Examination of hemostasis Stanislav Matoušek

Hemostatic disorders + thrombosis and embolism (e.g. DVT or MI or stroke) Virchow’s trias – Endothelium dysfunction, stasis, blood factors Procoagulational changes in the blood = HYPERCOAGUABILITY DISORDERS - excessive bleeding hemorrhagic diatheses = bleeding states //Endothelium and vessel wall – purpuras/vasculopathies //Platelets – thrombocytopenias and -pathias (common) //Plasmatic coagulation system – coagulopathies

Causes and signs of bleeding disorders Lack or defect of platelets Blood vessel disorder Lack of plasmatic factors Mostly bruises and bleeding to joints Excessive bleeding Mostly petechiae

Disorders of primary hemostasis Platelets and vessels

- - - - Endothelium Bleeding Platelets activated + + + Vessel injury + Platelet thrombus - Exposition of procoagulation phospholip. (pf3) Coagulation cascade fibrin web - thrombin + Vasoconstriction in injured vessel - adrenalin + serotonin + Bleeding Vessel injury myocytes & fibroblasts PDGF ADP + prostacycline PGI2 - membrane phospholipids Arachidonic acid eicosanoids PGG2 PGH2 Collagen Exposal Exposal of von Wilebrand’s factor Desquamated epithelium Platelet adhesion Platelets activated thromboxan A2 + kys. arachidonová endoperoxidy PGG2 PGH2 membr. fosfolipidy Platelet aggregation PAF granulocytes, baso-, macrophages + von Wilebrand’s factor Endothelium

Thrombocytopenia 1) Production decreased↓ 2) Consumption increased ↑ A) with increased activity of thrombin B) imuno-thrombocytopenia C) other 3) Combination of both mechanisms

Platelet count 200 – 400 x 103/µL (109/L) = 200 000 – 400 000 /µL The risk of spontaneous bleeding is low if the number of platelets is > 30 000 /µL and blood vessels and coagulation system are intact

I) Thrombocytopenia from insufficient production Aplastic disorder Congenital Acquired Bone marrow damaged by medicaments (chemotherapy), chemicals (benzene), radiation, infection (HIV), autoantibodies Infiltration of bone marrow (carcinomas, lymphomas, leukemia) Myelodysplastic syndrome, Myelofibrosis Defect in megakaryocyte maturation vit. B12 and folate deficiency Bone marrow aspiration (less megakaryocytes in aplastic)

2) Thrombocytopenia from increased consumption A) Increased activity of thrombin Disseminated intravascular coagulation – DIC activation in infections/malignities (extracellular proteases)-chronic DIC B) Imunothrombocytopenia (e.g. autoantibodies) Idiopathic thrombocytopenic purpura- autoantibodies produced with unknown cause Acute vs. chronic Secondary ( SLE, lymphomas, HIV) After medicaments …. Heparin induced C) Other Hypersplenism, artificial valve TTP = Thrombotic thrombocytopenic purpura Activity of thrombin, clotting tests PT and aPTT

Thrombotic thromboplastic purpura ? Cause is probably damage to endothelium - release of von Willebrand factor - decreased degradation of vWf by its protease (antibodies against the protease, congenital?)

Purpura

3) Combination of low production and increased consumption Liver cirrhosis

platelet dysfunction = thrombocytopathy Congenital Acquired aggregation inhibitors therapy (aspirin, ticlopidin) platelets covered by IgA or IgM (multiple myeloma) Dextran (infusion, often given in bleeding) Uremia (Kidney failure) Essential Thrombocytosis (one of myeloproliferative diseases analogy of polycythemia vera) Normal number of platelets, bleeding time ↑ Clinically: Generally, it is difficult to stop bleeding (e.g. during surgery),

Bleeding time (Duke) Standardized puncture of the earlobe, or standardized incision to the forearm with cuff inflated to 40 Torr . We measure the time until the bleeding stops 2-5 min (2-9 min in forearm) Increased in platelet dysfunction and in vonWillebrand disease (but not in Hemophilia)

vonWillebrand disease Missing or defective vonWillebrand factor The factor is important in platelet adhesion (primary hemostasis) and serves also as a carrier for factor VIII (decrease of concentration of factor VIII leads to defect in coagulation) Combined bleeding of petechial and hemophilic type Congenital or acquired Prevalence 1%, but generally mild symptoms (heterozygotes) Prolonged bleending time (Duke) and aPTT

Vasculopathies / purpuras congenital e.g. Ehlers-Danlos syndrom (defect of collagen) Acquired scurvy (vitamin C deficiency) glucocorticoid excess Purpura senilis Henoch-Schoenlein purpura (children after an upper respiratory infection xx DD DIC in meningococcal infection!)

Tourniquet test (Rumpel-Leede capillary-fragility test) A blood pressure cuff is inflated to a point little bit above diastolic blood pressure during 5 min (10 min). Positive, if more that 5 petechiae per square inch are found. Vasculopathies, dengue fever diagnosis (more than 20 petechiae).

Scurvy

Secondary hemostasis disorders Plasma coagulation system

only by collagen contact Prekallikrein Kallikrein + Intrinsic pathway Intrinsic activation only by collagen contact HMWK Subendothelium (contact with collagen surface) XII (Hageman) - - - - - - - - - - - - - - - - - - - - - - - + XI + XIIa (Hageman) XIa + IX IXa + VIII von Willebrand factor Extrinsic pathway Exogenous activ. by tissue factor Ca++ VII VIIa + VIIIa von Willebrand factor + { + + } X Xa + Platelet phospholipides III Tissue thromboplastin Platelet phospholipides (pf.3) Ca++ II prothrombin Ca++ Platelet phospholipides } IIa thrombin + V Va + XIII (fibrin stab. f.) Ca++ + Platelet phospholipides (pf.3) Ca++ XIIIa (fibrin stab. f.) + Fibrin web Weak bonds I fibrinogen + Fibrin web Covalent bonds +

(contact with collagen) Intrinsic pathway (contact with collagen) XIIa (Hageman) + Extrinsic pathway (tissue factor) Anti Vit. K + XIa (PTA) Anti Vit. K { + } Hemophilia B VIIa (konvertin) IXa (Christmas) Hemophilia A Membrane phospholipides III (tissue tromboplastin) Ca++ VIIIa (AF) Platelet factor 3 (platelet phospholipides) Ca++ + Anti Vit. K II prothrombin Ca++ Platelet phospholipides } IIa thrombin + } Anti Vit. K Xa (Stuart-Prower) Va (accelerin) Ca++ + IIa (thrombin) Fibrin web + XIIIa (fibrin stab. f.) fibrin monomer I (fibrinogen) +

Queen Victoria of Britain Carrier of Hemophilia X-linked Great- grandson Tsar Nicolai II of Russia Tsarevich Alexei Sufferer of Hemophilia

Partial Tromboplastin Time (activated) Partial Tromboplastin Time PTT,aPTT, APTT Intrinsic pathway (contact with collagen) XIIa (Hageman) + Extrinsic pathway (tissue factor) + XIa (PTA) { + } VIIa (konvertin) IXa (Christmas) Prothrombin Time, PT, (Quick) Membrane phospholipides III (tissue tromboplastin) Ca++ VIIIa (AF) Platelet factor 3 (platelet phospholipides) Ca++ + } Xa (Stuart-Prower) Va (accelerin) + IIa (thrombin) Fibrin web + XIIIa (fibrin stab. f.) fibrin monomer I (fibrinogen) +

(contact with collagen) Intrinsic pathway (contact with collagen) XIIa (Hageman) + Extrinsic pathway (tissue factor) + XIa (PTA) { + } VIIa (konvertin) IXa (Christmas) Membrane phospholipides III (tissue tromboplastin) Ca++ VIIIa (AF) Platelet factor 3 (platelet phospholipides) Ca++ + } Xa (Stuart-Prower) Va (accelerin) + IIa (thrombin) Thrombin time Fibrin web + XIIIa (fibrin stab. f.) fibrin monomer I (fibrinogen) +

aPTT Citrate or oxalate plasma, We add phospholipids, Ca++, caolin or other negatively charged surface Time until clot formation (depends on the lab) ~ 25 - 39 s Monitoring of heparin therapy, intrinsic and common pathway, hemophilia

Prothrombin Time (Quick test) Citrate or oxalate plasma, We add phospholipids, Ca++, and tissue thromboplastin Time until clot formation (depends on the used thromboplastin) ~ 12-15 s INR = PTtest/ PTnormal (norm 0.8 – 1.2) Monitoring of anti vitamin K anticoagulant therapy (warfarin), extrinsic and common pathway

Evaluation of clotting screen aPPT PT(Quick) trombo-cytes Bleedin time Common Causes Vessel disorder, f. XIII deficiency ↑ Thrombocytopathy ↓ thrombocytopenia Heparin, deficiency of VIII, IX, XI, XII, HMWK, prekallikrein Factor VII deficiency Warfarin, vit. K deficiency, I, II, V,X vonWillebrand disease Liver disease, DIC, sepsis

Inhibition of coagulation

(contact with collagen) Intrinsic pathway (contact with collagen) Antithrombin 3 XIIa (Hageman) + Antithrombin 3 Extrinsic pathway (tissue factor) + XIa (PTA) { + + } Antithrombin 3 VIIa (konvertin) IXa (Christmas) Protein C Membrane phospholipides III (tissue tromboplastin) Ca++ VIIIa (AF) Platelet factor 3 (platelet phospholipides) Ca++ + } Antithrombin 3 Xa (Stuart-Prower) Protein C Va (accelerin) + Antithrombin 3 IIa (thrombin) Fibrin web + XIIIa (fibrin stab. f.) fibrin monomer I (fibrinogen) +

Antitrombin 3 Protein C Protein C Antithrombin 3 Antitrombin 3 Antithrombin 3 Protein C Antithrombin 3 Protein C Antithrombin 3

Antithrombin 3 Protein C Antithrombin 3

Heparin Heparin Heparin Platelet factor 4 Non-active Antithrombin 3 IXa (Christmas) Xa (Stuart-Prower) XIa (PTA) XIIa (Hageman) Platelet factor 4 Heparin Non-active Antithrombin 3 Antithrombin 3 Antithrombin 3 IIa (trombin)

(contact with collagen) Intrinsic pathway (contact with collagen) Antithrombin 3 XIIa (Hageman) + Antithrombin 3 Extrinsic pathway (tissue factor) + XIa (PTA) { + + } Antithrombin 3 VIIa (konvertin) IXa (Christmas) Protein C Membrane phospholipides III (tissue tromboplastin) Ca++ VIIIa (AF) Platelet factor 3 (platelet phospholipides) Ca++ + } Antithrombin 3 Xa (Stuart-Prower) Protein C Va (accelerin) + Antithrombin 3 IIa (thrombin) Fibrin web + XIIIa (fibrin stab. f.) fibrin monomer I (fibrinogen) +

Fibrinolysis =thrombolysis

Plasminogen Activator Thrombolysis Plasminogen activator Plasminogen Activator Inhibitor of Inactivates Inhibitor of Plasminogen Activator Inhibitor of Plasminogen Activator Aktivátor plazminogenu Protein S Protein C + non-active protein C Protein S Protein S Protein C Protein C VIIIa (AF) Va (akcelerin) protein C activator + IIa (thrombin) Thrombomodulin Endothelium

Hypercoaguability (thrombophilic) disorders

Most common hypercoagubility disorders Mutation of factor V – Leiden mutation / insensitivity to protein C Defect in Antithrombin III Defect in Protein C Defect in Protein S