associated with bleeding disorders. · XII interacts with the plasminogen system (see below), and paradoxically, a deficiency predisposes to thromboembolism (remember Mr. Hageman!). Abnormalities of any of the following clotting factors produce an abnormal activated partial thromboplastin time(APTT): high molecular weight kininogen, prekallikrein, factors XII, XI, X, IX, VIII, V, II or fibrinogen. Abnormalities of any of the following clotting factors produce both an abnormal APTT and an abnormal prothrombin time: Factors X, V, II, or fibrinogen (Factor I)  

7. TISSUE FACTOR PATHWAY
TISSUE FACTOR PATHWAY % of FVII circulates as FVIIa. Injury exposes Tissue Factor (TF). TF binds to FVIIa 2.TF-VIIa complex activates X to Xa and IX to IXa (more X than IX) FXa activates V to Va and combines with the co-factor FVa on the TF bearing cell. 3.FXa/Va complex converts a small amount of prothrombin to thrombin. This small amount of thrombin does the following: a. Causes platelet activation that sets the stage for tenase and prothrombinase complex formation. b. Converts FXI to XIa that leads to the activation of FIXa. c. Activate and release FVIII from von Willebrand factor (vWF) resulting in FVIIIa . This combines with the IXa (originally formed ) and activates plasma X to Xa. Assembly of this 'tenase' (FVIIIa +FIXa +Xa) complex occurs on the platelet surface. d. Activates FV (some of which comes from the platelet alpha granule). It is a critical cofactor in the prothrombinase complex (Xa+ Va+ II) that assembles on the platelet surface FXa cannot move to the platelet surface because of the presence of normal plasma inhibitors, but instead remains on the TF-bearing cell. 4.The IXa moves to the platelet surface as it is not destroyed quickly by circulating antithrombin. 5. The tenase and prothrombinase lead to enough FIIa formation , additional platelet activation conversion of fibrinogen to fibrin. Hemophilia is a Failure of Platelet Surface Thrombin Generation, and therefore a lack of thrombin burst.  

8. PLATELET
The Platelet info Platelets are an important component of hemostasis. Platelet Production Platelets are cytoplasmic fragments of megakaryocyte (precursor cells of platelets) that contain no nucleus. They number about 150,000 to 400,000/mm3 in the blood and range in diameter from 1-4 microns and a volume of 5-12 femtoliters (fl) 12; however, the average platelet is about 7.3 fl. Platelet production is regulated to meet the demand for circulating platelets by growth factors. One of these growth factors is "thrombopoietin". Megakaryocytes mature by endomitosis, i.e., nuclear division without cell division. As the megakaryocytes mature, the cytoplasm becomes demarcated into platelet subunits. Platelets are released into the circulation through a process of megakaryocyte fragmentation, and have a life span of days. Normally, two-thirds of the platelets released from the bone marrow stay in the general circulation. The remainder of the platelets are in a pool in the spleen that is freely exchangeable with circulating platelets. With progressive splenomegaly, a larger percentage of the body's platelets are pooled in the spleen and, as a result, peripheral thrombocytopenia may develop unless the bone marrow can increase platelet production sufficiently to compensate. Splenomegaly by itself does not lead to a shortened platelet survival. In general, there is a direct relationship between the megakaryocyte mass in the bone marrow and the rate at which platelets are added to the circulation. This relationship breaks down when megakaryocyte development is faulty or when megakaryocytes are destroyed within the bone marrow. In these circumstances platelet delivery to the circulation is impaired, and thrombopoiesis is said to be ineffective. Ineffective thrombopoiesis is a frequent finding in patients with vitamin B12 or folate deficiency and is analogous to the production problem of red cells, which occurs in these conditions and gives rise to ineffective erythropoiesis. When maximally stimulated, the bone marrow can increase platelet production 6-fold. However, when platelets are destroyed very rapidly, increased delivery to the peripheral blood will not occur for approximately five days. Structure In vivo the platelet is shaped like a porous disc and resembles a microscopic sponge. The invaginations of the surface membrane into the center of the platelet provide canals, through which components within granules of the platelet escape into the surrounding plasma during the release reaction. The platelet is covered by a plasma protein coat that is the mediator for the platelet membrane's adhesion to surfaces and allows coagulation proteins or factors to adhere to it. Neither adhesion of platelets to surfaces nor adhesion to proteins requires energy, but each process requires fibrinogen. The membrane beneath the surface coat contains phospholipids and selectively absorbs certain coagulation factors. Below the coat and membrane are submembranous filaments made of actomyosin that cause the platelet to contract. The equator of the disc-shaped platelet contains a prominent structure composed of microtubules that maintain the platelet's disc-like shape. After the release reaction begins, the microtubules constrict concentrically. This process moves the clusters of organelles and granules toward the center of the platelet. The granules are important because they contain several constituents that take part in the release reaction, i.e., ADP, catecholamine, and serotonin. Energy for these reactions is derived primarily from glycogen storage granules, and the platelet is capable of both aerobic and anaerobic metabolism. Glycoproteins (Gp) on the surface of the platelets mediate platelet adhesion and aggregation. These glycoproteins trigger reactions of the platelet during these processes. They also act as receptors for extracellular ligands, various proteins, and chemicals that affect platelet function. Glycoproteins have two other very important functions: they stabilize the platelet surface by contributing to the cell surface charge; they also give the platelet its antigen specificity. Immune problems arise when these antigens are directed against certain glycoproteins. Properties and Functions of Membrane Glycoproteins of Platelets *Mediate platelet adhesion and aggregation *Act as receptors for extracellular ligands *Participate in recognition phenomena and phagocytosis · *Bind complement · *Give platelet its antigenic specificity *Mediate membrane transport processes · *Act as enzymes or anti-proteases · *Stabilize platelet surface and contribute to cell surface charge Role of Platelets in Hemostasis: In hemostasis, platelets play three main roles: 1. They maintain the endothelial surface. Loss of circulating platelets quickly results in morphologic changes in the endothelial cells of the capillaries. These changes cause intravascular material to leak into the capillary bed. 2. They initially arrest bleeding in severed blood vessels. 3. They provide phospholipid, which acts as the catalytic surface for initiation of the coagulation process. When platelets encounter a break in the endothelial surface, several important actions cause the bleeding to cease. A.Adhesion occurs when they encounter collagen, membranes or non-collagenous microfibrils beneath the basement membranes. Adhesion is mediated by GP-Ib and von Willebrand Factor (vWF). a.Receptors (especially for glycoprotein-Ib) bind vWF and facilitate adhesion. b.vWF is an extremely large molecule (a multimer) that bridges the gap between different cells, the platelet, and subendothelial surfaces. c.vWF is a very “sticky” protein and binds readily. d.Besides binding GP-Ib, vWF also binds to GP-IIb-IIIa to facilitate adhesion. e.Fibrinogen, binding to the GP IIb-IIIa complex on two separate platelets, can bridge the gap between those two platelets (this is important to a subsequent function—aggregation). f.In the absence of these factors (or their receptors) both adhesion and aggregation are abnormal and certain types of bleeding problems can occur. B.Platelets then undergo a "release reaction". This process involves change from a disc shape to a spherical shape, constriction of the microtubules toward the center of the platelet, and the release of contents of the granules (primarily ADP, catecholamine, and serotonin) into the open canalicular system. These platelets thus become “activated”. a.During the release reaction, the granules within the platelets release their contents into the canicular system. b.These granule components then leak into the plasma around the platelet. c.The released ADP binds other circulating platelets in close proximity to activated platelets and this binding to surface receptors initiates the release reaction in these “recruited” platelets. d.The release reaction is mediated by means of Thromboxane A2. Arachidonate is integrated in the phospholipids in the cell wall and is freed by a phospholipase, activated during the process of adhesion or by binding of certain ligands to receptors on the platelet surface. e.Cyclo-oxygenase converts arachidonate to an intermediate, prostaglandin H2. f.In the platelets PGH2 is acted upon by thromboxane synthetase to form thromboxane A2. Thromboxane A2 promotes the release reaction, change in shape, and aggregation. In the endothelial cell, the pathway is different from that of the platelet. g.Following the formation of PGH2, prostacyclin synthetase produces PGI2, which inhibits adhesion, aggregation and the release reaction, forces that oppose those of Thromboxane A2. h.Aspirin blocks cyclo-oxygenase and therefore the pathway that leads to both Thromboxane A2 and PGI2. In the platelet, the block is permanent for the life of the platelet, because the platelet does not have a nucleus to direct the formation of more cyclo-oxygenase. This yields a platelet that cannot function. Since the life of the platelet is about 7-10 days, the effect of the aspirin on bleeding will gradually decrease over a week, as new platelets replace those that were exposed to aspirin. In the endothelial cell, however, cyclo-oxygenase is regenerated. C.Platelet aggregation occurs as platelets are "recruited" from the immediate area by the released contents, for example, ADP. This process is accomplished by fibrinogen, binding to the GP IIb-IIIa complex on separate platelets, and bridging the gap between platelets When the release of ADP, or other aggregating agents, is minimal, the local concentration of these agents do not reach a high level, this aggregation may be reversible; with higher concentrations, aggregation is irreversible. Associated with the change of shape of the platelet and the release reaction, is the appearance of clotting promoting sites (historically referred to as platelet factor 3) on the platelet membranes. The receptor sites for the coagulation proteins serve as a catalytic site for the clotting proteins and assists in initiating the clotting mechanism. Important coagulation proteins that are bound to the surface include factors V and VIII among others. E.Clot retraction occurs when platelets are trapped within the enlarging blood clot. During the release reaction, pseudopodia like structures are extended some distance from the surface of the platelet, and attach to similar structures on adjacent platelets. With time, these structures retract, pulling the body of the clot together, and sealing the vessel wall at the site of injury.  

9. PLATELET PRODUCTION

10. THE PLATELET MEMBRANE
PLATELET MEMBRANE RECEPTORS Platelet membrane receptors are embedded in the platelet phospholipid bi-layer and allow interaction of the platelet via proteins with the blood vessel wall and with other platelets. Glycoproteins (Gp) are also called integrins Glycoprotein mediators of platelet adhesion The most important ones are, Gp-Ib-IX that interacts with the von Willebrand factor (vWF), and GpIa-IIa that interacts with collagen. These glycoproteins tether the platelet to the blood vessel wall and prevent it from being detached by flowing blood. Other adhesive proteins include fibronectin and laminin. Glycoprotein mediators of aggregation GpIIb-IIa binds to fibrinogen that acts as a bridge between two platelets. It can also bind to vWF. Other receptors Protease activated receptor (PAR-1) binds to thrombin, a powerful aggregating agent. Other receptors include receptors for Prostaglandin, Adenosine Di-phosphate (ADP), epinephrine, thromboxane A2.  

11. THE PLATELET BODY
The platelet body The platelet consists of: 1. Cytoskeleton or peripheral zone containing the plasma membrane, receptors and the open canalicular system. 2. Sol-Gel zone that is centrally located and contains the cytoplasm and contractile proteins 3. Organelle zone made up of granules, lysosomes, mitochondria, Granules and cytoplasmic organelles The platelet membrane is a phospholipid bi-layer and responds to stimuli. It communicates with the interior via channels called the open canalicular system (OCS). The latter are invaginations of the surface membrane and provide a channel for entry of plasma proteins and exit of platelet chemicals. The membrane also has glycoproteins (Gp) receptors for various proteins such as collagen, von Willebrand factor. The glycoproteins participate in shape change and adhesion. Contractile proteins of the microtubules and microfilaments in the sol-gel zone are responsible for the discoid shape of the platelets. The microtubule lies as an circular coil underneath the membrane. They dissociate into subunits during platelet shape change. Actin is the predominant contractile protein in the cytoplasm. Actin-binding proteins bind actin and Gp1b-IX  

12. THE PLATELET GRANULES
Platelet granules The platelet consists of: 1. Cytoskeleton or peripheral zone containing the plasma membrane, receptors and the open canalicular system. 2. Sol-Gel zone that is centrally located and contains the cytoplasm and contractile proteins 3. Organelle zone made up of granules, lysosomes, mitochondria, Granules and cytoplasmic organelles The main feature of the organelle zone is the granules. The granule contents are released during platelet activation. There are three types of granules: Dense granules appear as dark bodies on electron microscope. There are 2-7 dense granules per platelet. They contain Adenosine-Di-phosphate (ADP) and ATP, serotonin, calcium and magnesium, and cyclooxygenase,. The serotonin is a powerful vasoconstrictor. Alpha granules number approximately per platelet. They contain proteins some of which are specific to the platelets while other are absorbed from the plasma. Some of the contents include platelet derived growth factor (PDGF), Platelet factor 4 (PF-4), high molecular weight kininogen, fibronectin, thrombospondin and protein stores (absorbed from the plasma) of FV, von Willebrand Factor (vWF), plasminogen activator inhibitor 1 (PAI-1), fibrinogen etc. Lysosomal granules containing hydrolyses that dissolve phagocytosed debris  

13. THE PLATELET ROLE IN HEMOSTASIS
The platelet Role in Hemostasis In hemostasis, platelets play three main roles: 1. They maintain the endothelial surface. Loss of circulating platelets quickly results in morphologic changes in the endothelial cells of the capillaries. These changes cause intravascular material to leak into the capillary bed. 2. They initially arrest bleeding in severed blood vessels. 3. They provide phospholipid, which acts as the catalytic surface for initiation of the coagulation process. When platelets encounter a break in the endothelial surface, several important actions cause the bleeding to cease. A. Adhesion occurs when they encounter collagen, membranes or non-collagenous microfibrils beneath the basement membranes. Adhesion is mediated by GP-Ib and von Willebrand Factor (vWF). a. Receptors (especially for glycoprotein-Ib) bind vWF and facilitate adhesion. b. vWF is an extremely large molecule (a multimer) that bridges the gap between different cells, the platelet, and subendothelial surfaces. c. vWF is a very “sticky” protein and binds readily. d. Besides binding GP-Ib, vWF also binds to GP-IIb-IIIa to facilitate adhesion. e. Fibrinogen, binding to the GP IIb-IIIa complex on two separate platelets, can bridge the gap between those two platelets (this is important to a subsequent function—aggregation). f. In the absence of these factors, (or their receptors) both adhesion and aggregation are abnormal and certain types of bleeding problems can occur. B. Release reaction: Platelets then undergo a "release reaction". This process involves change from a disc shape to a spherical shape, constriction of the microtubules toward the center of the platelet, and the release of contents of the granules (primarily ADP, catecholamine, and serotonin) into the open canalicular system. These platelets thus become “activated”. a. During the release reaction, the granules within the platelets release their contents into the canalicular system. b. These granule components then leak into the plasma around the platelet. c. The released ADP binds other circulating platelets in close proximity to activated platelets and this binding to surface receptors initiates the release reaction in these “recruited” platelets. d. The release reaction is mediated by means of Thromboxane A2. Arachidonate is integrated in the phospholipids in the cell wall and is freed by a phospholipase, activated during the process of adhesion or by binding of certain ligands to receptors on the platelet surface. e. Cyclo-oxygenase converts arachidonate to an intermediate, prostaglandin H2. f. In the platelets, PGH2 is acted upon by thromboxane synthetase to form thromboxane A2. Thromboxane A2 promotes the release reaction, change in shape, and aggregation. In the endothelial cell, the pathway is different from that of the platelet. g. Following the formation of PGH2, prostacyclin synthetase produces PGI2, which inhibits adhesion, aggregation and the release reaction, forces that oppose those of Thromboxane A2. h. Aspirin blocks cyclo-oxygenase and therefore the pathway that leads to both Thromboxane A2 and PGI2. In the platelet, the block is permanent for the life of the platelet, because the platelet does not have a nucleus to direct the formation of more cyclo-oxygenase. This yields a platelet that cannot function. Since the life of the platelet is about 7-10 days, the effect of the aspirin on bleeding will gradually decrease over a week, as new platelets replace those that were exposed to aspirin. In the endothelial cell, however, cyclo-oxygenase is regenerated. C. Platelet aggregation occurs as platelets are "recruited" from the immediate area by the released contents, for example, ADP. This process is accomplished by fibrinogen, binding to the GP IIb-IIIa complex on separate platelets, and bridging the gap between platelets When the release of ADP, or other aggregating agents, is minimal, the local concentration of these agents do not reach a high level, this aggregation may be reversible; with higher concentrations, aggregation is irreversible. Associated with the change of shape of the platelet and the release reaction, is the appearance of clotting promoting sites (historically referred to as platelet factor 3) on the platelet membranes. The receptor sites for the coagulation proteins serve as a catalytic site for the clotting proteins and assists in initiating the clotting mechanism. Important coagulation proteins that are bound to the surface include factors V and VIII among others. E. Clot retraction occurs when platelets are trapped within the enlarging blood clot. During the release reaction, pseudopodia like structures extend some distance from the surface of the platelet, and attach to similar structures on adjacent platelets. With time, these structures retract, pulling the body of the clot together, and sealing the vessel wall at the site of injury.  

14. END