Hemostasis and Blood Coagulation

• Homeostasis is the body's balanced equilibrium

• The coagulation process ensures appropriate clotting to prevent excessive bleeding or clotting disorders

• Hypercoagulable states involve excessive clot formation, increasing thrombosis risk like deep vein thrombosis or stroke

• Hypocoagulable states entail insufficient clot formation, leading to uncontrolled bleeding such as hemophilia or platelet disorders

• Blood vessel injury initiates a series of steps called blood coagulation

• Primary hemostasis/platelet function, is controlled by platelets, plasma proteins, and blood vessel contents

• Produces a white clot/platelet clot

• The steps of platelet function

  • Platelet adhere
  • Release of products
  • Platelet aggregation, which prevents blood leakage

• Secondary hemostasis occurs when primary hemostasis isn't enough

• Red clots form, stopping bleeding via plasma/coagulation proteins and platelets

• Tissue repair involves the lysis of the clot (fibrinolysis)

Ultrastructure of Platelets

• Platelets are observable only under an electron microscope

Resting Plasma Membrane

• Is a bilayer of proteins and lipids
• It will activate once it is performing its functions
• Resting platelets have a round/discoid shape
• Activated platelets have an irregular margin and pseudopods to accelerate aggregation, and a negative charge

Lipids

• Phospholipids form the basic structure and barrier of the membrane
  • Outer membrane contains phosphatidylcholine and sphingomyelin, which are neutral
  • Inner membrane contains, phosphatidylinositol, phosphatidylethanolamine & phosphatidylserine, which supply arachidonic acid, support platelet activation, and are anionic and polar
• Cholesterol, esterified (inner) and unesterified (outer), is distributed asymmetrically throughout the phospholipids, serves as a passage, stabilizes the membrane, maintains fluidity, and controls transmembranous passage of materials

Proteins

• Glycoproteins and proteoglycans are anchored in the membrane
• They support surface glycosaminoglycans, oligosaccharides, glycolipids, and essential plasma surface-oriented glycosylated receptors
• Respond to cellular and humoral stimuli and bind to stimuli/ligands for platelet activation
  • ADP binds to platelet, enhancing the release of platelet contents once ligands bind to the platelet surface via receptors
• Glycocalyx is the platelet membrane surface
• It's smooth, contains pore-like indentations, absorbs albumin, fibrinogen, and other plasma proteins from the plasma
• Transports plasma proteins to internal storage organelles via endocytosis which are alpha granules and dense granules
• The thickness is 20-30 nm
• It's adhesive and responds to hemostatic demands
• Located outside the phospholipid bilayer
• It helps the plasma membrane accelerate adhesion to the injury surface

Surface-Connected Canalicular System (SCCS)

• Is an invagination from the platelet exterior
• Stores additional quantities of the same hemostatic proteins found on the glycocalyx
• It allows enhanced access to the environment to platelets' interior and enhanced release of platelet products
• It is a route for endocytosis and secretion of alpha-granule contents upon platelet activation
• The release process sees alpha granules with protein contents bind to SCCS and release contents via SCCS

Dense Tubular System

• The system is parallel and aligned with the SCCS
• Remnant of rough ER which sequesters Ca2+
• Contains phospholipase A2, cyclooxygenase, and thromboxane synthetase, enzymes that support platelet activation
  • Supports thromboxane A2 production
  • Phospholipase C also supports inositol triphosphate (IP3) and diacylglycerol (DAG)

Cytoskeleton

• Microtubules surround the platelet in a 25 nm diameter
• Maintains the platelet's discoid shape, with 8-20 tubules disassembling at ref. temperature or with colchicine treatment, making round platelets
• Microtubules move inward during platelet activation, enabling alpha-granule content expression and reassembling in long parallel bundles during shape change to provide rigidity to pseudopods
• Microfilaments, made of of actin make anchor PM glycoproteins and proteoglycans
  • Consists of 8-12 nm intermediate filaments which connect with actin and the tubules to maintain shape
• Functions of cytoskeleton: Platelet shape change, extension of pseudopods, secretion of granule contents

Platelet Granules

• Alpha (α)-granules
  • There are 50-80 in each platelet
  • Stain medium-gray in osmium dye TEM and are filled with endocytosed plus synthesized proteins
  • During platelet activation they fuse with SCCS, releasing contents to the microenvironment where they enable adhesion and support plasma coagulation

Alpha Granule Contents

• The cytoplasm & a-granules contain:
  • Endocytosed coagulation proteins such as fibronectin, fibrinogen, Factor V, and VWF
  • Megakaryocyte synthesized proteins such as Thrombospondin
• A-granules also contain megakaryocyte synthesized B-thromboglobulin, EGF, HMWK, Multimerin, PAI-1, PDCF, Plasmionogen, PF4, and TGF-β

Platelet Membrane-Bound Proteins

• Restricted to a-granules are P-selectin and GMP33
• In a-granules and plasma membrane are GP Ilb / Illa, Osteonectin, capl, GPIV, GP Ib/IX/V, CD9 and PECAM-1
• Dense granules
  • Have 2-7 in each platelet
  • Stain black in osmium dye TEM
  • Appear later than a-granules in megakaryocyte differentiation
  • Stores ADP, ATP, Serotonin, Ca2+ & Mg2+ in small molecules
  • Release their contents directly into the plasma during platelet activation
    • ADP is nonmetabolic
  • Supports neighboring platelet aggregation by binding to P2Y1 & P2Y12 ADP receptors
  • ATP's function is unknown, but its release is detectable on platelet activation
  • Serotonin is a vasoconstrictor that binds endothelial cells and platelet membranes
  • Ca2+ & Mg2+ ions support platelet activation and coagulation
• Lysosomes
  • Platelet granules are digesting enzymes
  • Few in platelets and 300 nm in diameter similar to those in neutrophils
    • Stain (+): arylsulfatase, b-glucuronidase, acid phosphatase, and catalase
  • Contents digest vessel wall matrix components during in vivo aggregation and may also digest autophagic debris, removing materials that impede aggregation before platelet aggregation
  • Digest nonfunctional contents not needed in the process, like mitochondria

Megakaryocytes

• Are the largest cell in bone marrow that are less than 0.5%
• Nucleus include multiple chromosome copies termed multilobulated or polyploid
• Cytoplasm is granular
• Growth factor is thrombopoietin

Locations

• Cluster in bone marrow with hematopoietic stem cells, near venous sinusoid endothelial cells and have proplatelet processes that release platelets into the circulation
• They undergo endomitosis which is multiple mitotic divisions without cytoplasmic divison
• This generates a giant multinucleated cell
• The main cytokine that influences platelet production is TPO

Stages

• Megakaryoblast is the earliest recognizable maturation stage
• Cytoplasm shows irregular membrane, polyribosomes, and clear central ER vacuoles that diffusely stain blue
• The nucleus is predominant with nucleoli but fine occupying most of the cell volume, surrounded by the Golgi complex
• Staining is (+): alpha granules & DMS, only in bone marrow (1-4 per 1000 cells)
• Promegakaryocyte cytoplasm shows irregular membrane, polyribosomes, and it stains dark blue
• Nucleus shows barely detectable chromatin margination and increasing/indented lobes
• Staining (+): alpha granules & DMS Megakaryocyte
• Cytoplasm has polyribosomes that stain reddish-blue or lavender, contains spread alpha granules and DMS
• Nuclus has corase+ linear chromatin
• Mulit-lobulated

Thrombocytopoiesis

• Also known as "platelet shedding"
  • 1 megakaryocyte = 2000-4000 platelets
  • 10^8 megakaryocytes produce 10^11 platelets per day
  • Platelets lifespan of 8-10 days with a naked megakaryocyte released to be consumes by marrow macrophages

Platelets

• Are nonnucleated cells with irregular and granular cytoplasm of about 2.5 um diameter and 8-10 fL volume
• They are fragments of megakaryocytes shaped biconvex when resting and round when circulating
• Normal value isNV: 150-400 x 10^9/L, women greater than men and women/men >65 years old
• 2/3 of platelets are circulating and 1/3 in spleen
• They arise from megakaryocytes, with the cytokine TPO influencing differentiation and the hormone IL-3 having various influences

Reticulated Platelets:

• Referred to: stress platelets
• Appears in compensation for thrombocytopenia
• Markedly larger than ordinary mature circulating platelets; 6um, 12-14fL and appear round via EDTA and cylindrical/beaded via Citrated
• Resemble reticulocytes through the transport of free ribosomes and fragments of rough ER

Basic Concepts of Hemostasis

• Hemostasis from the greek 'stoppage of blood flow'
• Normal Hemostasis: delicate balance between coagulation and fibrinolysis, where coagulation must happen followed by fibrinolysis

Hemostatic Components

• Extravascular component: tissue surrounding blood vessel

• Vascular component: blood vessels

• Platelets & plasma proteins: active players in the primary and secondary phases of hemostasis

• When local blood vessels are injured it engages

• Back pressure for injured vessles, it traps the blood

  • Depends on the type and amount of surrounding tissues
  • Bone is more absorbent than elastic tissue, more tissue= less bleeding

• The abiltiy of hemostasis depends on amount smooth muscle to constrict vesse; also the cell type

  • Vasocontriction is essential for hemostatis
  • Integrut=iy of endothelial lining
  • BV divded into 3 laters with enothelial lning

• Intravascular Component: platelets and biochemicals in the blood clot formation/lysis, platelets= primary, coagulation=secondary

  • Occurs without balance of stimulus BV damaged causing coagulation in the present of of Fibrinolysis
  • More blood production when ↑COAGULATION > FIBRINOLYSIS, called hypercoagulable state Excessive Bleeding, vasocclusion

Hemostatic Mechanism: In BV injury

Primary Hemostasis - Small Injures in blood vessels with vascular intima, platelets, Rapid short response - Requires protien for pro-coagulates, adhesion, secreetion, aggregation - Involves collagen exposure

Secondary Hemostasis - Large Injures in blood vessels, tissues - Has playes and Coag system - Involves tissue factor for cell membranes Basic sequence - Vasocontriction, platelet adhesion, platelet release - Fibern Formation and stablization

Roles in BV

Intact Vessels - Carry blood and structered on 3 laters: inner laters -