Overview of hemostasis

INTRODUCTION — Hemostasis is the process of blood clot formation at the site of vessel injury. When a blood vessel wall is disrupted, the hemostatic response must be quick, localized, and carefully regulated. Abnormal bleeding or thrombosis (ie, nonphysiologic blood clotting not required for hemostatic regulation) may occur when specific elements of these processes are missing or dysfunctional.

●The pathways of thrombin-mediated fibrin clot formation and plasmin-mediated clot lysis are linked and carefully regulated (figure 1 and figure 2 and figure 3) [1]. When they work in coordinated harmony, a clot is laid down initially to stop bleeding, followed by eventual clot lysis and tissue remodeling.

●Abnormal bleeding can result from diminished thrombin generation (eg, due to factor VIII deficiency) or enhanced clot lysis (eg, due to alpha-2-antiplasmin deficiency). Conversely, excessive production of thrombin (eg, due to an inherited thrombophilia) can lead to thrombosis.

The elements responsible for normal hemostasis will be reviewed here. Approaches to the patient with abnormal bleeding or abnormal thrombosis are discussed separately. (See "Approach to the adult with a suspected bleeding disorder".)

The uses of platelet function testing and coagulation assays to diagnose hemostatic abnormalities are discussed separately. (See "Platelet function testing" and "Clinical use of coagulation tests".)

PHASES OF THE HEMOSTATIC PROCESS — Although the clotting process is a dynamic, highly interwoven array of multiple processes [2], it can be viewed as occurring in phases, which are discussed in detail in the following sections:

●Endothelial injury and formation of the platelet plug. (See 'Formation of the platelet plug' below.)

●Propagation of the clotting process by the coagulation cascade. (See 'Clotting cascade and propagation of the clot' below.)

●Termination of clotting by antithrombotic control mechanisms. (See 'Control mechanisms and termination of clotting' below.)

●Removal of the clot by fibrinolysis. (See 'Clot dissolution and fibrinolysis' below.)

FORMATION OF THE PLATELET PLUG — Platelets are activated at the site of vascular injury to form a platelet plug that provides the initial hemostatic response to stop bleeding. (See "Inherited platelet function disorders (IPFDs)", section on 'Conceptual framework' and "Platelet biology and mechanism of anti-platelet drugs".)

Injury to the endothelium leads to exposure of the circulating blood to subendothelial elements from which it would normally be protected, and endothelial cell activation may further promote recruitment of platelets, other cell types, and procoagulant factors. (See "The endothelium: A primer".)

The functional response of activated platelets involves four different processes:

●Adhesion – The deposition of platelets on the subendothelial matrix

●Aggregation – Platelet-platelet cohesion

●Secretion – The release of platelet granule proteins

●Procoagulant activity – The enhancement of thrombin generation

Platelet activation — There are a number of physiologic platelet stimuli including adenosine diphosphate (ADP), epinephrine, thrombin, and collagen. ADP and epinephrine are relatively weak platelet activators, while collagen and thrombin are the most potent platelet activators.

The spatial hierarchy of platelet agonists under flow conditions begins with thrombin, which activates a core of platelets in the core of the hemostatic plug [3]. ADP activates more loosely packed platelets in a shell overlying the core, and thromboxane provides a critical activation in the shell region. (See 'Platelet secretion' below and 'Prostacyclin and thromboxane' below.)

Collagen — The intact endothelium prevents the adherence of platelets by the production of nitric oxide and prostacyclin. Intimal injury impairs these processes and exposes subendothelial elements such as microfibrils, laminin, and collagen. These factors lead to the adherence of platelets, platelet activation, and secretion.

Integrins are a superfamily of adhesive protein receptors that are found in many different cell types. They typically exists as a heterodimer composed of an alpha subunit and a beta subunit. The integrin glycoproteins GPIa/IIa (also known as alpha2 beta1) and GPVI are the two major platelet collagen receptors, playing critical roles in platelet adhesion and activation, respectively [4]. Patients with GPIa/IIa deficiency generally have mild bleeding diathesis, while severe spontaneous bleeding has been reported with platelet GPVI deficiency.

Thrombin — Thrombin activation of cells is mediated by a family of G-protein coupled protease-activated receptors (PARs) [5-8]. Platelets have a dual receptor system for thrombin, with two distinct receptors (PAR-1 and PAR-4). Thrombin cleaves the amino-terminal exodomain of PAR, exposing a new amino-terminus, which then serves as a tethered ligand that binds intramolecularly to the body of the receptor to initiate transmembrane signaling [9]. PAR-1 is a high-affinity receptor that mediates the effect of thrombin at low concentrations; PAR-4 is a low-affinity receptor that requires high levels of thrombin for activation [5]. Vorapaxar is an oral PAR-1 antagonist developed as an antiplatelet agent [10,11].

In an animal model of laser-induced vascular injury, it was shown that thrombin, rather than collagen, is the major agonist leading to platelet activation [12].

ADP — ADP binds to two G-protein coupled purinergic receptors, P2Y₁ and P2Y₁₂. Activation of P2Y₁ leads to calcium mobilization, platelet shape change, and rapidly reversible aggregation; activation of P2Y₁₂ leads to platelet secretion and more stable aggregation [13]. ADP is released from platelets upon platelet activation and functions in a paracrine/autocrine fashion to recruit additional platelets and amplify platelet aggregation. Clopidogrel blocks the activation of P2Y₁₂.

Platelet adhesion — Following activation, platelets undergo significant shape changes, producing elongated pseudopods that make the platelets extremely adhesive. Platelet adhesion is primarily mediated by the binding of platelet surface receptor GPIb/IX/V complex to von Willebrand factor (VWF) in the subendothelial matrix [14]. Deficiency of any component of the GPIb/IX/V complex or VWF leads to congenital bleeding disorders: Bernard-Soulier disease [15] and von Willebrand disease, respectively. (See "Pathophysiology of von Willebrand disease" and "Inherited platelet function disorders (IPFDs)", section on 'Conceptual framework'.)

In addition, there are other adhesive interactions that contribute to platelet adhesion. One example is binding of the platelet collagen receptor GPIa/IIa to collagen fibrils in the matrix [16].

Platelet aggregation — Platelet activation results in both exposure of and conformational changes in the GPIIb/IIIa receptor on the platelet surface, leading to binding of VWF and fibrinogen, resulting in platelet-platelet cohesion or aggregation [17-19].

GPIIb/IIIa is also a member of the integrin superfamily. The GPIIb/IIIa complex (integrin alphaIIb beta3) is the most abundant receptor on the platelet surface, with about 80,000 complexes per platelet. GPIIb/IIIa does not bind fibrinogen on nonstimulated platelets. However, following platelet stimulation (eg, by thrombin, collagen, or ADP), GPIIb/IIIa undergoes a conformational change and is converted from a low-affinity to a high-affinity fibrinogen receptor, a process referred to as "inside-out" signaling (since the conformational change in the external cell surface exposed GPIIb/IIIa complex is mediated by changes in the intracellular cytosolic portion ["cytoplasmic tail"] of the complex). Fibrinogen is a divalent symmetrical molecule, able to bind to two activated GPIIb/IIIa complexes on two different platelets, thus crosslinking them. (See "Disorders of fibrinogen" and "Platelet biology and mechanism of anti-platelet drugs", section on 'Integrin alphaIIbbeta3 (GPIIb/IIIa) binding to fibrinogen, activation, and platelet aggregation'.)

In addition to mediating platelet aggregation, when GPIIb/IIIa binds to immobilized VWF (on exposed subendothelial matrix), the cytosolic portion of the activated GPIIb/IIIa complex binds to the platelet cytoskeleton, resulting in platelet spreading and clot retraction, which has been referred to as "outside-in" integrin signaling. Thus, the GPIIb/IIIa complex integrates receptor-ligand interactions that occur on the external face of the membrane with cytosolic events in a bidirectional fashion [20,21]; it is the final common pathway for platelet aggregation, irrespective of the mode of platelet stimulation.

The importance of GPIIb/IIIa is illustrated by the congenital bleeding disorder Glanzmann thrombasthenia, which is characterized by mutations in the gene for either the alphaIIb or the beta3 subunit [22], as well as the clinical utility of GPIIb/IIIa antagonists in the treatment of coronary artery disease. (See "Acute ST-elevation myocardial infarction: Antiplatelet therapy", section on 'Glycoprotein IIb/IIIa inhibitors' and "Acute non-ST-elevation acute coronary syndromes: Early antiplatelet therapy", section on 'Glycoprotein IIb/IIIa inhibitors' and "Inherited platelet function disorders (IPFDs)", section on 'Glanzmann thrombasthenia'.)

Platelet secretion — Platelets contain two types of granules: alpha granules and dense granules (which appear dense on transmission electron micrographs). The alpha granules contain many proteins including fibrinogen, von Willebrand factor, thrombospondin, platelet-derived growth factor (PDGF), platelet factor 4, and P-selectin. Dense granules contain ADP, ATP, ionized calcium, histamine, and serotonin. Platelets secrete a variety of substances from their granules upon cell stimulation:

●ADP and serotonin stimulate and recruit additional platelets [23]. Platelet-released serotonin normally causes vasodilation; however, it can induce vasoconstriction in the presence of damaged or abnormal (dysfunctional) endothelium. ADP-activated platelets increase the surface expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells [24].

●Fibronectin and thrombospondin are adhesive proteins that may reinforce and stabilize platelet aggregates.

●Fibrinogen is released from platelet alpha granules, providing a source of fibrinogen at sites of endothelial injury in addition to that present in plasma [25].

●Thromboxane A2, a prostaglandin metabolite, promotes vasoconstriction and further platelet aggregation.

●Growth factors, such as PDGF, have potent mitogenic effect on smooth muscle cells. The release of PDGF from platelets at the site of vascular injury probably mediates tissue repair physiologically and, at a site of repeated injuries, may contribute to the development of atherosclerosis and coronary reocclusion following angioplasty. (See "The role of platelets in coronary heart disease".)

●Release of the thiol isomerase, protein disulfide isomerase (PDI), by platelets and disrupted vessel wall cells may serve to activate tissue factor and enhance the generation of fibrin and platelet thrombus formation at sites of vascular injury [26,27].

Procoagulant activity — Platelet procoagulant activity is an important aspect of platelet activation and involves both exposure of procoagulant phospholipids, primarily phosphatidylserine, and the subsequent assembly of the enzyme complexes in the clotting cascade on the platelet surface [28]. These complexes are an important example of the close interrelationship between platelet activation and activation of the clotting cascade that has been referred to as "cell-based" coagulation [29]. (See 'Multicomponent complexes' below.)

CLOTTING CASCADE AND PROPAGATION OF THE CLOT

Overview of clot propagation — The central feature of the clotting cascade is the sequential activation of a series of proenzymes or inactive precursor proteins (zymogens) to active enzymes, resulting in significant stepwise response amplification. As an example, the generation of a small number of factor VIIa molecules will activate many molecules of factor X, which in turn generates even larger numbers of thrombin molecules, which then converts fibrinogen to fibrin (figure 2). The resultant local generation of fibrin, in turn, enmeshes and reinforces the platelet plug.

The function of the active enzymes is markedly facilitated by the formation of multiple component macromolecular complexes, such as the tenases (X-ase) that activate factor X and the prothrombinase which produces thrombin from prothrombin. (See 'Multicomponent complexes' below.)

All of the procoagulants are synthesized in the liver except for von Willebrand factor (VWF), which is synthesized in megakaryocytes and endothelial cells, and factor VIII, which is produced in endothelial cells in the liver as well as other tissues such as lymphatics and renal glomeruli [30-32]. VWF and factor VIII are co-expressed in postcapillary high endothelial venules but not in most other endothelial cells. VWF stabilizes factor VIII. (See "Biology and normal function of factor VIII and factor IX" and "Pathophysiology of von Willebrand disease", section on 'VWF protein'.)

Post-translational modification of these factors is known to occur [33]. The best characterized modifications occur in the vitamin K-dependent procoagulants (ie, prothrombin, factors VII, IX, and X) and anticoagulants (ie, protein C and protein S). For each of these factors, vitamin K-dependent carboxylated glutamic acid residues function as calcium-binding sites that are important in the assembly of the membrane-bound macromolecular procoagulant complexes [34]. (See "Vitamin K and the synthesis and function of gamma-carboxyglutamic acid".)

Traditionally, the clotting cascade is depicted as consisting of an intrinsic and extrinsic pathway (figure 1). This view of coagulation is useful for interpreting in vitro tests of coagulation. (See "Clinical use of coagulation tests".)

●The intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (such as celite, kaolin, or silica in the in vitro activated partial thromboplastin clotting time [aPTT]).

●The extrinsic pathway is activated by tissue factor exposed at the site of injury or tissue factor-like material (thromboplastin, TPL in the in vitro prothrombin clotting time [PT]).

●Both pathways converge on the activation of factor X which, as a component of prothrombinase, converts prothrombin to thrombin, the final enzyme of the clotting cascade. Thrombin converts fibrinogen from a soluble plasma protein into an insoluble fibrin clot (tested in the in vitro thrombin time [TT]).

Thrombin generation — While the classical view of the clotting cascade based on intrinsic and extrinsic pathways has been useful in the interpretation of in vitro coagulation tests (eg, PT and aPTT), it may not be physiologically accurate.

It is now established that the generation or exposure of tissue factor at the wound site and its interaction with activated factor VII (factor VIIa) (figure 2) is the primary physiologic event in initiating clotting [35]. The small initial amount of thrombin generated then activates factor XI in a feedback manner, leading to amplification of thrombin generation. This was shown via the time course of thrombin generation, which demonstrated an initiation phase with only a small amount of thrombin being generated, followed by a propagation phase consisting of the bulk of thrombin generation, and finally cessation of thrombin generation [36].

Standard laboratory clotting tests, which detect initial fibrin clot formation, primarily measure the initiation and not the propagation phase of clotting. The initiation phase is largely mediated by the activation of factor X by tissue factor/factor VIIa, giving rise to a small amount of thrombin (figure 4), which in turn activates factor V, factor VIII, factor XI, and platelets, exposing anionic phospholipids on the platelet surface to support the assembly of the multi-component enzyme complexes (intrinsic tenase and prothrombinase) [37]. Thus, the initial small amount of generated thrombin primes the clotting cascade and activates platelets, which then leads to explosive thrombin generation. This is illustrated in the Amplification and Propagation phases in the figure (figure 5).

At least two cell types play important roles in the initial phases of thrombin generation.

●Activated platelets in the initial platelet plug adherent to the endothelium provide a phospholipid surface that promotes assembly of multicomponent complexes. (See 'Multicomponent complexes' below.)

●Endothelial cells at the site of injury provide binding sites for coagulation factors and other procoagulant effects.

The prominent role of endothelial cells rather than platelets to thrombin generation was illustrated in a study using a mouse model of arteriolar injury in which accumulation of fluorescently labeled activated clotting factors was imaged in vivo [38]. Fluorescently labeled factors Xa and Va bound to the endothelium outside the edges of the initial platelet plug and did not bind to platelets a small distance from the site of injury. When platelet thrombus formation at the injured site was blocked extensively, using an inhibitor of platelet aggregation or an animal lacking the platelet thrombin receptor PAR4, accumulation of factors Xa, Va, and fibrin at the site of injury was virtually unaffected.

These findings may explain why patients with severe thrombocytopenia or inherited platelet defects do not have typical findings associated with impaired coagulation such as tissue hematomas and joint bleeding (table 1).

Multicomponent complexes — Four multicomponent macromolecular complexes play a major role in the coagulation pathways: three procoagulant complexes (extrinsic and intrinsic X-ase and prothrombinase) and one anticoagulant complex. These complexes consist of the enzyme, a cofactor protein, the enzyme substrate, assembled on cellular membrane components (anionic phospholipid surfaces) in the presence of calcium (figure 1):

●Extrinsic X-ase (ten-ase) – Consists of activated factor VII (factor VIIa) as the protease, tissue factor as the cofactor, and factor X (Stuart factor) as the substrate [39-41]. Tissue factor/FVIIa activate both factor X and factor IX [42,43].

●Intrinsic X-ase (ten-ase) – Consists of factor IXa as the protease, activated factor VIII (factor VIIIa) as the cofactor, and factor X as the substrate [44]. Factor IXa can be generated by tissue factor/FVIIa or via activation of the intrinsic pathway, either directly or indirectly via thrombin-induced activation of factor XI.

●Prothrombinase – Consists of factor Xa as the protease, factor Va as the cofactor, and prothrombin (factor II) as the substrate. Data from cryo-electron microscopy (cryo-EM) demonstrate that the role of the factor Va cofactor is a structural one, serving as a scaffold to allow efficient cleavage (activation) of prothrombin by factor Xa [45]. This is analogous to the activation of protein C by the thrombin/thrombomodulin complex, in which thrombomodulin serves as a scaffold to align protein C with thrombin.

●Protein C anticoagulant complex – Consists of thrombin (factor IIa) as the enzyme, thrombomodulin as the cofactor, and protein C as the substrate. In addition, endothelial protein C receptor (EPCR), which is primarily expressed on the endothelium of large blood vessels, functions as a receptor for protein C and further accelerates the activation of protein C by the thrombin/thrombomodulin complex [46].

All the activated clotting factors in the clotting cascade (ie, factors VIIa, XIa, IXa, Xa, and thrombin [IIa]) are trypsin-like serine proteases (with serine as a major component of their catalytic site). Despite their structural relatedness, they display remarkable specificity towards their substrates (eg, FIXa activates factor X but not prothrombin (factor II) or fibrinogen). This substrate specificity results from the structural requirement of the substrate to be aligned properly ("docked") with the enzyme before proteolytic cleavage of the substrate can take place. This docking is mediated by an exosite on the enzyme surface that lies outside the active catalytic site (figure 4) [47].

The advantages of these multicomponent enzyme complexes can be illustrated by the prothrombinase complex [48]. When platelets are activated, anionic lipids – primarily phosphatidylserine – become exposed on the platelet surface, and factor V, stored in platelet granules, is released and bound to these anionic lipids. Factor V is activated to factor Va by the initial trace amount of thrombin generated from interactions between tissue factor and factor VIIa at the wound site. Together with the appropriate anionic membrane phospholipids and calcium, this activated factor Xa and its cofactor, factor Va, form the prothrombinase complex, which cleaves prothrombin (factor II) to thrombin (factor IIa).

Because of its extremely favorable local proximity, thrombin generation by the prothrombinase complex is approximately 300,000 times more efficient compared with that generated by factor Xa and prothrombin alone. In addition, factor Xa bound to factor Va is also relatively protected from inhibition by circulating inhibitors such as antithrombin. The net effect is that thrombin generation is dramatically enhanced on the surface of activated platelets and restricted to sites of vascular injury.

The importance of the procoagulant effect of platelets in the assembly of multicomponent complexes is illustrated by congenital platelet function disorders in which the platelet membrane phospholipids do not change in response to platelet activation. Scott syndrome is an autosomal recessive disorder caused by mutation of TMEM16F, a protein partly responsible for lipid scramblase activity [28,48-52]. This protein forms a calcium-activated channel that promotes the "scrambling" (ie, disrupting the membrane gradient) of phosphatidylserine, moving it from the inner leaflet of the platelet plasma membrane to the outer leaflet [52]. Activated platelets with phosphatidylserine on their surface bind factor Va and initiate assembly of the prothrombinase complex.

Patients with Scott syndrome have impaired thrombin generation, clinically prolonged bleeding, and a reduced tendency to form thromboses.

Extrinsic pathway

Tissue factor — Vessel wall damage leads to expression of tissue factor (TF, tissue thromboplastin), an integral membrane glycoprotein [53]. Tissue factor is not normally expressed on vascular endothelial cells or monocytes but is constitutively expressed on certain biological surfaces, such as skin, organ surfaces, vascular adventitia, and many of their malignant counterparts. Normally, TF is exposed to blood flow only after endothelial damage [54]. The mechanism may be either via direct exposure of the subendothelial matrix and/or via cytokine- (especially interleukin [IL]-6-) induced expression of TF on activated monocytes and endothelial cells [55].

The majority of the TF is in a functionally inactive ("encrypted") state. Upon cell lysis or certain in vitro conditions, such as stimulation by calcium ionophore, TF will become activated ("de-encrypted") and support FVIIa binding and activation of factor X. The encrypted state may be due to a pair of free cysteine residues (Cys186 and Cys209) which would become linked into a disulfide bond upon cell perturbation, leading to an allosteric change in the conformation of TF resulting in its active de-encrypted state [56,57]. Protein disulfide isomerase (PDI), glutathione, and nitric oxide have all been implicated in mediating this process [57]. However, not all of the data are consistent and whether Cys186 and Cys209 are involved in the de-encryption of TF remains controversial [58-60].

TF may also circulate in the blood, associated with cell-derived membrane microvesicles as well as in a soluble, alternatively-spliced form [61,62]. These microvesicles derive from lipid rafts on the surface of stimulated monocytes/macrophages, and are capable of fusing with, and initiating coagulation on, activated platelets [63]. (See 'White blood cell contributions' below.)

In addition to assembling clotting reactions on their surface, platelets may also synthesize their own tissue factor in order to generate thrombin in a timely and spatially circumscribed process [64], although this has been called into question [65].

Activation of factors VII, X, and IX — TF serves as the cofactor required for the production of activated factor VII (FVIIa) [66,67]. The TF-FVIIa complex activates factors X [39,40] and IX [42,43]. Activation of factor X occurs at the Arg52-Ile53 peptide bond in the heavy chain of factor X, leading to the formation of the serine protease Xa [41]. Factor IXa in complex with its cofactor factor VIIIa also activates factor X (the intrinsic X-ase as noted above) (figure 1) [40].

This dual pathway of factor X activation (ie, directly and indirectly via activation of factor IX) is necessary because of the limited amount of TF generated in vivo and the presence of the tissue factor pathway inhibitor (TFPI) which, when complexed with factor Xa, inhibits the TF/FVIIa complex [68]. Thus, sustained generation of thrombin depends upon the activation of factor IX and its cofactor factor VIII. This process is amplified because factor VIII is activated by both factor Xa and thrombin [39,69] and factor IXa by thrombin-induced activation of factor XI [70-74]. As a result, there is a progressive increase in factor VIII and factor IX activation as factor Xa and thrombin are formed.

In vitro studies using human plasma have also demonstrated a feed-forward loop in which the TF/FVIIa complex can activate factor VIII, a component of the intrinsic (contact activation) pathway [75]. (See 'Intrinsic or contact activation pathway' below.)

Intrinsic or contact activation pathway — The initial phase of the intrinsic or contact activation pathway consists of several plasma proteins including factor XII (Hageman factor), prekallikrein (Fletcher factor) and high molecular weight kininogen (HMWK, Fitzgerald factor). These factors are activated by contact with negatively charged surfaces, leading to the term "contact activation pathway." Contact with negatively charged surfaces initiates the following sequence:

●Enhanced auto-activation of factor XII. Activated factor XII (factor XIIa) in conjunction with HMWK activates factor XI, which in turn, activates factor IX.

●Factor IXa in complex with factor VIIIa forms the intrinsic X-ase, leading to the formation of factor Xa (see 'Multicomponent complexes' above). Factor VIII is activated by both factor Xa and thrombin [39,69]. Thus, there is a progressive increase in factor VIII activation as factor Xa and thrombin are formed. Thrombin also increases the generation of factor IXa via the activation of factor XI [70,71,73,74].

●Factor XIIa activates plasma prekallikrein (PPK) to plasma kallikrein (PK), which liberates bradykinin (BK) from HMWK. BK is a major inflammatory peptide that induces pain and vasodilation (see 'Blood coagulation as part of the host defense system' below). PK also activates factor XII, thus forming a positive feedback loop of activation [76].

The remainder of the intrinsic pathway uses the same cascade as the extrinsic pathway (the common pathway), which involves factors V, prothrombin, and fibrinogen.

As noted above, in vitro studies suggest that this pathway can also be activated by the TF/FVIIa complex. (See 'Extrinsic pathway' above.)

Critical role of polyphosphate in initiating blood clotting via the intrinsic contact pathway — Inorganic polyphosphate (polyP) is a linear highly anionic polymer of orthophosphates linked by high-energy phosphoanhydride bonds. It is ubiquitous in biology and can vary from a few to several thousand phosphate units in polymer length, depending on the organisms and tissues from which it is derived. In microorganisms, polyP is synthesized from ATP and may serve as an energy store allowing the bacteria to resynthesize ATP in times of starvation [77]. PolyP is also stored in human platelet dense granules and is efficiently released upon platelet activation [78]. In contrast to microbial polyP, platelet polyP is smaller, consisting of 60 to 100 phosphate units.

With its multiple anionic charges, polyP is a potent procoagulant, and it likely represents the "physiological" or "pathological" negatively charged surface that triggers blood coagulation via the intrinsic pathway. The source of polyP in this setting may derive from injured tissues at the wound site or a microbial source in the case of an infection. In addition to triggering intrinsic pathway activation, polyP has other effects on blood clotting. It accelerates factor V activation, abrogates the inhibitory effect of tissue factor pathway inhibitor (TFPI), enhances fibrin polymerization (making the fibrils thicker), and accelerates factor XI activation by thrombin. The potency of polyP is dependent on its chain length. Long-chain polyP (from a microbial source) is efficient in carrying out all four functions, while medium-size polyP (from platelets) is less effective in triggering the intrinsic pathway but effective in accelerating blood clotting once it is initiated [79].

Deficiencies of intrinsic contact pathway proteins — The physiologic relevance of the initial complex of the intrinsic contact pathway is not fully established. Deficiencies in these proteins (prekallikrein, HMWK, and factor XII) are not associated with bleeding tendencies, suggesting that the initiation portion of the intrinsic pathway (the contact phase) is not very important in vivo [80,81].

Mutations in factor XII have been seen in a subset of patients with hereditary angioedema (HAE) and normal C1 inhibitor levels, an autosomal dominant disease characterized by recurrent attacks of upper respiratory tract edema, although the bleeding phenomena and coagulation defects in these patients have not been well characterized. (See "Hereditary angioedema with normal C1 inhibitor".)

However, injury-related bleeding is seen with deficiency of factor XI [82], suggesting that factor XI plays an important hemostatic role, independent of contact activation and factor XII. One likely mechanism is that thrombin feedback activates factor XI, with polyP, released from activated platelets or derived from injured tissues, serving as a cofactor [70-72,83]. Factor XIa then activates factor IX, which leads to further thrombin formation after the clot has been formed [71].

This interpretation is also consistent with the phenotype observed in mice deficient in either factor XII or factor XI. These mice do not exhibit any bleeding problems under normal circumstances, similar to patients deficient in factor XII or factor XI, but surprisingly, they are protected from arterial thrombosis in experimental thrombosis models [84,85]. In this regard, it is notable that patients severely deficient in factor XI (<15 percent), are reported to be protected from ischemic stroke but not myocardial infarction [86]. Further evidence for the lack of spontaneous bleeding with factor XI deficiency comes from a trial in which antisense therapy was used to lower the activity of factor XI to approximately 20 percent of normal in patients undergoing knee replacement surgery; this was able to reduce the incidence of venous thrombosis without increasing bleeding [87]. Genetic polymorphisms of factor XII have also been linked to an increased incidence of thrombosis [88-90]. These data suggest that the additional thrombin generated via the tertiary amplification pathway, involving factor XII or factor XI, may play more of a role in pathological thrombosis rather than physiological hemostasis.

This mechanism also provides a rationale by which bleeding can occur in patients with factor XI deficiency: increased fibrinolysis due to diminished activation of thrombin-activatable fibrinolysis inhibitor (TAFI) [71,91,92]. The relatively high concentration of thrombin required within the clot for activation of TAFI appears to come from thrombin-mediated factor XI activation after the clot has been formed [91]. Thus, in the absence of factor XI, sufficient TAFI is not generated in order to protect the clot from lysis. (See "Factor XI (eleven) deficiency" and 'Carboxypeptidase B2 (thrombin-activatable fibrinolysis inhibitor)' below.)

Continuation of the coagulation cascade — Once factor X has been activated, the coagulation pathway proceeds as follows:

●Factor V, released from platelet alpha granules during platelet activation and cleaved by thrombin to form activated factor V (factor Va), binds factor Xa [93-96]. Platelet factor V appears to be more important for assembly of the prothrombinase complex than circulating factor V [97]. Subsequent factor Va inactivation by activated protein C (aPC) and protein S is an important step in preventing excessive coagulation. (See 'Activated protein C and protein S' below.)

●Factor Xa linked to factor Va on the platelet phospholipid surface forms the prothrombinase complex, which converts one molecule of prothrombin (factor II) to one molecule of its activated form, thrombin (factor IIa), with the concomitant release of the prothrombin activation fragment F1+2 (F1.2) (figure 4) [98,99].

●Thrombin converts fibrinogen to fibrin [100], which undergoes polymerization. (See "Disorders of fibrinogen", section on 'Biology'.)

●Activated factor XIII (factor XIIIa) stabilizes and crosslinks overlapping fibrin stands [101]. Factor XIIIa (along with fibrinogen) also controls the volume of red blood cells (RBCs) trapped within a thrombus, which in turn controls clot size. Mice lacking factor XIII (or harboring a fibrinogen mutation that abrogates factor XIIIa binding) formed clots approximately one-fifth the size of those in wild type mice, due to extrusion of RBCs during clot retraction; similar findings were seen in the plasma from a factor XIII-deficient patient [102]. The formation of factor XIIIa is promoted by the complexing of thrombin, fibrin, and factor XIII [103].

As noted above, thrombin also accelerates the activation of factors XI and VIII, in addition to factor V [39,69-72,94]. Activated factor XI in turn activates factor IX. Thus, factor XI activation by thrombin may function as an adjunctive activation pathway, the absence of which may account for delayed bleeding episodes in factor XI deficient patients, usually in a postoperative setting. (See 'Carboxypeptidase B2 (thrombin-activatable fibrinolysis inhibitor)' below.)

CONTROL MECHANISMS AND TERMINATION OF CLOTTING — The interactions between activated platelets and the clotting cascade, with its built-in amplification, give rise to a hemostatic response that is rapid and localized to the injury site. It is also potentially explosive, and, if unchecked, could lead to thrombosis, vascular inflammation, and tissue damage. This does not usually happen because coagulation is modulated by a number of mechanisms: dilution of procoagulants in flowing blood, removal of activated factors through the reticuloendothelial system, especially in the liver, and control of the activated procoagulants and platelets by natural antithrombotic pathways [1]. Most of these antithrombotic pathways are anchored on vascular endothelial cells, which play a major and active role in maintaining the fluidity of blood (figure 6).

The physiologic (endogenous) inhibitors of the coagulation pathways are:

●Tissue factor pathway – tissue factor pathway inhibitor (TFPI)

●Contact activation pathway – C1 esterase inhibitor (C1-inh)

Tissue factor pathway inhibitor — TFPI circulates in plasma, but at very low concentrations, in contrast to AT [104]. TFPI is a Kunitz-type protease inhibitor. It inhibits factor X activation in two ways: it directly inhibits factor Xa; and it complexes with factor Xa and the TFPI-FXa complex inhibits TF/FVIIa, thereby regulating the triggering mechanism of the extrinsic pathway (figure 5) [105-107].

TFPI is primarily synthesized by the microvascular endothelium. TFPI circulates in plasma in a free form (TFPI-alpha) that accounts for 20 percent of total plasma TFPI and in a slightly truncated form in association with low-density lipoproteins that accounts for 80 percent of total plasma TFPI. The majority of TFPI remains associated with the endothelial surface (as TFPI-beta), bound to cell surface glycosaminoglycans [108]. The plasma concentration of TFPI is greatly increased following intravenous heparin administration; this release of endothelial TFPI may contribute to the antithrombotic effects of heparin and low-molecular-weight-heparin.

TFPI gene disruption leads to intrauterine lethality in mice; TFPI deficiency has not been reported in humans [109]. Conversely, TFPI levels and activity are markedly increased in two inherited bleeding disorders involving factor V mutations (factor V Amsterdam and factor V East Texas) [110-112]. Both of these mutations lead to production of a truncated form of factor V via alternative splicing, and the factor V-short forms a complex with TFPI-alpha, leading to a 10-fold increase in circulating levels of TFPI-alpha. Individuals with these mutations manifest an autosomal dominant bleeding disorder. (See "Rare inherited coagulation disorders", section on 'Genetics'.)

Recombinant TFPI is being evaluated for its potential role as an anticoagulant [113]. (See "Investigational anticoagulants", section on 'Recombinant TFPI and anti-TF antibodies'.)

C1 esterase inhibitor — C1-inh is a member of the family of serine protease inhibitors (SERPINs). It is synthesized in the liver. C1-inh inhibits FXIIa and PK as well as the complement proteases C1r, C1s, and FXIa. Deficiency of C1-inh results in an angioedema syndrome known as C1INH-AAE; this is not a clinical hypercoagulable state. (See "Acquired C1 inhibitor deficiency: Clinical manifestations, epidemiology, pathogenesis, and diagnosis" and "Acquired C1 inhibitor deficiency: Management and prognosis".)

Control of the termination phase of coagulation — The regulation of the termination phase of the coagulation process involves another circulating serine protease inhibitor (SERPIN), antithrombin (previously called antithrombin III), and a clotting-initiated inhibitory process, the protein C pathway. In addition, prostacyclin, thromboxane, and nitric oxide (NO) modulate vascular and platelet reactivity.

This termination phase is critical in mediating the extent of clot formation, as demonstrated by the thrombotic disorders present in individuals with deficiencies in these pathways and bleeding disorders in individuals with activating mutations. Examples include:

●Deficiencies of antithrombin, protein C, and protein S. (See "Antithrombin deficiency" and "Protein C deficiency" and "Protein S deficiency".)

●Activating mutations in factor V and thrombomodulin. (See "Rare inherited coagulation disorders" and "Approach to the adult with a suspected bleeding disorder" and 'Activated protein C and protein S' below and 'Tissue factor pathway inhibitor' above.)

White blood cell contributions — Activated macrophages and monocytes are able to express tissue factor (TF) on their surfaces or within microparticles, which contributes to physiologic hemostasis as well as pathologic thrombosis [114]. Regulation of TF messenger RNA levels by a poly(adenosine 5’-diphposphate [ADP]-ribose) polymerase (PARP) protein, PARP-14, may prevent excessive thrombosis during periods of inflammation [115].

Antithrombin, heparin, and heparan — Antithrombin (AT) is a circulating serine protease inhibitor (SERPIN). It neutralizes most of the enzymes in the clotting cascade, especially thrombin (factor IIa), factors Xa, and IXa; as well as factor XIIa and factor XIa, by forming equimolar, irreversible complexes with them [116]. AT has two active functional sites, the reactive center, Arg393-Ser394, and the heparin binding site located at the amino terminus of the protein [116].

The role of AT is enhanced by endogenous heparan sulfate (a glycosaminoglycan found on endothelial surfaces), which contain a pentasaccharide sequence that mediates its binding to AT [117-119]. The binding of endogenous (or exogenous) heparins to the heparin binding site on AT produces a conformational change in AT, thus "activating" it and accelerating its inactivation of clotting factors by 1000 to 4000-fold (figure 7) [116,117]. Fondaparinux, a form of low-molecular-weight heparin, is the pharmacologic version of this pentasaccharide sequence. (See "Heparin and LMW heparin: Dosing and adverse effects", section on 'Mechanisms of action' and "Fondaparinux: Dosing and adverse effects", section on 'Mechanism of action'.)

The entire luminal surface of the vascular system is covered by endothelial cells that are coated with activated AT and poised to rapidly inactivate any excess thrombin in the general circulation (figure 6). The potential of this system is magnified by the surface-to-volume geometry of the microcirculation, in which 1 mL of blood can be exposed to as much as 5000 cm² of endothelial surface.

Activated protein C and protein S — As clot formation progresses, thrombin (factor IIa) binds to thrombomodulin (TM), an integral membrane protein on the endothelial cell surface (figure 6) [120].

Binding of thrombin to TM induces a drastic change in thrombin substrate specificity such that it loses all of its procoagulant functions (eg, platelet activation, fibrin clot formation) and instead acquires the ability to activate protein C (figure 8) [121-123]. Thus, binding to TM functions like a molecular switch, transforming thrombin from a procoagulant to an anticoagulant via its activation of protein C. This remarkable transformation is not due to a conformational change in thrombin's active site, as initially proposed, but rather due to the binding of TM to the exosite I of thrombin, the major docking site for thrombin's procoagulant substrates, and preventing them from binding to thrombin [124]. On the other hand, protein C binds to TM complexed with thrombin, allowing it to be cleaved to become activated. This activation step is enhanced by an endothelial receptor for protein C (EPCR) [125-127]. As a testament to this TM-induced change of function for thrombin, a knockout mouse model in which the TM gene was ablated was associated with unfettered activation of the coagulation system and widespread thrombosis [128]. Conversely, a naturally-occurring TM mutation (C1611A) that causes TM to be shed from the endothelial surface and circulate at very high levels in the plasma is associated with a bleeding phenotype. (See "Rare inherited coagulation disorders", section on 'Genetics'.)

Activated protein C (aPC), in association with protein S on phospholipid surfaces, proteolytically inactivates factors Va and VIIIa, thereby inactivating the prothrombinase and the intrinsic X-ase, respectively [129-132].

●Factor Va is first cleaved at Arg506 and then at Arg306 and Arg679 by aPC. The peptide bond cleavage at Arg506 is essential for the exposure of cleavage sites at Arg306 and Arg679 [130].

Factor V Leiden, in which the arginine at position 506 is replaced by glutamine, is not susceptible to cleavage at position 506 by aPC and is therefore inactivated more slowly, resulting in a hypercoagulable state. (See "Factor V Leiden and activated protein C resistance".)

●Factor V cleaved at position 506 is also thought to be a cofactor, along with protein S, in supporting the role of aPC in the degradation of factors Va and VIIIa [133]. Thus, lack of this cleavage product decreases the anticoagulant activity of aPC, further increasing the hypercoagulable state in patients with factor V Leiden [134].

●Factor VIIIa is first cleaved at Arg336 and then Arg562 by aPC [132]. The rate of aPC-mediated inactivation is increased several fold by protein S, an effect that is impaired by factor IXa.

Protein S circulates in two forms. In the free form, it is active as an anticoagulant; in the bound form, it is complexed to C4b binding protein of the complement system and is functionally inactive. C4b binding protein is an acute-phase reactant that increases in concentration in inflammatory states; as a result, the activity of free protein S is reduced in these conditions, enhancing the likelihood of thrombosis.

Prostacyclin and thromboxane — Intact endothelial cells in proximity to disrupted endothelium release arachidonic acid from cell membrane phospholipids by phospholipase A2. The enzyme cyclooxygenase-1 (COX-1 or prostaglandin endoperoxide H synthase-1) converts arachidonic acid into thromboxane A2 (TxA2) in platelets, while prostacyclin (PGI2), a dominant product of the endothelium, appears to largely derive from COX-2, the production of which is induced by laminar blood flow under physiologic conditions [135,136].

TxA2 is a potent stimulator of platelet aggregation and produces vasoconstriction, while PGI2, via activation of adenylate cyclase, blocks platelet aggregation and antagonizes TxA2-mediated vasoconstriction [137]. (See "NSAIDs: Pharmacology and mechanism of action", section on 'Cyclooxygenase enzymes'.)

●Low dose aspirin irreversibly acetylates and inhibits COX-1 and only weakly inhibits COX-2 [138]. Since platelets cannot make new COX-1, the inhibition of TxA2 is permanent for the life of the platelet.

●In comparison, endothelial cells can make new COX-1 as well as COX-2 and higher doses of aspirin are required for inhibition of PGI2 production [139].

This distinction underlies the hypothesized mechanism for the benefit of low-dose aspirin in cardiovascular disease, as well as the increased cardiac toxicity of the selective COX-2 inhibitors. (See "Aspirin in the primary prevention of cardiovascular disease and cancer".)

Nitric oxide — Nitric oxide (NO) (also known as endothelium-derived relaxing factor, EDRF) is formed from L-arginine in endothelial cells, catalyzed by the enzyme nitric oxide synthase (NOS) (figure 9). Acting through soluble guanylate cyclase in the vascular smooth muscle cells with the production of cGMP, it causes vasodilatation. NO also inhibits platelet adhesion and aggregation [140].

Platelets are also capable of synthesizing NO, with enhanced production in the setting of platelet adhesion to fibrillar collagen under shear flow [141]. This may provide negative feedback to limit excessive platelet adhesion and vasoconstriction at sites of vessel injury.

NO diffuses freely across cellular membranes and is rapidly destroyed by hemoglobin in nearby red blood cells; it therefore functions as a local (paracrine) hormone with a very short half-life. Release of free hemoglobin into the circulation (eg, during intravascular hemolysis) causes greater local depletion of NO.

Intravenous infusion of an arginine analog that blocks NO production leads to an immediate and substantial rise in blood pressure, suggesting that there is a continual, basal release of NO to regulate vascular tone [142].

Release of NO by endothelial cells can be upregulated (eg, by exercise) and downregulated (eg, by oxidative stress, smoking, and oxidized low-density lipoprotein [LDL]). NO levels are also reduced with aging and in the course of vascular disease (eg, diabetes and hypertension) as part of the manifestation of endothelial dysfunction [143].

Thrombospondin 5 — Thrombospondin 5, also called cartilage oligomeric matrix protein (COMP), is an extracellular matrix protein that can regulate vascular tone. COMP is expressed in a variety of cartilage and smooth muscle tissues.

In a mouse model, COMP was shown to inhibit thrombin and act as a natural anticoagulant [144]. COMP is expressed in platelets and released upon platelet activation. It causes a dose-dependent prolongation of the thrombin time (see "Clinical use of coagulation tests", section on 'Thrombin time (TT)') and inhibits thrombin-dependent platelet aggregation.

CLOT DISSOLUTION AND FIBRINOLYSIS — To restore vessel patency following hemostasis, the clot must be organized and removed by the proteolytic enzyme plasmin in conjunction with wound healing and tissue remodeling.

Plasmin — Plasminogen, the precursor molecule to plasmin, binds fibrin and tissue plasminogen activator (tPA). This ternary complex leads to conversion of the proenzyme plasminogen to active, proteolytic plasmin [145,146].

Plasmin has broad substrate specificity and, in addition to fibrin, cleaves fibrinogen and a variety of plasma proteins and clotting factors [147]. Plasmin cleaves the polymerized fibrin strand at multiple sites and releases fibrin degradation products (FDPs). One of the major FDPs is D-dimer, which consists of two D domains from adjacent fibrin monomers that have been crosslinked by activated factor XIII (figure 10). Plasmin also cleaves factor XIIIa, but not factor XIII, leading to reduced fibrin crosslinking [148].

The plasminogen/plasminogen-activator system is complex, paralleling the coagulation cascade (figure 3) [149]. Plasmin activity is regulated by vascular endothelial cells that secrete both serine protease plasminogen activators (tissue-type plasminogen activator and urokinase-type plasminogen activator) and plasminogen activator inhibitors (PAI-1 and PAI-2). (See "Thrombotic and hemorrhagic disorders due to abnormal fibrinolysis".)

Plasminogen deficiency (PLGD) is a rare disorder that presents with poor wound healing and formation of pseudomembranes in the eye, nose, mouth, respiratory, gastrointestinal, and genitourinary systems. (See "Plasminogen deficiency".)

Tissue-type plasminogen activator — The tPA molecule is predominantly an endothelial cell enzyme. Its release is stimulated by a variety of substances including thrombin, serotonin, bradykinin, cytokines, and epinephrine [150,151]. In plasma it circulates as a complex with its natural inhibitor PAI-1 and is rapidly cleared by the liver.

Analogous to the prothrombin complex, the rapid generation of plasmin by tPA optimally takes place on a surface, the fibrin clot. Both tPA and fibrinogen bind to fibrin via recognition of lysine residues in the fibrin clot. When bound to fibrin, the binding interaction aligns tPA and plasminogen on the fibrin surface [152] so that the catalytic efficiency of tPA is increased several hundred-fold [146].

Urokinase — Urokinase is the second physiologic plasminogen activator. It is present in high concentration in the urine. While tPA is largely responsible for initiating intravascular fibrinolysis, urokinase is the major activator of fibrinolysis in the extravascular compartment. Urokinase is secreted by many cell types in the form of prourokinase, also termed single-chain urokinase-type plasminogen activator (scu-PA). Prourokinase is converted to urokinase by plasmin; it has a low level of proteolytic activity unless it is exposed to fibrin.

Plasminogen activator inhibitors and alpha-2-antiplasmin — The PAIs inhibit tPA [153,154], while alpha-2-antiplasmin inhibits plasmin [155].

●PAI-1 is synthesized by endothelial cells and platelets which regulate its release during fibrinolysis. Patients deficient in PAI-1 have a bleeding diathesis usually related to trauma or surgery [156,157]. The release of PAI-1 by activated platelets may contribute to the relative resistance of platelet-rich arterial thrombi to thrombolysis.

●PAI-2 is synthesized by white blood cells and the placenta and its levels are greatly increased during pregnancy. PAI-2 is less effective as a plasminogen inhibitor [158] and its biologic importance is uncertain.

Alpha-2-antiplasmin is secreted by the liver and is also present within platelets [159]. It can be crosslinked into the fibrin clot by factor XIIIa, and plays an important role in making thrombi resistant to plasmin by complexing with it. Plasmin released into the circulation is rapidly inactivated by alpha-2-antiplasmin. However, alpha-2-antiplasmin is present in lower concentrations than is plasminogen and therefore can become depleted while plasmin is continuing to be generated.

Carboxypeptidase B2 (thrombin-activatable fibrinolysis inhibitor) — When fibrin is degraded by plasmin, new carboxy-terminal lysines are exposed in the partially digested clot. These residues provide additional sites for plasminogen and tPA incorporation into the clot, creating a positive feedback loop in clot lysis. The carboxy-terminal lysines are susceptible to removal by carboxypeptidases [160,161].

A proenzyme form of carboxypeptidase-B2 (CPB2), also called thrombin-activatable fibrinolysis inhibitor (TAFI), circulates in plasma. Pro-CPB2 is a physiologic substrate for the thrombin-thrombomodulin (TM) complex [162,163]. Similar to protein C, the activation of pro-CPB2 by the thrombin-TM complex is approximately 1000-fold faster than with free thrombin alone [164,165]. Activated CPB2 functions as a fibrinolysis inhibitor by cleavage of C-terminal lysines from partially digested fibrin, thereby diminishing the incorporation and activation of plasminogen, leading to delayed clot lysis.

Factor XIIIa may crosslink CPB2 to fibrin, helping to protect the newly formed fibrin from premature plasmin degradation. Crosslinking may also facilitate the activation of pro-CPB2, stabilize enzymatic activity, and protect the active enzyme from further degradation [166].

From a physiologic standpoint, one can envisage that upon thrombin binding to TM, the thrombin-TM complex will activate protein C to dampen the clotting cascade, and will also activate pro-CPB2, thereby protecting the clot already formed at the vascular wound from premature degradation. The concentration of thrombin required for the activation of pro-CPB2 is substantially higher than that for fibrinogen clotting. Thus, CPB2 activation requires amplification and acceleration of thrombin generation through feedback activation of factors V, VIII, and XI by the small amount of initially generated thrombin (figure 1).

This requirement for adequate activation of pro-CPB2 can have important clinical consequences in a number of acquired and congenital disorders of coagulation:

●The bleeding diathesis encountered in people with hemophilia A (factor VIII deficiency) and factor XI deficiency may be partly related to suboptimal activation of pro-CPB2, leading to enhanced and premature clot lysis (figure 11) [91,92,167-170].

●The low levels of pro-CPB2 seen in patients with chronic liver disease may be responsible, in part, for the low-grade fibrinolysis seen in this condition [171].

●Enhanced thrombin generation in patients with the G20210A prothrombin gene mutation may lead to increased activation of pro-CPB2, resulting in inhibition of fibrinolysis and increased thrombotic risk [172]. (See "Prothrombin G20210A".)

BLOOD COAGULATION AS PART OF THE HOST DEFENSE SYSTEM — Interactions between coagulation factors and the immune system have been identified at several stages of the hemostatic process. These interactions are complex and include contributions of hemostatic factors to host defense as well as effects of immune mediators on hemostasis [173-175].

●Platelets may be capable of killing microorganisms. In a series of blood samples from individuals living in malaria-endemic areas, platelets were shown to bind to several plasmodial species within red blood cells and to kill the parasites [176]. An in vitro assay demonstrated that parasite killing was dependent on platelet factor 4.

●Activated protein C (aPC), in addition to its well-established anticoagulant role in inhibiting activated factor V (FVa) and FVIIIa, also has cellular cytoprotective effects. This is dependent on aPC binding to endothelial protein C receptor (EPCR). EPCR-bound aPC (but not free aPC) cleaves endothelial protease activated receptor (PAR)-1, resulting in cell signaling and the initiation of a cell protective program that includes antiinflammatory and anti-apoptotic activities and stabilization of endothelial barriers to prevent vascular leakage [177]. The finding that this cytoprotective effect of aPC is dependent on cleavage of PAR-1 is surprising and puzzling since thrombin cleavage of endothelial PAR-1 leads to a prothrombotic phenotype and disrupts the endothelial barrier integrity, the opposite of the aPC/EPCR-PAR-1 signaling effect. The mechanisms for this remarkable change in signaling profile are under investigation. The aPC/EPCR-PAR-1 signaling may involve different coupling with other G protein-coupled receptors (GPCRs) [177]. There is also evidence that aPC/EPCR cleaves PAR-1 at Arg46 rather than a Arg41 (which is cleaved by thrombin), resulting in a different signaling program from that of thrombin [178].

The role of carboxypeptidase B2 (CPB2) is also not limited to dampening fibrinolysis. CPB2 can inactivate activated complement components C3a and C5a, the major anaphylatoxins resulting from complement activation, and bradykinin (BK), potent inflammatory mediators found at sites of inflammation; inactivation is achieved by removing the carboxy-terminal arginine residue from these inflammatory mediators [179].

Thus the activation of protein C and pro-CPB2 by the thrombin-TM complex on endothelial cells may serve as a negative homeostatic mechanism to down-regulate thrombin's inflammatory activities in the cross-talk between coagulation, inflammation, and innate immunity [180]. (See 'Thrombin generation' above.)

●Inflammatory mediators may alter the normal thromboresistant phenotype of vascular endothelial cells, and proinflammatory cytokines can induce the expression of tissue factor on a number of surfaces [181]. (See "The endothelium: A primer", section on 'Procoagulant properties' and 'Tissue factor' above.)

●Monocytes and neutrophils are recruited to the vessel wall and incorporated into the growing clot [175]. When pathogen-associated molecular patterns or damage associated molecular patterns (PAMPs or DAMPs) are recognized by monocytes, the monocytes may deliver tissue factor to sites of pathogen exposure. Neutrophil extracellular traps (NETs) can activate factor XII and bind von Willebrand factor, leading to platelet recruitment. (See "An overview of the innate immune system", section on 'Microbial detection through pattern recognition'.)

●Deposition of fibrin at sites of infection prevents the dissemination of microorganisms from the initial site of inoculation. Virulence factors in bacteria such as Yersinia and Streptococcus have been identified as bacterial plasminogen activators that exploit the host's plasmin-mediated fibrinolysis process to support microbial dissemination [182]. (See 'Continuation of the coagulation cascade' above.)

●The complement protein C4b, an acute phase reactant, sequesters protein S. Thus, in infections and inflammatory states, free protein S is reduced, shifting the hemostatic balance toward clotting. Factor V appears to have a role in inflammation that is independent of its proteolytic activity against factors Va and VIIIa [183]. (See 'Activated protein C and protein S' above.)

●Excessive activation of the alternative pathway of complement, due to mutations or autoantibodies affecting complement regulators or effectors, can cause a thrombotic microangiopathy (TMA) associated with microvascular thrombosis. (See "Pathophysiology of TTP and other primary thrombotic microangiopathies (TMAs)", section on 'Complement-mediated TMA pathogenesis'.)

SUMMARY

●Phases of coagulation and fibrinolysis – When the pathways of clot formation and clot lysis are appropriately linked, a clot is laid down initially to stop bleeding, followed later by clot lysis and tissue remodeling (figure 2 and figure 3). Abnormal bleeding can result from diminished clot generation or enhanced clot lysis, while excessive clot formation or reduced clot lysis can lead to excessive thrombosis. The following phases coordinate this overall process. (See 'Phases of the hemostatic process' above.)

•Platelet plug – Platelets are activated at the site of vascular injury to form a platelet plug that provides the initial hemostatic response, including exposure of procoagulant phospholipids on the platelet surface and the assembly of components of the clotting cascade. (See 'Formation of the platelet plug' above.)

•Fibrin deposition – Generation or exposure of tissue factor at the wound site, its interaction with factor VIIa and the subsequent generation of activated factor X, are the primary physiologic events in initiating clotting, while components of the intrinsic pathway (ie, factors VIII, IX, XI) are responsible for amplification of this process. (See 'Clotting cascade and propagation of the clot' above.)

•Termination – The termination phase of coagulation involves antithrombin, tissue factor pathway inhibitor and the protein C pathway (figure 6). This phase is critical in mediating the extent of clot formation, as demonstrated by the thrombotic disorders present in individuals with abnormalities in this pathway. (See 'Control mechanisms and termination of clotting' above.)

•Fibrinolysis – To restore vessel patency, the clot must be organized and removed. Plasminogen and tissue plasminogen activator (tPA) bind to fibrin and tPA converts plasminogen to active, proteolytic plasmin, which cleaves fibrin, fibrinogen, and a variety of plasma proteins and clotting factors. The plasminogen/plasminogen-activator system is complex, paralleling the coagulation cascade (figure 3). (See 'Clot dissolution and fibrinolysis' above.)

●Connections to the immune system – Several connections have been identified between the hemostatic system and the immune system. (See 'Blood coagulation as part of the host defense system' above.)