HEMATOLOGY PRIMARY HEMOSTASIS OUTLINE • Primary Hemostasis o Overview of Hemostasis o Vasoconstriction o Platelet Adhesion o Platelet Aggregation o Platelet Plug Consolidation and Stabilization o Laboratory Assessment of Platelets o Vascular Disorders o Abnormal Platelet Morphology o Platelet Disorders • PRIMARY HEMOSTASIS • Hemostasis is a complex process that retains the blood within the vascular system after injury. The mechanism localizes the reactions involved in the site of injury. What follows is repair and re-establishment of blood flow through the vessel which was injured. It is a system in dynamic balance that in the presence of deficiencies (congenital or acquired) in the coagulation factors favoring coagulation or there is increase of the factors favoring fibrinolysis, there will be resulting uncontrolled bleeding (hemorrhage), when tipped by deficiencies (congenital or acquired) of the fibrinolytic portion or uncontrolled activation of the procoagulant portion, the result is excessive clot formation or persistence of clot (thrombosis). • The mechanism of hemostasis requires the interaction of numerous components: blood vessels, endothelium, platelets, coagulation factors, inhibitors of coagulation, and fibrinolytic factor/substances. • Knowledge of hemostasis (controls bleeding) and fibrinolysis (clot dissolution) is necessary in order to understand of principle sof testing in the laboratory. • In primary hemostasis, constriction of the damaged blood vessels, which decrease the blood flow through the injured blood vessel. Platelets aggregate and adhere to the site of injury to form a PLATELET PLUG and inhibits bleeding. • Knowledge of the primary hemostasis mechanism is important for understanding the laboratory test to assess it. Example are bleeding time, platelet count, clot retraction tests, platelet function tests, capillary fragility, platelet closure time and others. • The laboratory tests are useful in the assessment and management of the disorders involving the platelet and blood vessels, the two main player in the mechanism. The laboratory tests guide the physician in management and institution of therapy to the patient. • Blood vessels have peripheral nervous system responsible for functioning and irritability • Vasoconstriction - neurologic response on vessel spasm o Lessening of lumen to reduce blood loss o Constriction of the vessel to prevent blood loss o Takes at least 1 minute o Nervous response takes care of this • Prolonged vasoconstriction is due to substances (serotonin) secreted by platelets when they adhere upon contact surface (subendothelium) o Serotonin - from dense/delta bodies of platelets (5-8) ▪ Prolong neurologic response to preserve blood o Adhesion is facilitated by von Willebrand Factor that serves as a bridge between GP1b/V/IX complex and contact surface of collage upon injury • After adhesion, platelet secretion/release mechanism/platelet activation where platelets from the disc-shape become spherical causing activation of organelles to release their contents • Platelets adhered to subendothelium will tend to aggregate or lay side-by-side (platelet aggregation; third stage of hemostasis) o Platelet to platelet aggregation is facilitated by ligand fibrinogen o GPIIb/IIIa - most abundant GP; where fibrin will connect during aggregation o Aggregation will produce platelet plug (product of primary hemostasis) to seal wounds o Platelet plug - loose mass, not compact ▪ May cause leak of blood ▪ Shall be stabilized for efficient blood sealing o Shall immediately move forward to secondary hemostasis to have stable fibrin clot ▪ Fibrin Polymer - In vitro clot o End product of this mechanism is FTP (fibrin, fibrinogen products) → very dangerous to the body so that they should be eaten by the mononuclear phagocyte system (reticuloendothelial system – old name) o A normal blood should be in the liquid state inside the vessels o Thrombus – aggregate of platelet o Platelet plug → automatic secondary hemostasis or coagulation • Fibrinolysis - elimination and lysis of stable fibrin clot after fibrin clot served its purpose o Plasmin/fibrinolysin - main enzyme from plasminogen/ proplasminolysin synthesized from the liver o Stable fibrin clot are eliminated by mononuclear phagocyte system (MPS) OVERVIEW OF HEMOSTASIS • Hemostasis - or cessation of bleeding o occurs within the intravascular compartment lined with endothelium o occurs naturally • Physiologic Hemostatic System o A tightly regulated balance between formation and dissolution of hemostatic plugs modulated by a series of enzymes and scaffolding proteins.
• Factors involved in Normal Hemostasis and Thrombosis o Platelets, Granulocytes & Monocytes o Coagulation Protein System (Clot Forming) ▪ Coagulation factors/Procoagulants o Fibrinolysis Protein System (Clot Lysing) ▪ Plasmin o Anticoagulation Protein System (Regulating) ▪ Anti-plasmin, anti-thrombin, thrombin S • Factors involved in Normal Hemostasis and Thrombosis o Cellular Components ▪ Platelets, Granulocytes & Monocytes) o Protein Systems ▪ Coagulation System (Clot Forming) → serves to form thrombin that initiates the proteolysis of fibrinogen, leading to fibrin clot formation ▪ Fibrinolytic Sytem (Clot Lysing) → functions to lyse the clot formed by thrombin ▪ Anticoagulation System (Regulating) → regulates all enzymes of the coagulation and fibrinolytic systems, so that no inappropriate excess of clotting or bleeding occurs. • Arteriolar vasoconstriction o Mechanism is applicable to small and medium sized vessels, if large, then surgery is the key o LS – longitudinal section of blood section o Non-contact surface – endothelium, and below it is the basement membrane o If the vessel is large, then it is tunica media o Tunica intima – osmosis and diffusion will happen o Collagen is produced by fibroblasts which feeds on Vitamin C • Platelet aggregation o Adhesion – once they are disc shaped, they will be activated o Shape changes - disc to sphere o Hallmark of platelet activation - release of granules/contents ▪ ADP - very potent aggregator ▪ Thromboxane A2 (TxA2) - potent platelet stimulator; production is engendered by cyclooxygenase (neutralized by aspirin) o Aspirin – blood thinner o Potent platelet stimulator (thromboxane A2) ▪ Thromboxane A2 is produced in the thromboxane pathway sometimes called prostaglandin pathway or eicosanoid pathway o Platelet plug sometimes called hemostatic plug o vWF - from Weibel-Palade bodies ▪ marker of ** o megakaryocytes also produce vWF when we have dysfunctional o Bernard-Soulier Syndrome – problem in platelet adhesion o Glanzmann Disease – problem in aggregation, lack in glycoprotein IIb/ IIIa o Hemorrhagic dialysis – problem in platelet adhesion and aggregation o vWF is the bridge • Platelet clot o Fibrinopolymerization – another term for fibrin clot; acted upon stable fibrin A (stable fibrin clot) o Thrombin activation – extrinsic factor o Tissue factor – intrinsic factor o PTL (platelet factor tree) – big role in secondary hemostasis, serves as the sync • Fibrinolysis - Happen after clot formation VASOCONSTRICTION • Upon vessel injury (in a large or medium sized artery or veins), contraction occurs to control bleeding. This contraction of the blood vessel wall is called vasoconstriction. • Vasoconstriction is a short-lived reflex reaction of the smooth muscle in the vessel wall produced by the sympathetic branches of the autonomic nervous system. PLATELET ADHESION • If vascular injury exposes the endothelial surface and underlying collagen, platelets adhere to the subendothelial collagen fibers, spread pseudopods along the surface, and clump together (aggregate). • Platelet adhesion to subendothelial connective tissues, especially collagen, occurs within 1 • Epinephrine and serotonin promote vasoconstriction. • ADP increases the adhesiveness of platelets.
• The transformation of the platelet from a disc to a sphere with pseudopods produces surface membrane reorganization. • Internal contraction of the platelet results in release of granular contents of the alpha and dense granules and the lysosomal contents. • Platelets adhere at sites of mechanical vascular injury and then undergo activation and express functional glycoprotein IIb/IIIa receptors (also referred to as integrin alpha IIb beta 3 ) for circulating adhesive ligand proteins (primarily fibrinogen). o These functional glycoprotein IIb/IIIa receptors mediate the recruitment of local platelets by forming fibrinogen bridges between platelets —a process called platelet cohesion. o Why mechanical vascular injury? Something hurt the vessel then undergo activation • Glycoprotein IIb/IIIa is specific for platelets. o Most abundant platelet membrane protein (with approximately 50,000 receptors per platelet). PLATELET AGGREGATION • Platelet aggregation is the gold standard test to determine platelet function. o Gold standard – best test o Platelet aggregometer – machine used • Agents capable of producing platelet aggregation in vitro (an energy dependent process): o Collagen o Proteolytic enzymes (thrombin) o Biological amines (epinephrine and serotonin) • Aggregation - results from bridges formed by fibrinogen in the presence of calcium produce a sticky surface on platelets. o If these aggregates are reinforced by fibrin, they are referred to as a thrombus. o Energy-dependent process • Aggregation of platelets by at least one pathway can be blocked by substances such as: o Prostaglandin E (PGE), o Adenosine o Non-steroidal anti-inflammatory agents (eg. aspirin) PLATELET PLUG CONSOLIDATION & STABILIZATION • Fibrinogen (under the influence of small amounts of thrombin) - provides basis for platelet consolidation & stabilization. o This process involves the precipitation of polymerized fibrin around each platelet o The result is fibrin clot that produces an irreversible platelet plug. LABORATORY ASSESMENT OF PLATELETS • Quantitative Determination of Platelet o Electronic Particle Counter o Examination of stained blood film - count the platelets directly using pipettes or indirect using blood film among 1000 RBC ▪ Platelet Estimation – you examine a blood smear and then count platelets amongst 10 Oil Immersion Field then get the mean ▪ Cannot be reported because it is only an estimation ▪ Multiply by 20,000 • Qualitative Assesment of Platelet - done if the platelet count is normal → assessment of platelet function o Peripheral Blood Smear (PBS) - are there dwarf (microthrombocytes) or giant (macrothrombocytes) platelets o Platelet Count - thrombocytopenia if it’s low, thrombocytosis is it is high, thrombocytopenia if it is over a million o Template Bleeding Time o Petechiometer - to count petechiae ▪ Petechia 1-3mm discoloration, pinpoint o Platelet Aggregation - can make use of these substances that may form into clumps ▪ Adenosine diphosphate (ADP) ▪ Epinephrine ▪ Collagen ▪ Ristocetin ▪ Arachidonate ▪ Thrombin o Platelet lumiaggregation (release) - to measure platelet release mechanism or platelet secretion o Platelet Antibodies (IgM and IgG) - to determine what antibody present, they coat the platelets in the rocess of immune complex; end point of complement system is lysis o Platelet Membrane Glycoproteins o Platelet factor IV o (Beta)-thromboglobulin - to detect if you have platelet IV (heparin neutralizing factor) o Thromboxanes • Bleeding Time with and without Aspirin o In vivo measurement of platelet adhesion and aggregation on locally injured vascular subendothelium. o Provides an estimate of the integrity of the platelet plug and thereby measures the interaction between the capillaries and platelets o If there’s no aspirin, no prolongation o Can differentiate Von Willebrand’s disease to who is normal
• Clot Retraction o Reflects the number and quality of platelets, fibrinogen concentration, fibrinolytic activity, and packed red cell volume. o The degree of clot retraction is directly proportional to the number of platelets & inversely proportional to the hematocrit and the level of the blood coagulation factor fibrinogen. o When there is expression of serum to clotted o Test primarily to platelets • Platelet Aggregation o Agents such as ADP, collagen, epinephrine, snake venom, thrombin, and ristocetin can be used to aggregate platelets. o The principle of the test is that platelet-rich plasma is treated with a known aggregating agent. If aggregated, cloudiness or turbidity can be measured using a spectrophotometer. • Antiplatelet Antibody Assays o Available techniques can include complement fixation methods, lysis of chromium 51 –labeled platelets, assays of platelet-bound immunoglobulins, and competitive inhibition assays. o To determine if there is IgM or IgG against complement fixation VASCULAR DISORDERS • Purpura associated with direct endothelial cell damage. • Purpura associated with an inherited disease of the connective tissue. • Purpura associated with decreased mechanical strength of the microcirculation. • Purpura associated with mechanical disruption of small venules. • Purpura associated with microthrombi (small clots). • Purpura associated with vascular malignancy. ABNORMAL PLATELET MORPHOLOGY • Wiskott-Aldrich syndrome o Demonstrates the smallest platelets seen o Very rare in the world • May-Hegglin anomaly o Characterized by the presence of large platelets and the presence of Döhle-like bodies in the granulocytic leukocytes • Alport syndrome B o Disorder that exhibits giant platelets and thrombocytopenia • Bernard-Soulier syndrome (giant platelet syndrome) o Demonstrates the largest platelets. In this disorder, it has been demonstrated that the giant platelets are probably an artifact of the slide o Deficiency in GP Ib/V/IX PLATELET DISORDERS QUANTITATIVE PLATELET DISORDERS • Issue in number of platelet • Thrombocytopenia o <150 x 10 9 /L / <100 x 10 9 /L o Disorders of Production ▪ Nutritional cause (vit. B12 deficiency) ▪ Idiopathic cause o Disorders of Destruction or Utilization ▪ Destruction caused by Immune Mechanisms, Antigens, Antibodies or Complement • Immune mechanism - ab & ag • Complement - attach to immune complex o Cascade process o Terminate when platelet’s surface is attacked & presence of holes o End product - membrane attack unit o Causes leakage • Increased destruction of platelets causes decreased number ▪ Heparin-Induced Thrombocytopenia • Heparin - anticoagulant (therapeutic for management of thrombosis) • May be caused by Heparin therapy due to thinning of blood ▪ Increased Utilization of Platelets • Disseminated intravascular coagulopathy (DIC) - both intrinsic & extrinsic are active o Too much platelet utilized o Widespread systemic coagulation ▪ Immune Thrombocytopenia • Involves complement • New name for idiopathic thrombocytopenic purpura ▪ Thrombocytopenia • Misc condition that lessens plt count ▪ Thrombotic Thrombocytopenic Purpura • Lacks ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type motifs) o Disorders of Platelet Distributions ▪ Platelet sequestration - distribution of plts • 2/3 plts - blood • 1/3 plts - spleen ▪ Splenomegaly ▪ Splenectomy - increases platelets in the systemic circulation • Thrombocytosis o Hereditary or familial thrombocytosis associated with ger_x0002_mline mutations of the thrombopoietin (THPO) gene in the THPO receptor (MPL) gene ▪ mutation to induce increased production o Thrombocytosis associated with myeloproliferative neoplasms and/or myelodysplastic disorders (clonal thrombocytosis associated with somatic mutations of JAK2[V617F], MPL, and additional currently unknown genes) ▪ Neoplastic cause - cancerous (thrombocythemia) ▪ Approaches millions of platelets ▪ Exhibited in platelet cancer o Reactive (secondary thrombocytosis) ▪ Over prodcution QUALITATIVE CHARACTERISTICS OF PLATELETS: THROMBOCYTOPATHY • Some platelets can be examined in HPO • Types of Platelet Dysfunction o Acquired ▪ Myeloproliferative Syndromes • Cancerous
▪ Uremia • Increased toxins in blood ▪ Paraprotein Disorders • Abnormal protein production • Multiple myeloma • Increased plasma cell in bone marrow ▪ Cardiopulmonary Bypass and Platelet Function ▪ Miscellaneous Disorders Associated with Platelet Dysfunction o Drug-Induced o Platelet Membrane Receptors o Hereditary ▪ Bernard-Soulier Syndrome - manifests in GPIB • Platelet adhesion ▪ Glanzmann Thrombasthenia and Essential Athrombia - parts of issues in platelet aggregation • Glanzmann’s - GP IIB/IIIA complex o Platelet aggregation ▪ Hereditary Storage Pool Defect • Dense bodies, alpha bodies cannot store much elements they should be storing