Platelets, blood & HemostasisPrimary hemostasis is when your body forms a temporary plug to seal an injury.

Primary hemostasis is when your body forms a temporary plug to seal an injury. To accomplish that, platelets that circulate in your blood stick to the damaged tissue and activate. That activation means they can “recruit” more platelets to form a platelet “plug” to stop blood loss from the damaged area.

1. Hemostasis

2. Platelets It seems like you're describing the process of platelet production and regulation in the body. Here's a summary of the key points from the provided text: 1. Platelet Production Platelets are produced in the bone marrow through fragmentation of the cytoplasm of megakaryocytes, which are derived from precursor cells called megakaryoblasts. 2. Megakaryocyte Maturation: Megakaryocytes undergo endomitotic synchronous replication, enlarging the cytoplasmic volume as the number of nuclear lobes increases. They develop a highly branched network of plasma membrane called the demarcation membrane. The cytoplasm becomes granular as the megakaryocyte matures. 3. Platelet Formation: Platelets are formed by fragmentation from the tips of cytoplasmic extensions of megakaryocyte cytoplasm. Each megakaryocyte can produce approximately 1000–5000 platelets. Platelets are released through the endothelium of the vascular niches of the marrow.

3. 4. Thrombopoietin (TPO): TPO is the major regulator of platelet formation, with 95% produced by the liver. TPO levels are regulated based on platelet destruction. Thrombopoietin increases the number and rate of maturation of megakaryocytes via the c‐MPL receptor. 5. Platelet Count: The normal platelet count is approximately 250 × 109 /L (range 150–400 × 109 /L), and the normal platelet lifespan is 10 days. Platelet count regulation involves the balance of apoptotic BAX and anti‐apoptotic BCL‐2 proteins. 6. Spleen's Role: Up to one-third of the marrow output of platelets may be trapped in the spleen at any one time, rising to 90% in cases of massive splenomegaly. This process ensures a steady supply of platelets in the bloodstream for proper hemostasis and clotting function.

4. Hemostasis The stoppage of bleeding or hemorrhage through a blood vessel is termed as hemostasis. This term comes from the Greek roots: Heme means blood whereas Stasis means halt or stopping. Components of hemostasis Vasculature Platelets Proteins Fibrin-forming proteins (coagulation factors) Coagulation inhibiting proteins Fibrinolytic proteins

5. Components of haemostatic response Vascular system The vascular system prevents bleeding through Vessel contraction Diversion of blood flow from damaged vessels Initiation of contact activation of platelets with aggregation Contact activation of the coagulation system The vessel wall contains varying amounts of fibrous tissue such as collagen and elastin as well as endothelium.

6. Functions of Endothelial cells In an undisturbed state they contribute to maintain Blood in a fluid state In a disrupted state they release substances to Promote coagulation Activate inhibitors & Fibrinolysis

7. Functions of Endothelial Coagulation Production and release of Von Willebrand Factor (VWF) Production of tissue factor Anticoagulant Cell surface protein thrombomodulin Production of Protein C Production of Protein S Fibrinolytic Production and regulated secretion of tissue Plasminogen activator ( tPA ) Production of inhibitors of tissue Plasminogen activator ( tPA )

8. Platelets Platelet production Platelets are produced in the bone marrow by fragmentation of the cytoplasm of megakaryocytes. Approximately each megakaryocyte gives rise to 1000-5000 platelets. The time interval from differentiation of the human stem cell to the production of platelets averages approximately 10 days.

9. Platelets Platelet production Thrombopoietin is the major regulator of platelet production and is produced by the liver and kidneys. The normal platelet count is 150-450 x 10 9 /L and the normal platelet lifespan is 7-10 days. Up to one-third of the platelets reside in the normal spleen but this may rises to 90% in cases of massive splenomegaly.

10. Platelet structure Adhesion to collagen is facilitated by glycoprotein Ia ( GPIa ). Glycoproteins Ib (defective in Bernard- Soulier syndrome) and GP IIb / IIIa (defective in thrombasthenia ) are important in the attachment of platelets to von Willebrand factor (VWF) and hence to vascular endothelium.

11. Storage granules of platelets The platelet contains three types of storage granules: Dense granules Alpha (α) granules Lysosomes During the release reaction, the contents of the granules are discharged into the open canalicular system.

12. Storage granules of platelets Dense granules: Dense granules contain ATP, ADP, serotonin, and calcium that are the mediators of platelet function and hemostasis. Lysosomal granules: Lysosomal granules contain several hydrolytic enzymes, similar to lysosomes found in other cells. Alpha (α) granules: Fibrinogen, Von Willebrand factor, Platelet-derived growth factor, Factor V, High molecular weight Kininogen , Factor XI

13. Platelet function The main function of platelets is the formation of mechanical plugs during the normal haemostatic response to vascular injury. In the absence of platelets, spontaneous leakage of blood through small vessels may occur.

14. Tissue injury causes platletes to adhere to subendothelial collagen. Shape change, secretion of granules contents, and aggregation follows

15. Additional platletes become activated by the secreted substances and clump togethe , eventually forming a mechanical barrier that stop the flow of blood from the wound.

16. Platelet adhesion and activation Following blood vessel injury, platelets adhere to exposed endothelial matrix proteins via special adhesive glycoprotein-Ia (GP-Ia). The exposed endothelial matrix is initially coated with VWF multimers. The platelets than make contact with VWF via the GPIb

17. Platelet adhesion and activation This receptor binding results in a complex cascade of signals which result in platelet activation which eventually results in the shape change of platelets and platelets become more spherical and develop spiny projections which enhance platelet to vessel and platelet to platelet interaction.

18. Platelet adhesion. The binding of glycoprotein (GP) Ib (which consists of four proteins: GPIbo :, GPIb ~, GPIX, GPV) to von Willebrand factor leads to adhesion to the subendothelium and also exposes the GPIIb / IlIa binding sites to fibrinogen and von Willebrand factor leading to platelet aggregation. The GPIa site permits direct adhesion to collagen.

19. Platelet aggregation It is characterized by cross linking of platelets through activated GP IIb / IIIa receptors with fibrinogen bridges. Binding brings about molecular changes in platelets and resulting in a strong connection and further activation of the platelet.

20. Platelet release reaction and amplification Primary activation leads to the release of granules. Alpha Granules contents play an important role in platelet aggregate formation and stabilization and, in addition, the ADP released from dense granules plays a major positive feedback role in promoting platelets activation.

21. Clot formation and retraction The highly localized enhancement of ongoing platelet activation by ADP and Thromboxane A 2 (TXA 2 ) results in a platelet plug formation, large enough to plug the area of endothelial injury. In this platelet plug the platelets are completely degranulated and adherent to each other.

22. Platelet procoagulant activity After platelet aggregation and release, the exposed membrane phospholipid (platelet factor 3) is available for two reactions in the coagulation cascade. The first reaction involves factors IXa , VIlla and X which results in the formation of factor Xa . The second reaction results in the formation of thrombin from the interaction of factors Xa , Va and prothrombin (II).

23. Von Willebrand factor (VWF) Von willebrand is involved in platelet adhesion to the vessel wall and to other platelets (aggregation). It also carries factor VIII and used to be referred as factor VIII related antigen. VWF is encoded by a gene on chromosome 12 and is synthesized both in endothelial cells and megakaryocytes

24. Blood coagulation

25. Coagulation cascade Blood coagulation involves a biological amplification system in which some initiation substances are activated by proteolysis and a cascade of circulating precursor proteins (the coagulation factor enzymes) is started, which finally results in the generation of thrombin; this, in turn, converts soluble plasma fibrinogen into fibrin.

26. Coagulation cascade Fibrin stabilizes the platelet aggregates at the sites of vascular injury and converts the unstable primary platelet plugs to firm, definitive and stable haemostatic plugs. The coagulation factors are either enzyme precursors or cofactors.

27. The reactions involved in coagulation were originally described as occurring in a cascade or waterfall-like fashion in which circulating, inactive coagulation factor precursors, called zymogens , are sequentially activated to their active enzyme forms. Each zymogen serves first as the substrate for the preceding enzyme in the cascade and, when activated, serves as an enzyme for the subsequent zymogen.

28. Coagulation Proteins Factor Number Factor Name I Fibrinogen II Prothrombin III Tissue Factor IV Calcium V Labile Factor VII Pro-Convertin VIII Anti-Hemophilic Factor IX Christmas Factor X Stuart-Prower Factor XI Plasma Thromboplastin antecedent XII Hageman (Contact) Factor XIII Fibrin Stabilizing Factor Prekallikrein (Fletcher Factor) High Molecular Weight Kininogen (Fitzgerald Factor)

29. Coagulation Cascade The coagulation cascade is traditionally separated into 3 pathways: Intrinsic pathway Extrinsic pathway Common pathway

30. Intrinsic pathway requires enzymes and protein cofactors that are present in plasma extrinsic pathway requires enzymes and protein cofactors present in plasma as well as an activator—tissue factor—not found in blood under normal conditions. common pathway Both converge in a third path the to generate the fibrin clot

31. A simplified diagram of the cascade

32. The endothelial cell forms a barrier between platelets and plasma clotting factors and the subendothelial connected tissues. Endothelial cells produce substances that can initiate coagulation, cause vasodilatation, inhibit platelet aggregation or haemostasis , or activate fibrinolysis.

33. The concept of the three pathways, combined with available tests for in vitro evaluation of the intrinsic system (activated partial thromboplastin time [APTT]) and the extrinsic system ( prothrombin time [PT]) are important in diagnosing clinical hemorrhagic disorders.

34. Initiation of Coagulation The coagulation cascade is initiated by the extrinsic pathway with the generation/exposure of tissue factor (Factor III). Tissue factor is expressed by endothelial cells. Tissue factor then binds to factor VII and this complex activates factor X. Factor X, in the presence of factor V, calcium and platelet phospholipid (" prothrombinase complex") then activate Prothrombin to thrombin.

35. Initiation of Coagulation This pathway is rapidly inhibited by a lipoprotein-associated molecule, called tissue factor pathway inhibitor . However, the small amount of thrombin generated by this pathway (before inhibition) activates factor XI of the intrinsic pathway, which amplifies the coagulation cascade.

36. The pathway of blood coagulation initiated by tissue factor (TF) on the cell surface. When plasma comes into contact with TF, factor VII binds to TF. The complex of TF and activated VII (VIla) activates Xand IX. TF pathway inhibitor (TFPI) is an important inhibitor of TF/ VIIa . VIIIa-IXa complex greatly amplifies Xa production from X

37. The generation of thrombin from prothrombin by the action of Xa-Va complex leads to fibrin formation. Thrombin also activates XI (dashed line), Vane XIII. Thrombin cleaves VIII from its carrier von Willebrand factor (VWF), greatly increasing the formation of VIIIa-IXa and hence, of Xa-Va.

38. Amplification of coagulation The coagulation cascade is amplified by the small amounts of thrombin generated by the extrinsic pathway. This thrombin activates the intrinsic pathway by activation of factors XI and VIII. Activated factor IX, together with activated factor VIII, calcium and phospholipid (" tenase complex"), amplify the activation of factor X, generating large amounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form the soluble fibrin polymers.

39. Alternate pathway A second route of stimulation of the intrinsic pathway (called the alternate pathway) is the direct activation of factor IX by the tissue factor-factor VII complex. However, this is a minor pathway and the major stimulator of the intrinsic pathway is thrombin, through activation of factor XI.

40. Functions of Thrombin in Coagulation Activation of Fibrinogen to Fibrin Activation of FXI to XIa Activation of FVIII to VIIIa Activation of FV to Va Activation of FXIII to XIIIa

41. Coagulation factor inhibitors It is important that the effect of thrombin is limited to the site of injury. The first inhibitor to act is tissue factor pathway inhibitor (TFPI) which is synthesized in endothelial cells and is present in plasma and platelets and accumulates at the site of injury caused by local platelet activation. This inhibits Xa and VIla and tissue factor to limit the main extrinsic pathway.

42. Coagulation factor inhibitors There is direct inactivation of thrombin and other serine protease factors by other circulating inhibitors of which antithrombin is the most important. Another protein, heparin cofactor II, also inhibits thrombin. Many more other inhibitors are also involved in limiting the coagulation process exclusively at the site of injury.

43. Protein C and Protein S These are inhibitors of coagulation cofactors V and VIII. Thrombin binds to an endothelial cell surface receptor, thrombomodulin . The resulting complex activates the vitamin K-dependent serine protease protein C which is able to destroy activated factors V and VIII, thus preventing further thrombin generation. The action of protein C is enhanced by another vitamin K-dependent protein S.

44. Fibrinolysis Fibrinolysis (like coagulation) is a normal haemostatic response to vascular injury. Plasminogen a β-globulin pro-enzyme in blood and tissue fluid is converted to the serine protease plasmin by activators either from the vessel wall (intrinsic activation) or from the tissues (extrinsic activation). The most important route follows the release of tissue plasminogen activator ( tPA ) from endothelial cells. This tPA is a serine protease that convert plasminogen into plasmin .

45. Fibrinolysis Plasmin is capable of digesting fibrinogen, fibrin, factors V and VIII and many other proteins. Cleavage of peptide bonds in fibrin and fibrinogen produces a variety of split (degradation) products. Large amounts D- Dimers and Fibrin degradation products (FDPs) can be detected in the plasma of patients with disseminated intravascular coagulation (DIC).

46. Hemostasis Response

47. Hemostasis Response Hemostasis response to bleeding is divided in 3 categories. Primary Hemostasis Secondary Hemostasis Tertiary Hemostasis

48. 1. Primary Hemostasis Vaso-constriction An immediate vasoconstriction of the injured blood vessel results in initial slowing of blood flow to the area of injury. The reduced blood flow allows contact activation of platelets and coagulation factors.

49. Platelet Adhesion Following vessel injury endothelial collagen exposes. The vWF becomes a “bridge” connecting the platelets to the collagen fibers also platelets to other platelets.

50. Platelet Activation Platelet adhesion triggers a series of morphological and functional changes known as platelet activation. The process includes changes in: Metabolism Shape Surface receptors Membrane phospholipid orientation

51. Agonist Agent that induces platelet activation is known as Agonist. e.g , Collagen ADP Thrombin Epinephrine Thromboxane A2 Arachidonic acid The shape change in platelets membrane also allows the platelet to function in secondary hemostasis. Fibrin-forming proteins (coagulation factors) bind to the surface of the activated platelets known as platelet procoagulant activity.

52. Platelet Aggregation Platelet aggregation is the attachment of platelets to one another.   Platelet Secretion After the adhesion and shape change, platelets begin to discharge granules content into the surrounding area. This is known as platelet secretion or release reaction.

53. Formation of primary hemostatic plug Eventually the platelets form the barrier (primary hemostatic plug) that seals the injury and prevents further loss of blood. The unstable primary haemostatic plug produced by these platelet reactions in the first few minutes following injury is usually sufficient to provide temporary control of bleeding.

54. 2. Secondary Hemostasis Stabilization of the platelet plug by fibrin Secondary hemostasis is achieved when fibrin formed by blood coagulation is added to the platelet plug. Following vascular injury, the formation of extrinsic Xase ( VIla , TF, PL and Ca 2+ ) initiates the coagulation cascade. Platelet aggregation and release reactions accelerate the coagulation process by providing abundant membrane phospholipid .

55. Stabilization of the platelet plug by fibrin Thrombin generated at the injury site converts soluble plasma fibrinogen into fibrin. After a few hours the entire haemostatic plug is transformed into a solid mass of cross-linked fibrin.

56. 3. Tertiary Hemostasis Plasminogen in blood and tissue fluid is converted to serine protease plasmin by activators in vessel wall or from the tissues. Plasmin digests Fibrinogen, Fibrin, V, VIII and many other proteins. This makes possible the removal of hemostasis plug after the wound gets healed.

57. Tests of Hemostatic Function: Bleeding Time (BT): Principle: Bleeding time assesses the primary hemostatic mechanism involving platelet plug formation. It measures the time taken for bleeding to stop after standardized skin puncture. Clinical Implications: Prolonged bleeding time may indicate platelet dysfunction or qualitative platelet disorders. Platelet Count: Principle: Platelet count measures the number of platelets in a microliter of blood and provides insights into platelet production and consumption.. Clinical Implications: Thrombocytopenia (low platelet count) or thrombocytosis (high platelet count) may indicate various hematologic disorders, including immune thrombocytopenic purpura or essential thrombocythemia, respectively. Prothrombin Time (PT) and International Normalized Ratio (INR): Principle: PT assesses the extrinsic and common coagulation pathways by measuring the time taken for fibrin clot formation after the addition of tissue factor and calcium. Clinical Implications: Prolonged PT/INR may indicate deficiencies in factors involved in the extrinsic and common pathways, such as factor VII deficiency or liver dysfunction.

58. Activated Partial Thromboplastin Time (APTT): Principle: APTT evaluates the intrinsic and common coagulation pathways by measuring the time taken for fibrin clot formation after the addition of activators and calcium. Clinical Implications: Prolonged APTT may indicate deficiencies in factors involved in the intrinsic and common pathways, such as hemophilia or von Willebrand disease. Thrombin Time (TT): Principle: TT assesses the final step in the coagulation cascade, converting fibrinogen to fibrin, by measuring the time taken for fibrin clot formation after the addition of thrombin. Clinical Implications: Prolonged TT may indicate abnormalities in fibrinogen levels or function, such as afibrinogenemia or dysfibrinogenemia .

59. Tests of Platelet Function: Platelet Aggregation Studies: Principle: Platelet aggregation studies assess the ability of platelets to aggregate in response to various agonists, such as ADP, collagen, epinephrine, or arachidonic acid. Clinical Implications: Abnormal platelet aggregation responses may indicate qualitative platelet disorders, such as von Willebrand disease, Bernard- Soulier syndrome, or Glanzmann thrombasthenia. Bleeding Time (BT): Principle: Bleeding time measures the time taken for bleeding to stop after standardized skin puncture, primarily assessing primary hemostasis mediated by platelet plug formation. Clinical Implications: Prolonged bleeding time may indicate platelet dysfunction or qualitative platelet disorders.

60. Platelet Function Analyzer-100 (PFA-100): Principle: PFA-100 evaluates primary hemostasis by simulating high shear conditions and measuring the time taken for platelet plug formation within a capillary. Clinical Implications: Prolonged closure time on the PFA-100 may indicate platelet dysfunction or qualitative platelet disorders. Flow Cytometry-Based Assays: Principle: Flow cytometry-based assays assess various aspects of platelet function, including platelet activation, surface receptor expression, and platelet-leukocyte interactions.. Clinical Implications: Flow cytometry-based assays provide insights into platelet activation pathways and may aid in the diagnosis of platelet function disorders.

61. Tests of Fibrinolysis: D-Dimer Assay: Principle: D-Dimer assay measures the concentration of D-dimer, a fibrin degradation product formed during fibrinolysis, in the blood. Clinical Implications: Elevated D-dimer levels are indicative of increased fibrinolytic activity and are commonly used in the diagnosis and exclusion of thrombotic disorders, such as deep vein thrombosis (DVT) and pulmonary embolism (PE). Fibrinogen Degradation Products (FDP) Assay: Principle: FDP assay measures the levels of fibrinogen degradation products, including fibrinogen split products (FSP) and fibrin split products (FSP), which are generated during fibrinolysis. Clinical Implications: Elevated FDP levels are indicative of increased fibrinolytic activity and may be observed in conditions associated with fibrinogenolysis , such as disseminated intravascular coagulation (DIC) or liver disease.

62. Euglobulin Clot Lysis Time (ECLT): Principle: ECLT evaluates the overall fibrinolytic activity by measuring the time taken for a fibrin clot to dissolve in a euglobulin fraction of plasma. Clinical Implications: Prolonged ECLT may indicate impaired fibrinolysis and is observed in conditions such as primary fibrinolysis or hyperfibrinogenemia. Global Fibrinolytic Capacity (GFC) Assays: Principle: GFC assays assess the overall fibrinolytic potential of plasma by evaluating the ability of plasminogen activators to lyse exogenous fibrin clots. Clinical Implications: GFC assays provide insights into the fibrinolytic capacity of plasma and may aid in the diagnosis and monitoring of fibrinolytic disorders.

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