MDL (242 & 430) - Coagulation

Identify the enzyme that degrades fibrin

*Plasmin* o Plasminogen is activated by tissue plasminogen activator to produce plasmin which then degrades fibrin into x monomers. o Plasminogen is produced by the liver

Prolonged aPTT causes

*Presence of heparin* *Liver disease, other liver conditions* Vitamin K deficiency Hemophilias DIC *von Willebrand disease* *Lupus anticoagulant* Inadequate levels of Factors I, II, V, VIII, IX, X, XI, and/or XII

INR Normal Values

0.9-1.1 normal, 2.0- 3.5 therapeutic

vWF classification

3 major subclasses Type 1: Partial quantitative deficiency of vWF Mild-moderate disease 60%-80% Type 2: Qualitative deficiency of vWF Mild to moderate disease 15%-30% Type 3: Total or near total deficiency of vWF Severe disease 5%-10% Additional subclass Acquired vWD

Venous thromboembolisms (VTE)

30 % mortality rate; clot fragment travels to lungs causing a pulmonary embolism (PE)

When to preform a mixing study

A mixing study should be considered when a patient has a prolonged PT and/or APTT along with: No history of heparin or warfarin therapy No history of liver disease, DIC, or Vit K deficiency No pre-analytical errors with specimen collection Differentiate between a factor deficiency and a coagulation inhibitor Performed by mixing the patient plasma that has a prolonged PT or aPTT with normal pooled plasma. A PT and/or aPTT is then retested on the mixed sample.

Von Willebrand Disease

A platelet disorder - essential for platelet binding. vWF is an adhesive plasma glycoprotein. Roles in both primary and secondary hemostasis. 1/3rd is within circulation and the rest is stored in the endothelial. Binds GPIb and GPIIb-IIIa on activated platelets. Functional defect in Von Willebrands factor (vWF) itself. Binds to Factor VIII, Platelets and connective tissue. Affects primary and secondary hemostasis In secondary: Carrier protein for Factor VIII Protects FVIII from proteolytic degradation Brings FVIII to the site of vascular injury. Stabilizes the hemostatic plug against shear forces Often clinically manifests with a concurrent deficiency of factor VIII, but will present with a normal platelet count. Both men and women are affected equally. Treatment - administration of cryoprecipitate

Prolonged aPTT interpretation

A prolonged aPTT suggests a deficiency in one or more of the associated factors. An aPTT mixing study may be ordered in this case. Since heparin inhibits the activity of multiple coagulation factors in the aPTT test, it is advisable to perform a thrombin time (TT) to rule out heparin contamination prior to performing a mixing study.

Fibrinogen Assay

A quantitative measure of factor I. Used to determine whether there is enough fibrinogen present to allow for normal clotting. Performed in cases of an unexpected, prolonged bleeding event, or an unexpected abnormal PT and/or APTT. Aids in the diagnosis of disseminated intravascular coagulation (DIC). A normal reference range is typically around 200-400 mg/dl. That range is significant because fibrinogen levels <100 mg/dl are often associated with free bleeding, or impaired hemostatic function.

Platelet Function Assay

A screening test for the evaluation of platelets/primary hemostasis. Common clinical applications include the following: Preoperative evaluation of platelet function Determining the presence of drug-induced platelet dysfunction Determining platelet functionality in high-risk pregnancy Evaluation of patients with suspected inherited or acquired platelet disorders such as von Willebrand disease Evaluation of a bleeding patient Replaced the bleeding time test.

Tests for Lupus anticoagulant

APTT - most commonly used for initial screening Although LA causes the blood to clot, coagulation test will be prolonged due to the difference between in vivo and in vitro processes. No single screening test can detect all lupus anticoagulant-positive (LA-positive) patients. Several tests are available and at least two should be employed to verify the presence of LA. Dilute Russell's Viper Venom time (dRVVT) Platelet neutralization procedure (PNP) Hexagonal Phospholipid Neutralization Assay Anti-Phospholipid Antibody Assays

Extrinsic Pathway

Activated by tissue factor which is released after vessel damage Factor VII combines with Ca+ and tissue factor forming activated factor VII (VIIa) VIIa converts factor X to Xa which begins the 'common pathway' Process takes 10-15 seconds

Protein C and Protein S

Activated protein C inhibits Va and VIIIa activity Protein C is activated by Thrombin and by Thrombomodulin from the endothelial cell Protein S is also necessary to activate Protein C Protein C's activity is enhanced by Protein S Protein C is Vitamin K dependent

Bleeding Time Test

An incision is made Time to stop bleeding is measured Normal clotting time is 7.5 minutes Lacks standardization between persons performing Not reproducible Used to assess platelet function, but may be affected by platelet quantity. Cannot be used to identify patients who may have recently ingested aspirin or non-steroidal anti-inflammatory drugs or patients who may have a platelet defect attributable to these drugs. Aspirin, and some other drugs, may falsely prolong bleeding times. Patients must be asked about aspirin use, and be aspirin free for 7-10 days prior to testing, for valid results.

Anticoagulation Therapy

Anticoagulant therapy is employed in a number of clinical situations which include: After an episode of thrombosis, such as deep venous thrombosis (DVT) in the veins of the legs, to prevent reoccurrence. Prophylactically after some surgeries, especially those involving vascular repair such as coronary bypass surgery to prevent clots from blocking newly formed vasculature. In heart valve and chamber disorders where there is an increased risk of thrombosis occurring.

Another name for Factor VIII & pathway

Antihemophilic factor Procoagulant protein Carrier protein Von Willebrand factor & Intrinsic

Lupus anticoagulant

Antiphospholipid antibody Falsely named Is not only see with Lupus disease. Only 50% of cases are in those with Lupus Increases clotting not bleeding, as the name would suggest.

Review and understand the general mechanism of the other natural anticoagulants (ATIII, Protein C and S)

Antithrombin III o Main inhibitor of thrombin, factor Xa, factor IXa, factor XIa, factor XII, activated protein C and kallikren. o Cofactor of heparin. Heparin increases ATIII's inhibiting ability by as much as 1000X. Protein C & S o Activated protein C inhibits Va and VIIIa activity o Protein C is activated by Thrombin and by Thrombomodulin from the endothelial cell o Protein S is also necessary to activate Protein C o Protein C's activity is enhanced by Protein S o Protein C is Vitamin K dependent

Common Pathway

Begins with activation of Factor X by either intrinsic or extrinsic pathway. Factor Xa + factor V, calcium and phospholipids converts prothromin (II) to its active form, thrombin (IIa) Thrombin (IIa) activates Factors VIII and V to convert Fibrinogen (I) to fibrin monomers. Fibrin clot- unstable Clot is stabilized by converting Factor XIII to XIIIa which helps create fibrin polymers.

Coagulation test requirements for specimen collection

Blood collected in Na Citrate tubes Blood to anticoagulant ratio is CRITICAL: 9:1 - overfill /underfill not acceptable Na Citrate binds with all the Ca+ Ca+ necessary at steps in Coagulation Cascade, so blood will not clot Analyzers test how long blood takes to clot following each pathway by adding back Ca+ and other needed chemicals to begin cascade

Vitamin K Deficiency

Can cause a loss of functionality in Vitamin K dependent coagulation factors, specifically, factors II, VII, IX and X. Most often associated with a diet lacking in Vitamin K, it may also present in situations of broad spectrum antibiotic use, where normal flora in the gut have been eliminated. Treatment involves a diet rich in Vitamin K containing foods, and judicious use of broad spectrum antibiotics.

Another name for Factor IX & pathway

Christmas factor & intrinsic

Thrombin

Circulates in its inactive form, Prothrombin Proteolytic enzyme converts Fibrinogen to Fibrin Activates factor XIII Provides positive feedback in clotting process by activating factor VIII, enhancing factor V and aggregating more platelets. Activates Protein C as a negative feedback.

Explain the role of Factor XIII.

Clot is stabilized by converting Factor XIII to XIIIa which helps create fibrin polymers.

Activated Clotting Time (ACT)

Clotting time of whole blood in the presence of silica based activator. Normal clotting times = 90 to 170 sec. Used to monitor heparin doses from 1 to 10 U/mL (APTT is sensitive to heparin at 0.2 to 1 U/mL). Used with invasive procedures that require on-site adjustment of heparin and protamine dosage. (ex. Cardiopulmonary bypass surgery). Not amenable for use with an optical instrument, too cloudy. Point-of-care test

Interpretation of Mixing Study results

Correction of both the immediate and incubated PT/aPTT tests, the patient most likely has a factor deficiency (or multiple factor deficiencies). No correction in either the immediate or incubated PT/aPTT, the patient may have a coagulation inhibitor, most likely a lupus anticoagulant. Correction for the immediate PT/aPTT results, but no correction in the incubated PT/aPTT, the patient may have a slow-acting inhibitor such as anti-factor VIII

Tests for Fibrinolysis

D-Dimer - ELISA or latex-agglutination Euglobulin Lysis Time: Measures time it takes for a clot to dissolve in the absence of plasmin inhibitors A ↓ ELT suggests increased fibrinolytic potential Fibrin Degradation Products (FDP): ↑ amounts associated with thrombotic episodes With an elevated PT, APTT and ↓ PLT, suggestive of DIC

Explain why D-Dimer and FDP are important and how they are formed.

D-dimer has a Negative predictive value test (rules out) DVT and PE. o Specific to the D fragments of FDP's o Result of plasmin's action on cross¬linked fibrin FDP

vWF Treatment

DDAVP (Desmopressin acetate) - Causes the vWF in storage to be released into circulation Not effective in all patients Possible side effects prevent it from being used in children Factor concentrates Cryoprecipitate - in ermegency situations

Hemophilia B

Deficiency of factor IX. Sex-linked recessive, most commonly seen in males Clinical symptoms - almost identical to Hemophilia A Treatment - administration of Factor IX concentrates.

Hemophilia A

Deficiency of factor VIII. The most commonly encountered hereditary based coagulation disorder. Sex-linked recessive, most commonly seen in males Clinical symptoms - mild to severe . Classic symptom - spontaneous bleeding into their joints Treatment - administration of commercial factor VIII products.

Describe what DIC is, different causes of DIC, clinical presentation of DIC, and how it is diagnosed in the laboratory.

Disseminated intravascular coagulation o It is the consumption of factors and platelets, with thrombus being formed where it isn't needed. Micro clots occur in capillaries. Causes of DIC o TF release from monocytes from sepsis or tissue necrosisis is the primary initiator o Obstetrical: amniotic fluid, TF-like substance initiates extrinsic pathway o Tumors: thromboplastin like materials released Direct activation of factors X or II. o Snake venom: causes prothrombin to convert to thrombin Clinical Representation o Excess thrombin and plasmin activity o Depends on stage of DIC, patient clinical picture important o Confirm procoagulant and fibrinolytic activy o PTT and aPTT can be prolonged because of consumption of clotting factors o Platelets are consumed and see possible thrombocytopenia o RBC's under pressure pass through blood vessels partly closed due to fibrin strands. Result: RBC's are shredded into fragments (schistocytes). o Large increase in FDP's and D-dimers

Thrombosis Causes:

Disturbances of regulatory mechanisms: Protein S, Protein C, Antithrombin III, plasminogen Hereditary: factor V Leiden mutation, Prothrombin gene mutation; ATIII, Protein S, and Protein C deficiencies Acquired Antiphospholipid antibody, malignacy, increased factor VIII and increased fibrinogen

Secondary Hemostasis

Enzymatic activation of coagulation proteins (factors) to an end-product of fibrin, used to stabilize the platelet plug. Involves enzymes, substrates, phospholipid surfaces and cofactors

Afibrinogenemia

Extremely low fibrinogen <10 mg/dl, sometimes unmeasurable profuse bleeding with slightest trauma. Treat with cryoprecipiate or FFP

Why are Mixing studies performed?

Factor deficiency, or multiple factor deficiencies The normal pooled plasma will increase the factor levels to the point of correcting the prolonged test result. Coagulation inhibitor The normal pooled plasma will not correct the prolonged test result

Acquired vWF

Fewer than 300 cases reported Usually encountered in adults with no personal or family bleeding history Laboratory work-up most consistent with Type II vWD Mechanisms: Autoantibodies to vWF Absorption of HMW vWF multimers to tumors and activated cells Increased proteolysis of vWF Defective synthesis and release of vWF from cellular compartments Myeloproliferative disorders, lymphoproliferative disorders, monoclonal gammopathies, CVD, and following certain infections

What is the substrate for plasmin?

Fibrin & Fibrinogen

Another name for Factor XIII & pathway

Fibrin-stabilizing factor & Common

Another name for Factor 1 & pathway

Fibrinogen & Common pathway

Explain the consequence of a Factor V Leiden gene mutation.

Genetic mutation causes an impaired degradation of factor V by activated protein C (APC) • Also know as Activated Protein C Resistance • Causes recurrent thrombosis

Another name for Factor XII & pathway

Hageman factor (contact factor) & Intrinsic

Inherited Coagulation Disorders

Hemophilia A Hemophilia B Von Willebrands Disease

Two types of Coagulation Disorders

Hemorrhage - bleeding external to the vasculature, most often stems from physical vessel trauma, but may also arise from a wide variety of disease states. Thrombosis does not require physical trauma, and is the activation of hemostatic processes at an inappropriate time in an inappropriate place, and may arise from a number of inherited or acquired disease states.

Identify heparin's mechanism of action, what LMWH is and how it differs.

Heparin: • UFH is enhanced by binding with ATIII • UFH/ATIII complex inactivates thrombin and factor Xa LMWH: • Subcutaneous; patient can self administer • cannot be monitored by aPTT -is monitored by a target concentration strategy, direct test • acts to inactivate Xa. Little effect on factor IIa.

Tests to Assess Hereditary Thrombotic Risk

Hereditary risks for DVT or PE Tests include APC Resistance/Factor V Leiden Clot based assays available PCR method used to confirm

Factor Deficiency Testing

In order to do this, you must first identify which factors are possibly involved. This can be done by referring back to the principles of the PT and aPTT test and their respective factor involvements. When you have narrowed down the possible factors involved in the prolongation, the next step should be individual factor assays. These assays use a similar protocol to the original mixing study; however, pooled normal plasma is substituted with an individual factor-deficient plasma. A factor-deficient plasma is used for each factor that is suspected to be causing the prolongation. If the aPTT or PT is still prolonged after mixing the patient sample with the specific factor-deficient plasma, this is most likely the factor that is deficient in the patient sample.

What is the INR?

International Normalization ratio o standardization of test results worldwide o 0.9-1.1 normal, 2.0- 3.5 therapeutic o INR= (patient's PT/geometric population mean PT)isi

The three major pathways in the cascade theory of coagulation

Intrinsic, Extrinsic and Common Pathway

Another name for Factor IV & pathway

Ionized Calcium (Ca++) & Extrinsic

Another name for Factor V & pathway

Labile factor & Common

Hypofibrinogenemia

Low fibrinogen 20 - 100 mg/dL Bleeding mild and not spontaneous

Antithrombin III

Main inhibitor of thrombin, factor Xa, factor IXa, factor XIa, factor XII, activated protein C and kallikren. Cofactor of heparin. Heparin increases ATIII's inhibiting ability by as much as 1000X.

Coagulation Instrumentation

Manual methods Tilt-tube Wire device may be used to look for fibrin strand Semi-automated Still need to manually add reagents and samples Fully automated

Expected PT Values

Mean Normal Plasma = 10 to 14 seconds. Mean Normal Plasma value varies with PT sensitivity. A high sensitivity (Low ISI) PT will give a high normal PT value (13 to 15 seconds). Oral anticoagulant monitoring = Target INR of 2.0 to 3.0. INR of greater than 5 or 5.5 = unacceptable high risk of bleeding.

What does aPTT measure?

Measures the adequacy/functionality of factors that comprise the intrinsic and common pathways (I, II, V, VIII, IX, X, XI, XII) Used to determine therapeutic effectiveness of heparin administration.  Results are presented to the clinician in seconds- the actual time elapsed until a clot was detected.

Methods of Endpoint Detection

Mechanical: Electromechanical Steel ball movement Photo-Optical, turbidometric Chromogenic - uses color-producing substance Immunologic - antigen/antibody reactions

vWF Type 1

Mild to moderate Quantitative deficiency (20-40% of normal levels) vWF is functionally normal May see a deficiency of Factor VIII

Expected aPTT values

Normal Range: 26 to 40 seconds Slightly Elevated: 45 to 65 seconds Extremely Elevated = > 70 seconds

Fibrinogen

Normal range 200-400 mg/dL Principal substrate of coagulation system 5 grams /day produced by the liver

Describe the common laboratory tests for evaluation of hemostasis and what pathways each test detects.

PT - tests the effectiveness of the Extrinsic pathway (VII) o Used to measure coumadin/warfarin therapy o Detects Vitamin K deficiency aPTT - tests the effectiveness of the Intrinsic and common pathways o Used to monitor heparin anticoagulant therapy

Fibrinolysis

Physiologic removal of fibrin deposits through a gradual enzymatic cleavage of fibrin to soluble fragments to reestablish blood flow Plasminogen is an inactive plasma protein produced by the liver When plasminogen is activated by tissue plasminogen activator (t-PA), found in the vascular endothelium, it is converted to Plasmin, the active enzymatic form The substrate for Plasmin is fibrinogen and fibrin Plasmin breaks fibrin (or fibrinogen) to the X monomer X monomer is split into Y and D fragments Y monomer is split into D and E fragments which are known as fibrin split products (FSP) or fibrin degradation products (FDP)

Another name for Factor XI & pathway

Plasma thromboplastin antecedent (PTC) & Intrinsic

Differentiate primary and secondary hemostasis.

Primary hemostasis - Vasoconstriction - formation of a platelet plug o Forms a temporary platelet plug very weak o Sensitive to decreased platelets • Secondary hemostasis - The coagulation cascade stabilized fibrin clot o Stronger repair of the broken blood vessel o Disorders can originate from decreased clotting factors or clotting inhibitors o More serious disorders

Heparin Therapy

Prophylactic, either to prevent thromboembolism (a condition in which a blood clot forms inside a vessel), or used to limit a previous thromboembolism. Heparin inhibits thrombin. The degree of inhibition is dosage dependent. Low doses of heparin inhibit initial thrombin formation in the coagulation cascade, and act to slow down overall thrombin generation. At higher doses, heparin can inhibit thrombin entirely, making blood coagulation impossible. Heparin is a potent anticoagulant. Accurate monitoring is essential. The activated partial thromboplastin time (APTT) and/or activated clotting time is used to monitor unfractionated heparin therapy.

Fibrin Formation

Proteolysis- Thrombin cleaves Fibrinogen into fibrin monomers and peptides Polymerization- monomers join end to end to form polymers with weak hydrogen bonding Stabilization- Factor XIIIa links the monomers into polymers covalently, forming insoluble fibrin clot

Another name for Factor II & pathway

Prothrombin & Common pathway

vWF Type 3

Quantitative deficiency (little to no vWF) Symptoms are severe with spontaneous episodes of bleeding Deficiency of Factor VIII also present Patient is unable to make a platelet plug and stabilize it

Use of aPTT

Sensitive to 30 to 40% deficiencies of all factors except VII and XIII. Heparin inhibits the activity of clotting factors in the Intrinsic Pathway. A standard curve (APTT time vs Heparin concentration) is prepared using a heparin standard. APTT is also sensitive to other non-specific Factor inhibitors such as Lupus Anticoagulant. Can be influenced by Vitamin K deficiency and Coumadin therapy. *Normal APTT result usually rules out Hemophilia*

Another name for Factor VII & pathway

Stable factor & Extrinsic

Another name for Factor X & pathway

Stwart-Prowler & Common

vWF Type 2

Symptoms are mild to moderate but usually more severe than Type 1 Qualitative deficiency Consists of 4 Subtypes - categorization depends on presence and behavior of multimers 2A - platelets cannot make a good plug 2B - vWF is too active and binds platelets within circulation 2M - vWF unable to bind to platelets 2N - vWF unable to be carrier and protector of Factor VII

PT/INR Calculation

The INR calculation is INR= (PT of patient/PT of geometric mean of the normal population)ISI

D-Dimer test

The presence of D-dimers in plasma or whole blood indicates that fibrin has been formed and degraded (fibrinolysis). Can be useful in the diagnosis of deep venous thrombosis (DVT) or pulmonary embolism (PE), two forms of venous thromboembolism (VTE). If a negative result is reported then it practically rules out DVT or PE. A positive result would require further testing. A sensitive, but not specific, diagnostic test for disseminated intravascular coagulation An indicator of increased risk of future myocardial infarction in patients evaluated for chest pain. *Negative predictive value test (Rules OUT)* -Tests for DVT and PE (pulmonary embolism) -Don't confuse with FSP and FDP (non-specific) -Specific to the D fragments of FDP's -Result of plasmin's action on crosslinked fibrin *Normal range is < 0.5ug/mL*

Oral Anticoagulant Therapy (OAT)

The therapeutic use of oral anticoagulants is typically the long-term solution for the patient in terms of managing situations of thrombosis. Warfarin, a dicumarol derivative, is one of the most popular oral anticoagulants used today. While heparin is administered intravenously and acts to inhibit thrombin, warfarin is given orally, taken in pill form, and functions as a Vitamin K antagonist. Certain clotting factors are considered to be vitamin K dependent. They require vitamin K molecules for their action to occur. Vitamin K dependent factors include factor II, VII, IX, and X. Vitamin K dependent metabolic processes involved with these coagulation factors are inhibited by drugs such as warfarin. The chemical structure of warfarin and similar anticoagulants enables them to bind competitively with free vitamin K. The prothrombin time (PT/INR) is used to monitor oral anticoagulant therapy.

Factor Assays

Think of mixing studies as being the screening test, while factor assays are confirmatory tests for specific factor deficiencies. Involves performing a PT and aPTT, except that plasma known to be deficient in a specific factor type is combined with the patients plasma, comparing the resultant time to a standard curve. This assay involves mixing various dilutions of a patient sample with the specific factor-deficient reagent plasma. Plot clotting time vs sample dilution and compare to a standard curve A standard curve is created utilizing known commercial standards When developing the standard curve, at least 3 points must be plotted to ensure accuracy when utilizing the curve for patient factor activity assays. Once the standard curve has been constructed and all points plotted, the approximate amount of factor contained in the patient sample can be interpreted. The patient sample must have two or more points plotted against the standard curve, which enhances accuracy and allows for the recognition of inhibitors in the patient sample. These points must fall within the acceptable range of the standard curve to be considered valid.

Negative Feedback Pathways

Thrombin- enhances factor V and VIII activities until levels are too high, then destroys them by proteolysis Factor Xa- enhances factor VII activity, but limits VII from activating more factor X and tissue factor through tissue factor pathway inhibitor (TFPI) Negative feedback pathways by these enzymes self-limit their own ability to activate the coagulation cascade.

Thrombin Time (TT)

Time required for thrombin to convert fibrinogen to insoluble fibrin Measures levels of fibrinogen Thrombin is the reagent added to the plasma specimen Normal is 10-15 seconds

Another name for Factor III & pathway

Tissue thromboplastin (tissue factor) & Extrinsic

Heparin Function

Unfractionated heparin (UFH) is a naturally occuring anticoagulant in human tissue Commerical UFH is from bovine, lung or porcine sources UFH is enhanced by binding with ATIII UFH/ATIII complex inactivates thrombin and factor Xa Fast acting, potent. Half-life is dose dependent Efficacy can be affected by low ATIII or heparin resistance

Intrinsic Pathway

Vascular trauma initiates factor cascade Presence of prekallikrein activates XII to XIIa XIIa activates XI to XIa XIa activates IX to IXa IXa + VIII + Ca + PF3 activates X to Xa beginning the 'common pathway' Takes longer than extrinsic pathway

Acquired Coagulation Disorders

Vit K deficiency Disseminated Intravascular Coagulation (DIC) Liver disease Anticoagulant therapy Lupus anticoagulant

Prolonged PT causes

Warfarin therapy Liver disease Disseminated intravascular coagulation (DIC) Vitamin K deficiency Liver conditions such as cirrhosis or hepatitis Inadequate level of Factors I, II, V, VII, and/or X

Inhibitor Studies

When considering the presence of a coagulation factor inhibitor other than the common lupus anticoagulant, specific inhibitors such as anti-Factor VIII should be considered. Titers can be performed for the antibody in question to quantify the inhibitor and determine the extent of the antibody proliferation. These tests are called Bethesda titer assays. These tests use a universal inhibition unit in measuring specific-factor antibody activity. The appropriate dosing of medications may be measured based on these titer results.

vWF Testing

aPTT - Mildly prolonged in approximately 50% of patients Normal PTT does not rule out vWD PT - Usually within reference ranges Prolongations of both the PT and the aPTT signal a problem with acquisition of a proper specimen or a disorder other than or in addition to vWD Historically, bleeding time is a test used to help diagnose vWD Lacks sensitivity and specificity Subject to wide variation Not currently recommended for making the diagnosis of vWD Von Willebrand Antigen - Quantitative Ristocetin Cofactor Assay - Qualitative, ability to bind platelets Von Willebrand Factor Multimers Measures the different parts of vWF Used to determine type Performed after the confirmation of vWD Collagen Binding Assay - Qualitative

Dysfibrinogenemia

altered fibrinogen molecule Fibrinogen clotting assay normal and TT increased

Coumadin/Warfarin affect what vitamin K dependent factors?

limits vitamin K dependent factors (II, VII, IX,X,PC, PS) by blocking recycling of vitamin K • Takes 5-7 days to achieve stable anticoagulation

Arterial thrombosis

stroke, heart attack (Ml), and Peripheral Vascular Disease (PVO)

vWR diagnostic difficulties

vWF levels can increase in certain situations: Pregnancy Estrogen Stress Exercise Infection vWF levels vary with blood type - lowest seen in O blood type Thyroid issues can cause vWF levels to increase or decrease

Explain various causes of thrombosis

• Disturbances of regulatory mechanisms: - Protein S, Protein C, Antithrombin III, plasminogen • Hereditary - factor V Leiden mutation, Prothrombin gene mutation; ATIII, Protein S, and Protein C deficiencies • Acquired Antiphospholipid antibody, malignacy, increased factor VIII and increased fibrinogen

What is secondary hemostasis? What does it involve?

• Enzymatic activation of coagulation proteins (factors) to an end-product of fibrin, used to stabilize the platelet plug. • Involves enzymes, substrates, phospholipid surfaces and cofactors

Explain the similarities and differences between Hemophilia A and B.

• Hemophilia A and Hemophilia B are both disorders that are due to a deficiency in a clotting factor. The clinical symptoms are almost identical and range from unexplained/excessive bleeding to pain/swelling in the joints. Both Hemophilia A & B are sex-linked recessive disorders that predominantly affect males. The difference between Hemophilia A and B lies in the treatment of the disorder due to the deficient coagulation factor. Hemophilia A patients are deficient in factor VIII which can be resolved by the administration of commercial factor VIII products. Hemophilia B patients are deficient in factor IX and are treated by the administration of factor IX concentrates.

Describe how heparin functions to inhibit clotting.

• Heparin: unbranched polysaccharide that is heavily sulfated from cow lungs or pig intestines • Give IV or subcutaneous, binds with ATIII • Inactivates factors IIa and Xa • Increases ATIII's anticoagulant properties by 1000x • Cleared by reticuloendotheial cells and by kidneys • Monitor 6 hours after dose for effectiveness and changes using APTT test • Function: • Unfractionated heparin (UFH) is a naturally occurring anticoagulant in human tissue • UFH is enhanced by binding with ATIII • UFH/ATIII complex inactivates thrombin and factor Xa • Fast acting, potent. Half-life is dose dependent • Efficacy can be affected by low ATIII or heparin resistance

Know the basic tests for hemostasis in the lab and what can interfere or cause incorrect test results.

• PT - tests the effectiveness of the Extrinsic pathway (VII) o Used to measure coumadin/warfarin therapy o Detects Vitamin K deficiency • aPTT - tests the effectiveness of the Intrinsic and common pathways o Used to monitor heparin anticoagulant therapy • Fibrinogen o Normal range 200-400 mg/dL o Principal substrate of coagulation system • D-Dimer o Tests for DVT and PE (pulmonary embolism) • Thrombin Time o Time required for thrombin to convert fibrinogen to insoluble fibrin o Measures levels of fibrinogen o Thrombin is the reagent added to the plasma specimen • Clotting Factor Testing o Not direct measurement, test for factor activity % o >50% activity is normal range o Reasons for low factor activity o Low factor production (Vit. K, congenital) o Factor inhibitors o Non-Parallelism- Upon consecutive dilutions factor results do not correlate, are diluting out factor inhibitor. • Testing Interferances: o Tube underfilled Too much anticoagulant for blood: high PT and aPTT o Tube overfilled Too much blood for anticoagulant: clotting o Must be tested within 4 hrs. Factors V and VIII are liable. o High HCT (>55%) High rbc = low plasma: result is high PT, aPTT o Improper centrifuge time Must be platelet poor plasma o Falsely shortened results due to PF4 activation Low PT or aPTT results o Microclots, fibrin strands: already clotting before test, therefore shorter clotting times High PT or aPTT o Blood clotted (clotting factors used up) Overdosed heparin o Blood drawn from an arterial line (heparin contamination)

Describe the process of fibrinolysis, the enzymes involved, and the products of fibrinolysis.

• Physiologic removal of fibrin deposits through a gradual enzymatic cleavage of fibrin to soluble fragments to reestablish blood flow • Plasminogen is an inactive plasma protein produced by the liver • When plasminogen is activated by tissue plasminogen activator (t-PA), found in the vascular endothelium, it is converted to Plasmin, the active enzymatic form • The substrate for Plasmin is fibrinogen and fibrin

Describe how plasminogen functions in the clotting process.

• Plasminogen is activated by tissue plasminogen activators and then is turned into plasmin. Plasmin is the enzyme that degrades fibrin (and fibrinogen) into fibrin degradation products (X monomers). • Part of the Fibrinolysis process

Review coagulation cascade and focus on fibrin's many roles

• Proteolysis- Thrombin cleaves Fibrinogen into fibrin monomers and peptides • Polymerization- monomers join end to end to form polymers with weak hydrogen bonding • Stabilization- Factor XIIIa links the monomers into polymers covalently, forming insoluble fibrin clot

Which factors are vitamin K dependent and why is it important?

• vitamin K dependent factors (II, VII, IX,X,PC, PS) • Deficiencies can reduce clotting ability