plt

Hemostasis:

Hemostasis Vessel wall Platelet Plasma coagulation factors. Inhibitors of coagulation. Fibrinolysis .

Platelet Endothelial Interaction:

Platelet Endothelial Interaction Done by: Dr.Sameera AlSafwani

Vessel wall:

Vessel wall intema media adventitia Elastic lamena interna glycocalyx

Vessel wall: :

Vessel wall: The endothelium contains three thromboregulators: Nitric oxide, Prostacyclin Ectonucleotidase CD39 Collagen Accumulates & activates Plt Tissue factor Initiates Thrombin generation

The Endothelium Thromboregulators:

The Endothelium Thromboregulators Endothelial cells convert arachidonic acid into prostacyclin wit with the help of COX-1 or COX-2 and prostacyclin synthase. Prostacyclin inhibits platelet function by elevating intracellular cyclic AMP levels. Nitric oxide diffuses into platelets, stimulates: -The production of cyclic guanosine monophosphate (GMP) -Regulates cyclic GMP–dependent protein kinases, causing decrease in intracellular Ca2+ flux. Ecto-ADPase, limits the plasma level of nucleotides (ADP and ATP).

In resting phase:

In resting phase

Role of endothelium in hemostasis:

Role of endothelium in hemostasis Express Thrombomodulin. Express Endothelial protein C receptors. Express Ecto - ADPase CD39. Produce Plasminogen activator. Produce VWF. Synthesize protein S. Release nitric oxide (NO) and generate prostacyclin.

Platelet:

Platelet Small and discoid in shape. Dimentions 3.0 × 0.5 μ m and a volume of 7 – 11 fL. Marginated to the edge of the vessel. Normal range (150 – 400 × 109 / L). Normal haemostasis (20 × 10 9 /L).

Slide 14:

Platelets lack a nucleus. Platelets exhibit a very limited and specific degree of protein synthesis. Recent evidence suggests that activated platelets can synthesize a small number of key proteins that may be of functional significance. The presence of low levels of platelet mRNA also provides an opportunity to perform a limited number of genetic studies.

Platelet cytoskeleton:

Platelet cytoskeleton

Platelet cytoskeleton:

Platelet cytoskeleton Consists of a spectrin - based membrane skeleton, Circumferential bands of a single microtubule that lie beneath the plasma membrane Rigid actin filament network that fills the cytoplasm of the cell (2 million copies of actin per platelet). Actin polymers connect with each other and with the cyosolic tail of the membrane glycoprotein (GP)Ib α via filamin in a lattice – like structure that gives the platelet its discoid shape.

Platelet granules:

Platelet granules Dense Granules 5-9 per platelet Content: ADP, ATP, Polyphosphate, Serotonin, Ca++ Lysosomes Peroxisomes Alpha Granules 80 per platelet Content: PF4, vWF, Fibrinogen, Factor V, Protin S, TFPI, SDF-1 α , β - thromboglobulin, ENA – 78, RANTES, PDGF, VEGF, integrin GPIIb/IIIa, P- selectin (CD62) and CD40L.

Slide 18:

Platelets are enriched in signalling proteins, which facilitate the rapid switch from quiescence to activation, and have a high cytoskeletal protein content that gives rise to the dramatic changes in morphology and which enables the platelet aggregate or thrombus to withstand the very high shear forces that exist in the arteriolar system. Platelets have a network of intracellular membranes known as the dense tubular system (part of the endoplasmic reticulum) that release intracellular Ca 2+ in response to the second messenger inositol 1,4,5 – trisphosphate (IP3). Platelets have a network of invaginations of the surface membrane, known as the surface - connected canalicular system, which increase the surface area of the plasma membrane during platelet spreading and thereby provide more sites for release of intracellular granules. The surface - connected canalicular system also gives rise to membrane tethers that play a vital role in supporting adhesion. Platelets contain several mitrochondria that generate energy during their short lifespan.

Megakaryopoeisis:

Megakaryopoeisis Endomitosis

Platelet formation:

Platelet formation Platelet HSC Megakaryocytes c-MPL c-MPL c-MPL TPO (JAKII/STAT, TF) eltrombopag romiplostim

Platelet formation:

Platelet formation Platelets are formed from megakaryocytes, one of the largest cells in the bone marrow, reaching more than 50 μ m in diameter. The nucleus of the megakaryocyte undergoes a process known as endomitosis that involves nuclear replication without cellular division, giving rise to DNA ploidy values that range from 4 n to 128 n . The reason why endomitosis occurs is not fully understood, but it may simply reflect the need to increase the DNA content to enable the cell to expand its protein synthetic capacity to generate 2000 – 3000 platelets per megakaryocyte. it allows cell growth and differentiation to occur without interruption by nuclear and cell divisions.

:

The differentiation of bone marrow progenitor cells into megakaryocytes is regulated by the cytokine thrombopoietin (TPO). The TPO receptor, c - Mpl, expressed on stem cells, megakaryocytes and platelets, signals via the JAK/STAT family of kinases and transcription factors, respectively. TPO is synthesized at a constant rate in the liver and its circulating free levels are largely controlled by binding and internalization mediated through c - Mpl on the platelet surface. This provides a simple and elegant means of tightly controlling the platelet count. Thus, if the platelet count decreases, the free level of TPO rises and there is an increase in megakaryocytopoiesis and platelet formation. Two TPO mimetics (eltrombopag and romiplostim) have recently entered clinical use for treatment of immune thrombocytopenia.

Slide 26:

There is now a growing consensus that megakaryocyte differentiation occurs in a defined compartment of the bone marrow known as the osteoblastic niche and that megakaryocytes subsequently migrate and adhere to sinusoidal endothelial cells through a process that is regulated by the chemokine SDF - 1 α and its receptor CXCR4. At this vascular niche, the megakaryocytes generate long thin processes known as proplatelets, which form platelets at their terminals. The proplatelet arms protrude between bone marrow sinusoidal endothelial cells and release platelets directly into the bloodstream. This carefully orchestrated process ensures that platelets are not released into the bone marrow. Megakaryocytes also undergo chemotaxis into the bloodstream where they can also form platelets. To maintain a normal platelet count, approximately 1 million new platelets are released into the blood every second but this can dramatically increase by a factor of 10 in severe haemorrhage.

Thrombus formation:

Thrombus formation

Slide 30:

Exposure of VWF to cllagen and or elevated shear conformational change in VWF VWF & GPIb interaction plt translocation on the surface of VWF in stop start manner through sliding membrane tethers to sustain the adhesion. Capturing & recruitment of plt to agreggate mediated by ADP & TXA2+ thrombin Binding of VWF to GPIIb/IIIa mediate tethering of non-activated plt through GP1b.

Platelet activation:

Platelet activation Initial phase: discoid shape, reversible & independent on the ADP& TXA2 and serve to recruit plt to growing aggregate. Irreversible phase; morphology change, plt activation, dependant on ADP & TXA2 and thrombin.

Low vs High shear rate:

Low vs High shear rate shear forces of less than 500/s, platelets are able to adhere directly to exposed subendothelial matrix proteins and undergo stable adhesion independent of VWF and GPIb – IX – V Release of secondary mediators and generation of thrombin is also sufficient to enable aggregate growth to occur independent of VWF and GPIb – IX – V. At shear rates of 10,000/s and above adhesion and aggregation are mediated entirely by the interaction of soluble and immobilized VWF with GPIb – IX – V, and are independent of platelet activation and integrin GpIIb/lIIa

Spreading:

Spreading Initial shape change or rounding is followed by the generation of finger - like projections known as filopodia, which grow from the periphery of the cell, and the subsequent formation of lamellipodia, which fill in the areas between adjacent filopodia. granules and organelles are squeezed into the centre of the cell, resulting in a characteristic ‘ fried egg ’ appearance. These dramatic changes in morphology are brought about by a powerful severing and reassembly of the actin cytoskeleton. The spreading of platelets helps to secure the platelet and thrombus against the flow and shear forces of the vascular system.

Granule secretion and TxA2 formation:

Granule secretion and TxA2 formation The secretion of ADP from dense granules and the de novo generation of TxA 2 from arachidonic acid, liberated by the action of cytosolic phospholipase (PL) A 2, play a critical feedback role in mediating platelet activation. Activation of platelets by low concentrations of agonists is potentiated by the release of the two feedback agonists, and only high concentrations of powerful stimulants such as thrombin are able to induce full aggregation in the presence of inhibitors of the two feedback stimuli. The major physiological role of the two feedback mediators, along with thrombin, is to generate a platelet aggregate on the monolayer of cells that have adhered to subendothelial collagen.

Slide 37:

Secretion from platelet α - granules usually occurs concomitantly with that from dense granules. Fusion of α - granules with the platelet plasma membrane has the potential to increase the expression of GPIIb/IIIa to over 120,000 copies per platelet, also leads to expression of P - selectin on the platelet surface, which is the major ligand for P - selectin glycoprotein ligand (PSGL) - 1 on circulating tissue factor - rich microparticles and leucocytes. The capture of microparticles provides a mechanism for further activation of the coagulation cascade on a growing thrombus, while binding to leucocytes contributes to inflammatory events at the vessel wall.

Aggregation:

Aggregation Aggregation is the specific term used for the cross - linking of activated platelets through binding of bivalent or multivalentligands to integrin GPIIb/IIIa. The integrin GPIIb/IIIa is present in a low - affinity form on non - stimulated platelets but undergoes a conformational change to a high - affinity form in response to ‘ inside- out ’ signals. The subsequent binding of bivalent ligands to platelets mediates platelet aggregation and adhesion. These interactions lead to clustering of GPIIb/IIIa on the platelet surface and the generation of ‘ outside- in ’ signals that regulate actin polymerization (and therefore platelet spreading) and other responses in synergy with other platelet receptors.

Slide 40:

Fibrinogen, VWF and fibronectin have similar affinities for integrin GPIIb/IIIa but under low shear conditions, fibrinogen is considered to be the major ligand because of its much higher concentration in plasma. Recent studies in fibronectin- deficient mice and in in vitro flow assays have emphasized an important role for fibronectin in the initiation, growth and stabilization of platelet aggregates. The importance of fibronectin may be related to its ability to bind to additional proteins such as fibrinogen and thrombospondin, and in this way contributes to the stability of the platelet aggregate. Overall, however, we have a relatively poor understanding of the interplay between fibrinogen, VWF and fibronectin, and are still unable to explain why a combination of GPIIb/IIIa ligands is more effective in supporting aggregation and thrombus formation.

Thrombus stabilization:

Thrombus stabilization Events involve in stabilization of the platelet aggregate or thrombus : Remodelling of the actin cytoskeleton. The interaction of several platelet membrane proteins with themselves or with each other. Clot retraction.

Slide 42:

The ability of blood clots to retract over a course of minutes to hours is termed clot retraction. The platelets are the force - generating components of this response, with integrin GPIIb/IIIa playing a fundamental role both by linking cytoplasmic actin filaments to surface – bound fibrin polymers and by generating intracellular signals that, together with those from other receptors, enable myosin to serve as a motor and drive the process. A novel actin - dependent, but fibrin - independent, mechanism of retraction has been recently described that brings newly captured platelets into the aggregate and presumably also strengthens the aggregate by reducing the shear forces at the aggregate edge.

Slide 43:

Through the work of Brass and others, it is now recognized that the aggregate is consolidated by the binding of a number of platelet membrane proteins to themselves (homophilic interactions) or to other surface receptors on adjacent platelets (heterophilic interactions). Many of the adhesion proteins that mediate platelet formation become shed during activation, including GPVI, GPIb α , GPV, P - selectin, and semaphorin 4D, and potentially this may serve to limit thrombus growth. On the other hand, in the case of CD40L, which becomes exposed on platelet activation, there is evidence that its shedding and subsequent binding to integrin GPIIb/IIIa via a KGD motif stabilizes thrombus formation.

Procoagulant activity:

Procoagulant activity Activated platelets provide a negatively charged phospholipid surface for the assembly of two multiprotein complexes that form part of the intrinsic and amplication pathway of coagulation, namely the tenase and prothrombinase complexes. A complex of FIXa and FVIIIa on the negatively charged lipid surface converts FX to FXa (tenase complex), which in turn forms a complex with FVa on the same surface to efficiently convert prothrombin to thrombin (prothrombinase complex). A large amount of thrombin is generated in the vicinity of the growing aggregate that serves to enhance platelet activation and convert fibrinogen to fibrin.

Slide 45:

The platelet procoagulant response is elicited only by very powerful platelet agonists or combinations of agonists, and requires sustained entry of Ca 2+ across the plasma membrane. Five patients have been described with a mild bleeding disorder linked to defective procoagulant activity, a condition known as Scott syndrome . Procoagulant activity can be monitored by flow cytometry in the presence of external Ca 2+ through the binding of fluorescently labelled annexin A5, FXa and other ligands. The activation of platelets by powerful agonists and entry of Ca 2+ is associated with formation of microparticles and these too have been proposed as providing a massive increase in the surface area for binding of the tenase and prothrombinase complexes.

Two Independent Pathways to Platelet Activation :

Two Independent Pathways to Platelet Activation Subendothelial collagen initiates platelet activation: Platelet GlycoproteinVI (independent of Thrombin). Platelet Glycoprotein Ib-V-IX with VWF bound collagen. Tissue factor triggers a second pathway: Independent of VWF and GP VI. Forms a complex with F VIIa. Initiates a proteolytic cascade that generates thrombin. Thrombin cleaves protease- activated receptor 4 (Par4). activating platelets release ADP, serotonin, and TXA2.

Propagation of the Platelet Thrombus :

Propagation of the Platelet Thrombus The platelet integrin GPIIb/IIIa: mediates recruitment of plt to the thrombus as well as plt-plt interactions. GPIIb/IIIa activated by protein disulfide isomerase (PDI) Activation of platelets conformational transition in GPIIb/IIIa that increases the affinity of the integrin for its ligands, fib and VWF. Late signaling events enhance platelet–platelet affinity. CD40 ligand, Ephrin. Platelet activation releases the contents of plt alpha & dense granules.

CD40 L :

CD40 L CD40 ligand released from platelets induces inflammatory responses in the endothelium. Transmembrane protein in the tumor necrosis factor family Stored in the cytoplasm of resting platelets and rapidly appears on the surface after platelet activation. Platelet derived CD40 ligand can induce endothelial cells to produce reactive oxygen species, adhesion molecules, chemokines and tissue factor.

Stimulatory receptors and their signalling pathways:

Stimulatory receptors and their signalling pathways single transmembrane proteins that regulate tyrosine kinases. e.g. adhesion molecules collagen, VWF and fibrinogen seven - transmembrane – spanning proteins that are coupled to heterotrimeric G proteins. e.g. thrombin, ADP and TxA 2.

Inhibitory agonists and their receptors:

Inhibitory agonists and their receptors The prevention of platelet activation in the intact circulation is ordinarily achieved by the antithrombotic nature of the endothelial cell surface by: Removal of platelet stimulatory agonists, most notably ADP and thrombin. The release of NO and prostacyclin. Activation of the fibrinolytic pathway. Additional mechanisms including; shedding of platelet surface glycoprotein receptors, cleavage of intracellular proteins, and removal of intracellular messengers and reversal of phosphorylation by the action of tyrosine and serine/threonine phosphatases .

Blood Coagulation:

Blood Coagulation Tissue factor is expressed: Fibroblasts and pericytes in the adventitia Medial smooth muscle of vessel wall Monocytes Endothelial cells Circulating blood in a latent form (assoc. with microparticles) It has been postulated that the activation of encrypted TF by Protein disulphide isomerase (PDI) initiates coagulation .

Functional contacts between platelets and endothelial cell (the cross talk) :

Functional contacts between platelets and endothelial cell (the cross talk) The maintenance of vascular integrity proangiogenic cytokines growth factors These molecules are stored within plt Granules. Elicit intracellular signaling that stabilizes the vascular-endothelium cadherin complex at intercellular adherens junctions.

Platelets influence the development and fate of endothelial-cell progenitors:

Platelets influence the development and fate of endothelial-cell progenitors Promoting the migration and adherence of bone marrow–derived cells to sites of angiogenesis. Induce differentiation of endothelial-cell progenitors into mature endothelial cells. Under the influence of platelets, endothelial-cell progenitors down-regulate expression of c-kit and increase the synthesis of CD31 (platelet–endothelial cell adhesion molecule, or PECAM1) Aggregation and activation of platelets at sites of exposed subendothelium cause them to release stromal cell–derived factor1. Platelets participate in the recruitment of dendritic cells to atherosclerotic plaques.

Platelets and the VEGF System :

Platelets and the VEGF System VEGF, discovered 25 years ago Vascular permeability factor Four members of the VEGF family: VEGF-A : Interacts with two tyrosine kinase receptors (VEGF-R1) and (VEGF-R2). Contributes to vascular integrity VEGF-B: Form heterodimers with VEGF-A. Brown fat, myocardium, and skeletal muscle. VEGF-C & VEGF-D they pair with (VEGF-R3) & regulate lymphangiogenesis.

Platelets and the VEGF System :

Platelets and the VEGF System Platelets contain the three major isoforms of VEGF-A VEGF121. VEGF165. VEGF189. After exposure to thrombin in vitro, they release VEGF-A. VEGF-A alters the endothelial-cell phenotype by: markedly increasing vascular permeability. up-regulating expression of: Urokinase. Tissue plasminogen activator. Connexin. The Vascular- Cell Adhesion Molecule.

BIOLOGIC CHARACTERISTICS OF JUNCTIONS :

BIOLOGIC CHARACTERISTICS OF JUNCTIONS The major anatomical sites of bleeding in patients with thrombocytopenia are the intercellular gaps in the postcapillary venular bed. Adherens junctions are abundant in the postcapillary venular bed. They consist of clusters of the highly specialized membrane spanning vascular endothelium cadherin molecules. Vascular endothelium cadherins are coupled intracellularly to catenins.

Slide 59:

VEGF-A induced signaling directly controls the interactions of the junctional machinery. The cytoplasmic domain of VEGF- R2 is a part of a macromolecular vascular endothelium cadherin-β-catenin complex. The binding of intracellular catenin to vascular endothelium cadherin is increased by a factor of 700 after phosphorylation of specific cadherin serine residues. Platelet derived VEGF can induce the phosphorylation and activation of endothelial VEGF R2.

Adherens Junction at the Postcapillary Venular Bed :

Adherens Junction at the Postcapillary Venular Bed - Brain-derived neutrotrophic factor -Epidermal growth factor -Platelet activating factor -Sphingosne 1-phosphate -Angiopetin

Other contributors to microvascular stability :

Other contributors to microvascular stability Platelet derived epidermal growth factor increases production of Bcl-xL by endothelial cells.(Bcl-xL up regulates VEGF A production). Platelet activating factor, a phospholipid proinflammatory mediator, induces expression of VEGF A by endothelial cells. Paracrine VEGF A, delivered to the vascular lining by platelets and other cells, increases endothelial cell production of Bcl 2, another inhibitor of the cell death pathway. Phosphorylated sphingosine phosphate released from platelets supports endothelialcell integrity and survival, inhibits apoptosis, and stabilizes endothelial cell junctions.

Thrombocytopenia, Disassembly of the Adherens Junction. :

Thrombocytopenia, Disassembly of the Adherens Junction.

Anti VEGF medications (Antiangiogenic):

Anti VEGF medications (Antiangiogenic) Bevacizumab (humanized monoclonal antibody against VEGF) VEGF-R2 tyrosine kinase inhibitors such as sorafenib and sunitinib. Potential inhibitors of platelet–endothelial cell interactions. Associated with hemorrhagic side effects

Summary :

Summary Platelets and endothelial cells are intimately related In the physiologic steady state, platelets maintain the stability of the vasculature by means of several mechanisms. Preserve the structural and functional integrity of the vascular-endothelium cadherin zipperlike machinery at the intercellular gaps. It remains to be determined how much the induced signaling pathways overlap and whether the VEGFA–VEGF-R2 receptor loop is the final common pathway.

Platelet function testing:

Platelet function testing Affected by the collection and processing Most current testing is performed on citrated blood within a few hours of sampling. Global tests of platelet function are often initially used as screening tests. For initially screening: the template bleeding time, light transmission aggregometry (LTA) and the closure time within the platelet function analyser (PFA) - 100.

Screening for platelet fonction:

Screening for platelet fonction BT. PFA-100. CPA. CBC; plt count, MPV & plt size distribution. PBF; plt size and granulation, neutrophils inclusion and red cell schistocytes.