[Hemostasis] General – Diagnostic Approach to the Bleeding Disorders

November 21, 2016 Clinical Skills, Hematology No comments , , , , , , , , , , , , , ,

screen-shot-2016-10-10-at-10-38-16-amClinical Presentations and Clinical Distinction Between Platelet- or Vessel-Induced Bleeding and Coagulation-Induced Bleeding

Certain signs and symptoms are virtually diagnostic of disordered hemostasis. They can be divided arbitrarily into two groups: those seen more often in disorders of blood coagulation and those most commonly noted in disorders of the vessels and platelets. The latter group is often called purpuric disorders because cutaneous and mucosal bleeding usually are prominent. The clinical findings that are most valuable in distinguishing between these two broad categories are summarized in Table 45.1. Although these criteria are relative, they provide valuable clues to the probable diagnosis if they are applied to the predominant clinical features in a given patient.

Bleeding into Skin and Soft Tissues

Petechiae are characteristic of an abnormality of the vessels or the platelets and are exceedingly rare in the coagulation disorders. These lesions are small capillary hemorrhages ranging from the size of a pinhead to much larger. They characteristically develop and regress in crops and are most conspicuous in areas of increased venous pressure, such as the dependent parts of the body and areas subjected to pressure or constriction from girdles or stockings. Petechiae must be distinguished from small telangiectasias and angiomas. Vascular structures such as telangiectasias or angiomas blanch with pressure, whereas petechiae do not.


Hemorrhage into synovial joints is virtually diagnostic of a severe inherited coagulation disorder, most commonly hemophilia A or hemophilia B, and is rare in disorders of the vessels and platelets or in acquired coagulation disorders. This disabling problem often develops with pain and swelling as chief symptoms but without discoloration or other external evidence of bleeding. Subperiosteal hemorrhages in children with scurvy and swollen painful joints that may developin some patients with allergic purpura occasionally may be confused with hemarthrosis.

Traumatic Bleeding

The unavoidable traumas of daily life and even minor surgical procedures are a greater challenge to hemostasis than any test yet developed in the laboratory. In contrast to "spontaneous" bleeding manifestations, bleeding after trauma in a person with a hemorrhagic diathesis differs in a quantitative way from that which would normally be expected in terms of the amount, duration, and magnitude of the inciting trauma. Such variables are extremely difficult to assess accurately by taking the patient's history. The need for transfusions and the number administered may serve as a rough guide. The patient's statement concerning the duration of bleeding is more reliable.

In individuals with a coagulation disorder, the onset of bleeding after trauma often is delayed. For example, bleeding after a tooth extraction may stop completely, only to recur in a matter of hours and to persist despite the use of styptics, vasoconstrictors, and packing. The temporary hemostatic adequacy of the platelet plug despite defective blood coagulation may explain this phenomenon of delayed bleeding, as well as the fact that patients with coagulation disorders seldom bleed abnormally from small superficial cuts scuh as razor nicks. In contrast, posttraumatic or postoperative surgical bleeding in thrombocytopenic patients usually is immediate in onset, as a rule responds to local measures, and rarely is as rapid or voluminous as that encountered in patients with coagulation disorders. However, it may persist for hours or days after surprisingly small injuries.

Anyway, the response to trauma is an excellent screening test for the presence of an inherited hemorrhagic disorder, and a history of surgical procedures or significant injury without abnormal bleeding is equally good evidence against the presence of such a disorder.

Clinical Features of Inherited and Acquired Bleeding Disorders

An inherited bleeding disorder is suggested by the 1) onset of bleeding symptoms in infancy and childhood, 2) a positive family history (particularly if it reveals a consistent genetic pattern), and 3) laboratory evidence of a single or isolated abnormality, most commonly the deficiency of a single coagulation factor.

Birth and the neonatal period provide unique challenges to the hemostatic mechanism, and bleeding during the first month of life often is the first evidence of an inherited disorder of hemostasis. Small cephalohematomas and petechiae are common in tne newborn as a result of the trauma of delivery. Large cephalohematomas that progressively increase in size may result from hemophilia but are more common in association with acquired bleeding disorders such as hemorrhagic disease of the newborn. Bleeding from the umbilical stump and after circumcision is common in the acquired coagulation disorders, and it also occurs in the inherited coagulation disorders, with the exception of hypofibrinogenemia, dysfibrinogenemia, and factor XIII deficiency. The onset of bleeding from the umbilical cord may be delayed in these latter disorders. In the evaluation of bleeding in the neonate, the clinician should remember that hematochezia and hematemesis may originate from swallowed blood of maternal origin. Simple tests to distinguish such maternal blood from fetal blood have been described. It must be paid attention that manuy infants with inherited coagulation disorders do not bleed significantluy in the neonatal period. In such patients, the disorder may become clinically silent for a time. Hematomas may first be seen only when the child becomes active. Hemarthrosis commonly does not develop until a child is 3 or 4 years of age.

The family history is of great importance in the evaluation of bleeding disorders. In disorders inherited as autosomal dominant traits with characteristic symptoms and high penetrance, such as hereditary hemorrhagic telangiectasia, an accurate pedigree spanning several generations can often be obtained. The presence of typical bleeding manifestations in male siblings and maternal uncles is virtually diagnostic of X-linked recessive inheritance, which characterizes hemophilia A and hemophilia B. But, the limitations of the family history, however, are greater than is commonly realized. Hearsay history is difficult to evaluate, and it is often impossible to assess the significance of easy bruising or to differentiate between manifestations of a generalized bleeding disorder and more common localized lesions, such as peptic ulcer and uterine leiomyomas. A negative family history is of no value in excluding an inherited coagulation disorder in an individual patient. As many as 30% to 40% of patients with hemophilia A have a negative family history. The family history usually is negative in the autosomal recessive traits, and consanguinity, which is commonly prsent in these kindreds, is notoriously difficult to document or exclude.

Approach to the Patient W/ Excessive Bleeding

Excessive bleeding may occur in both male and female patients of all ages and ethnicities. Symptoms can begin as early as the immediate newborn period (uncommonly even in utero) or anytime or anytime thereafter. The bleeding symptoms experienced are related in large part to the specific factor and level of deficiency.

  • [Spontaneous or induced] Bleeding can be spontaneous; that is, without an identified trigger, or may occur after a hemostatic challenge, such as delivery, injury, trauma, surgery, or the onset of menstruation.
  • [Anatomic location(s)] Furthermore, bleeding symptoms may be confined to specific anatomic sites or may occur in multiple sites.
  • [Family history] Finally, bleeding symptoms may be present in multiple family members or may occur in the absence of a family history. All of this information is important to arrive at a correct diagnosis rapidly and with minimal yet correctly sequenced laboratory testing.

Thus, a detail patient and family history is a vital component of the approach to each patient with a potential bleeding disorder.


Obtaining a detail patient and family history is crucial regardless of prior laboratory testing. The history includes a detailed discussion of specific bleeding and clinical symptoms. Information regarding bleeding symptoms should include location, frequency, and pattern as well as duration both in terms of age of onset and time required for cessation.


The location may suggest the part of the hemostatic system affected; patients with disorders of primary hemostasis (platelets and vWF) often experience mucocutaneous bleeding, including easy bruising, epistaxis, heavy menstrual bleeding, and postpartum hemorrhage in women of child-bearing age; whereas patients with disorders of secondary hemostasis (coagulation factor deficiencies) may experience deep-tissue bleeding, including the joints, muscles, and central nervous system.

Pattern and Duration

The bleeding pattern and duration of each episode, particularly for mucus membrane bleeding, assist in the determination of the likelihood of the presence of an underlying bleeding disorder.


The onset of symptoms can suggest the presence of a congential versus acquired disorder. Although congenital conditions can present at any age, it is more likely that patients with a long history of symptoms or symptoms that begin in childhood have a congenital condition, whereas patients whose onset occurs at an older age are more likely to have an acquired condition. Congenital clotting factor deficiencies that do not present until later in life do occur and include mild factor deficiencies and coagulation factor deficiencies associated with variable bleeding patterns, most notably FXI deficiency.


Additional important information to be collected includes the current use of medications and herbal supplements, as these may affect the hemostatic system; the presence or absence of a family history of bleeding; a history of hemostatic challenges, including surgery, dental procedures, and trauma; and a menstrual history in females.

The goal at the end of the history is to establish the likelihood of a bleeding disorder, as this will guide the direction of the laboratory investigation. Quantification of clinical bleeding is a challenge, particularly in the outpatient setting. In recent years, several bleeding assessment tools (BAT) have been developed to more accurately differentiate bleeding phenotypes in healthy individuals and in patients with bleeding disorders. These tools, which were originally designed for assessing bleeding in von Willebrand disease (vWD) do not appear to be diagnostic and are in the process of being validated for the ability to screen other bleeding disorders. However, it is increasingly clear that a normal bleeding score rules out the presence of a bleeding disorder. Therefore, if the bleeding score is indicative of excessive bleeding, it should be followed by an evaluation of a hematologist to evaluate the need for further laboratory tests.

Screening Tests

  • Platelet count, PT, aPTT

The laboratory evaluation for bleeding includes performance of initial screening tests. The most common screening tests utilized include the platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). When the PT or aPTT is prolonged, mixing studies are required via a one-to-one mix of patient plasma with known normal standard plasma. Test correction in the mixing study indicates a deficiency state, whereas lack of correction indicates an inhibitor, either one directed against a specific factor or a a global inhibitor as best exemplified by a lupus anticoagulant. Specific factor analyses are performed after mixing studies reveal a correction of prolonged coagulation screening test(s) indicative of a deficiency state or in the face of normal screening tests with a positive history. Screening tests are not sensitive and do not evaluate all abnormalities associated with bleeding including vWF, FXIII, PAI-1, and 𝛼2AP deficiencies and may be insensitive to mild FVIII and FIX deficiencies; therefore, a patient history strongly suggestive of a bleeding disorder may warrant testing for such deficiencies, including rare abnormalities regardless of screening test results. The most common screening tests utilized include the platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). When the PT or aPTT is prolonged >10 seconds, mixing studies are required via a one-to-one mix of patient plasma with known normal standard plasma. Test correction in the mixing study indicates a deficiency state, whereas lack of correction indicates an inhibitor.

  • Platelet function

Screen tests also are utilized to identify individuals with a high likelihood of vWD or platelet disorders. The bleeding time, once widely used, has become obsolete because of the lack of sensitivity and specificity. The PFA-100 (platelet function analyzer) has been proposed to have a role in screening individuals with suspected platelet dysfunction or vWD. Initial studies demonstrated the efficacy of the PFA-100 in the evaluation of patients with known severe platelet disorders or vWD. The PFA-100 induces high shear stress and simulates primary hemostasis by flowing whole blood through an aperture with a membrane coated with collagen and either ADP or epinephrine. Platelets adhere to the collagen-coated surface and aggregate forming a platelet plug that enlarges until it occludes the aperture, causing cessation of blood flow. The time to cessation of flow is recorded as closure time (CT). The sensitivity and spcificity of the CT of the PFA-100 were reported as 90% for severe platelet dysfunction or vWD, with vWD plasma levels below 25%. The utility of the PFA-100 as a screening tool, however, has been challenged based on the reported low sensitivity (24%-41%) of the device in individuals with mild platelet secretion defect, mild vWD or storage pool disorders. Additionally, a significant limitation of the PFA-100 is the fact that the platelet count and hemoglobin levels affect the CT. The CT will be abnormal if the platelet count is less than 100,000/𝜇L and the hemoglobin is <10 g/dL.

Hemostasis Mechanism – Platelet Structure and Function

August 24, 2016 Cardiology, Hematology No comments , , , , , , , , , , , , , , , , , , , , , ,

Platelet Granules and Organelles

Platelets possess secretory granules and mechanisms for cargo release to amplify responses to stimuli and influence the surrounding environment. Platelet granule structures include 𝛼- and dense granules, lysosomes, and peroxisomes. 𝛼-Granules and the dense bodies are the main secretory granules that release cargo (e.g., fibrinogen and adenosine diphosphate [ADP]) upon platelet activation.

Platelet granule secretion begins with a dramatic increase in platelet metabolic activity, set off by a wave of calcium release and marked by increased adenosine triphosphate (ATP) production. After platelet stimulation by agonists, a "contractile ring" develops around centralized granules, the granules fuse with the surface membranes, and then they extrude their contents. Granule secretion in platelets is a graded process that depends on the number, concentration, and nature of the original stimulus/stimuli, either strong (e.g., thrombin and collagen) or weak (e.g., ADP and epinephrine).


What the 𝛼-granules have include:

  • 𝛽-thromboglobulin
  • PF4
  • thrombospondin
  • vWF
  • Fibrinogen
  • Other plasma proteins (small amount)
  • Growth factors
  • 𝛼IIb𝛽3 [platelet receptor]

𝛼-Granules, with a cross-sectional diameter of approximately 300 nm and numbering approximately 50 per platelet, are the predominant platelet granules. They are approximately spherical in shape, with an outer membrane enclosing two distinct intragranular zones that vary in electron density. The larger, electron-dense region is often eccentrically placed and consists of a nucleoid material that is rich in platelet-specific proteins such as 𝛽-thromboglobulin. The second zone, of lower electron density, lies in the periphery adjacent to the granule membrane and contains tubular structures with adhesive GPs such as von Willebrand factor (vWF) and multimerin, along with factor V. Platelets take up plasma proteins and store them in their 𝛼-granules.

Three proteins, 𝛽-thromboglobulin, PF4, and thrombospondin, are synthesized in megakaryocytes and highly concentrated in 𝛼-granules. The first two, 𝛽-thromboglobulin and PF4, show homology in amino acid sequence and share the additional features of localization in the dense nucleoid of 𝛼-granules, heparin-binding properties, and membership in the CXC family of chemokines. Together, they constitute approximately 5% of total platelet protein, and they can serve as useful markers for platelet activation in serum or plasma. Thrombospondin may comprise up to 20% of the total platelet protein released in response to thrombin, and likely participates in multiple biologic prcesses.

vWF is also synthesized by megakaryocytes and is present in the tubular structures of the 𝛼-granule peripheral zone, similar to its localization within Weibel-Palade bodies of vascular endothelial cells. Factor V and multimerin, a factor V/Va-binding protein, co-localize with vWF in platelets but not in endothelial cells. Fibrinogen is also found in 𝛼-granules, but is incorporated actively from plasma and not synthesized by megakaryocytes. In fact, small amounts of virtually all plasma proteins, such as albumin, immunoglobulin G (IgG), fibronectin, and 𝛽-amyloid protein precursor, may be taken up into the platelet 𝛼-granules. 𝛼-Granules also contain many growth factors, including platelet-derived growth factor, transforming growth factor-𝛽1 (TGF-𝛽1), and vascular endothelial growth factor. These signaling molecules may contribute to the mitogenic activity of platelets.

Platelet 𝛼-granules serve as an important reservoir for 𝛼(IIb)𝛽3 that contributes significantly to the surface fibrinogen receptors present on activated platelets. The 𝛼-granule membrane protein, P-selectin (granule membrane protein-140) is translocated to the plasma membrane after platelet activation. Finally, a number of additional proteins have been located to the surface of 𝛼-granules alone, including CD9, platelet endothelial cell adhesion molecule-1 (PECAM-1), Rap 1b, GPIb-IX-V, and osteonectin.

The platelets and megakaryocytes of patients with gray platelet syndrome have decreased numbers of 𝛼-granules and reduced levels of some proteins. It is proposed that there is incorrect targeting of 𝛼-granule proteins to the 𝛼-granule in the megakaryocyte in this disease.

Dense Bodies

Dense bodies contain a large reservoir of ADP, a crtitical agnoist for platelet activation that amplifies the effect of other stimuli. In addition to this nonmetabolic pool of ADP, the  dense bodies are rich in ATP, pyrophosphate, calcium, and serotonin (5-hydroxytryptamine), with lesser amounts of guanosine triphosphate (GTP), guanosine diphosphate (GDP), and magnesium. The adenine nucleotides are synthesized and segregated by megakaryocytes, whereas serotonin is incorporated into dense granules from the plasma by circulating platelets. There is more ADP than ATP in dense bodies, and both can lead to adenosine monophosphate (AMP). In turn, AMP can be dephosphorylated to adenosine or cyclized to produce cyclic AMP, an inhibitor of the platelet-stimulatory response. The dense granule membrane contains P-selectin and granulophysin.

Compared ADP/ATP within the metabolic/cytoplasmic pool (at least two different pools: metabolic pool and cytoplasmic pool), ADP/ATP in storage pool (dense bodies) contains approximately two-thirds of the total platelet nucleotides, mainly in the form of ADP and ATP, and is metabolically inactive, does not rapidly incorporate exogenous adenine or phosphate, and equilibrates slowly with the metabolic pool. Nucleotides in this pool (storage pool) are extruded fromt the platelet during the release reaction and cannot be replenished after release.

The ATP (metabolic pool) that is broken down to provide energy for the release reaction is not rephosphorylated; rather, it is irreversibly degraded to hypoxanthine, which diffuses out of the cell.


Lysosomes are small, acidified vesicles, approximately 200 nm in diameter, that contain acid hydrolases with pH optima of 3.5 to 5.5, including 𝛽-glucuronidase, cathepsins, aryl sulfatase, 𝛽-hexosaminidase, 𝛽-galactosidase, heparitinase, and 𝛽-glycerophosphatase. Additional protein found in lysosomes include cathepsin D and lysosome-associated membrane proteins (LAMP-1/LAMP-2, which are expressed on the plasma membrane after activation). Lysosomal constituents are released more slowly and incompletely (maximally, 60% of the granules) than 𝛼-granules or dense-body components after platelet stimulation, and their release also requires stronger agonists such as thrombin or collagen.


Peroxisomes are rare, small granules, demonstrable with alkaline diaminobenzidine as a result of their catalase activity. The structure may participate in the synthesis of platelet-activating factor.

Mitochondria in platelets are similar, with the exception of their smaller size, to those in other cell types. There are approximately seven per human platelet, and they serve as the site for the actions of the respiratory chain and the citric acid cycle. Glycogen is found in small particles or in masses of closely associated particles, playing an essential role in platelet metabolism.

Platelet Kinetics

Approximately one-third of the total platelet mass appears to pool in the spleen. The splenic pool exchanges freely with the platelets in the peripheral circulation. Administration of epinephrine, which evacuates platelets from the spleen, increases the peripheral platelet count 30% to 50%, and platelet counts in asplenic patients are not affected by epinephrine. Some studies suggest that the splenic pool consists of the youngest, largest platelets. Pathophysiologi states can result in 80% to 90% of platelets being sequestered in the spleen, resulting in thrombocytopenia.

Other organs that have pool of platelets (accounting for about 16% of total platelets) include the lungs and the liver and so on.

The life span of platelet has been estimated to be 8 to 12 days in humans. In steady state, when platelet production equals destruction, platelet turnover has been estmated at 1.2 to 1.5 x 1011 cells per day.

PS: Details of various platelet products can be found in thread "Platelet Transfusion for Patients w/ Cancer" at http://www.tomhsiung.com/wordpress/2013/04/platelet-transfusion-for-patients-with-cancer-part-one/

Platelet Adhesion and Activation

Part I – Adhesion

  • Adhesive ligands: vWF, collagen, fibronectin, thrombospondin, laminin (perphaps)
  • Platelet surface receptors: GPIb/V/IX complex, GPVI, 𝛼IIb𝛽3, ð›¼2𝛽1, ð›¼5𝛽1, ð›¼6𝛽1
  • Interaction of GPVI with collagen activates platelet intergrins
  • At low shear conditions, fibrinogen is the primary ligand (interacting with 𝛼IIb𝛽3), but other ligands may also be involved

Platelet adhesion to exposed subendothelium is a complex multistep process that involves a diverse array of adhesive ligands (vWF, collagen, fibronectin, thrombospondin, and perphaps laminin) and surface receptors (GPIb/V/IX, GPVI, integrins 𝛼IIb𝛽3, 𝛼2𝛽1, 𝛼5𝛽1, and 𝛼6𝛽1). The specific ligand/receptor palyers in primary platelet adhesion are largely dependent on the arterial flow conditions present. In high shear conditions, platelet tethering is dependent on the unique shear-dependent interaction between GPIb/V/IX and subendothelial vWF, derived either from plasma or released by endothelial cells and/or platelets. A tether forms between GPIb and vWF that either halts platelet movement or reduces it such that other interactions can proceed. Subsequent interactions are mediated by GPVI binding to glycineproline-hydroxyproline sites on collagen and perhaps to exposed laminin. The interaction of GPVI with collagen strongly activates platelets such that 𝛼IIb𝛽3, can engage in high-affinity interactions with ligands. At low shear conditions, fibrinogen is thought to be the primary ligand supporting platelet plug formation through its interaction with 𝛼IIb𝛽3, although thrombus formation can take place in the absence of vWF and fibrinogen, so other ligands may also be involved.

Following initial platelet adhesion, subsequent platelet-platelet interactions are intially mediated by two receptors, GPIb/V/IX and 𝛼IIb𝛽3, and their respective contributions are dependent on the flow conditions present. In high shear stress conditions, GPIb/V/IX receptor and vWF ligand action are predominant, with fibrinogen playing a stabilizing role.

Platelet Glycoprotein Ib Complex-von Willebrand Factor Interaction and Signaling

Screen Shot 2016-08-18 at 4.01.30 PMThe interaction of the platelet GPIb "complex" (the polypeptides GPIb𝛼, GPIb𝛽, GPIX, and GPV) with its primary ligand. vWF, is the receptor-ligand pairing that initiates platelet adhesion followed by a cascade of events leading to pathologic thrombosis or physiologic hemostasis. A unique aspect of this receptor-ligand interaction is that it requires the presence of high arterial shear rates to take place, thus explaining the predisposition of platelet-rich "white clots" in the arterial circulation over clots found in the venous circulation, with its relatively lower shear forces, in which clot formation takes place independent of the GPIb complex.

The binding site for vWF is present in the N-terminal 282 residues of GPIb. Important to the interactions are a cluster between residues Asp 252 and Arg 293 containing sulfated tyrosine residues and important anionic residues, a disulfide loop between Cys 209 and Cys 248, and an N-terminal flanking sequence of the leucine-rich repeats (LRG). Mutations involving single amino acid residues within these LRGs account for some cases of the congential bleeding disorder Bernard-Soulier syndrome, in which the GPIb complex binds poorly, or not at all, to vWF.

Unlike other receptors, GPIb does not require platelet activation for its interactions with vWF. In vitro, the interactions of vWF and binding with the GPIb complex occur with generally very low affinity in the absence of shear. The presence of the vancomycin-like antibiotic ristocetin or viper venom proteins, such as botrocetin, promotes the interactions. Mobilization may uncoil vWF to promote interactions with GPIb. The addition of shear, in a parallel-plate flow system, results in platelet interaction with subendothelial vWF that occurs in a biphasic fashion. Likewise, the rate of translocation of platelets from blood to the endothelial cell surface, which is dependent, increases linearly up to wall shear rates of 1,500 s-1, whereas the translocation rate remains relatively constant with the wall shear rate between 1,500 and 6,000 s-1. Thus, the presence of shear is important for promoting the interactions between the GPIb complex and vWF. Studies of real-time thrombus formation in the absence of platelet GPIb complex and in blood from individuals with severe (type 3) von Willebrand disease indicate that GPIb and vWF interaction are required for platelet surface interaction at high shear rates (>1,210 s-1), whereas GPIb deficiency results in poor platelet adhesion at lower shear. Shear accelerates thrombus formation likely by promoting this receptor-ligand interaction.

When the GPIb complex interacts with its vWF ligand under conditions of elevated shear stress, signals are initiated that activate integrin 𝛼IIb𝛽3. The pathways involved lead to a) elevation of intracellular calcium; b) activation of a tyrosine kinase signaling pathway that incorporates nonreceptor tyrosine kinases such as Src, Fyn, Lyn, and Syk, phospholipase C(gamma)2, and adaptor protein such as SHC, LAT, and SLP-76; c) inside-out signaling through the 𝛼IIb𝛽3 integrin followed by platelet aggregation; and d) activation of protein kinase C (PKC), protein kinase G (PKG), and phosphoinositide 3-kinase (PI3K), …… and so on.

Once vWF binds to GPIb-V-IX, signaling complexes form in the vicinity of the GPIb𝛼 cytoplasmic tail consisting of cytoskeletal proteins such as 14-3-3ζ  as well as signaling protein like Src and PI 3-kinase. This process leads to Syk activation, protein tyrosine phosphorylation, and recruitment of other cytoplasmic proteins with pleckstrin homology domains that can support interactions with 3-phosphorylated phosphoinositides and ultimately activation of integrin 𝛼IIb𝛽3.

Glycoprotein Ib Complex Interaction with Thrombin and Other Molecules

The GPIb complex serves as an 𝛼-thrombin binding site on platelets, although the physiologic relevance of the interactions is not clear. The density of GPIb complexes (~20,000/platelet) far exceeds the number of thrombin binding sites reported on platelets (~6,000/platelet). Studies have identified interaction of the GPIb complex with ligands other than vWF. These include a study a reversible association of GPIb with P-selectin, which is examined in more detail in the section "Platelets and Endothelium." The interaction of platelet GPIb with the neutrophil adhesion receptor 𝛼(M)𝛽2 (Mac-1) is discussed in the section "Platelets and White Blood Cells." Additionally, GPIb reportedly interacts with high-moecular weight kininogen, factor XII, and factor XI.

Platelet-Collagen Interaction and Signaling

  • Receptors: GPVI, ð›¼2b𝛽1
  • Ligands: collagens

Collagens, one of the most thrombogenic substance in vessels, are very important activators of platelets in the vascular subendothelium and vessel wall, and thus are prime targets for therapeutic intervention in patients experience a pathologic arterial thrombotic event such as MI or stroke. Platelets have two major surface receptors for collagen, the immunoglobulin superfamily member GPVI and the integrin 𝛼2𝛽1. The former is considered to be the primary palyer in platelet adhesion. In additon to these two surface receptors, the GPIb complex can also be considered an indirect collagen receptor because its subendothelial vWF ligand essentially acts as bridging molecule between platelets and collagen by fixing itself to the latter, which, in turn, acts as scaffolding for the multimers. Collagen adhesion also results in indirect activation of the protease-activated receptor 1 via MMP-1. Other molecules, such as CD 36, may also sustain collagen interaction.

Glycoprotein VI receptor

GPVI is the main receptor involved in collagen-mediated platelet activation. Studies of mice lacking platelet GPVI show that they lose collagen-induced platelet activation due to a defect in platelet adhesion. Thus, GPVI appears to serve as teh initial receptor involved in platelet adhesion, and it activates integrin binding. GPVI alone supports adhesion to insoluble collagens, and works with 𝛼2𝛽1 to promote platelet adhesion to soluble collagen microfibrils. GPVI can also be engaged by collagen-related peptides (arranged in triple helical structures with sequences similar to collagen) and teh snake venom convulxin, which elicit signals through GPVI.

Synergism between GPVI pathways and those related to other adhesion receptors such as GPIb-V-IX and soluble agonists released by activated platelets are likely necessary for the full repertoire of platelet-collagen signaling. Exposure of platelets to collagen surfaces likely results in GPVI clustering that in turn triggers the tyrosine phosphorylation of the FcRγ chain. The GPVI/FcRγ-chain complex leads to platelet activation through a pathway that has many aspects in common with signaling by immune receptors, such as the Fc receptor family and the B- and T-cell antigen receptors.

α2β1 receptor

The first platelet collagen receptor identified was the integrin 𝛼2𝛽1 receptor, also known as platelet GPIa/IIa and lymphocyte VLA-2.

When compared to vWF, collagen is a more efficient substrate when it comes to supporting stable platelet adhesion and thrombus formation. The fact that initial platelet tethering to collagen under high shear flow first requires interaction between vWF and platelet GPIb serves to underscore the importance of the two major collagen receptors, GPVI and 𝛼2𝛽1, in promoting platelet adhesion and activation under shear conditions.

In addition to GPVI, the α2β1 receptor also propagates signaling. The use of α2β1 selective ligands has demonstrated calcium-dependent spreading and tyrosine phosphorylation of several proteins when interaction with platelets takes place.

Physiologic Inhibition of Platelet Adhesion

Negative regulation of platelets is essential to set the stimulus threshold for thrombus formation, determine final clot size and stability, and prevent uncontrolled thrombosis. The mechanisms behind the negative regulation of platelet activation are described later, and in this respect, roles of players such as nitric oxide and prostacyclin have been well characterized. Platelet activation can also be inhibited by signaling through the adhesion moleculde PECAM-1 (CD31). Expressed on a number of blood cells and endothelial cells, PECAM has a wide array of regulatory functions in processes such as apoptosis and cell activation. Following homophilic interactions and/or clustering, PECAM-1 is tyrosine phosphorylated in its cytoplasmic tail ITIM domain. Phosphorylation of PECAM-1 recruits and activates the SH2 domain-containing protein-tyrosine phosphatase, SHP-2. Studies suggest that the PECAM-1/SH-2 complex functions to counteract platelet activating, most particularly for collagen by inhibiting GPVI/FcRγ chain signaling.

Part II – Activation

PAR Thrombin Interactions

  • See Figure 16.9

Platelet thrombin receptors/platelet protease-activated receptors/PARs  and signaling

Screen Shot 2016-08-21 at 12.46.55 PMPARs are G-protein-coupled receptors that use a unique mechanism to convert an extracellular protein cleavage event into an intracellular activation signal. In this case, the ligand is already part of the receptor per se, by virtue of the fact that it is represented by the amino acid sequence SFLLRN (residues 42 through 47) and is unmashed as a new amino terminus after thrombin cleaves the peptide bond between Arg 41 and Ser 42 (Figure 16.9). This "tethered ligand" then proceeds to irreversibly dock with the body of its down receptor to effect transmembrane signaling, as shown in Figure 16.9.

Thrombin signaling in platelet is mediated, at least in part, by four members of a family of G-protein-coupled PARs (PAR-1, -2, -3, and -4). Human platelets express PAR-1 and PAR-4, and activation of either is sufficient to trigger platelet aggregation. PAR-1, -3, and -4 can be activated by thrombin, whereas PAR-2 can be activated by trypsin, tryptase, and coagulation factors VIIa and Xa. Presumably, other proteases are capable  of recognizing the active sites of these receptors and can thus also trigger PAR signaling.

Once activated, PAR-1 is rapidly uncoupled from signaling and internalized into the cell. It is then transported to lysosomes and degraded. Platelet presumably have no need for a thrombin receptor recycling mechanism, becuase once activated, they are irreversibly incorporated into blood clots. Conversely, in cell lines with characteristics similar to megakaryocytes, new protein synthesis is needed for recovery of PAR-1 signaling, and in endothelial cells, sensitivity to thrombin is maintained by delivery of naive PAR-1 to the cell surface from a preformed intracellular pool.

Platelet ADP (Purinergic) Receptors and Signaling

  • P2Y1
  • P2Y12
  • P2X

Evidence that ADP plays an important role in both the formation of the platelet plug and the pathogenesis of arterial thrombosis has been accumulating since its initial characterization in 1960 as a factor derived from red blood cells that influences platelet adhesion. ADP is present in high concentratons (molar) in platelet-dense granules and is released when platelet stimulation takes place with other agonists, such as collagen; thus, ADP serves to further amplify the biochemical and physiologic changes associated with platelet activation and aggregation. Inhibitors of this ADP-associated aggregation include commonly used clinical agents, including ticlopidine, clopidogrel, prasugrel, and ticagrelor, proven to be very effective antithrombotic drugs.

Adenine nucleotides interact with P2 receptors that are ubiquitous among different cell types and have been found to regulate a wide range physiologic processes. They are divided into two groups, the G-protein-coupled superfamily named P2Y and the ligand-gated ion channel superfamily termed P2X. Two G-protein-coupled (P2Y) receptors contribute to platelet aggregation. The P2Y1 receptor initiates aggregation through mobilization of calcium stores, and the P2Y12 receptor is coupled to inhibition of adenylate cyclase and is essential for a full aggregation response to ADP with stabilization of the platelet plug.

PS: ADP >>> P2Y12 >>> inhibition of adenylate cyclase >>> decreased cAMP production >>> decreased intensity of aggregation

Inhibition of either P2Y1 or P2Y12 receptors is sufficient to block ADP-mediated platelet aggregation, and coactivation of both receptors is therefore necessary, through the G proteins Gq and Gi, respectively, for ADP to activate and aggregate the platelet.

Although considered a weak agonist in comparison to collagen or thrombin, ADP clearly palys an important role in thrombus stabilization, likely by contributing to the recruittment of additional platelets to growing thrombi. Aggregation is often reversible when platelets are stimulated by ADP alone. In addition, low concentrations of ADP serve to amplify the effects of both strong and weaker agonists, the latter inlcuding serotonin and adrenaline, among others.

Platelet Activation by Soluble Agonists


Epinephrine is unique among platelet agonists because it is considered to be  capable of stimulating secretion and aggregation, but not cytoskeletal reorganization responsible for shape change. Platelet responses to epinephrine are mediated through 𝛼2-adrenergic receptors, and these responses have been found to vary among individuals, with some donors with otherwise normal platelets manifesting delayed or absent responses.

Arachidonic acid, thromboxane A2, and thromboxane receptors

After platelet stimulation by a number of agonists, arachidonic acid is generated directly by phospholipase A from its membrane phospholipid precursors (PC, PS, and PI) and indirectly by PLC generation of DAG followed by DAG lipase action. Most platelet agonists are believed to activate this pathway. Three known eicosanoid subsetsl of biochemical compounds are known to be derived from the formation of arachidonic acid – the prostanoids, leukotrienes, and epoxides. Although all three of these pathways are present in platelets, most arachidonic acid ends up being metabolized to thromboxane A2 (TxA2).

TxA2 is produced in platelets from arachidonic acid through the generation of PGH2 by the enzyme cyclo-oxygenase, which is irreversibly inhibited by aspirin through acetylation of a serine residue near its C terminus. PGE2 and PGI2 act to inhibit platelet activation by generating intracellular cAMP, whereas TxA2 activates platelets. Platelets primarily synthesize thromboxane, and endothelial cells mainly synthesize prostaglandins such as PGI2.

Like ADP and epinephrine, TxA2 is also capable of activating nearby platelets after its release into plasma. It has a very short half-life of 30 seconds before its conversion to the inactive metabolite thromboxane B2 prevents widespread platelet activation beyond the vicinity of thrombus formation. Both arachidonic acid and analogs of TxA2 have been found to activate and aggregate platelets by mediating shape change and phosphorylation of signaling enzymes. The thromboxane receptor (TP) is a member of the seven-transmembrane G-protein-coupled receptor family and has been localized to the plasma membrane. Two isoforms of the receptor have been identified in platelets TP𝛼 and TP𝛽 – and they activate platelets through ghe Gq pathway.

Physiologic Inhibition of Platelet Activation

One of the many remarkable features of platelets is their ability to remain in a physiologic resting state and resist becoming activated while navigating the heart, arterial, and venous circulations. Indeed, the pathologic consequences associated with widespread inappropriate platelet activation are life- and limb-threatening in the settings of well-characterized clinical disorders, such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia. The mechanisms responsible for maintaining the fine balance of keeping platelets in a resting state until they encounter a genuine need  to undergo adhesion, activation, and aggregation at the site of vascular injury are nearly as diverse as those responsible for mediating these physiologic phenomena.

Some general mechanisms involved in physiologic inhibition of platelet activation include phenomena such as a) generation of negative-regulating molecules by the platelet (e.g., cAMP), endothelium (e.g., PGI2, nitric oxide, heparan sulfate), and at distant sites (e.g., antithrombin); b) barrier of endothelial cells that prevents direct contact of circulating platelets with collagen; c) ecto-ADPase (CD39) expression by endothelial cells that metabolizes ADP secreted from platelets; d) tendency for blood flow to wash away unbound thrombin and other soluble mediators from the site of platelet plug formation; e) brief half-life of certain key platelet activators such as TxA2; f) tight regulation of the affinity state of receptors such as 𝛼IIb𝛽3; g) downregulation of signaling receptors to limit their actions; and h) inhibitory pathways mediated by ITIM-containing and/or contact-dependent adhesion receptors, such as PECAM, CECAM-1, JAM-A, and potentially others.

Receptor downregulation and desensitization

Signaling through G-protein-coupled receptors present on the surface of platelets is limited by their phosphorylation, which triggers desensitization, that is, uncoupling from G proteins, and internalization via Claritin-mediated endocytosis (for detail about G-protein coupled receptors please refers to thread "G Protein-Coupled Receptors and Second Messengers" at http://www.tomhsiung.com/wordpress/2014/09/g-protein-coupled-receptors-and-second-messengers/). G-protein kinases and 𝛽-arrestin are central to these processes. In addition, G-protein-coupled receptors interact with a myriad of other molecules that finely tune their signaling, including regulators of G-protein signaling (RGS) and GPCR-associated sorting proteins.

Inhibitory prostaglandins

Generation of the prostaglandins from arachidonic acid metabolism, such as PGI2 and PGE2 (at high concentrations), results in inhibition of platelet activation and aggregation, and counterbalances the actions of thromboxanes derived from the same pathway. While PGI2 and PGD2 inhibit platelet function at low doses, PGE2 displays a biphasic reponse, and inhibits platelet function only at higher concentrations, likely via the EP4 receptor. The inhibitory effects are mediated via G-protein-coupled receptors (IP and EP receptors, respectively) that couple to the 𝛼 subunits of Gs to regulate adenylate cyclase-mediated generation of cAMP. cAMP levels in platelets are also governed by the activity of phosphodiesterase, the enzyme responsible for cAMP metabolism. This enzyme activity is inhibited drugs such as the weak antiplatelet agent dipyridamole, the bronchodilator theophylline, and sildenafil, used to treat erectile dysfunction in men.

Nitric oxide

NO is generated by endothelial cells and platelets from L-arginine in response to shear stress forces and other platelet agonists, such as thrombin and ADP. The bulk of the evidence suggests that at high concentrations NO functions to inhibit platelet activation through the cyclic guanosine monophosphate (cGMP) second messenger generated by guanylate cyclase activation. Elevations in cGMP, by modulating phosphodiesterase activity, can raise intraplatelet cAMP. Paradoxically, low levels of NO may elicit platelet activation pathways. Endothelial NO synthase activity is enhanced during platelet activation, presumably as an additional means for limiting platelet aggregation.

Platelet Aggregation: 𝛼IIb𝛽3 (GPIIb/IIIa) Receptor and Its Signaling Mechanisms

Platelet aggregation is a complex phenomenon that is the end result of a series of adhesion- and activation-related processes. Essential components of this process include an agonist, calcium, and the adhesive proteins fibrinogen and vWF. Divalent cations, such as calcium and magnesium, are required for platelet aggregation in trace amounts, and these alter the specificity of the integrin ð›¼IIb𝛽3 for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to ð›¼IIb𝛽3, and also by the ability of the former to generate polymerized fibrin as support for the platelets in a thrombus.

The signaling pathways of 𝛼IIb𝛽3 are complex. Central concepts of the signaling pathway include inside-out signaling, which involves the processes termed affinity and avidity modulation, and outside-in signaling, in which messages are transmitted to the inside of the platelet via the events occurring outside the membrane through 𝛼IIb𝛽3 activation. Primary platelet agonists such as ADP, thrombin, and matrix proteins collagen and vWF affect platelet aggregation through a process known as inside-out signaling. In the inside-out signaling, agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails. This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, vWF, and fibronectin. In the outside-in signaling, extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tail. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.

Brief Review of Physiology of Platelet

Following injury to the blood vessel, platelets interact with collagen fibrils in the exposed subendothelium by a process (adhesion) that involves, among other events, the interaction of a plasma protein ,vWF, and a specific glycoprotein (GP) complex on the platelet surface, GP Ib-IX-V (GPIb-IX). This interaction is particularly important for platelet adhesion under conditions of high shear stress. After adherence to the vessel wall via vWF and the long GP Ib-IX-V receptor, other platelet receptors interact with proteins of the subendothelial matrix. Hereby collagen provides not only a surface for adhesion but also serves as a strong stimulus for platelet activation.

Activated platelets release the contents of their granules (secretion), including ADP and serotonin from the dense granules, which causes the recruitment of additional platelets. These additional platelets form clumps at the site of vessel injury, a process called aggregation (cohesion). Aggregation involves binding of fibrinogen to specific platelet surface receptors, a complex composed of GPIIb-IIIa (integrin 𝛼IIb𝛽3), an integrin that normally exists in a resting (low-affinity) state but that transforms into an activated (high-affinity) state when stimulated by the appropriate signal transduction cascade. GPIIb-IIIa is platelet specific and has the ability to bind vWF as well. Although resting platelets do not bind fibrinogen, platelet activation induces a conformational change in the GPIIb-IIIa complex that leads to fibrinogen binding.

Moreover, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane lipoprotein surface. During platelet activation, the negatively charged phospholipids, especially PS, become exposed on the platelet surface, and essential step for accelerating specific coagulation reactions by promoting the binding of coagulation factors involved in thrombin generation.

A number of physiologic agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 production. Other agonists, such as prostacyclin, inhibit these responses. Binding of agonists to platelet receptors initiates the production or release of several intracellular messenger molecules, including products of hydrolysis of phosphoinositide (PI) by phospholipase C, TxA2, and cyclic nucleotides. These induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and thromboxane production.

[Physiology][Hematology] Coagulation Factors, Anticoagulation Factors, and Pathways of Hemostasis and Thrombosis

July 14, 2016 Hematology, Molecular Biology No comments , , , , , , , , , , , , , , , , , ,

The role of surfaces in coagulation and coagulation inhibition and fibrinolysis

Functionally, the relationship between clotting and surface is striking. For a process that is typically termed humoral, most of the coagulation reactions take place on biologic surfaces.

  • The presence of a phospholipid surface increases the rate of activation of prothrombin by several orders of magnitude.
  • The presence of a phospholipid surface also localizes the reaction to the site of injury and may protect the reaction from inhibitors.
  • The activation of protein C by thrombin is a reaction that occurs on a cellular surface.
  • Coagulation inhibitors such as antithrombin and heparin cofactor II are also more efficient when the reactions occur on surfaces. The typical surface in these reactions is glycosaminoglycans like heparin, heparin sulfate, and derma tan sulfate.

The Vitamin K-dependent Zymogens


Tenase/Intrinsic tenase complex: factor VIIIa-factor IXa complex

Extrinsic tenase complex: tissue factor-factor VIIa complex

Prothrombinase compelx: factor Va-factor Xa complex

These zymogens all a similar domain structure of a C-terminal serine protease domain and an N-terminal γ-carboxy glutamic acid (Gla) domain, which are connected by two epidermal growth factor (EGF)-like domains or kringle domains. The Gla domain mediates the binding of zymogens to a negatively charged lipid surface (in a calcium-dependent manner), a domain that is characteristic to the vitamin K–dependent proteins.

Screen Shot 2016-08-11 at 7.36.50 PMThe Gla domain refers to the 42-residue region located in the N-terminus of the mature protein that comprises 9 to 12 glutamic acid residues that are posttranslationally γ-carboxylated into Gla residues by a specific γ-glutamyl carboxylase in the endoplasmatic reticulum of hepatocytes. This γ-carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action, hence the name vitamin K–dependent proteins. For each Glu residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form. Warfarin inhibits the activity of vitamin K epoxide reductase, thereby preventing vitamin K recycling and hinibiting γ-carboxylation, which results in a heterogeneous population of circulaing undercarboxylated forms of the vitamin K-dependent proteins with reduced activity. Recognition by and interaction with γ-carboxylase is facilitated by the propeptide sequence that is located C-terminal to the signal peptide.

The serine protease domains of the vitamin K-dependent proteins are highly homologous, as they bear a chymotrypsin-like fold and display trypsin-like activity.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Activates TAFI, platelet, fibrinogen, FV, FVIII, FXI, FXIII, and protein C

Activated by prothrombinase complex

Inhibited by serpins (enhanced by glycosaminoglycans like heparin)

Screen Shot 2016-05-20 at 2.19.12 PMProthrombin is composed of fragment 1 (F1: Gla and kringle 1), fragment 2 (F2: kringle 2), and the serine protease domain. The primary function of kringle 1 and kringle 2 domain is to be bound by prothrombinase complex. PS: Gla and kringle, kringle 2, and serine protease.

Prothrombin is proteolytically activated by the prothrombinase complex that cleaves at Arg271 and Arg320, both of which are necessary to generate procoagulant α-thrombin (IIa). Thrombin's main function is to induce the formation of a fibrin clot by removing fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize. The dynamic structural conformation of thrombin allows for binding to diverse ligands, and the subsequent ligand-indued conformational stabilization, known as thrombin allostery, regulates and controls thrombin activity. Thrombin also is able to cleave a wide variety of substrates with high specificity (TAFI, platelet, FV, FVIII, FXI, FXIII, protein C).

The physiologic inhibitors of thrombin are the serine protease inhibitors (serpins) antithrombin, heparin cofactor II, protein C inhibitor, and protease nexin 1, with antithrombin being the primary plasma inhibitor. For all four serpins, the rate of thrombin inhibition can be accelerated by glycosaminoglycans, such as heparin, through mutual binding to the serpin and thrombin, which ensures rapid inhibition of thrombin at the intact endothelial cell surface where heparin-like glycosaminoglycans are found.


Activates FIX, FX (in the form of extrinsic tenase)

Activated by Xa, thrombin, IXa, and XIIa

Inhibited by TFPI; antithrombin (only in the presence of heparin)

Screen Shot 2016-05-20 at 2.38.51 PMFactor VII consists of a Gla domain with 10 Gla residues, two EGF-like domains, a connecting region, and the serine protease domain.

Factor VII is proteolytically activated once it has formed a high-affinity complex with its cofactor tissue factor (there are small amount of VIIa in the circulation by unknown mechanism). A number of coagulation proteases including factor Xa, thrombin, IXa, and XIIa are capable of cleaving factor VII at Arg152 to generate factor VIIa, with factor Xa being considered the most potent and physiologically relevant activator of factor VII. Autoactivation can also occur, which is initiated by minute amounts of preexisting factor VIIa.

The extrinsic tenase complex activates both FIX and X.

The extrinsic tenase complex is inhibited by the tissue factor pathway inhibitor (TFPI). Antihrombin (only in the presence of heparin) also can inhibits the extrinsic tenase complex.


Aactivates FX in the form of intrinsic tenase

Activated by extrinsic tenase, factor XIa

Inhibited by antithrombin (enhanced by heparin)

Screen Shot 2016-05-20 at 3.08.29 PMFactor IX consists of a Gla domain, two EGF-like domains, a 35-residue activation peptide, and the serine protease domain. The Gla domain contains 12 Gla residues, of which the 11th and 12th Gla (Glu36 and Glu40) are not evolutionary conserved in other vitamin K-dependent proteins and are not essential for normal factor IX function.

Limited proteolysis of factor IX at both Arg145 and Arg180 by either the extrinsic tenase or factor XIa results in the release of the activation peptide and generation of factor IXa.

Factor IXa has a low catalytic efficiency as a result of impaired access of substrates to the active site that results from steric and repulsion. Reversible interaction with the cofactor VIIIa on anionic membranes and subsequent factor  X binding leads to rearrangement of the regions surrounding the active site and proteolytic factor X activation.

The primary plasma inhibitor of factor IXa is the serpin antihrombin, and this inhibition is enhanced by heparin, which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor IXa.


Activates prothrombin (prothrombinase complex); FV, VII, and VIII

Activated by extrinsic tenase; intrinsic tenase

Inhibited by antithrombin (enhanced by heparin); TFPI

Factor X is a two-chain zymogen consisting of a light chain which comprises the Gla domain with 11 Gla residues and the EGF domain, and a heavy chain that consists of a 52-residue activation peptide and the serine protease domain. The two chains are linked via a disulfide bond.

Factor X is activated by intrinsic tenase or extrinsic tenase, following cleavage at Arg194 in the heavy chain. After activated, Xa reversibly associates with its cofactor Va on an anionic membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Factor Xa is also involved in the proteolytic activation of FV, FVII, and VIII.

Further autocatalytic cleavage at Arg429 near the C-terminus of the factor Xa heavy chain leads to release of a 19-residue peptide, yielding the enzymatically active factor Xaβ. Plasmin-mediated cleavage of factor Xa at adjacent C-terminal Arg or Lys residues also results in the generation of factor Xaβ and factor Xaβ derivatives. While the coagulation activity is eliminated in the factor Xaβ derivatives, they are capable of interacting with the zymogen plasminogen and enhance its tissue plasminogen activator-mediated conversion to plasmin, thereby promoting fibrinolysis.

A primary plasma inhibitor of factor Xa is the serpin antithrombin, and this inhibition is enhanced by heparin. Another potent factor Xa inhibitor is TFPI, which inhibits both the extrinsic tenase-factor Xa complex as well as free factor Xa, for which protein S function as a cofactor.

Protein C

Inactivates FVa, FVIIIa (cofactor protein S, with calcium and surface) 

Activated by thrombin-thrombomodulin complex (enhanced by EPCR and PF4)

Inhibited by heparin-dependent serpin protein C inhibitor and by PAI-1

Protein C is synthesized as a single-chain precursor and during intracellular processing amino acids Lys146-Arg147 are excised. The zymogen consists of a light chain comprising the Gla domain and the EGF domains, which is linked via a disulfide bond to the heavy chain that consists of the activation peptide and the serine protease domain.

Screen Shot 2016-05-24 at 8.45.18 PMProtein C is proteolytically activated by alpha-thrombin in complex with the endothelial cell surface protein thrombomodulin following cleavage at Arg169. The activation peptide is released and the mature serine protease activated protein C (APC) is formed. Activation of protein C is enhanced by its localization on the endothelial surface through association with the endothelial cell protein C receptor (EPCR). Also, protein C activation is accelerated by platelet factor 4 (PF4), which is secreted by activated platelets. Upon interaction with the Gla domain of protein C, PF4 modifies the conformation of protein C, thereby enhancing its affinity for the thrombomodulin-thrombin complex.

APC consists of the disulfide-linked light chain comprising the Gla and EGF domains and the catalytic heavy chain. In complex with its cofactor protein S, APC proteolytically inactivates factors Va and VIIIa in a calcium- and membrane-dependent manner. Intact factor V has been reported to function as a cofactor for the inactivation of factor VIIIa in the presence of protein S.

APC is primarily inhibited by the the heparin-dependent serpin protein C inhibitor and by plasminogen activator inhibitor-1 (PAI-1).

The Procoagulant Cofactors V and VIII

Factors V and VIII both function as cofactors in coagulation and dramatically enhance the catalytic rate of their macromolecular enzyme complexes, resulting in the generation of thrombin (via prothrombinase) and factor Xa (via intrinsic tenase), respectively. Apart from their functional equivalence, they also share similar gene structure, amino aacid sequences, and protein domain structures.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Accelerates the ability of FXa to rapidly convert prothrombin to thrombin

Activated by thrombin (principal activator), FXa (primarily in initiation phase)

Inhibited by APC

FV and factor V-short interact with full-length TFPI

Approximately 20% percent of the total factor V in blood is stored in the alpha-granules of platelets. Although it was originally thought that megakaryocytes synthesize factor V, studies in humans indicate that platelet factor V originates from plasma through endocytic uptake. Platelet factor V is modified intracellularly such that it is functionally unique compared to its plasma-derived counterpart. It is partially activated, more resistant to inactivation by APC, and has several different posttranslational modifications.

Screen Shot 2016-05-29 at 8.22.28 PMFactor V has an A1-A2-B-A3-C1-C2 domain structure. The two C-type domains belong to the family of discoidin domains, which are generally involved in cell adhesion, and share approximately 55 percent sequence identity with the factor VIII C domains. The C domain mediate binding to the anionic phospholipid surface, thereby localizing factor V to the site of injury and facilitating interaction with factor Xa and prothrombin. In contrast, large central B domain of factor V shows weak homology to the factor VIII B domain or to any other known protein domain.

Factor V undergoes extensive postranslational modifications, including sulfation, phosphorylation, and N-linked glycoslation. Sulfation at sites in teh A2 and B domain are involved in the thrombin mediated proteolytic activation of factor V. Phosphorylation at Ser692 in the A2 domain enhances the APC-dependent inactivation of the cofactor Va.

Sequential proteolytic cleavage of the procofactor factor V at Arg709, Arg1018, and Arg1545 in the B domain results in release of the inhibitory constraints exerted by the B domain and in the generation of the heterodimeric cofactor Va, where maximal cofactor activity correlates with cleavage at Arg1545. Thrombin has generally been recognized to be the principal activator of factor V. However, recent findings suggest that in the initiation phase of coagulation factor V is primarily activated by factor Xa. Factor Xa initially cleaves factor V at Arg1018, followed by proteolysis at Arg709 and Arg1545.

Factor Va is composed of a heavy chain comprising the A1-A2 domains and the A3-C1-C2 light chain, which are noncovalently associated via calcium ions. Factor Va is a nonenzymatic cofactor within the prothrombinase complex that greatly accelerates the ability of factor Xa to rapidly convert prothrombin to thrombin.

APC catalyzes the inactivation of factor Va by cleavage at the main sites Arg306 and Arg506, upon which the cleaved A2 fragment dissociates and factor Va can no longer associated with factor Xa.

Both factor V and an alternativel spliced isoform of factor V (factor V-short), which lacks the major part of the B domain (residues 756 to 1458) and normally circulates in low abundance, interact with full-length TFPI (TFPIalpha), most likely through the acidic B domain region. The linkage of factor V and TFPIalpha is considered to attenuate the bleeding phenotype in factor V-deficient patients, as the low TFPIalpha levels in these patients allow the residual platelet factor to be sufficient for coagulation. Conversely, increased factor V-short expression caused by an A2440G mutation in the factor V gene leads to a dramatic increase in plasma TFPIalpha, resulting in a bleeding disorder.


Accelerates the ability of FIXa to rapidly convert FX to FXa

Activated by thrombin (principal) and FXa (also principal)

Activity downregulated spontaneously, by APC, FXa or FIXa

Factor VIII (antihemophilic factor) was first discovered in 1937, but it was not until 1979 that its purification by Tuddenham and coworkers led to the molecular identification of the protein. The mature factor VIII procofactor comprises 2332 amino acids and circulates in a high-affinity complex with its carrier protein VWF at a concentration of approximately 0.7 nM and a circulatory half-life of 8 to 12 hours. Complex formation with VWF protects factor VIII from proteolytic degradation, premature ligand binding, and rapid clearance from the circulation.

The primary source of factor VIII is the liver, but extrahepatic synthesis of factor VIII also occurs. While contradictory evidence exists on the cellular origin of both hepatic and extrahepatic factor VIII synthesis, recent studies in mice support that endothelial cells from many tissues and vascular beds synthesize factor VIII, with a large contribution from hepatic sinusoidal endothelial cells. This is consistent with observations on factor VIII expression in human endothelial cells from the liver and lung.

Screen Shot 2016-05-29 at 8.22.42 PMThe A1-A2-B-A3-C1-C2 domain structure of factor VIII shares significant homology with factor V except in the B domain region. In contrast to factor V, factor VIII B domain is dispensable for procoagulant activity. The C-terminal regions of the A1 and A2 domians and the N-terminal portion of the A3 domain contain short segments of 30 to 40 negatively charged residues known as the a1, a2, and a3 regions. Interaction with VWF is faciliated by the a3 region and C1 domain. The C domains mediate binding to the anionic phospholipid surface, thereby localizing factor VIII to the site of injury and facilitating interaction with factor IXa and factor X.

Factor VIII is heavily glycosylated and the majority of the N-linked glycosylation sites are found in the B domain, which mediate interaction with the chaperons calnexin and calreticulin and, in part, with the LMAN1-MCDF2 receptor complex. Sulfation of tyrosin residues is required for optimal activation by thrombin, maximal activity in complex with factor IXa, and maximal affinity of factor VIIIa for VWF.

Thrombin and factor Xa are the principal activators of the procofactor VIII and generate the cofactor VIIIa through sequential proteolysis at Arg740, Arg372, and Arg1689. The heterotrimeric factor VIIIa is composed of the A1, A2, and the A3-C1-C2 light chain subunits. The A1 and A3-C1-C2 subunites are noncovalently linked through calcium ions, whereas A2 is associated with weak affinity primarily by electrostatic interactions. Once activated, factor VIIIa functions as a cofactor for factor IXa in the phospholipid-dependent conversion of factor X to factor Xa. The rapid and spontaneous loss of factor VIIIa activity is attributed to A2 domain dissociation from the heterotrimer. Additional proteolysis by APC, factor Xa, or factor IXa also results in the downregulation of factor VIIIa cofactor activity.

The Soluble Cofactors Protein S and Von Willebrand Factor

Protein S

Protein S is a vitamin K-dependent single-chain GP of 635 amino acids that circulates with a plasma half-life of 42 hours. Part of the total protein S pool circulates in a free form at a concentration of 150 nM, whereas the majority (~60%; 200 nM) circulates bound to the complement regulatory protein C4b-binding protein (C4BP). Protein S is primarily synthesized in the liver by hepatocytes, in addition to endothelial cells, megakaryocytes, testicular Leydig cells, and osteoblasts.

The protein structure of protein S differs from the other vitamin K-dependent proteins as it lacks a serine protease domain and, consequently, is not capable of catalytic activity. Protein S is composed of a Gla domain comprising 11 Gla residues, a thrombin-sensitive region (TSR), four EGF domains, and a C-terminal sex hormone-binding globulin (SHBG)-like region that consists of two laminin G-type domains. The SHBG-like domain is involved in the interaction with beta-subunit of C4BP.

Apart from gamma-carboxylation of Glu residues, protein S is posttranslationally modified via N-glycosylation in the second laminin G-type domain of the SHBG-like region. beta-Hydroxylation of Asp95 or Asn residues in each EGF domain allows for calcium binding that orients the four EGF domains relative to each other.

Free protein S serves as a cofactor for APC in the proteolytic inactivation of FVa and FVIIIa. Interaction of protein S with APC on a negatively charged membrane surface alters the location of the APC active site relative to FVa, which accounts for the selective protein S-dependent rate enhancement of APC cleavage at Arg306 in FVa. C4BP-bound protein S also exerts a similar stimulatory effect on Arg306 cleavage, albeit to lower extent, whereas it inhibits the initial APC-mediated FVa cleavage at Arg506, resulting in an overall inhibition of FVa inactivation. Cleavage of the TSR by thrombin and/or FXa results in a loss of APC-cofactor activity. Protein S also functions as a cofactor for TFPIalpha in the inhibition of factor Xa, which is mediated by the SHBG-like region in protein S.


VWF is a large multimeric GP that is required for normal platelet adhesion to components of the vessel wall and that serves as a carrier for factor VIII. It is exclusively synthesized in megakaryocytes and endothelial cells and stored in specialized organelles in platelets and endothelial cells. VWF multimers circulate at a concentration of 10 nM with a half-life of 8 to 12 hours. Clearance of VWF multimers is mainly mediated by macrophages from the liver and spleen.

Large VWF multimers are cleaved by the plasma protease ADAMTS-13. This cleavage produces the smaller size VWF multimers that circulate in plasma. Reduced ADAMTS-13 activity is linked to various microangiopathies with increased platelet activity.

The precursor protein of VWF is composed of a 22-residue signal peptide and of a proVWF protein comprising 2791 amino acids that has 14 distinct domains. Upon translocation to the ER, the signal peptide is cleaved off, and the proVWF dimerizes in a tail-to-tail fashion through cysteines in its cysteine knot (CK) domain. During transit through the Golgi apparatus, proVWF dimers multimerize in a head-to-head fashion through the formation of disulfide bonds between cysteine residues in the D3 domain. At the same time, D1 and D2 domains are cleaved off as a single fragment to form the VWF propeptide (741 amino acids), while the remaining domains comprising 2050 amino acid residues and up to 22 carbohydrate chains form mature VWF. In the trans-Golgi network, the VWF propeptide promotes mature VWF to assemble into high-molecular-weight multimers. These multimers subsequently aggregate into tubular structures that are packaged into alpha-granules in megakaryocytes and into Weibel-Palade bodies in endothelial cells.

Upon exocytosis from Weibel-Palade bodies and at high shear rates, multimeric VWF unrolls from a globular to a filamental conformation, up to many microns long, which becomes a high-affinity surface for the platelet GPIb-V-IX complex. Large VWF multimers are more active than smaller multimers, which is explained by the fact that the former contain multiple domains that support the interactions between platelets, endothelial cells, and subendothelial collagen. VWF binds to matrix collagens via its A1 and A3 domains. The A1 domain also mediates binding to platelet GPIb, which is required for the fast capture of platelets. Platelet adhesion to VWF is further supported by VWF immobilization on surface and by high shear stress.

VWF complexes with factor VIII through the first 272 residues in the N-terminal region of the mature VWF protein subunit, thereby protecting factor VIII from proteolytic degradation, premature ligand binding, and rapid dlearance from the circulation.

Factor XI and The Contact System


Activates FIX with cofactor HK (calicum-dependent, phospholipid-independent)

Activated by FXIIa, thrombin, and FXIa

Inhibited by nexin 1, nexin 2, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin

Screen Shot 2016-06-11 at 2.57.05 PMFactor XI is synthesized in the liver and secreted as a single-chain zymogen of 607 amino acids. In the circulation, FXI is found as a homodimer at a concentration of 30 nM with a plasma half-life of 60 to 80 hours. All FXI homodimers circulate in complex with high-molecular-weight kininogen (HK). HK is thought to mediate binding of factor XI to negatively charged surfaces, thereby facilitating factor XI activation.

Each FXI subunit comprises four apple domians and a serine protease domain. The apple domains are structured by three disulfide bonds and form a disk-like platform on which the serine protease domain rests. The dimerization of two FXI subunits is mediated by interactions between the two apple 4 domains that involve a disulfide bond between the Cys321 residues, hydrophobic interactions, and a salt bridge, of which only the latter two are required for dimerization. The domain structure of FXI is highly similar to that of the monomer prekallikrein (PK), the zymogen of the protease kallikrein, which also circulates in complex with HK.

FXI does not bear a Gla domain and thus does not require gamma-carboxylation to exert its procoagulant activity.

Activation of a FXI subunit to FXIa proceeds through proteolysis at Arg369 in the N-terminal region of the serine protease domain and yields two-chain activated factor XIa. There are several catalysts capable of FXI activation, which include the contact factor XIIa, thrombin, or factor XIa itself in the presence of negatively charged surfaces. FXI must be a dimer to be activated by FXIIa, whereas thrombin and factor XIa lack this requirement.

Following activation of FXI, binding sites for the substrate FIX become available in the apple 3 domain and serine protease domian of factor XIa. FXIa proteolytically activates FIX to factor FIXa in a calcium-dependent but phospholipid-independent manner. Both forms of the FXIa dimer as well as monomeric FXIa activate FIX in a similar manner.

Accumulating evidence supports the notion that FXIIa-dependent activation of FXI is not essential to normal hemostasis, but is important in pathologic thrombus formation. Thrombin-mediated activation of FXI, on the other hand, seems most significant under conditions of low tissue factor and is assumed to enhance clot stability thorugh thrombin-activation of TAFI.

FXIa function is regulated by the serpins protease nexin 1, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin. Platelets also contain a FXIa inhibitor, the Kunitz-type inhibitor protease nexin 2.

The Contact System: FXII, Prekallikrein, and High-Molecular Weight Kininogen

FXII, HK, and PK are part of the contact system in blood coagulation, which is triggered following contact activation of FXII mediated via negatively charged surface.


Activates FXII

Activated by FXIIa

PK is synthesized in the liver, circulates as a zymogen, and is highly homologous to FXI. Conversion into the serine protease proceeds through limited proteolysis by FXIIa, and the generated kallikrein reciprocally activates more FXII.


HK, which is also synthesized in the liver, is a nonenzumatic cofactor that circulates in complex with FXI, which then activates FIX.

Contact system

The contact system is at the basis of the activated partial thromboplastin time (APTT) assay that is widely used in clinical practice. In this clinical laboratory test, the negatively charged surface is provided by reagents. FXIIa activates FXI, which then activates FIX. Despite HK and PK being required for a normal APTT, they appear to be dispensable for coagulation in vivo. Individuals who are deficient in any of these factors do not have a bleeding tendency, even after significant trauma or surgery.


Activates FXI, PK

Activated by negatively charged surface (platelet polyphosphate, microparticles derived from platelets and erythrocytes, RNA, and colalgen), kallikrein

Inhibited by serpin C1 inhibitor, antithrombin, and PAI-1

FXII, which is homologous to plasminogen activators, consists of an N-terminal fibronectin type I domain, an EGF-like domain, a fibronectin type II domain, a second EGF-like domain, a kringle domain, a proline-rich region, and a C-terminal serine protease domain. The proline-rich region is unique to FXII, as it is not found in any of the other serine proteases.

Limited proteolysis by kallikrein at Arg353 in FXII yields the activated two-chain alpha-FXIIa. Once activated, alpha-FXIIa activates FXI to FXIa. Furthermore, alpha-FXIIa activates PK, thereby contributing to its own feedback activation. Also FXI is known to acquire alpha-FXIIa activity upon contact with a negatively charged surface, the latter inducing a conformation change in FXII. This conformational change induces a limited amount of proteolytic activity in FXII, known as auto-activation. Furthermore, the surface-induced active conformation of FXII is suggested to enhance the proteolytic conversion to alpha-FXIIa. The fibronectin type I and II domains, EGF-2, the kringle domain, and the proline-rich region are reported to conribute to interaction with a negatively charged surface.

Further cleavage of alpha-FXIIa by kallikrein at Arg334 and Arg343 in the light chain results in the generation of beta-FXIIa, which comprises a nine-residue heavy-chain fragment that is disulfide-linked to the light chain. Given the absence of the heavy chain, beta-FXIIa does not interact with anionic surfaces. Even though beta-FXIIa is still capable of activating PK, it is no longer activates FXI.

The serpin C1 inhibitor is the main plasm inhibitor of alpha-FXIIa and beta-FXIIa. In addition, antithrombin (AT) and PAI-1 also inhibit FXIIa activity.

Revised Model of Coagulation

Screen Shot 2016-07-14 at 6.11.20 PM


Hemostasis is the process through which bleeding is controlled at a site of damaged vascular endothelium and represents a dynamic interplay between the subendothelium, endotheliumcirculating cells, and plasma proteins. The hemostatic process often is divided into three phases: the vascular, platelet, and plasma phases. Although it is helpful to divide coagulation into these phases for didactic purposes, in vivo, they are intimately linked and occur in a continuum.

The vascular phase is mediated by the release of locally active vasoactive agents that result in vasoconstriction at the site of injury and reduced blood flow. Vascular injury exposes the underlying subendothelium and procoagulant proteins, including von Willebrand factor (vWF), collagenn and tissue factor (TF) that then come into contact with blood.

During the platelet phase, platelets bind to vWF incorporated into the subendothelial matrix thorugh their expression of glycoprotein Iba (GPIbalpha). Platelets bound to vWF form a layer across the exposed subendothelium, a process termed platelet adhesion, and subsequently are activated via receptors, such as collagen receptors integrin alpha2beta1 and glycoprotein (GPVI), resulting in calcium mobilization, granule release, activation of the fibrinogen receptor, integrin alpha(IIb)b3, and subsequent platelet aggregation.

The plasma phase of coagulation can be further subdivided into initiation, priming, and propagation. Initiation begins when vascular injury also leads to exposure of TF in the subendothelium and on damaged endothelial cells. TF binds to the small amounts of circulating activated factor VII (FVIIa), resulting in formation of teh TF:FVIIa complex (extrinsic tenase complex); this complex binds to and activates factor X (FX) to activated FX (FXa). The TF:FVIIa:FXa complex converts a small amount of prothrombin (factor II/FII) to thrombin (activated factor II/FIIa). This small amount of thrombin is able to initiate coagulation and generate an amplification loop by cleaving factor VIII (FVIII) from vWF, activating clotting factors FVIII, XI (FXI), and platelets, which result in exposure of membrane phospholipids and release of partially activated factor V (FVa). At the end of the initiation and priming phases, the platelet is primed with an exposed phospholipid surface with bound activated cofactors (FVa and FVIIIa).

During the propagation phase, FIXa, generated either by the action of FXIa on the platelet surface or TF-VIIa complex on the TF bearing cell, bind to its cofactor, FVIIIa, to form the potent intrinsic tenase complex. FX is then bound and cleaved by the intrinsic tenase complex (FIXa:FVIIIa) leading to large amounts of FXa, which in association with its cofactor, FVa, forms the prothrombinase complex on the activated platelet surface. The prothrombinase complex (FXa:FVa) then binds and cleaves prothrombin leading to an ultimate burst of thrombin sufficient to convert fibrinogen to fibrin and to result in subsequent clot formation. The formed clot is stabilized by the thrombin-mediated activation of factor XIII (FXIII), which acts to cross-link fibrin, and thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to remove lysine residues from the fibrin clot, thereby limiting plasmin binding. Utimately, the clot undergoes fibrinolysis, resulting in the restoration of normal blood vessel architecture. The fibrinolytic process is initiated by the release of tissue plasminogen activator (tPA) near the site of injury. tPA converts plasminogen to plasmin, which (via interactions with lysine and arginine residues on fibrin) cleaves the fibrin into dissolvable fragments.


Hemostasis – Plasma Phase – Initiation

  • FX > FXa via TF:FVIIa complex
  • FII > FIIa (small amount) via TF:FVIIa:FXa complex

Hemostasis – Plasma Phase – Priming

  • FVIII:vWF complex > FVIII + vWF via FIIa (small amount)
  • FVIII > FVIIIa via FIIa (small amount)
  • FXI > FXIa via FIIa (small amount)
  • Platelet > activated platelet via FIIa (small amount)
  • Activated platelet > partially activated FV via release

Hemostasis – Plasma Phase – Propagation

  • FIX > FIXa via FXIa or TF:VII complex
  • FX > FXa (large amount) via FIXa:FVIIIa complex
  • FII > FIIa (large amount) via FXa:FVa complex
  • FXIII > FXIIIa via FIIa (large amount)

Regulation of Hemostasis

Both the hemostatic and fibrinoglytic processes are regulated by inhibitors that limit coagulation at the site of injury and quench the reactions, thereby preventing systemic activation and pathologic propagation of coagulation. The hemostatic system has three main inhibitory pathways: antihrombin (AT), the protein C:protein S complex, and tissue factor pathway inhibitor (TFPI).

AT/Antithrombin target at thrombin and FXa

AT (antithrombin) released at the margins of endothelial injury binds in a 1:1 complex with thrombin, inactivating thrombin not bound by the developing clot. Antithrombin also rapidly inactivates FXa; thus, any excess FXa generated by the TF:VIIa complex during initiation is inactivated and unable to migrate to the activated platelet surface.

Thrombomodulin, protein C and S, target at FVa and FVIIIa

Excess free thrombin at the clot margins binds to thrombomodulin, a receptor expressed on the surface of intact endothelial cells that when complexed with thrombin activates protein C; activated protein C complexes with its cofactor protein S and inactivates FVa and FVIIIa.

Tissue factor pathway inhibitor/TFPI, target at TF:FVIIa and FXa

TFPI is a protein produced by endothelial cells that inhibits the TF:FVIIa complex and FXa. Binding to FXa is required for the inhibitory effect on TF:FVIIa. This negative feedback results in reduced subsequent thrombin generation and quenching of fibrin generation. The action of both AT and TFPI inhibits FXa during the initiation phase leading to dependence of platelet-surface FXa generation during the propagation phase for adequate hemostasis.


The fibrinolytic system also includes two inhibitors, principally plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin (alpha2AP), which inhibit tPA and plasmin, respectively.

Thrombotic Thrombocytopenic Purpura and Its Management

December 18, 2012 Hematology, Therapeutics No comments , , ,

Thrombotic thrombocytopenic purpura (TTP) is the common name for adults with microangiopathic hemolytic anemia, thrombocytopenia, with or without neurologic or renal abnormalities, and without another etiology for thrombotic microangiopathy (TMA).

In 10 years before, we believed that the classic clinical features of TTP which was called “classic pentad” were thrombocytopenia, microangiopathic hemolytic anemia, neurologic abnormalities, renal function abnormalities, and fever. I gave these symptoms a abbreviation name that is “TMNRF”. However now the “classic pentad” is not considered to be the classic clinical features of TTP anymore. In a study of 58 TTP patients, only 5% (3) of TTP patient had the old “classic pentad” symptoms, in whom two were subsequently discovered to have systemic infections as the cause of their clinical features; one had a previously established diagnosis of systemic lupus erythematosus (SLE).

Now the characteristic of all TTP is TMA which is called thrombotic microangiopathy. TTP is the name used for adults with microangiopathic hemolytic anemia and thrombocytopenia, with or without renal failure or neurologic abnormalities, and without another cause for TMA. These were the inclusion criteria for the randomized clinical trial that documented the benefit of plasma exchange treatment, and they have become the definition and diagnostic criteria for TTP.

Before the plasma exchange era, survival of patients with TTP was only 10%. When plasma exchange was reported to increase survival to 78%, compared with 51% survival for patients treated with plasma infusion.

VWF-Cleaving Protease (ADAMTS13)

Platelets in flowing blood adhere transiently to exposed, immobilized VWF. Transient adhesion is followed by the engagement of other adhesive and signaling receptors, which causes platelet activation, immobilization, and spreading. This platelets surface can recruit more VWF and more platelets by the same mechanism. Under the influence of fluid shear stress, the VWF is recognized by ADAMTS13, which cleaves VWF multimers, releases the platelets, and limits the growth of the thrombus. Without ADAMTS13, this feedback inhibitory mechanism fails, and microvascular thrombi continue to grow, causing tissue ischemia and infarction.

However, measurements of ADAMTS13 activity are not required and may not be appropriate for the critical initial management decision to begin or not begin plasma exchange. But severe acquired ADAMTS13 deficiency dose define a subgroup of patients who appear to benefit from treatment with corticosteroids and other immunosuppressive agents in addition to plasma exchange but who have a high risk for relapse.

Management of TTP

For acute episodes of TTP, plasma replacement is essential;replacement with one plasma volume is appropriate;all plasma products (fresh-frozen plasma, 24-hour plasma, cryoprecipitate-poor plasma) appear to have equivalent efficacy. Plasma infusion can provide temporary benefit until plasma exchange can be begun.

Management of TTP

The assumption that plasma exchange may work by replacing ADAMTS13 and removing autoantibodies that inhibit its activity may not apply to all patients because response to plasma exchange may be the same in patients without a severe deficiency of ADAMTS13.

The complications of plasma exchange treatment are list in the table below, including central venous catheter-related sepsis, hemorrhage caused by catheter insertion, and cardiac arrest with pulseless electrical activity, which can cause fatal consequence.

For patients with severe ADAMTS13 deficiency, corticosteroids treatment may be valuable because their suppression effects on autoantibodies that inhibit ADAMTS13 activity. Patients who are unlikely to have severe ADAMTS13 deficiency are not treated with corticosteroids.

The response to plasma exchange, with or without corticosteroids, is judged by the platelet count. Once the platelet transfusions in patients with TTP is dangerous several decades ago. Now a systematic review of published case reports and case series did not document a risk from platelet transfusions. In most patients with severe thrombocytopenia and anemia, the platelet transfusion were given as part of common initial care. And no adverse events were identified.

After a remission occurs, patients need gradually fewer routine blood counts over several months;after this, they need only routine care from their primary physician. A platelet count is absolutely necessary when symptoms of any illness occur, to immediately diagnose a possible recurrence of TTP.

Many patients have persistent or intermittent ADAMTS13 deficiency after recovery. Among 41 patients who initially had severe ADAMTS13 deficiency and who have had one to 4 measurements of ADAMTS13 activity during remission, 7 (17%) have had ADAMTS13 activity less than 50% and 19 (46%) have had ADAMTS13 less than 50% at some time during their remissions;9 (22%) have had an ADAMTS13 inhibitor. However, severe ADAMTS13 deficiency during remission was not associated with clinical signs of TTP, and its clinical importance related to risk for relapse is uncertain.

Risk for relapse

The estimated risk for relapse is 41% at 7.5 years;relapses are most common during the first year after recovery. Relapse rarely occurs in patients without severe ADAMTS13 deficiency.