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Hemostasis - when there is damaged vessel o constricts (release of endothelin), and o platelets aggregate at the site of puncture (and attract more platelets) to seal the leak by a platelet plug or white clot. The time required for sealing (ca. 2 to 4 min) is called the bleeding time. o Subsequently, the coagulation system produces a fibrin meshwork. Due to covalent cross-linking of fibrin, it turns to a fibrin tangle or red thrombus that retracts afterwards, thus reinforcing the seal. o Later recanalization of the vessel can be achieved by fibrinolysis

Platelets half-life ≈10 days) When an endothelial injury occurs, - platelets adhere to subendothelial collagen fibers bridged by von Willebrand’s factor (vWF), which is formed by endothelial cells and circulates in the plasma complexed with factor VIII.

Weibel–Palade bodies are the storage granules of endothelial cells, the cells that form the inner lining of the blood vessels and heart. They store and release two principal molecules, von Willebrand factor and P-selectin (A receptor for leukoyctes)

- Glycoprotein complex GP Ib/IX on the platelets are vWF receptors. - This adhesion activates platelets. They begin to release substances, o some of which promote platelet adhesiveness (fibronectin, vWF, fibrinogen). o Others promote vasoconstriction like serotonin, platelet- derived growth factor (PDGF) and thromboxane A2 (TXA2).  platelet-derived growth factor stimulates proliferation of vascular smooth muscle cells and skin fibroblasts and plays a role in wound healing  TxA2 causes shape change and stimulates release reaction from alpha and dense granules. TxA2 also induces aggregation of other platelets and local vasoconstriction. o Mediators released by platelets enhance platelet activation and attract and activate more platelets: ADP, TXA2, platelet-activating factor (PAF).  ADP released from dense granules promotes platelet aggregation by inducing a configurational change in the membrane so that receptors for fibrinogen (GpIIb/IIIa) are exposed.  Platelet factor 4 released from alpha granules neutralises the anticoagulant activity of heparin - The shape of activated platelets change drastically. Discoid platelets become spherical and exhibit pseudopodia (formation of microvilli) that intertwine with those of other platelets. during which the glycoproteins IIb/IIIa (among others) are exposed on the platelet surface - Once a platelet changes its shape, GP IIb/IIIa is expressed on the platelet surface, leading to o fibrinogen binding that links the platelets together in a net-like structure = platelet aggregation. o GP IIb/IIIa also increases the adhesiveness of platelets, which makes it easier for them to stick to subendothelial fibronectin. - This platelet aggregation is further enhanced by ADP,TxA2 & thrombin (generated from activation of coagulation system) and stabilized by GP IIb/IIIa. vWF bind GP Ib = platelet adhesion

fibrinogen bind GP IIb/IIIa = plt aggregation

 Congenital absence of glycoprotein receptor GpIb (Bernard-Soulier syndrome”AR”)  Congenital absence of vWF (vWD)  result in defective platelets adhesion

 Congenital absence of glycoprotein receptor IIb/IIIa (Glanzmann thrombasthenia”AR”)  result in defective platelets aggregation

Bernard-Soulier Syndrome - rare autosomal recessive (AR) congenital bleeding disorder. - In this disease, adhesion of platelets to subendothelium is defective due to congenital absence of glycoprotein Ib receptor complex (which consists of GpIb, V and IX) on platelet surface. - This receptor is essential for binding of platelets to subendothelium via von Willebrand factor Platelet procoagulant activity: - When platelets are activated, negatively charged phospholipids (phosphotidylserine and phosphatidylinositol) located in the inner half of the lipid bilayer become exposed on the outer surface. These phospholipids play active role in coagulation by providing surface for interaction of some coagulation factors. - Platelets may play a role in the activation of F XII in the presence of ADP and kallikrein. Platelets also can directly activate F XI independent of F XII. This may explain the absence of bleeding diathesis in persons with F XII deficiency

- In addition platelets also secrete calcium, FV, fibrinogen, and FXII and contribute to the coagulation system Endothelial Prothrombotic-Antithrombotic Balancea Prothrombotic Antithrombotic Platelet-activating factor Prostacyclin Tissue factor Thrombomodulin von Willebrand factor Tissue plasminogen activator Plasminogen activator Urokinase Inhibitor-1 Heparin-like molecules Other coagulation factors - Synthesis of factor V - Binding of factors V, IXa, Xa - Activation factor XII * These various endothelial-associated factors and functions contribute to a dynamic physiological antagonism or “balance” that determines the status of local hemostatic/thrombotic activity coagulation factors - Coagulation proteins can be divided into following categories: o (1) Fibrinogen (F I); o (2) Serine proteases:  (a) Vitamin K-dependent Factors—II, VII, IX, X,  (b) Contact factors—XI, XII, high molecular weight kininogen, prekallikrein; o (3) Cofactors—V, VIII (do not develop enzymatic activity but become modified and are called as cofactors), tissue factor (F III); and o (4) Transglutaminase: F XIII.

- coagulation factors are involved in the clotting process. Except for Ca2+, they are proteins formed in the liver (I, IIK, V, VIIK, IXK, XK, XIII, kininogen). - Factors labeled with a “K” as well as protein C and protein S) are produced with vitamin K, an essential cofactor in post-translational γ-carboxylation of a number of glutamyl residues at the N-terminal of the peptide chains. These γcarboxyglutamyl groups are chelators of Ca2+. They are required for Ca2+mediated complex formation of factors on the surface of phospholipids layers (PL), particularly on the platelet membrane (platelet factor 3) “in other word Calcium in turn, is necessary for binding of these coagulation factors to phospholipid surface. Attachment of coagulation factors to phospholipid is essential for coagulation reactions to occur” - Vitamin K is oxidized in the reaction and has to be re-reduced by liver epoxide reductase (vitamin K recycling). Ca2+ ions are required for several steps in the clotting process. When added to blood samples in vitro, citrate, oxalate, and EDTA bind with Ca2+ ions, thereby preventing the blood from clotting. This effect is desirable when performing various blood tests. Activation of blood clotting. Most coagulation factors normally are not active, proenzyme or zymogenic. Their activation requires a cascade of events (An “a” added to the factor number means its converted to serine protease “activated”). Thus, even small amounts of a trigger factor lead to rapid blood clotting. The trigger can be endogenous (within a vessel) or exogenous (external). - Endogenous activation o occurs at an endothelial defect. o XII is activated to XIIa by the contact with negative charges of subendothelial collagen and sulfatide groups. o This stimulates the conversion of prekallikrein (PKK) to kallikrein (KK), which enhances XII activation (positive feedback). o Next, XIIa activates XI to XIa, which converts IX to IXa and, subsequently, VIII to VIIIa. o Complexes formed by conjugation of IXa and VIIIa with Ca2+ on phospholipid (PL) layers activate X. - Exogenous activation now merges with endogenous activation (!B1).

o In relatively large injuries, tissue thrombokinase (factor III), present on of nonvascular cells, is exposed to the blood, o resulting in activation of VII. VII forms complexes with Ca2+ and phospholipids, o thereby activating X (and IX). Fibrin formation (!B3). - After activation of X to Xa by endogenous and/or exogenous activation (the latter is faster), o Xa activates V and conjugates with Va and Ca2+ on the surface of membranes. This complex, called prothrombinase, o activates prothrombin (II) to thrombin (IIa). In the process, Ca2+ binds with phospholipids, and the N-terminal end of prothrombin splits off. The thrombin liberated in the process now activates  (a) fibrinogen (I) to fibrin (Ia) by cleavage of fibrinopeptide A and B  (b) fibrin-stabilizing factor (XIII), and  (c) V, VIII and XI (positive feedback).  (d) Thrombin is a powerful platelet agonist.  (e) Thrombin activates protein C, a natural anticoagulant

o The single (monomeric) fibrin threads spontaneously polymerize by forming end-to-end and side-to-side non-covalent bonds with each other. This is called as fibrin polymer (fibrinS; “s” for soluble meshwork). F XIIIa which is a transamidase (generated from F XIII by thrombin), in the presence of calcium, mediates the formation of covalent bonds between adjacent polypeptide chains so ultimately stabilizes soluble fibrin to insoluble fibrin (fibrini).

There is no haemorrhagic tendencies in patients of F XII, prekallikrein, or HMWK deficiency. In vivo, blood clotting seems to be initiated primarily by tissue factor

Clotting factors : Mnemonic : "Foolish People Try Climbing Long Slopes After Christmas Some People Have Fallen" No.


Factor I


Factor II Factor III

Prothrombin Tissue thromboplastin = tissue factor

½ life Note (h)


Deficiency AR, called Afibrinogenaemia (Rare) AR (Rare)

Vit-K dependent Cofactor composed of 2 parts: protein & phospholipid it activate 1- in addition to F VII of extrinsic pathway, 2- it has also been shown to activate F IX to F IXa in intrinsic pathway There is no haemorrhagic tendencies in patients of F XII, prekallikrein, or HMWK deficiency. In vivo, blood clotting seems to be initiated primarily by tissue factor Factor IV Ionized calcium (Calcium 2+) Factor V Labile factor = Ac globulin = 20 cofactor AR called Proaccelerin Parahaemophilia (Rare)

Factor VI

Factor VI Does not exist as it was named initially but later on discovered not to play a part in blood coagulation Factor VII Stable factor = serum prothrombin conversion accelerator (SPCA) = Proconvertin Factor AntiHemophilic Factor A (AHF) VIII AntiHemophilic Globulin (AHG)

Factor IX

Factor X Factor XI


Vit-K dependent


Cofactor its synthesis is Xlinked activated by thrombin deactivated by Further cleavage by thrombin & activated ptn C Vit-K dependent

Antihemophilic factor B = plasma 24 thromboplastin component (PTC) or Christmas factor (named after the first patient in whom the factor deficiency was documented) Stuart Prower factor Antihemophilic factor C = Plasma thromboplastin antecedent (PTA)

Factor XII Hageman factor

AR Incidence: 1:500,000 XR Called Classical haemophilia, haemophilia A Incidence: 1:10,000 XR Haemophilia B, Christmas disease

30 48

Vit-K dependent Contact factor


Contact factor plays a role in

Incidence: 1:60,000 AR (Rare) AR Called: Haemophilia C (Rare)

Factor XIII Fletcher factor

Fibrin stabilizing factor = transamidase 250 = transglutaminase. Prekallikrein (PKK)

Fitzgerald High-molecular-weight (HMW) factor kininogen; vWF = Factor VIII–related antigen

1-contact activation of coag system, 2- inflammatory response, 3- complement system, 4- fibrinolysis, 5formation of kallikrein & kinin Synthesized by AR (Rare) megakaryocytes Contact factor Kallikrein plays a role in 1- chemotaxis 2- in activation of fibrinolysis. 3- converts HMW kininogen to bradykinin, a chemical mediator of inflammation. Contact factor Synthesized by megakaryocytes

AR or AD

(15%) and endothelial cell is autosomal in inheritance Fibronectin Heparin cofactor II Fibrinolytic system Plasminogen, plasmin tPA plasma kallikrein FDPs Fibrinolytic Inhibitory factors Îą2-macroglobulin Îą1-antitrypsin PAI-1, PAI-2. thrombin-activated fibrinolytic inhibitor (TAFI) Coagulation inhibitory factors (anticoagulant) antithrombin III protein CK, and protein SK

Called von Willebrand disease Incidence : 1:100

The probable cause of a more serious HD can be elucidated with a few simple clotting tests - Quick test (PT) o plasma is transiently made incoagulable with substances that form complexes with Ca2+ (citrate, oxalate, or EDTA); an excessive amount of Ca2+ and tissue thrombokinase (Thromboplastin) are then added to test extrinsic system o normal value is 50% (normal range: 70– 125% = 12–13 seconds). International Normalized Ratio (INR) normal range = 0.9–1.2] o used to test for abnormalities in factors VIIK “exogenous system”, XK, I (fibrinogen), IIK (prothrombin), V “common pathway”. o Low/prolonged Quick values by warfarin, vitamin K deficiency, liver disease, DIC. - partial thromboplastin time (PTT), o kephalin, kaolin (substitute for contact activation), and Ca2+ are added to the citrated plasma o normal value: 25–38 s. o If it is increased, the disorder lies  either in the endogenous activation  or in the common final pathway from factor X onward Warfarin exo monitored by PT = INR Heparin endo monitored by PTT or TT mnemonic : Waiting OUTside so PiTy - (plasma) thrombin time: o thrombin is added to the citrated plasma and the clotting time determined o normal values: 18–22 s o the result can reveal fibrinogen deficiency o or can be used to monitor treatment with heparin, which enhances the inhibitory action of antithrombin III on thrombin - bleeding time o normal value 2 to 4 min

o prolonged due to Any abnormality in the platelets (bleeding > 5 min),

Fibrinolysis and Thromboprotection - To prevent excessive clotting and occlusion of major blood vessels (thrombosis) and embolisms due to clot migration, ďƒ fibrinS is re-dissolved (fibrinolysis) and inhibitory factors are activated as soon as vessel repair is initiated. - Fibrinolysis is mediated by plasmin o Plasminogen is converted to plasmin by plasminogen activators. This reaction occurs on the surface of fibrin.

Oxford : The fibrinolytic system : The process starts with the release of t-PA from endothelial cells, a process stimulated by fibrin formation >> t-PA converts inactive plasminogen to plasmin which can then cleave fibrin >> t-PA and plasminogen both bind fi brin thus localizing fibrinolysis to the area of the clot. o Plasmin can cause cleavage of both fibrinogen as well as fibrin.  Plasmin initially attacks α chains of the fibrinogen molecule to release fibrinogen degradation products (liberating two fragments D and )  Plasmin digests insoluble or cross-linked fibrin to release fibrin degradation products or FDPs (the characteristic fragments are oligomers of X and Y, D-dimer, D2E complex, and Y-D complex)  FDP are then cleared from the circulation by macrophages of the mononuclear phagocytic system  However, when FDPs increase they have a potent anticoagulant action in the form of inhibition of polymerisation of fibrin, antithrombin activity, and impairment of platelet function. o Various activating factors in blood  plasma kallikrein formed from prekallikrein by the action of F XIIa   tissues (tissue plasminogen activator, tPA, endothelial etc.)  tPA synthesized by endothelial cells and is the most important physiological plasminogen activator  urine (urokinase) activate plasminogen to plasmin.  Streptokinase, staphylokinase and tPA are used therapeutically to activate plasminogen. This is useful for dissolving a fresh thrombus located, e.g., in a coronary artery. o Fibrin is split into fibrinopeptides which inhibit thrombin formation and polymerization of fibrin to prevent further clot formation. - inhibitors of fibrinolysis o (1) Alpha 2-antiplasmin is an endogenous inhibitor of fibrinolysis  it combines rapidly with plasmin in circulation to form plasmin-antiplasmin complex o (2) α2-macroglobulin which inhibits plasmin; o (3) plasminogen activator inhibitors PAI-1 and PAI-2 released from endothelial cells which neutralize tPA and

o (4) thrombin-activated fibrinolytic inhibitor (TAFI) which cleaves specific fibrin lysine residues, thus removing binding sites for plasminogen and tPA. o Tranexamic acid is administered therapeutically for the same purpose.

Primary fibrinolysis is associated with conditions in which gross activation of the fi brinolytic mechanism with subsequent fibrinogen and coagulation factor consumption occurs. The important characteristic of primary fibrinolysis

is that no evidence of fibrin deposition occurs. Primary fibrinolysis occurs when large amounts of plasminogen activator enter the circulatory system as a result of trauma, surgery, or malignancies. Although the same clinical conditions may also induce secondary fibrinolysis or DIC, the distinction between the two is essentially in the demonstration of fibrin formation. In secondary fibrinolysis, excessive clotting and fibrinolytic activity occur. Increased amounts of fibrin split (degradation) products (FSPs) and fibrin monomers are detectable because of the action of thrombin on the fibrinogen molecule. This fibrinolytic process is only caused by excessive clotting; therefore, it is a secondary condition. Distinguishing between primary and secondary fi brinolysis (Table 24.14) is important in treatment.

Thromboprotection/anticoagulation - 1- Antithrombin III, a serpin, is the most important thromboprotective plasma protein. o It inactivates the protease activity of thrombin and factors IXa, Xa, XIa and XIIa by forming complexes with them. o Antithrombin III is enhanced/activated by heparin and heparin-like endothelial glucosaminoglycans which present on the luminal surface of blood vessels. Heparin is produced naturally by mast cells and granulocytes, and synthetic heparin is injected for therapeutic purposes. o The importance of AT as a natural anticoagulant derives from the fact that AT deficiency is associated with increased risk of thrombosis - 2- Protein C o The binding of thrombin with endothelial thrombomodulin provides further thromboprotection. Only in this form does thrombin have

o o o o o

anticoagulant effects (negative feedback)  Thrombomodulin activates protein C to Ca Protein Ca, after binding to protein S, deactivates coagulation factors Va and VIIIa. The synthesis of proteins C and S is vitamin K-dependent and occur in the liver. Protein C also appears to enhance fibrinolysis. Deficiency of protein C or S is associated with risk of thrombosis An inhibitor of protein C is present in plasma; it is thought that deficiency of this inhibitor accounts for cases of combined deficiency of F V and FVIII.

- Other plasma proteins that inhibit thrombin are α2-macroglobulin and α1antitrypsin  by enhancing fibrinolysis system through plasmin & directly inh thrombin - Endothelial cells secrete o 3- tissue thromboplastin inhibitor, a substance that inhibits exogenous activation of coagulation  Tissue factor pathway inhibitor (TFPI) binds to FXa, and FXa-TFPI complex then attaches to tissue factor-VII complex to neutralize it “mainly inactivate FVII”

o prostacyclin (= prostaglandin I2), which inhibits platelet adhesion to the normal endothelium.

Anticoagulants - are administered for thromboprotection in patients at risk of blood clotting. - Injected heparin has immediate action. - Oral coumarin derivatives (phenprocoumon, warfarin, acenocoumarol) are vitamin K antagonists that work by inhibiting liver epoxide reductase, which is necessary for vitamin K recycling. ďƒ Therefore, these drugs do not take effect until the serum concentration of vitamin K-dependent coagulation factors has decreased. Cyclooxygenase inhibitors, - such as aspirin (acetylsalicylic acid), inhibit platelet aggregation by blocking thromboxane A2 (TXA2) synthesis

Hemorrhagic diatheses - can have the following causes: o Congenital deficiency of certain coagulation factors. Lack of VIII or IX, for example, leads to hemophilia A or B, respectively. o Acquired deficiency of coagulation factors. The main causes are liver damage as well as vitamin K deficiency due to the destruction of vitamin K-producing intestinal flora or intestinal malabsorption. o Increased consumption of coagulation factors, by disseminated intravascular coagulation. o Platelet deficiency thrombocytopenia) or platelet defect (thrombocytopathy). o Certain vascular diseases, and o excessive fibrinolysis. The hereditary coagulopathies - can affect practically each of the plasma factors, - but deficiency of some of the factors may produce relatively few symptoms (e.g., factors of the contact phase/endo pathway “Factor XII &KK”, factor XI). - Hemophilia o hemophilia A [deficiency of factor VIII]  The most common (one of 10 000 newborn boys)  inherited as X linked recessive  characterized by: decreased formation of thromboplastin, and diminished conversion of prothrombin  The most common bleeding sites are the muscles and the large joints of the leg, the latter becoming markedly deformed with time (hemophilic arthropathy). o hemophilia B [factor IX deficiency]  fivefold rarer than hemophilia A - The other rare disorders : homozygous hereditary deficiency of o factor I (afibrinogenemia) o of factor II (hypoprothrombinaemia) o of factors V, VII, and X leads especially to marked bleeding after severe injury or operations.

o Homozygous deficiency of α2-antiplasmin, an important inhibitor of fibrinolysis also results in a hemophilia-like bleeding tendency. o Factor XIII deficiency is characterized by fibrin instability so that bleedings occur only after a long interval (up to 11⁄2 days). The routine clotting tests are usually normal in factor XIII deficiency, because actual clotting is unchanged. - Acquired coagulopathies o Occur when formation of the various factors is reduced, when they are inhibited e.g., by  dministration of heparin  or by immune coagulopathies, e.g., factor VIII antibodies,  or if their consumption is high (consumption coagulopathy).  liver diseases through the following processes • As most of the clotting factors are formed in the liver, liver damage (in particular liver cirrhosis; results in clotting disorders. • Simultaneously occurring portal hypertension further increases the risk of hemorrhages (mainly from esophageal varices; • because platelets are sequestered in the enlarged spleen, resulting in thrombocytopenia  As several clotting factors are vitamin K–dependent a coagulopathy can also be caused by deficiency or inhibition of vitamin K Causes of vitamin K deficiency are: • obstructive jaundice, in which fat-soluble vitamins (e.g., vitamin K1 from green plants or synthetic vitamin K3) fail to be absorbed due to the lack of bile salts • generalized malabsorption • destruction by antibiotics of the intestinal flora, which through its synthesizing vitamin K2 contributes significantly to supplying the body with this substance. • The inhibition of the vitamin K effect by coumarin derivatives (phenprocoumon, warfarin, acenocumarol) is used for oral prophylaxis of thrombosis --------------DIC

- is a coagulation disorder caused by acute or chronic activation of thrombin with clot formation and platelet activation that secondarily results in hyperfibrinolysis. - It is caused by o large amounts of tissue thromboplastin entering the bloodstream, for example,  in amniotic fluid embolism,  extensive brain injury,  malignant disease (e.g., leukemia),  or sepsis (e.g., petechiae in meningococcal septicemia [Waterhouse– Friedrichsen syndrome]). o Vascular causes are seen, for example,  in aortic aneurysm  or in vascular malformations  as well as in ABO blood group mismatches,  and due to enzyme action with certain snake poisons.

hemorrhagic diathesis (HD) caused by platelet abnormalities are There are two groups of Platelets abnormalities - thrombocytopenias and - thrombocytopathies. Acquired thrombocytopenias (TCP) - are the most common HD. - TCP is due to o diminished platelet formation (aplastic TCP, e.g., in bone marrow tumors, radiation damage, or cobalamine or folate deficiency), o to increased platelet destruction (thrombocytoclastic TCP), o or platelet sequestration in an enlarged spleen. - Markedly increased bleeding tendency occurs when o the number of platelets falls below 20 × 103/μL. Idiopathic TCP (Werlhof’s disease) is relatively frequent, its acute form developing one to three weeks

after a viral infection (shortened platelet survival time due to immune complexes). The chronic form occurs as an autoimmune disease. - Drug allergy can produce TCP through the action of drugs (e.g., quinine or sulfonamides) as haptens A molecule that is incapable, alone, of causing the production of antibodies but can, however, combine with a larger antigenic molecule called a carrier. A hapten-carrier complex can stimulate production of antibodies, Acquired thrombocytopathies: occur in - uremia - and dysproteinemia An abnormality in plasma proteins, usually in immunoglobulins (platelet coating). - They can also be caused by such drugs as acetylsalicylic acid via their inhibitory effect on cyclo-oxygenase, an effect that is used in thrombosis prophylaxis. Congenital thrombocytic HDs : are the autosomal-dominant and autosomalrecessive hereditary thrombocytopenias (abnormal platelet production) with the following functional disorders: - Membrane defects such as o 1) deficiency of platelet glycoprotein Ib (!F1) that disturb adhesion (Bernard–Soulier syndrome); o 2) deficiency of glycoprotein complex IIa/IIIb (!F2), which inhibits aggregation and adhesion (Glanzmann–Naegeli thrombasthenia); o Diverse defects of storage or secretion, for example, deficiency of cyclooxygenase and thromboxane synthetase, in which ADP release is reduced (storage pool deficiency); (!F3). - Among the forms of HD of vascular cause are o the different kinds of hereditary von Willebrand’s (vW) disease, a defect of vascular endothelium in which the vW factor is reduced or defective (!F4). This weakens platelet adhesion and secondarily leads to factor VIII deficiency, because the vW factor acts as a kind of carrier for this factor (complex formation). - Finally, there are a number of functional disorders and tissue changes in the vascular wall and connective tissue that are

o either congenital (purpura simplex; Osler–Weber–Rendu disease; Schönlein–Henoch disease), o or acquired (scurvy in vitamin C deficiency; drug-mediated immune reactions).


hematology - hemostasis Data taken from color atlas (Thieme) + from other sources