Regulatory Strategies PDF

Regulatory Strategies  Four major Regulatory Strategies 1. Allosteric control: feedback inhibition 同位酵素 2. Multiple forms of enzymes (isozymes or isoenzymes) ─ provide an avenue for varying regulation of the same reaction at distinct loc...

Regulatory Strategies  Four major Regulatory Strategies 1. Allosteric control: feedback inhibition 同位酵素 2. Multiple forms of enzymes (isozymes or isoenzymes) ─ provide an avenue for varying regulation of the same reaction at distinct locations or times ─ located in a distinct tissue or organelles or at as distinct stage of development. 3. Reversible covalent modification: ─ Phosphoryation and dephosphorylation (PKAs vs. protein phosphatases) ─ Acetylation and deacetylation of histone proteins 4. Proteolytic activation: zymogens (blood coagulation and apoptosis)  Allosteric control = feedback inhibition E1 E2 E3 A B C D Feedback inhibition A, B, C, and D = substrates and/or product E1, E2, and E3 = Enzymes involved in each step  An allosteric enzyme, Aspartate transcarbamolyase (ATCase) ATCase CTP is final product! The enzyme activity of ATCase is inhibited by CTP ATCase Carbamoyl phosphate + Aspartate N-carbamoylaspartate Feedback inhibition CTP ATCase is composed of regulatory (r) and catalytic (c) subunits 3r2 + 2c3 → r6c6  Modification of cysteine residues of ATCase  p-hydroxymercuribenzoate 1. reacts with crucial cysteine residues in ATCase and 2. separates regulatory (r) and catalytic (c) subunits of the enzyme p-hydroxy- mercuribenzoate r6c6 3r2 + 2c3  The regulatory (r) and the catalytic (c) subunits of ATCase can be obtained by zonal centrifugation after treating the enzyme with p-hydroxymercuribenzoate Native enzyme p-hydroxymercuribenzoate- R2 dimer C3 treated enzyme trimer R6c6 (= 3 r2 + 2 c3 )  PALA is a specific inhibitor for ATCase! Carbamoyl phosphate Asp The active site of ATCase PALA The active site of ATCase. Some of the crucial active-site residues are shown binding to the inhibitor PALA (shaded gray). Notice that the active site is composed mainly of residues from one c chain, but an adjacent c chain also contributes important residues (boxed in green). The T-to-R state transition in ATCase PALA Tense state Relaxed state (T state) (R state) CTP stabilizes the T state by binding to regulatory subunits The T state and the R state are in equilibrium between the T state and the R state.  In the absence of substrate, almost all the enzyme molecules are in the T state because the T state is more stable than the R state. The ratio of the concentration of enzyme in the T state to that in the R state is called the allosteric constant (L). For most allosteric enzymes, L is on the order of 102 to 103. T L (allosteric constant) = R The T state has a low affinity for substrate and hence shows a low catalytic activity. The occasional binding of a substrate molecule to one active site in an enzyme increases the likelihood that the entire enzyme shifts to the R state with its higher binding affinity.  ATCase displays sigmodial kinetics ATCase is a typical allosteric enzyme! ATCase Carbamoyl phosphate + Aspartate N-carbamoyl-aspartate Basis of the sigmodial curve  CTP is an allosteric negative effector for ATCase  ATP is an allosteric positive effector for ATCase Comparison of the sequential model and the concerted model T T T T R T R R R R R R Binding affinity Sequential model Concerted model to substrate The binding of substrates to ATCase leads to a highly concerted allosteric transition  fR = R state에 있는 catalytic chain의 양(비율)  Y = 기질(S)과 결합한 catalytic chain의 양(비율) ▐ fR : the fraction of catalytic chains in the R state ▐ Y: the fraction of catalytic chains containing bound substrate ─ fR value can be determined by analyzing the sedimentation coefficient on the basis of the fact that T to R state transition leads to the change of the frictional coefficient of the enzyme on the sucrose gradient sedimentation ─ Y value is determined from the change in absorbance at 280 nm   fR = R state에 있는 catalytic chain의 양(비율)  Y = 기질(S)과 결합한 catalytic chain의 양(비율) 1. If sequential model is correct, ─ fR will be equal to the Y 2. If concerted model is right, ─ fR will increase more rapidly than Y as the substrate concentration is increased. Conclusion: The change in fR leads the change in Y on addition of substrate, as predicted by the concerted model.  ATCase is reacted with nitromethane to form a colored nitrotyrosine group (λmax = 430) The C3 trimer of ATCase is treated with nitromethane to label tyrosine residue.  This tyrosine-labeled C3 trimer cannot bind to substrate because of modified lysin residue during the labeling with nitromethane.  The labeled C3 trimer (Note that this is absolutely T state) in all subunits will be colored that can absorb a wavelength of 430 nm when it is changed from T state to R state!!  Key points: 1. Nitromethane에 의해 tyrosine이 표지된 C3 trimer는 lysine이 변형되어 기질과 결합하지 못함! 2. 이 표지된 C3 trimer는T state에 있음 (※ 430 nm의 빛을 흡수하지 못함) 3. 그러나 R state로 변환되면 430 nm의 빛을 흡수하는 특성을 지니게 됨  T state T state Nitromethane 처리 C3 trimer Labeled C3 기질과 결합: X trimer T state Nitrotyrosine & Modified lysine 430 nm 빛 흡수: X T state R state Labeled C3 T to R Labeled C3 기질과 결합: X trimer trimer R state로 변환 Nitrotyrosine & Nitrotyrosine & Modified lysine 430 nm 빛 흡수: O Modified lysine  Reconstituted ATCase can be made with the unlabelled (native) C3 trimers, labelled C3 trimers, and regulatory subunits Active C3 trimer that can bind to substrate, but in T state 430 nm 결론! r subunits +S C3 trimer labeled with nitromethane that cannot bind to substrate and also in T Concerted model is correct ! state. However, it will absorb 430 nm light when changed to R state!!  Reconstituted ATCase (native C3 trimers, labelled C3 trimers, and regulatory subunits) shows that the concerted model is correct! T R Reconstituted ATCase +S 430 nm r subunits 빛 흡수 T R The binding of ATP and CTP to the regulatory subunits of ATCase leads to global conformational changes Labeled trimer ATP or CTP R subunits Notice!  ATP and CTP can bind to only Labeled Reconstituted regulatory subunits trimer ATCase 결론! CTP ATP  Quantitative description of the MWC model  Concerted model [MWC model; 협주(協奏) 모델): In 1965, Monod, Wyman, and Changeux were proposed. ▬ Y: the fraction of active sites bound to substrate (→ directly proportional to reaction velocity) ▬ α: the ratio of [S] to the dissociation constant of S with the enzyme in the R state ▬ L: the ratio of the concentration of enzyme in the T state to that in the R state [T] L= [R] Covalent modification of protein (→ Post translational modification, PTM) Phosphorylation of protein Serine/threonine kinases & Tyrosine kinases  Dephosphorylation of protein  이 protein phosphatase(단백질 탈인산화 효소)들은.. Ptorein kinase(단백질 인산화효소)들에 의해 활성화된 세포 내 신호전달 경로(signaling pathway)들을 불활성화(turn-off)시키는 등, 매우 중요한 역할을 수행함. ex., PP2A phosphatase: proteon kinase들에 의한 암-촉진 활성 (cancer-promoting activity)을 억제  It is important noted that.. 1. 단백질의 인산화와 탈인산화 반응은 서로 가역적(reversible)이지 않다. 다시 말하면, " 인산화와 탈인산화는 생리조건에서 본질적으로 비가역적(essentially irreversible)이다". 2. 왜냐하면, 단백질 인산화는 오로지 특정 단백질 인산화효소(protein kinase)에 의해서만 일어나며, 탈인산화도 특정 단백질 탈인산화효소(protein phosphatase)에 의해만 일어나기 때문이다. 3. 따라서, 인산화와 탈인산화 반응은 한 방향 (unidirectional)으로만 일어나며, 인산화/탈인산화 주기율(the cycling rate of phosphorylation and dephosphorylation)은 kinase와 phosphatase의 상대적 활성(relative activity)에 의해 결정된다!  "Protein is irreversibly and unidirectionally phosphorylated and dephosphorylated by protein kinase and phosphatase, respectively" Dephosphorylated protein Protein kinase + ATP Protein phosphatase + H2O Phosphorylated + protein HOPO32- [Orthophosphate (Pi)]  Phosphorylation is a highly effective means of controlling the activity of proteins for structural, thermodynamic, kinetic, and regulatory reasons! 1. A phosphoryl group adds two negative charges to a modified protein, leading to change of electrostatic interactions and structure of the modified protein that result in altering substrate binding and catalytic activity. 2. A phosphoryl group can form three or more hydrogen bonds (→ tetrahydedral geometry) 3. The free energy of phosphorylation is large: Of the -50 kJ/mol provided by ATP, (1) about half is consumed in making phosphorylation irreversible; (2) the other half is conserved in the phosphorylated protein. 4. Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours. 5. Phosphorylation often evokes highly amplified effects. 6. ATP is the cellular energy currency.  Protein kinases vary in their degree of specificity 1. Dedicated protein kinases ─ phosphorylate a single protein or several related proteins 2. Multifunctional protein kinases ─ modify many different targets Multifunctional protein kinases phosphorylate the target amino acids in consensus sequences! 공유(공통)서 Arg-Arg-X-Ser-Z Arg-Arg-X-Thr-Z X, small residues Z, large hydrophobic residues ※ Lysine can substitute for one of the Arg residues but with some loss of affinity Short synthetic peptides containing a consensus motif are nearly always phosphorylated by serine-threonine protein kinases!  Thus, the primary determinant of specificity is the amino acid sequence surrounding the serine or threonine phosphorylation site! cAMP activates protein kinase A (= PKA) by altering the quaternary structure Protein kinases modulate the activity of many proteins, but what leads to the activation of a kinase? ─ Activation is often a multistep process initiated by hormones.  In some cases, the hormones trigger the formation of cyclic AMP (cAMP), a molecule formed by cyclization of ATP. → Hormone acts as a primary messenger cAMP serves as an intracellular messenger in mediating the physiological actions of hormones. → cAMPis second messenger Signal transduction pathway by a hormone Hormone (☞ Primary messenger) Activation of Receptor G protein activation Activation of adenylate cyclase Formation of cAMP (☞ Second messenger) Activation of PKA Phosphorylation of a protein  Primary messenger Gα-GTP Second G protein messenger G protein-coupled receptor (GPCR) (inactive) (active)  The striking finding is that most effects of cAMP in eukaryotic cells are achieved through the activation of a single protein kinase. ─ This key enzyme is called protein kinase A or PKA. ─ The kinase alters the activities of target proteins by phosphorylating specific serine or threonine residues. As we shall see, PKA provides a clear example of the integration allosteric regulation and phosphorylation PKA is activated by cAMP concentrations of the order of 10 nM! The activation mechanism is reminiscent of that of aspartate transcarbamoylase. PKA in muscle consists of two kinds of subunits: ─ a 49-kd regulatory (R) subunit, which has high affinity for cAMP, and ─ a 38-kd catalytic (C) subunit. In the absence of cAMP, the regulatory and catalytic subunits form an R2C2 complex that is enzymatically inactive.  Activation of PKA by cAMP 1. The binding of two molecules of cAMP to each of the regulatory subunits leads to the dissociation of R2C2 into an R2 subunit and two C subunits. 2. These free catalytic subunits are then enzymatically active. ※PKA and most other kinases exist in isozymic forms for fine-tuning regulation to meet the needs of a specific cell or developmental stage.  Activation of PKA by cAMP R2C2 complex Catalytically active (Catalytically inactive) C subunits Thus, the binding of cAMP to the regulatory subunit relieves its inhibition of the catalytic subunit. How does the binding of cAMP activate the kinase? 1. Each R chain contains the sequence Arg-Arg-Gly- Ala-lle (☞ pseudosubstrate sequence), which matches the consensus sequence for phosphorylation except for the presence of alanine in place of serine.  Pseudosubstrate sequence in R chain : Arg-Arg-Gly-Ala-Ile  Consensus sequences for PKA : Arg-Arg- X - Ser-Z Arg-Arg- X - Thr-Z 2. In the R2C2 complex, this pseudoaubstrate sequence of R occupies the catalytic site of C chain, thereby preventing the entry of protein substrates. 3. The binding of cAMP to the R chains allosterically moves the pseudosubstrate sequences out of the catalytic sites. 4. The released C chains are then free to bind and phosphorylate substrate proteins.  ATP and target protein bind to a deep cleft in the catalytic subunit of PKA X-ray crystallography was performed with 20-residue peptide inhibitor containing a pseudosubstate sequence (Arg-Arg-Asn-Ala-ile) The 350-residue catalytic subunit has two lobes (See the red dashed lines on the figure!) ATP and part of the inhibitor fill a deep cleft (substrate) between the lobes Binding of pseudosubstrate to protein kinase A Pseudosubstrate 1. The two arginine side chains of the pseudosubstrate form salt bridges with three glutamate 2. The isoleucine residue of the carboxylates. pseudosubstrate is in contact with a pair of leucine residues of the enzyme. 3. Hydrophobic interactions are also important in the recognition of substrate. Many enzymes are activated by specific proteolytic cleavage 1. The digestive enzymes are synthesized as zymogens in the stomach and pancreas * Zymogen = Proenzyme: 치모겐; 효소원(原) Secretion of zymogens by an acinar cell of the pancreas Acinar cell: 선포세포(腺胞細胞); 샘꽈리세포 2. Blood clotting is mediated by a cascade of proteolytic activation that ensures a rapid and amplified response to trauma 3. Some protein hormones are synthesized as inactive precursors. ex., insulin 4. The fibrous protein collagen, the major constituent of skin and bone, is derived from procollagen, a soluble precursor. 5. Many developmental processes are controlled by the activation of zymogens. ex., collagen absorption during the metamorphosis of tadpole and collagen breakdown in a mammalian uterus after delivery by the activated collagenase from procollagenase. 6. Programmed cell death, or apoptosis, is mediated by proteolytic enzymes called caspases, which are synthesized in precursor forms as procaspases. Chymotrypsinogen is activated by specific cleavage of a single peptide bond ile (14-15; 147-148) Proteolytic activation of chymotrypsinogen. The three chains of α-chymotrypsin are linked by two interchain disulfide bonds (A to B, and B to C). The approximate positions of disulfide bonds are shown.  Two dipeptides released by chymotrypsin: Ser 14–Arg 15 & Thr 147–Asp148 1. The newly formed amino-terminal group of isoleucine 16 turns inward and forms an ionic bond with aspartate 194 in the interior of the chymotrypsin molecule. The electrostatic interaction between the α- amino group of isoleucine 16 and the carboxylate of aspartate 194  Conformations of chymotrypsinogen and chymotrypsin Electrostatic interaction 16 1. Trypsin cleaves the 194 peptide bond between Arg 15 and ile 16 of chymotrypsinogen 2. Chymotrypsin makes two dipeptides (Ser 14–Arg 15 & Thr 147–Asp148) The electrostatic interaction between the α-amino group of isoleucine 16 and the carboxylate of aspartate 194, essential for the structure of active chymotrypsin, is possible only after cleavage of the peptide bond between isoleucine and arginine in chymotrypsinogen. 2. This electrostatic interaction triggers a number of conformational changes. ─ Methionine 192 moves from a deeply buried position in the zymogen to the surface of the active enzyme, and residues 187 and 193 become more extended. ─ These changes result in the formation of the substrate specificity site for aromatic and bulky nonpolar groups.  This cavity for binding part of the substrate is not fully formed in the zymogen! 3. The tetrahedral transition state in catalysis by chymotrypsin is stabilized by hydrogen bonds between the negatively charged carbonyl oxygen atom of the substrate and two NH groups of the main chain of the enzyme.  One of these NH groups is not appropriately located in chymotrypsinogen, and so the oxyanion hole is incomplete in the zymogen.  "Thus, the switching on of enzymatic activity in a protein can be accomplished by discrete, highly localized conformational changes that are triggered by the hydrolysis of a single peptide bond." Trypsin as the common activator of all the pancreatic zymogens secreted by duodenum cleaves K-I bond Duodenum: 십이지장 /djùːədíːnəm/ Interaction of trypsin with its pancreatic trypsin inhibitor  Lysine 15 of the inhibitor penetrates into the active site of the trypsin and forms a salt bridge with aspartate 189 in the active site  , Another important protease inhibitor, α1-antitrypsin (also called α1-antiproteinase) 53-kd plasma protein protects tissue from digestion by elastase (secreted by WBC) More tightly inhibits elastase than trypsin, so a more accurate name will be antielastase. Genetic disease: emphysema (肺氣腫) / / Smoking markedly can increase the rate of development of emphysema  The reason is that smoke oxidizes methionine 358 of the α1-antitrypsin, a residue essential for binding elastase. sulfoxide Met358 Blood coagulation cascade [Fibrinogen and fibrin] Fibrinogen is a 340 kDa glycoprotein consisted of six chains: ─ Two each of Aα, Bβ, and γ ─ The rode regions are triple-stranded α-helical coiled coils Thrombin cleaves four Arg-Gly bonds in the central globular region of fibrinogen  On thrombin cleavage, an A peptide of 18 residues is released from each of the two Aα chains, as is a B peptide of 20 residues from each of the two Bβ chains. ─ These A and B peptides are called fibrinopeptides. A fibrinogen molecule devoid of these fibrinopeptides is called a fibrin monomer and has the subunit structure (αβγ)2. Fibrin monomers spontaneously assemble into ordered fibrous arrays called fibrin. Structure of a fibrinogen molecule Thrombin B peptide (20 a.a) A peptide (18 a.a) Fibrinopeptides = A peptide + B peptide Thrombin cleaves fibrinopeptides A and B from the fibrinogen γ β α Fibrinogen Fibrin monomer [(αβγ)2] Fibrin ① Thrombin cleaves fibrinopeptides A and B from the central globule of fibrinogen ② Globular domains at the C-terminal ends of the β and γ chains interact with “knobs” exposed at the N-terminal ends of the β and α chains to form clots. Cross-linked fibrin is formed by transglutaminase called FXIIIa FXIIIa Electron microscope of fibrin. Prothrombin is readied for activation by a Vitamin K-dependent modification Thrombin is synthesized as a zymogen called prothrombin. The inactive molecule comprises four major domains, with the serine protease domain at its carboxyl terminus. ▬ The first domain is called a gla domain (= a γ-carboxyglutamate-rich domain), and the second and third domains are called kringle domains. [참고] Kringle = 스칸디나비아 파이와 비슷하게 생겨 붙여진 명칭.   Prothrombin is composed of four major domains Factor Xa (FXa) γ-carboxyglutamate-rich domain  These domains work in concert to keep prothrombin in an in-active form and to target it to appropriate sites for its activation by factor Xa (FXa, a serine protease) and factor Va (FVa, a stimulatory protein). Prothrombin activation is begun by ① proteolytic cleavage of the bond between arginine 274 and threonine 275 to release a fragment containing the first three domains. ② Cleavage of the bond between arginine 323 and isoleucine 324 (analogous to the key bond in chymotrypsinogen) yields active thrombin.  Prothrombin activation by FXa cleavage Arg274-Thr275 Arg323-Ile324  Activation of prothrombin by FXa FXa (protease) & Prothrombin FVa (stimulatory protein) Thrombin (Inactive) (Active) *FXa cleavage site: Ile(I)-Glu(E)/Asp(D)-Gly(G)-Arg(R)↓ (IEGR↓ or IDGR↓) Vitamin K has been known for many years to be essential for the synthesis of prothrombin and several other clotting factors… Indeed, it is called vitamin K because a deficiency in this vitamin results in defective blood koagulation (Scandinavian spelling). The results of studies of the abnormal prothrombin synthesized in the absence of vitamin K or in the presence of vitamin K antagonists, such as dicoumarol, revealed the vitamin's mode of action.  Antagonist [길항제(拮抗劑)/차단제/저해물질]: an agent that blocks or dampens a biological response by binding to and blocking a receptor  Agonist (작용제/작용물질): an agent capable of stimulating a biological response by occupying cell receptors.  Dicoumarol is found in spoiled sweet clover and causes a fatal hemorrhagic disease in 출혈성 질환 cattle fed on this hay. ─ This coumarin derivative is used clinically as an anticoagulant* to prevent thromboses* in patients prone to clot *anticoagulant: 항응고제(抗凝固劑) formation. Dicoumarol and such related vitamin K antagonists as warfarin also serve as effective rat poisons. *thrombosis: 혈전증(血栓症) Dicoumarol is found in spoiled sweet clover and causes a fatal hemorrhagic disease in cattle fed on this hay. Cows fed dicoumarol synthesize an abnormal prothrombin that does not bind Ca2+, in contrast with normal prothrombin. — This difference was puzzling for some time because abnormal prothrombin has the same number of amino acid residues as that of normal prothrombin and gives the same amino acid analysis after acid hydrolysis.  Nuclear magnetic resonance (NMR) Second studies revealed that… carboxyl ─ normal prothrombin contains group γ-carboxyglutamate, a formerly unknown residue that evaded detection because its second carboxyl group is lost on acid hydrolysis during amino acid analysis.  In fact, the first 10 glutamate residues in the ammo-terminal region of prothrombin are carboxylated to γ-carboxyglutamate by a vitamin K-dependent enzyme system.  The vitamin K-dependent carboxylation reaction converts glutamate, a weak chelator of Ca2+ into γ-carboxygtutamate, a much stronger chelator. The calcium-binding region of prothrombin ─ Prothrombin binds calcium ions with the modified amino acid γ-carboxyglutamate Prothrombin is thus able to bind Ca2+, but what is the effect of this binding? 1. The binding of Ca2+ by prothrombin anchors the zymogen (prothrombin) to phospholipid membranes derived from blood platelets after injury. 2. The binding of prothrombin to phospholipid surfaces is crucial because it brings prothrombin into close proximity to two clotting proteins (FXa and FVa) that catalyze its conversion into thrombin. 3. The calcium-binding domain is removed during activation, freeing the thrombin from the membrane so that it can cleave fibrinogen and other targets.  Prothrombin only synthesized in the presence of vitamin K can be activated to thrombin by FXa and FVa because it has γ-carboxyglutamates-containing Gla domain Ca2+Ca2+ Ca2+ 2+ Ca2+ Ca Gla 2+ Ca 2+ Ca FXa Phospholipid membrane Prothrombin FVa Prothrombin Vitamin K, Carboxylase Ca2+Ca2+ Thrombin Ca2+ 2+ ( = FIIa) γ-carboxy- Ca2+ Ca glutamates Gla Gla 2+ Fibrinogen Fibrin Ca 2+ Ca FXIIIa Prothrombin Prothrombin [※The first 10 glutamate residues in the N- Cross-linked Fibrin terminal region of prothrombin are Ca2+ carboxylated to γ- carboxyglutamates by a vitamin K-dependent enzyme system] Coagulation Prothrombin synthesized in the absence of vitamin K cannot be activated to thrombin by FXa and FVa because it has no γ-carboxyglutamates-containing Gla domain Ca2+Ca2+ Ca2+ 2+ Ca2+ Ca Gla FXa Ca2+ Ca2+ FVa Phospholipid membrane Prothrombin Prothrombin Vitamin K, Carboxylase Ca2+Ca2+ Thrombin Ca2+ 2+ Fibrinogen Fibrin γ-carboxy- Ca2+ Ca glutamates Gla Gla Ca2+ Ca2+ FXIIIa Prothrombin Prothrombin Cross-linked Fibrin Ca2+ Coagulation Hemophilia revealed an early step in clotting Some important breakthroughs in the elucidation of clotting pathways have come from studies of patients with bleeding disorders. Classic hemophilia, or hemophilia A, is the best- known clotting defect. ─ This disorder is genetically transmitted as a sex- linked recessive characteristic. ─ In classic hemophiha, factor VIII (antihemophilic factor) of the intrinsic pathway is missing or has markedly reduced activity hemophilia.  Although factor VIII is not itself Inactive factor 9 (zymogen) = IX a protease, it markedly Active factor 9 = IXa stimulates the activation of FX to FXa by FIXa (also called Christmas factor, related to hemophilia B), a serine protease. Positive feedback Thus, activation of the intrinsic (by thrombin & FXa) pathway is severely impaired in hemophilia.  It is interesting to note that the activity of FVIII is markedly increased by Iimited proteolysis by thrombin and FXa. → This positive feedback amplifies the clotting signal and accelerates clot formation after a threshold has been reached. FVIIIa defect & Hemophilia A in blood clotting X X X X X Hemophilia A  In the past, hemophiliacs were treated with transfusions of a concentrated plasma fraction containing factor VIII. This therapy carried the risk of infection. Indeed, many hemophiliacs contracted hepatitis and, more recently, AIDS. A safer preparation of factor VIII was urgently needed. — With the use of biochemical purification and recombinant DNA techniques, the gene for factor VIII was isolated and expressed in cells grown in culture. — Recombinant factor VIII purified from these cells has largely replaced plasma concentrates in treating hemophilia.  The blood clotting process must be precisely regulated! There is a fine line between hemorrhage and thrombosis. Clots must form rapidly yet remain confined to the area of injury. 혈전 (blood clot)은 신속히 형성되어야 하지만 부상 부위에 한정되어야 함! hemorrhage vs thrombosis 출혈(出血) vs 혈전증(血栓症)  What are the mechanisms that normally limit clot formation to the site of injury? The lability of clotting factors contributes significantly to the control of clotting. ─ Activated factors are short-lived because they are diluted by blood flow, removed by the liver, and degraded by proteases. ─ For example, the stimulatory protein factors Va and VIIIa are digested by protein C, a protease that is switched on by the action of thrombin. ─ Thus, thrombin has a dual function: (1) it catalyzes the formation of fibrin and (2) it initiates the deactivation of the clotting cascade. Cleavage Protein C Activation Specific inhibitors of clotting factors are also critical in the termination of clotting 1. For instance, tissue factor pathway inhibitor (TFPI) inhibits the complex of TF-VIIa-Xa. (Separate domains in TFPI inhibit VIIa and Xa). 2. Another key inhibitor is antithrombin III, a plasma protein that inactivates thrombin by forming an irreversible complex with it. ─ Antithrombm III resembles α1-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase.  ─ Antithrombin III also blocks other serine proteases in the clotting cascade - namely, factors IXa (9), Xa (10), XIa (11), and XIIa (12). ─ The inhibitory action of antithrombin III is enhanced by heparin, a negatively charged polysaccharide found in mast cells near the walls of blood vessels and on the surfaces of endothelial cells. → Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors.  Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors.  Mast cell: 비만(肥滿)세포/비반(肥胖)세포 Heparin과 histamine 등 분비  Histamine: 혈관 투과성에 영향  혈관팽창 유도  알러지반응 유발(Anaphylaxis 등) [Anaphylaxis (과민증; 아나필랙시스; 알러지반응)] [정의] 알러지성 반응(allergic reaction), 항원과 항체 면역 반응이 원인이 되어 발생하는 급격한 알러지성 전신 반응 [원인] 땅콩, 갑각류(새우 등), 곤충(벌, 개미 등에 쏘임), 항생제, 소염진통제 등 [발병기작] 알러젠(allergen; 알러지 유발물질)에 의해 B 세포에서 IgE 항체 생성  IgE가 mast cell 표면에 결합  면역 반응을 일으켰던 동일 allergen에 다시 노출  mast cell이 활성화되어 히스타민 등 분비  혈관팽창 등 아나필랙시스 증상 발현 [증상] 급성 전신 반응(두드러기, 혈관부종, 홍조, 기관지 근육의 수축에 의한 호흡곤란 등) 유발  보통, 발병시간이 매우 짧아 아주 소량의 allergen에 노출되더라도 수분 이내(보통 30분 이내) 증상이 나타남. Adopted from : https://healthlifemedia.com/healthy/what-is-anaphylaxis/ Adopted from : https://healthlifemedia.com/healthy/what-is-anaphylaxis/ Antithrombin limits the extent of clot formation, but what happens to the clots themselves? Clots are not permanent structures but are designed to dissolve when the structural integrity of damaged areas is restored. Fibrin is split by plasmin, a serine protease that hydrolyzes peptide bonds in the coiled coil regions. − Plasmin molecules can diffuse through aqueous channels in the porous fibrin clot to cut the accessible connector rods. − Plasmin is formed by the proteolytic activation of plasminogen, an inactive precursor that has a high affinity for the fibrin clots.  Plasmin is formed by the proteolytic activation of plasminogen This conversion is carried out by tissue-type plasminogen activator (TPA; tPA), a 72-kd protein that has a domain structure closely related to that of prothrombin. Modular structure of tissue-type plasminogen activator (tPA). tPA Plasminogen Plasmin  Overview of the fibrinolytic system Endothelial cells/ Monocyte/ Renal epithelium macrophages (腎臟上皮) sc-tPA sc-uPA · · PAI-1 tc-tPA tc-uPA PAI-1 Plasminogen XL-Fibrin α2-PI ·· Plasmin α2-MG FDP tPA, tissue plasminogen activator; uPA, urokinase; sc, single-chain; tc, two-chain; PAI, plasminogen activator inhibitor-1; α2-PI, α2-plasmin inhibitor; α2-MG, α2-macroglobulin. Sc- tPA and sc-uPA are secreted from endothelial cells/renal epithelium and monocyte/macrophages, respectively. tPA can dissolve the blood clots, as shown by X- ray images of blood vessels in the heart After 3 hrs