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Failure of C oagula tion

Failure of C oagula tion. MUDr. Tomáš Stopka Ph.D. and colleagues from the Institute of Pathophysiology, Charles University. Pl a n. Coagulation Methods DIC T h erap y Presentation. I. Coagulation. Initiating the Clotting Process

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Failure of C oagula tion

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  1. Failure ofCoagulation MUDr. Tomáš Stopka Ph.D. and colleagues from the Institute of Pathophysiology, Charles University

  2. Plan • Coagulation • Methods • DIC • Therapy • Presentation

  3. I. Coagulation Initiating the Clotting Process 1. Damaged cells (extrinsic pathway) display a surface protein called tissue factor (TF) that binds to activated Factor 7 (TF-7) to cleave: Factor 10 2. Factor 10 binds and activates Factor 5 (prothrombinase) convertíng prothrombin (also known as Factor II) to thrombin 3. Thrombin proteolytically cleave fibrinogen (Factor I) to fibrin. 4. Factor 13 forms covalent bonds between the soluble fibrin molecules converting them into an insoluble meshwork — the clot.

  4. I. Coagulation • Amplifying the Clotting Process • 1. The TF-7 complex also activates Factor 9. • 2. Factor 9 binds to Factor 8, a protein that circulates in the blood stabilized • by another protein, von Willebrand Factor (vWF). • 3. Complex 9-8-vW activates more factors: 5,8,10,11

  5. I. Coagulation LUMEN Blood clot WALL Endothelial demage Damaged endothelial cells display tissue factor (TF) that binds to activated Factor 7 (TF-7) to cleave: Factor 10 The intrinsic cascade is initiated when contact is made between blood and exposed endothelial cell surfaces.

  6. Heparin binds to and enhances antithrombin III. Warfarin (aka coumadin) is an effective vitamin K antagonist. I. Coagulation • Controlling Clotting Antithrombin III inactivates: prothrombin, factor 9, factor 10 Protein C and its cofactor Protein S together inhibit thrombin formation by inactivating Factor 5 and by inactivating Factor 8. Inherited deficiency(mutations) of Protein C or Protein S (or FV, Leiden)= thrombophilia Vitamin K is a cofactor needed for the synthesis (in the liver) of factors 2 (prothrombin), 7, 9, and 10, proteins C and S Deficiency of Vitamin K predisposes to bleeding. Conversely, blocking the action of vitamin K helps to prevent inappropriate clotting.

  7. I. Coagulation • Dissolving clots Plasma plasminogen to the fibrin molecules in a clot. Nearby healthy cells release tissue plasminogen activator (TPA), which also binds to fibrin and, activates plasminogen forming plasmin. Plasmin (serine protease) proceeds to digest fibrin, thus dissolving the clot.

  8. I. Coagulation HEMOCOAGULATION is INTEGRALPART ofINFLAMATORY RESPONSE VASCULAR WALL ENDOTHELIUM PLATELETS PLAZMATIC COAGULATION SYSTEM

  9. I. Coagulation DISORDERS OF HEMOCOAGULATION = THROMBOSIS AND EMBOLISM THROMBOSIS IN MICROCIRCULATION VENOUS ARTERIAL SIGNS -TISSUE ISCHEMIA - HEMODYNAMIC FAILURE LUNGS SYSTEMIC EMBOLISM

  10. I. Coagulation • Deep Venous Thrombosis (DVT) A) asymptomatic : > 50% Lung Embolism. B) symptomatic: pain (Homans’s sign), oedema, dicoloration and incr. temperature of the skin • Posthrombotic syndrome latent, 3 - 15 yafter DVT:distension of superf. veins, lipodermatosclerosis, varices, ulceration. • Lung embolism (LE) Dyspnoe, tachypnoe, tachykardia, pleuritic chest pain, distension of the jugular veins, hemoptysis, hemodynamic instability, hemodynamic failure or death.

  11. I. Coagulation BLEEDING SMALL, TRAUMATIC - sc., im.injections - Easy bruising DIFUSE MICROVASCULAR - purpura - petechia, ekchymosis (>3 mm) - organ apoplexia SURGERY A) trombocytopenia B) Desintegration of microvascular intima traumatic Failure of coagulation

  12. II. Laboratory BLEEDING TIME and RESISTANCE OF CAPILARS • Bleeding time (Duke, 1910) standard puncture of the ear lobe (Duke, 1910) 2 - 5 min. prolonged in thrombocytopenia (<20 000/uL)OR vonWillebrand disease • Capillary resistance ( Rumpel, Leede) pressure on the arm 10,5 kPa/10 min petechia > 5 = increased fragility of capillaries. (hereditary purpura e.g. Weber-Rendu-Osler).

  13. II. Laboratory Basic coagulation methods • Thrombin time -full blood is activated with thrombin to form fibrin fiber -used for measurement of fibrinogen levels (DIC)

  14. II. Laboratory Methods for measuring platelets and vWf • (PLT) – normal 150 - 300 000/uL, for surgery optimum > 100 000 /uL. Thrombocytopenia PLT < 20 000/ uL – spontaneous bleeding and purpura. • (MPV) - normal 6 - 9 fL, incr. hereditary trombocytopathy. • Agregometry – photometric, with addition of activator of platelet aggregation - ADP, thrombin, kolagen. Diagnosis of hereditary trombocytopaty • Flow cytometry - imunologic. • Anti PLT antibodies – diagnosis of imune-mediated trombocytopenia • vWf - imunologic or functional tests incl. ristocetin

  15. II. Laboratory Methods for measuring Coagulation factors Blood drown into citrate is centrifuged to obtain decalcified plasma • PT – prothrombin time PT (Quickův) • APTT - activated partial thromboplastin time Statim • FBG - fibrinogenu plasma levels (normal :2 - 4 g/L). ( FBG acute phase protein) • FDP - imunologic measurement of degradation products of fibri(noge)n (normal: < 1000 ug/L), ELISA or aglutination semiq. methods.

  16. II. Laboratory Methods for measuring Coagulation factors • D-dimer - imunologic measurement of FDP specific for stabilized fibrin (normal <500 ug/L). Increased D-dimer DVT/PE and DIC. • AT - function test to measure antithrombin activity in plasma (normal80 - 100% activity of the control plasma). With heparin part of the TAT inhibitory complex, deficiency predispose to thrombophilia or DIC.

  17. II. Laboratory Methods for measuring Coagulation • Ethanol test – FDP anti-polymeration effect on fibrin fiber is blocked by ethanol • Euglobulinmethod of measuring fibrinolytic activity Euglobulin fraction of plasma obtained with acetic acid conatins predominantly plasminogen, in DIC there is more plasmin and so the test is quicker (result of increased fibrinolysis).

  18. II. Laboratory Methods for measuring Coagulation • proteinu C- Act. Protein C resistence, mutationof FV, mutationof protein S • fibrinolytic system- tPA , inhibitor PAI-1, plazminogen, inhibitor alfa2AP • Antifosfolipid antibodies- lupus anticoagulans (LA) : modif. APTT • Individual factorshemofilia A (FVIII), B(FIX), C (FXI)

  19. II. Laboratory Protrombin time PT (Quick) • Principle:extrinsic pathway – tissue factor. Blood drawn to citrate and TF is added. with CaCl2. Time is measured until the fibrin fiber is formed. • Normal: PTN= 12 - 15 s • Prolonged PT:, deficiency of FV, vit. K dep: FII, VII, X, deficient FBG, high FDPs • International normalized ratio INR= (PTP/ PTN)ISI ISI = international index of used tromboplastin (usu > 1). (max. therapeutic INR = 4,5)

  20. II. Laboratory APTT • Principle :intrinsic pathway. Blood drawn to citrate and kaolin (activates inner system) is added with CaCl2.Time is measured until the fibrin fiber is formed. • NormalAPTTN = 27 - 35 s • Used:hemophilia, lupus anticoagulans, heparintherapy (1,5x - 2,5 x). • Prolonged APTT: deficient FII,V, X, - F XII, PreK, HMWK, - FXI, FIX , FVIII (hemofilia C, B,A), lupus anticoagulans, low FBG, high FDP. • Shortened APTT: thrombophilia

  21. PLT BT APTT PT TT FBG Disorder Trombocytopenia L P N N N N N N P N N N N N P N N N N N P N N N N P N/P N N N N N P N/P N N II. Laboratory Hemofilia A Hemofilia B Hemofilia C vW-disease LA

  22. II. Laboratory DisorderPLT BTAPTT PT TT FBG FV-def. FII-def. FVII-def. Vit.Kdef./OA FBG-def. Heparin N N P P N N N N P P N N N N N P N N N N P P N N N N P P P L N P/N P N/P P N

  23. III.DIC Definition Secondary Disorder of Coagulation with pro-thrombotic phase followed by severe bleeding phase (as a result of consumption of coagulation factors).

  24. III.DIC Ethiopathogenesis Intravascular coagulation

  25. Heat stroke Sepsis Viremia Pancreatitis Neoplasia (Diffuse and local) Parasitic Infections Intravascular Hemolysis Immune-mediated Diseases Exposure to venom/toxins Massive tissue injury (including burns, crush trauma, and surgical procedures) ObstetricComplications Insufficiency of major organs (Liver, Kidney) Diabetes mellitus Acidosis Polycythemia Severe prolonged hypotension (including shock) Severe volume depletion Impaired blood flow to a major organ Conditions Associated with DIC

  26. Activation of the coagulation cascade results in increased levels of circulating thrombin and plasmin. Thrombin cleaves fibrinopeptides A and B from fibrinogen, leaving soluble fibrin monomers as the end product (Figure 1). Activation of factor XIII results in polymerization of these fibrin monomers into insoluble cross-linked fibrin clots. DIC • Increased levels of circulating plasmin causes clot lysis and degradation of fibrinogen and the soluble fibrin monomers. • Plasmin cleaves fibrinogen into fragments X,Y,D, and E, known as fibrinogen degradation products (FDPs). • Plasmin also cleaves insoluble cross-linked fibrin polymers into x-oligomers. The main x-oligomers are known as d-dimers. What are FDPs and D-dimers and how do they relate to DIC?

  27. What are FDPs and D-dimers and how do they relate to DIC? FIBRIN • Monoclonal antibodies have been generated which recognize the cross-linked domain of d-dimers as an antigenic target. These antibodies are used in all available d-dimer assays. • Quantitative tests for d-dimers are available, including enzymatic immunoassays (ELISA) and immunoturbidometric systems.

  28. III.DIC NORM  • PLT   150 - 300 000 x 10 exp9 /l • APTT   30 - 35 s • AT    80 - 140 % • TT    14 - 16 s • FBG   2.5 - 5 g/l • FM (ethanol test)    • DD    < 500 ng/ml • FDP   

  29. III.DIC DIC  • PLT   low • APTT   short or prolonged • AT    low • TT    prolonged • FBG   low • FM (etanol test)  positive • plasminogen  low • DD    positive • FDP   positive • euglobulin lysis norm. - prolonged

  30. III.DIC • PLT • FBG • DD • AT Repeat every 3-4h

  31. III.DIC 1 Hypercoagulation Silent 2 Hypocoagulation Bleeding and thrombosis in microcirculation 3 Massive fibrinolysis Bleeding and multiorgan failure (MOF) 4 Death

  32. Thrombotic Thrombocytopenic Purpura Peripheral smear showing microangiopathic hemolytic features with numerous RBC fragments (helmet cells/schistocytes). Marked thrombocytopenia is evident. Renal biopsy showing hyaline thrombi in the glomerulus and small arterioles. von Willebrand factor protein multimer analysis on agarose gel electrophoresis. Lane 1. - normal plasma. Lane 2. - patient plasma when symptomatic. Multimer pattern is similar to the control plasma. Lane 3. - patient plasma after response to pheresis. Note the presence of ultra-large high molecular weight multimers.

  33. Researchers Pinpoint Cause of Deadly Blood-Clotting Disorder • Several earlier studies had implicated a clotting-related protein known as von Willebrand factor (VWF) in the disorder. These studies found that the blood of patients with TTP showed an abnormally large form of the VWF protein that had not been cleaved into two smaller sizes, as is normally the case. Thus, said Ginsburg, many scientists believed that a defect in a protein- clipping enzyme known as a protease might be responsible for the disorder. • One of the keys to identifying the gene mutations that underlie TTP was the development of a precise assay for detecting VWF protease activity. Han-Mou Tsai, a senior author of the Nature paper, and colleagues at Montefiore Medical Center and Albert Einstein College of Medicine developed the assay and applied it to blood samples that were provided by members of four families that had an inherited form of TTP. The assays clearly revealed that within these families, those who had TTP showed low VWF protease activity, while carriers of the disease showed medium levels of protease activity, and unaffected individuals showed normal levels. • Using results from the assay as a guide, Gallia G. Levy, lead author of the Nature article, performed linkage analyses of the family members and determined which of known genomic markers were inherited with the disease gene. These studies enabled her to narrow down the region containing the disease gene to a specific region of chromosome 9. • Levy then obtained the full gene sequence and proceeded to test the other patients for mutations in the gene, which they named ADAMTS13. Levy subsequently identified a dozen mutations in the gene among the patients, accounting for nearly all the cases of TTP. According to Ginsburg, Levy’s findings open the way to understanding how and why the ADAMTS13 protease cleaves VWF and how the failure to cleave the protein causes disease.

  34. III.DIC Therapy:  Blockade of activated coagulation  • 1 Heparin • 5-10 IU/kg/h • bolus 2500 IU, inf. Up to 10 000 IU/24h • LMWH

  35. III.DIC Therapy:  Blockade of activated coagulation  • 2 AT (Antitrombin III, Kybernin P) • If less 60%, target~ 100 - 150% • 500 - 1000 bolus • KI unknown • Half life 3-4 d, during sepsis hours

  36. III.DIC Therapy:  Substitution • 3 Fresh frozen plasma • 15 ml/kg if APTT more than 1.5 R • 4 Fibrinogen • If less than 1.0 g/l (maximally 2g/24h) • 2 - 4 g in infusion

  37. III.DIC Therapy:  Substitution • 5 Erythrocytes • 6  PLT • 1 unit/10kg

  38. III.DIC Therapy:  OTHER • 1 shock • 2  volume • 3 acidobasic and ionts • 4 ATB • 5 Surgical

  39. III.DIC

  40. Acute DICDIAGNOSIS • Clinical findings Multiple bleeding sites Ecchymoses of skin, mucous membranes Visceral hemorrhage Ischemic tissue • Laboratory abnormalities Coagulation abnormalities: prolonged prothrombin time, activated partial thromboplastin time, thrombin time; decreased fibrinogen levels; increased levels of FDP (eg, on testing for FDP, D dimer) Platelet count decreased as a rule but may be falling from a higher level yet still be normal Schistocytes on peripheral smear

  41. Chronic DIC DIAGNOSIS • Clinical findings Signs of deep venous or arterial thrombosis or embolism Superficial venous thrombosis, especially without varicose veins Multiple thrombotic sites at the same time Serial thrombotic episodes

  42. Chronic DIC • Laboratory abnormalities Modestly increased prothrombin time in some patients Shortened or lengthened partial thromboplastin time Normal thrombin time in most patients High, normal, or low fibrinogen level High, normal, or low platelet count Increased levels of FDP (eg, on testing for FDP, D dimer) Evidence of molecular markers* (eg, thrombin-antithrombin complexes, activation markers on platelet membranes, prothrombin fragment F1+2)

  43. Current Management of DIC • At present, diagnosis requires a set of blood tests; therapy focuses on reversing the underlying disorder and providing supportive treatment.

  44. Case 1 Presentation • A 56-year-old man was admitted to the emergency department after a car accident. • He had several bone fractures, a cerebral contusion, and hemodynamic instability caused by a ruptured spleen. • Emergency splenectomy and aggressive administration of fluids restored hemodynamic stability, and the patient was transferred to the intensive care unit (ICU). A few hours later, profuse extravasation was noted from the abdominal drains, endotracheal tube, and puncture sites of all intravascular lines.

  45. Case 1 Presentation • Laboratory tests showed a rapidly falling hemoglobin level and a platelet count of 25,000/µL. • The activated partial thromboplastin time (aPTT) was 44 sec (normal, <28) and the prothrombin time (PT) was 29 sec (normal, <12.5). • The level of fibrinogen degradation products was 360-520 g/L (normal, <40) and the plasma antithrombin III level was 28% (normal, 80-120).

  46. Case 1 Presentation • Based on these findings, the diagnosis was DIC secondary to severe trauma. Surgical exploration revealed diffuse oozing of blood at the site of the operation, but only partial surgical hemostasis could be achieved. • The patient was given supportive treatment with: • large infusions of fresh frozen plasma • platelet concentrates. The bleeding stopped 48 hours later. Coagulation parameters eventually returned to normal and the subsequent clinical course was uneventful.

  47. The pathogenesis of DIC

  48. Selected Disorders ThatMay Be Associated with DIC • Malignancy (solid tumors, myeloproliferative, lymphoproliferative) Obstetric emergencies (amniotic fluid embolism, abruptio placentae) • Organ destruction (severe pancreatitis) • Sepsis/severe infection (any microorganism) • Severe hepatic failure • Severe toxic or immunologic reactions (snake bites, recreational drugs, transfusion reactions, transplant rejection) • Trauma (polytrauma, neurotrauma, trauma resulting in fat embolism) • Vascular abnormalities (Kasabach-Merritt syndrome, large vascular aneurysms)

  49. Infection. • Bacterial infection, in particular septicemia, is commonly associated with DIC. However, systemic infections with other microorganisms, such as viruses and parasites, also may lead to DIC. • Components of the microorganism's cell membrane (lipopolysaccharide, or endotoxin) or bacterial exotoxins (e.g. staphylococcal alpha-toxin) may cause a generalized inflammatory response characterized by systemic production of cytokines, mainly by activated mononuclear cells and endothelial cells. • The cytokines are responsible for the derangement of the coagulation system in DIC.

  50. Trauma • Head trauma in particular is strongly associated with DIC; both local and systemic activation of coagulation may be detected after such an event. • The increased risk of DIC after head trauma is understandable in view of the relatively large amount of tissue factor in the cerebral compartment.

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