HEMOSTASIS

HEMOSTASIS COMPONENTS Vessel wall Platelets Coagulation enzymes Fibrinolytic system Control mechanisms, including inhibitors Normal hemostasis in involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors.Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would “clot up” at the slightest stimulus. NOTE: Normal hemostasis involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors. Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would “clot up” at the slightest stimulus.

Sequence of Changes With Vascular Injury Injury to vessel wall (endothelium) with resultant exposure of subendothelium Platelet adhesion mediated by HMW vWF Simultaneous activation of clotting enzymes Platelet aggregation via fibrinogen receptors on platelets Anchoring of platelet plug by cross-linked fibrin Let’s expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen. NOTE: Let’s expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen.

Normal Hemostasis Vessel Wall Exposed collagen vFW large multimers Contraction Tissue TPL Platelet adhesion Activation of Coagulation Platelet Aggregation Thrombin Definitive Hemostatic Plug 1º Hemostatic Plug This slide gives a diagrammatic representation of the whole process and the functional interrelationships. We will now go on to dissect the various components of this process in more detail. This slide gives a diagrammatic representation of the whole process and the functional interrelationships. We will now go on to dissect the various components of this process in more detail. Limiting Reactions

Platelet Components RECEPTORS vWF Fibrinogen Clotting Factors Canaliculus Actin RECEPTORS Alpha granules Dense Granules vWF Fibrinogen Clotting Factors

Platelet Granules Alpha granules vWF Fibrinogen PF4 Beta thromboglobulin PDGF Dense granules (delta) ADP Serotonin

Platelet Role in Hemostasis vFW binding sites- platelet adhesion Fibrinogen binding sites- platelet aggregation Multiple binding sites for coagulation factors - enhances appropriate steric relationships Production of multiple chemical mediators Binding sites for chemical mediators First, lets look more closely at the role of platelets. They play multiple roles in the hemostatic process, and, contrary to the view held 30 years ago, when they were thought to a small part of the process, many would now consider them to have a central role. We already have alluded to this by indicating that the first step after injury is platelet adhesion to the subendothelium. This is mediated by subendothelial HMW vWF binding to specific receptor sites on the platelet membrane. In, addition there binding sites that play a role in virtually every step of the process. Also. chemical mediators are synthesized by internal organelles, and they contain a contractile protein that is responsible for clot retraction. The coagulation enzymes are present in relative low concentrations in plasma. Binding of certain of these factors to specific receptors on the surface of activated platelets allows them to line up in appropriate steric configuration, catalyzing the process and limiting the reactions to the area of injury. First, lets look more closely at the role of platelets. They play multiple roles in the hemostatic process, and, contrary to the view held 30 years ago, when they were thought to a small part of the process, many would now consider them to have a central role. We already have alluded to this by indicating that the first step after injury is platelet adhesion to the subendothelium. This is mediated by subendothelial HMW vWF binding to specific receptor sites on the platelet membrane. In, addition there binding sites that play a role in virtually every step of the process. Also. chemical mediators are synthesized by internal organelles, and they contain a contractile protein that is responsible for clot retraction. The coagulation enzymes are present in relative low concentrations in plasma. Binding of certain of these factors to specific receptors on the surface of activated platelets allows them to line up in appropriate steric configuration, catalyzing the process and limiting the reactions to the area of injury.

von Willebrands Factor Synthesized in megakaryocytes and endothelial cells - approx. 230,000 M.W. Macromolecular multimer plasma: M.W. 1 x 106 - 10 X106. Plasma carrier of Factor VIII, stabilizes it Large molecular forms: a. Most effective in platelet adhesion b. Predominate in endothelial cells and subendothelium

Coagulation Cascade TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?) IX IXa TFPI VIII FACTOR X Xa V, Ca FACTOR II IIa FIBRINOGEN FIBRIN MONOMERS FACTOR XIII IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT) APTT PT PHOSPHOLIPID APTT CONTACT ACTIVATION FXII, FXI, FLETCHER, FITZGERALD FACTORS IX IXa INHIBITION OF CLOTTING VIII THROMBOMODULIN PROTEIN C PLATELET ACTIVATION

Coagulation Cascade- PT TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?) IX IXa TFPI VIII FACTOR X Xa V, Ca FACTOR II IIa FIBRINOGEN FIBRIN MONOMERS FACTOR XIII IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT) APTT PT PHOSPHOLIPID APTT CONTACT ACTIVATOR FXII, FXI, FLETCHER, FITZGERALD FACTORS IX IXa INHIBITION OF CLOTTING VIII PHOSPHOLIPID THROMBOMODULIN PROTEIN C PLATELET ACTIVATION (ENDPOINT)

Coagulation Cascade- APTT TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?) IX IXa TFPI VIII FACTOR X Xa V, Ca FACTOR II IIa FIBRINOGEN FIBRIN MONOMERS FACTOR XIII IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT) APTT PT PHOSPHOLIPID APTT CONTACT ACTIVATOR FXII, FXI, FLETCHER, FITZGERALD FACTORS IX IXa INHIBITION OF CLOTTING VIII THROMBOMODULIN PROTEIN C PLATELET ACTIVATION (ENDPOINT)

Control Mechanisms Intact endothelial cells Circulating inhibitors Chemical mediators Membrane bound receptors Synthesizes activators of fibrinolysis Circulating inhibitors Protein C “system” Fibrinolytic system Endothelial component plasma protein component

Intact Endothelium Limits Hemostasis INHIBITS PLATELET ACTIVATION INACTIVATES PAI FIBRINOLYSIS INACTIVATES Va + VIIIa PLASMINOGEN PLASMIN NO PGI2 ADPase INHIBITS Xa + THROMBIN PROTEIN S PROTEIN C PROTEIN Ca ATIII PLASMINOGEN ACTIVATORS (tPA) (uPA) THROMBIN + HEPARAN THROMBOMODULIN Subendothelium vFW multimers

Intact Endothelium Limits Hemostasis – Fibrinolysis INHIBITS PLATELET ACTIVATION FIBRINOLYSIS INACTIVATES PAI INACTIVATES Va + VIIIa PLASMINOGEN PLASMIN NO PGI2 ADPase INHIBITS Xa + THROMBIN PROTEIN S PROTEIN C PROTEIN Ca ATIII PLASMINOGEN ACTIVATORS (tPA) (uPA) THROMBIN + HEPARAN THROMBOMODULIN Subendothelium vFW multimers

Intact Endothelium Limits Hemostasis INHIBITS PLATELET ACTIVATION “Protein C System” INHIBITS PLATELET ACTIVATION INACTIVATES PAI FIBRINOLYSIS INACTIVATES Va + VIIIa PLASMINOGEN PLASMIN NO PGI2 ADPase INHIBITS Xa + THROMBIN PROTEIN S PROTEIN C PROTEIN Ca ATIII PLASMINOGEN ACTIVATORS (tPA) (uPA) THROMBIN + HEPARAN THROMBOMODULIN Subendothelium vFW multimers

Intact Endothelium Limits Hemostasis - Chemical Mediators INHIBITS PLATELET ACTIVATION INACTIVATES PAI FIBRINOLYSIS INACTIVATES Va + VIIIa PLASMINOGEN PLASMIN NO PGI2 ADPase INHIBITS Xa + THROMBIN PROTEIN S PROTEIN C PROTEIN Ca ATIII PLASMINOGEN ACTIVATORS (tPA) (uPA) THROMBIN + THROMBOMODULIN HEPARAN Subendothelium vFW multimers

Production of Coagulation Factors Synthesized in the liver- All except Factor VIII Vitamin K dependent II, VII, IX, and X Protein C, Protein S Factor VIII- unknown

Vitamin K Dependent Enzymes: Factors II, VII, IX, X Synthesized in liver Serine proteases, inactivated by AT3 Activation on surface of biologic membranes Have an affinity for binding calcium

Coagulation Cascade- PT TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?) IX IXa TFPI VIII FACTOR X Xa V, Ca FACTOR II IIa FIBRINOGEN FIBRIN MONOMERS FACTOR XIII IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT) APTT PT PHOSPHOLIPID APTT CONTACT ACTIVATOR FXII, FXI, FLETCHER, FITZGERALD FACTORS IX IXa INHIBITION OF CLOTTING VIII PHOSPHOLIPID THROMBOMODULIN PROTEIN C PLATELET ACTIVATION (ENDPOINT)

Coagulation Cascade- APTT TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?) IX IXa TFPI VIII FACTOR X Xa V, Ca FACTOR II IIa FIBRINOGEN FIBRIN MONOMERS FACTOR XIII IN VITRO CROSS-LINKED FIBRIN PT (STABLE FIBRIN CLOT) APTT PT PHOSPHOLIPID APTT CONTACT ACTIVATOR FXII, FXI, FLETCHER, FITZGERALD FACTORS IX IXa INHIBITION OF CLOTTING VIII THROMBOMODULIN PROTEIN C PLATELET ACTIVATION (ENDPOINT)

Prothrombin Time Poor reproducibility from lab to lab in US No good assayed standards Many manufacturers Many chemically different reagents Many different types of instruments Poor lot-to-lot reproducibility even from same manufacturer

Reporting Protime Results Each laboratory must establish it’s own normal range using the instrument and reagents that it is using It may have to be redone with each new lot of reagents, certainly, at least rechecked and verified- insist on it from the laboratory you use Results should be expressed in seconds, not INR Results should be compared to the normal range. The true “Control” value is meaningless for clinical use.

Prothrombin Time “The INR is the answer to our prayers- Hallelujah”- NOT!! Poor calibration by reagent manufacturers is the weak link in the chain Intended only for inter laboratory comparisons in patients who are on steady state anticoagulation with coumadin: at least two weeks of therapy, ambulatory, normal activity and diet Widely misapplied to express protime results in all other situations

Bleeding Time Widely misused as a screening test for platelet function abnormalities Can predict trends when used to study large populations Cannot predict bleeding risk in individual patients - use for this purpose has been discredited

Screening for Hemostatic Defects PT, APPT- sensitivity is too poor to pick up mild defects Bleeding time- poorly reproducible, too many false positives and false negatives, Most common cause of a prolonged bleeding time- improperly performed Best screen: good history

Screen for Platelet Abnormalities No good tests, history Immediate bleeding Mucous membrane bleeding Easy bruising Petechiae

Screen for Clotting Factor Deficiencies Delayed onset of bleeding Large ecchymoses or hematomas Bleeding into joints

Screening History for Bleeding Problems Do you think you have a bleeding problem? Does anyone in your family have a bleeding problem? Easy bruising? Previous hemostatic challenges: Major surgery Trauma Extraction of impacted teeth

Bleeding Problems Pre-operative screening Patient with suspicious history Actively bleeding patient

Pre-operative Screening Most common hereditary bleeding problems? Acquired bleeding problems? Sensitivity of screening tests?

Hereditary Bleeding Disorders von Willebrand’s disease - platelet function Storage pool disease (delta granule deficiency) - platelet function Factor VIII deficiency (Hemophilia A) Factor IX deficiency (Christmas disease) Factor XI deficiency

Patient with Suspicious History Refer to laboratory or specialist that specializes in bleeding disorders.

Actively Bleeding Patient Focal bleeding - catgut insufficiency Generalized bleeding - Thrombocytopenia Vitamin K deficiencies - common DIC - most over-diagnosed cause of bleeding in the acute care/ICU setting. Primary fibrinolysis - rare

Acquired Bleeding Problems drug-induced platelet function defects Thrombocytopenia vitamin K deficiency Liver disease Coagulation inhibitors Post viral Misc. others Idiopathic

Vitamin K Deficiency Appropriate clinical setting: a. Poor or no oral intake b. Broad spectrum antibiotics Prolonged PT, PTT with a normal platelet count and fibrinogen - presumptive diagnoses of Vitamin K deficiency

Acute DIC: A clinical-pathologic Dx Severely, acutely ill patient (not clinically stable). Decreasing platelet count and/or fibrinogen.

Acute DIC Principles Most over-diagnosed cause of bleeding in a hospital/ICU setting. Should be approached as a diagnosis of exclusion If it is the only diagnosis you can think of, you are over your head. GET HELP Vitamin K deficiency is much more common. Many other factors are more likely to be the cause of thrombocytopenia.

Possible DIC Run all tests on the same specimen: PT, PTT, Fibrinogen, FDP Platelets (Factor V, Factor VIII). It may take sequential testing to establish diagnosis.

Sources of Vitamin K Diet- Fresh, green leafy vegetables Synthesis by bacteria in the intestinal track Typical ICU/acutely ill, hospitalized patient No or poor oral intake Broad spectrum antibiotic therapy Increased vitamin K requirement because of illness Result: Acquired vitamin K deficiency within two to three days of admission

Vitamin K Dependent Factors II, VII, IX, X PT - II, VII, X APTT - II, IX, X

Vitamin K Deficiency vs. Acute DIC PT Prolonged N or prolonged APT Prolonged N or prolonged FDP Normal Usually elevated Fibrinogen Normal N or decreased *Platelets Normal Usually decreased

Elevated Levels of FDP Recent surgery Acute thromboembolic event Renal failure Hepatic failure Acute myocardial infarction DIC TTP/HUS Primary fibrinolysis

Suggested Approach to the Bleeding Hospitalized Patient Draw PT, APTT, FDP, fibrinogen and platelet count on same specimen as a panel. Do not try to use values drawn at different times. Immediately give Vitamin K Redraw panel in 4-6 hours. K deficiency should show some degree of correction of PT and APTT within this time period DIC should manifest itself by a decreasing fibrinogen without any significant correction of PT< APTT

Diagnostic Approach to Thrombocytopenia Good history; medication - don’t forget heparin; Acuteness of onset; Underlying diseases Physical - splenomegaly Examination of blood smear by an experienced individual; platelet morphology, Other hematologic abnormalities Bone marrow examination - almost never helpful in the absence of other hematologic abnormalities