Natural Anticoagulants Lecture 4 PDF

Natural Anticoagulants Dr Lisa Crawford BMS 3104  too much blood ? How do we stop clotting What is this lecture about? ▪ Natural or endoge...

Natural Anticoagulants Dr Lisa Crawford BMS 3104  too much blood ? How do we stop clotting What is this lecture about? ▪ Natural or endogenous anticoagulants: Regulate the activity of the coagulation system. ▪ Three are important: Protein C, Protein S and Antithrombin ▪ Protein C: Activated by Thrombin/Thrombomodulin complex and, along with Protein S, regulates coagulation by inactivating Factors V and VIII ▪ Activated protein C resistance: An inherited risk factor for thrombosis caused by the Factor V Leiden mutation. Can be detected by a PCR based test. ▪Antithrombin: regulates coagulation by inhibiting thrombin long with a number of other coagulation factors ▪ Key Features of Protein C, Protein S and Antithrombin deficiencies  Learning Objectives 1. To discuss the characteristics of the main natural anticoagulant proteins. 2. To describe how protein C is activated and how activated protein C acts in conjunction with Protein S to down-regulate coagulation through its effects on factors V and VIII. (negatively regulare) 3. To understand how the factor V Leiden mutation leads to activated protein C resistance. 4. To describe how antithrombin inhibits coagulation by its effect on thrombin and how the activity of antithrombin is affected by heparin. Learning Objective 1 Discuss the characteristics of the main natural anticoagulant proteins. Haemostasis requires a tightly controlled balance between clot formation and dissolution  Natural Anticoagulants Plasma proteins that regulate the activity of the Coagulation System need to have a Three predominant anticoagulants: good balance Protein C Protein S (deficiency) ↳ Antithrombin  Natural Anticoagulants Key features of the Coagulation System 1. Reacts rapidly to prevent blood loss (signal amplification) 2. Clot formation is limited to the area of blood vessel damage Natural Anticoagulants act to limit clot formation Coagulation System is finely balanced - too little activity -> bleeding - too much activity -> thrombosis Anticoagulant proteins maintain correct balance by regulating activity of coagulation factors Brief summary Natural Anticoagulants Three main endogenous anticoagulants: ▪ Protein C Serine protease, activated by the Thrombin/Thrombomodulin complex Regulates the activity of coagulation FVa and FVIIIa ▪Protein S - Notprotease itself Co-factor a. Co-factor for Protein C Regulates the activity of coagulation FVa and FVIIIa in conjunction with Protein C Can directly inhibit FXa ▪Antithrombin Serine protease inhibitor Regulates the activity of thrombin, FIXa, FXa, FXIa, FXIIa Inhibitory activity is enhanced by heparin specificand cofactor su Evilla r  Thrombophilia ▪ Anticoagulant protein deficiency leads to an increased risk of venous thrombosis ▪Can generally be categorised into 2 types ↳ excess Type I – low expression and low activity (deficiency) clotting Type II – normal expression and low activity (missense mutations) (deficiency) ↳ ie not functional  Learning Objective 2 DESCRIBE HOW PROTEIN C IS ACTIVATED AND HOW ACTIVATED PROTEIN C ACTS IN CONJUNCTION WITH PROTEIN S TO DOWN-REGULATE COAGULATION THROUGH ITS EFFECTS ON FACTORS V AND VIII.  Protein C Protein C synthesised in liver (requires vitamin K) serine protease secreted to circulate in plasma as inactive precursor (zymogen) to make Activated by partial proteolytic cleavage our section of protein > - cleaving it active Protein C activated by Thrombin/Thrombomodulin complex  Thrombomodulin ▪ Cell surface receptor on endothelial cells # on surface Of ECs. ▪ Binds thrombin > - circulating in blood vessels ▪ Converts thrombin from procoagulant to anticoagulant enzyme Thrombin loses ability to cleave fibrinogen Thrombin gains ability to activate Protein C Thrombin Thrombomodulin Endothelial Cells  Protein C Activation ▪ Protein C is activated to become Activated Protein C (APC) through partial proteolytic cleavage by the Thrombin-Thrombomodulin complex ▪ EPCR – endothelial Protein C receptor, expressed predominantly on endothelial cells of large blood vessels ▪ Can bind Protein C and present it to Thrombin-Thrombomodulin complex to enhance its activation by 20-fold more dangerous Protein C APC clos in to get a blood Thrombin larger EPCR Thrombomodulin vessels. Endothelial Cells  Activated Protein C (APC) breaks up protein to make it x ▪ APC inactivates Factors Va and VIIIa by proteolytic cleavage no longer functional ▪ These are the cofactors in the pro-coaguagulant tenase (FVIIIa) and prothrombinase (FVa) complexes ▪ Inactivation of FVa and FVIIIa greatly reduces production of thrombin and fibrin ↳ when they are no needed longer  Protein S alongside protein ↳ works ( ▪ Protein S is a cofactor for APC > - nor a protease ▪ Not a protease, but a vitamin K-dependent plasma protein ▪ APC activity is greatly increased when Protein S is bound ▪ Protein S is also important for regulation of the complement system ▪30-40% of Protein S circulates as free protein with the rest bound to C4b- binding protein, a regulator of the complement pathway ↳ ▪ Only free Protein S binds APC (the majority Protein S Protein S enhances APC phospholipid membrane binding affinity https://doi.org/10.1111/jth.15025  Protein C Anticoagulant Pathway Factor VIII (Inactive) Factor VIII (Active) Factor V (Inactive) Factor V (Active) Protein S Activated Protein C Protein C Thrombin EPCR J Thrombomodulin sidehou , Endothelial Cells Protein S as a co-factor for TFPI ↳ tissue factor pathway inhibitor ↳ inactivates factor Xa https://doi.org/10.1111/jth.15025  Protein C Deficiency ▪ Inherited disorder, autosomal dominant inheritance > one - copy of mutation Deep vein thrombosis/pulmonary embolism ▪ Genetic risk factor for Venous Thrombosis -> DVT/PE -> ▪ >160 PROC gene mutations, two outcomes 1. mutation results in reduced PC synthesis (type 1 deficiency) 2. mutation affects ↳biological activity of PC (type 2 deficiency) makes it non-functional a lo ▪ Mild/Severe forms : Mild,1:500 (heterozygous), Severe,1:4 million- raver (homozygous or compound heterozygous) ↳ one copy of muration ↳2 copies ↳ 2 different pC murations. ▪ Purpura Fulminans: life threatening, severe PC deficiency, develops in new- born, widespread thrombosis, tissue necrosis – treatment option: replace,ent therapy with a plasma-derived Protein C concentrate Ly homozygous or compound heterozygous  Protein S Deficiency ▪ Inherited disorder, autosomal dominant inheritance ▪ Similar features as protein C deficiency ▪ Genetic risk factor for venous thrombosis -> DVT/PE ▪ Risk of venous thrombosis increased in association with: age, surgery, immobility, pregnancy, other inherited VT risk factors ↳ venous thrombosis ▪ Mild PS deficiency:1:500; Severe PS deficiency is rare, prevalence is unknown ↳ as it is so rare ▪ Severe PS deficiency associated with Purpura Fulminans in new born infants ↳ life threatening thrombosis Learning Objective 3 UNDERSTAND HOW THE FACTOR V LEIDEN MUTATION LEADS TO ACTIVATED PROTEIN C RESISTANCE.  Activated Protein C Resistance APC Resistance (APC-R) Inherited trait; common in thrombosis patients 95% of APC-R caused by Factor V Leiden mutation ↳one mutation FV gene, c.1691G>A; p.Arg506Gln ↳G to A nucleotide replacement Mutated FV resistant to inactivation Prolonged FV activity leads to an increased tendency to clot ↳ because it can not be inactivated  Inactivation of FV APC destroys FVa in a series of sequential cleavages. The first cleavage at Arg506 exposes additional cleavage sites at Arg306 and Arg679. activated - when JVa is no longer "needed no arginine, apcanen break own JVa , prolonged Iva activity, enhanced thrombogeneration -  Factor V Leiden Mutation ▪ Inherited risk factor for venous thrombosis ▪ Carriers at increased risk of developing DVT/PE ▪ 5% population ▪ Increased risk of thrombosis - 5-10 fold heterozygotes - ~80 fold homozygotes  Laboratory testing for APC-R ▪ Can be tested for using activated partial thromboplastin time (APTT ) assay ▪If APC-R is detected, it would be appropriate to then test for Factor V Leiden mutation (PCR) ▪If present family members may be tested to determine carrier status  APTT > - does not you have tell you if IV Leiden mutation APTT can be used to aid diagnosis of APC resistance APC added to plasma: FV/FVIII inactivated APTT increases (+/- Ratio > 2.5) ↳ woulddefect clottinis Effect less pronounced in some thrombosis patients (APTT ratio DVT/PE ▪ Risk of venous thrombosis increased in association with: age, surgery, immobility, pregnancy, other inherited VT risk factors ▪ Prevalence: 1:2,000 >- common ▪ Mutations of SERPINC1 gene that encodes antithrombin protein ▪ Homozygous mutations usually incompatible with life ↳ only heterozygous mutations found in population n.  Antithrombin Deficiency ▪ SERPINC1 gene mutations, two outcomes 1. mutation results in reduced AT synthesis (type I deficiency) 2. mutation affects biological activity of AT (type II deficiency) Sub-types: IIa: Defect in AT active site (impaired thrombin binding) IIb: Defect in AT heparin binding site (impaired heparin binding)  Summary Natural anticoagulants limit the clotting process Deficiencies in these proteins can lead to thrombophilia Protein C is activated on endothelium by the thrombin–thrombomodulin–EPCR complex Activated protein C regulates the activity of FVIIIa and FVa Activated protein C resistance caused by the FV Leiden mutation leads to an increased risk of venous thrombosis FV Leiden mutation prevents cleavage of FV by APC at Arg 506 Antithrombin is an important inhibitor of thrombin and factor Xa (can also act on factors IXa, XIa, XIIab, kallikrein, and plasmin). Heparin binding to antithrombin alters its conformation and stimulates its activity.