Overview of Platelet Physiology Its Hemostatic

8/19/2019 Overview of Platelet Physiology Its Hemostatic

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Review ArticleOverview of Platelet Physiology: Its Hemostaticand Nonhemostatic Role in Disease Pathogenesis

Kakali Ghoshal and Maitree Bhattacharyya

Department of Biochemistry, University of Calcutta, Ballygunge Circular Road, Kolkata , India

Correspondence should be addressed to Maitree Bhattacharyya; [email protected]

Received August ; Accepted November ; Published March

Academic Editors: E. J. Benz, L. Olcay, and M. de F. Sonati

Copyright © K. Ghoshal and M. Bhattacharyya. Tis is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Platelets are small anucleate cell ragments that circulate in blood playing crucial role in managing vascular integrity and regulatinghemostasis. Platelets are also involved in the undamental biological process o chronic inammation associated with diseasepathology. Platelet indices like mean platelets volume (MPV), platelets distributed width (PDW), and platelet crit (PC) are useulas cheap noninvasive biomarkers or assessing the diseased states. Dynamic platelets bear distinct morphology, where and densegranule are actively involved in secretion o molecules like GPIIb , IIIa, brinogen, vW, catecholamines, serotonin, calcium, AP,ADP, and so orth, which are involved in aggregation. Differential expressions o surace receptors like CD, CD, CD andso orth have also been quantitated in several diseases. Platelet clinical research aces challenges due to the vulnerable nature o

platelet structure unctions and lack o accurate assay techniques. But recent advancement in ow cytometry inputs huge progressin the eld o platelets study. Platelets activation and dysunction have been implicated in diabetes, renal diseases, tumorigenesis,Alzheimer’s, and CVD. In conclusion, this paper elucidates that platelets are not that innocent as they keep showing and thusnumerous novel platelet biomarkers are upcoming very soon in the eld o clinical research which can be important or predictingand diagnosing disease state.

1. Introduction

Platelets were discovered by Giulio Bizzozero in [], butor many decades the dynamic and multiunctional nature o platelets remained a eld o interest only or biologists. Anu-cleate, discoid platelets are the smallest blood particles which

unveil their dynamicity through their morphology. Primarily they are associated with hemostasis, which is to initiate bloodcoagulation. Although very dynamic, they usually preer toremain in inactive state and get activated only when a blood

vessel is damaged. But hemostasis or blood coagulation is notthe sole unction o platelets; rather it is employed in severalmultiunctional attributes monitoring the homeostasis o thebody. Its high sensitivity to different disease states eventually assigned it to be one o the most accessible markers. Whilekeeping interactions with leukocytes and endothelial cells, itrestores its behaviour as an important inammatory marker[]. Platelet reactivity or different disease pathogenesis iswidely dependent upon some biologically active markers

like CD, CD, CDa, CDb, and CD. Tese includesome active surace receptors and platelet secretory products.Platelet tends to alter the expression and signaling o thesemarkers in different disease diagnosis and prognosis, provid-ing a huge eld to explore disease progression.

Primarily, platelet activity is associated with the initiation

o coagulation cascades. Damage in blood vessel makes thesubendothelial surace the primary target site o plateletaction, where it establishes the hemostasis. Various proag-gregatory stimuli also known as platelet agonists promotethe action o platelet adhesion to the subendothelial suraces.During this process, platelet changes its shape, releases itsgranule contents, and gradually orms aggregates by adher-ing with each other []. Tus its primary activity remainsassociatedwith minimizing blood loss.However, as discussedearlier plateletsare not only conned in regulating hemostasisand thrombosis, but they also play many pivotal roles in dis-ease pathophysiology. Platelet interaction and cardiovasculardisease progression remain an unsolved riddle ormany years

Hindawi Publishing Corporatione Scientific World JournalVolume 2014, Article ID 781857, 16 pageshttp://dx.doi.org/10.1155/2014/781857


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Te Scientic World Journal

granule

Densegranule

PAR 1

Pa

PAR 4

GPVI P2X1P2Y1

P2Y12

5H2A

IIb 3

IIb 3

IIb 3

Plateletactivation

Amplication

Aggregation

Fibrinogen

Trombingeneration

Shape change

Coagulation factorsinflammatory mediators

Coagulation

Trombin

Tromboxane A2

Collagen

5H5HADP

AP APADPADP

F : Platelet-activation mechanisms and role o the PY12

receptor. Platelet activation leads to dense-granule secretion o ADP, whichactivates PY

12, inducing amplication o aggregation, procoagulant, and proinammatory responses (adapted rom Storey, []).

[]. Platelet hyperaggregation among the diabetic patientswith CVD remains another striking area to explore. Platelethyperactivity in various diseases provokes adverse effectsin some cases, especially in coronary artery disease wherehyperaggregation obstructs blood circulation.

Expression o platelet markers can be well studied by ELISA or Western blot. However, till date ow cytometry isthe best standardized method to study platelet unction [, ].In this paper, we have tried to elucidate various aspects o platelets structure and unction and their potential role indisease pathophysiology.

2. Versatility of Platelets: Its Structuraland Functional Aspects

.. Ultrastructure. Platelet ultrastructure reveals its beha- vioural peculiarities. Megakaryocytes o the bone marrow are

site o platelet ormation. Diameter o a mature platelet is-m, which usually remains alive or – days. Approx-imately / o the platelets circulate in the blood and /is stored in the spleen. Te normal platelet count is (–) × 3 per microliter o blood. Each megakaryocyte canproduce – platelets. An average healthy adult can

produce 11 platelets per day; old platelets are destroyed by phagocytosis in the spleen and liver (Kupffer cells).

Platelets are unique in their structural assembly, thoughthey are anucleate but have distinct mitochondria. Plateletplasma membrane, composed o phospholipid bilayer, is thesite o expression o various surace receptors and lipid rafswhich helps in signalling and intracellular trafficking. Tese

markers include CD, CD, CD, GPCR, IIbIIIa, andGLU-. Tese surace receptors also trigger the releaseo granules which play a role in multiple unctions,namely, coagulation, inammation, atherosclerosis, antimi-crobial host deense, angiogenesis, wound repair, and tumori-genesis []. Among these surace receptors, GPCR has beenreported to play crucial role in ADP secretion rom densegranules which is its major secretory product []. Asym-metrically arranged phospholipids (e.g., phosphatidylserineand phosphatidylinositol) present in the inner layer o the plasma membrane maintain the stability o its suraceduring nonprocoagulant state. During activation (Figure )platelet surace gradually exposes aminophospholipids by AP-dependent oppases and scramblases to initiate coag-ulation cascades []. Te open canalicular system (OCS)is the “tunnel” system present throughout the platelet celland remains connected with the plasma membrane []. Temajor role o OCS is to give entry o external elements into

the platelets as well as to release its granule contents to theexterior. Other than being a major storage site or plasmamembrane glycoproteins, it acilitates the ormation o lopo-dia during platelet activation []. Dense tubular system o platelets is a closed-channel network o residual endoplasmicreticulum and primarily involved in calcium sequestrationwith the help o cascades o reactions involving the activationo G protein-coupled receptor PAR- [, ]. Te highly specialized cytoskeleton o platelets maintains its discoidstructures as well as protects the cell rom getting sheared inbloodstream. It has three major components: () the spectrin-based membrane skeleton, () the actin cytoskeleton, and ()the marginal microtubule coil.


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Platelets have two major storage granules, namely, anddense granules, whose unction is to store biologically activemolecules precisely involved in initiation o coagulation andrecruiting other cells during inammation []. Te moreprevalent granule contains proteins (e.g., GPIIbIIIa, b-rinogen, and vW) which initiate the coagulation cascades.

Numerous membrane proteins essential to platelet unctionare also packaged into granule which includes GPIIbIIIa,P-selectin (CDP), and CD. granules also have the bulk o cellular P-selectin in their membrane. P-selectin via P-selectin glycoprotein ligand (PSGL) has been reported torecruit neutrophils [, ]. Dense granules store a variety o hemostatically active molecules which are secreted duringplatelet activation; these include catecholamines, serotonin,calcium, adenosine -diphosphate (ADP), and adenosine -triphosphate (AP). ADP is a weak platelet agonist, triggeringplatelet shape change, granule release, and aggregation [].

.. Platelet Receptors. Platelet surace receptors have beenalways a eld o interest among scientists or many years andplatelets also can exert their granule contents during diseaseprognosis. A list o platelet receptors, also known as plateletagonists, have been summarized in (able ) according totheir activity [].

.. Platelet Endothelium Interaction, Hemostasis, and Platelet Aggregation. Platelets are completely different rom endothe-lial cells and can interact in multiple ways when exposedto endothelial surace (able ). Tese interactions can be o cross talk over a distance also known as paracrine signaling

via transient interactions or through receptor mediated cell-cell adhesion. Platelets are also able to release or transermany substances as discussed earlier that also interact withendothelial cell []. Although platelets and endothelial cellsare different in many ways, they do share some commoneatures, like both cell types are derived rom a common bonemarrow derived progenitor cell. Some o their transcriptionalnetworks and gene expression programs are also similar likeGAA-, vW, multimerin, and P-selectin. Both o them storetheir bioactive materials in their cytoplasmic granules. Froman evolutionary approach, endothelium can be consideredas sedentary in its way where platelets and megakaryocytescirculate in bloodstream [].

Endothelial cells with the help o COX-, COX-, andprostacyclin synthase can convert arachidonic acid intoprostacyclin, which in turn inhibits platelet unction by the

elevation o intracellular cyclic AMP levels [].An injury in the vessel wall activates platelets to initiate

coagulation, which is also known as hemostasis. Dynamicplatelets readily get activated/inhibited by several endoge-nous and exogenous stimuli. Tey initiate primary hemosta-sis by adhering themselves to the damaged vessel wall. GPIb-V-IX and GPIa-IIa receptors and subendothelial compoundslike vW and collagen interact with each other to mediate thisprocedure (Figure ). Binding o ligands to the GP receptorschanges platelet shape as well as triggers the release o itsgranule contents, which ultimately leads to the ormation o aggregates which are also known as “platelet plugs” or “whitethrombi” (Figure ).

Platelet starts to change its shape by the ormation o

pseudopods when intracellular Ca2+ concentration exceedsa specic threshold. During shape change, platelet brino-gen receptors (GPIIb/IIIa) are exposed and activated, andplatelet-platelet aggregation is initiated. Tis is also knownas primary aggregation which is reversible. However, resting

platelets are not able to bind brinogen. Arachidonic acidthromboxane pathway is an important platelet activationpathway (Figure ). Aspirin, also known as acetylsalicylicacid, a drug widely used in CVD, inhibits platelet aggregationthrough irreversible acetylation and inactivation o COX,resulting in blockage o xA

2 production [, ]. Mature

normal human platelets express only COX-, as the anucleateplatelet cannot synthesize enzyme de novo. As a result theeffect o aspirin on them is permanent and cumulative. Tus,the cardioprotective effect o aspirin is exerted through theirreversible and permanent impairment o thromboxane A

2-

dependent platelet unction, which reduces the developmento acute arterial thrombosis [].

ADP is another important platelet activator. PY12, anADP specic receptor, is present on the platelet membraneand is coupled to inhibitory G-proteins and mediates ADP-

induced release o Ca2+, inhibiting adenylate cyclase andactivating the GPIIb/IIIa receptor which leads to plateletaggregation. Te thienopyridines, ticlopidine, and clopido-grel inhibit platelet activation via blockage o the PY

12 recep-

tor []. Tromboxane A2, ADP, and other substances such

as serotonin, released rom the activated platelet, provideimportant positive eedback and strengthen the platelet-richclot initiating secondary aggregation which is irreversible(Figure ) [].

Platelet response is amplied via substances released by

platelet granules which recruit other platelets and blood cells.Te platelet plug initially ormed in primary hemostasis isrelatively unstable. Coagulation cascade and ormation o thrombin and brin prolong secondary hemostasis. Duringplatelet activation, platelet membrane phospholipids becomenegatively charged, which acilitates coagulation activation(e.g., FV, FVIIIa, FIXa, and FX). Binding o the prothrom-

binase complex (FXa, FVa, Ca2+, and prothrombin) to theplatelet membrane occurs in this step. Further platelet activa-tion is initiated by the ormation o thrombin. Tese cascadeslead to the ormation o “red thrombus” strengthening theblood clot [].

Intact vascular endothelium releases two major antiag-

gregants, prostacyclin (PGI2) and nitric oxide (NO), whichprevent the ormation o thrombus inside the blood vessel[].

.. Platelet Function Assay. Platelets are dynamic bloodparticles which can interact with each other as well as withleukocyte and endothelial cells. Previously, platelet unctionwas assessed using light transmission aggregometer []whose primary unction was to measure the increase in lighttransmission through a platelet suspension when plateletswere aggregated by an agonist. It has some major drawbacks.First o all, the result obtained may be affected by many

variables. Secondly, the accuracy and the reproducibility o


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: Platelet receptors in different activation stages (adapted rom Kauskot and Hoylaerts, []).

(a) Platelet receptors in recruitment, adhesion, and aggregation

Receptors Present in Family Ligands Comments

Initiation o platelet recruitment

GPIb-IX-V complex Platelet surace Leucine-rich repeat amily vW, thrombin, FXI, FXII,

P-selectin, HK, Mac-, SP-Bernard-Soulier

syndrome

Platelet adhesion and aggregation

GPVI Platelet surace Ig superamily Collagen, laminin

Platelet plasma

membrane Integrins Collagen

” ” Fibronectin

” ” Laminin

V ” ” Vitronectin, brinogen, vW,

osteopontin

IIb ” ” Fibrinogen, brin, vW, SP-,

bronectin, vitronectinGlanzmann

thrombasthenia

CD Platelet surace yrosine phosphatasereceptor Unknown Regulation o GPVI

CLEC- ” C-type lectin receptor Podoplanin (platelets? CLEC-?)

(b) Platelet receptors in the amplication phase


PYPlatelet plasma

membraneG protein-coupled

receptors ADP

PY ” ” ”

PAR ” ” Trombin High affinity

PAR ” ” ” Low affinity

tPA ” ” Tromboxane

PAF receptors ” ” -O-alkyl--acetyl-sn-glycero--phosphocholine

PAF: plateletactivating actor

PGE receptor (EP) ” ” PGE

Lysophosphatidic acid receptor ” ” Lysophosphatidic acid

Chemokine receptors ” ” Chemokines

Va vasopressin receptor ” ” Vasopressin

Aa adenosine receptor ” ” Adenosine

b adrenergic receptor ” ” Epinephrine

Serotonin receptor ” ” Serotonin (-hydroxytryptamin)

Dopamine receptor ” ” Dopamine

PX ” Ion channel AP

c-Mp ” yrosine kinase

receptor PO

Insulin receptor ” ” Insulin

PDGF receptor ” ” PDGF

Leptin receptor ” Cytokine Leptin

(c) Platelet receptors in the stabilization phase and in the negative regulation o platelet activation


Stabilization

Eph receptor Platelet plasma membrane yrosine kinase receptor Ephrin

Axl/yro/Mer ” ” Gas-


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(c) Continued.


P-selectinPlatelet

granule; comes in plasmamembrane upon activation

C-type lectin receptoramily

PSGL-, GPIb, F SolubleP-selectin: biomarker

SSC Platelet plasma membrane etraspanins —

CD ” ” —

CD ” Class B scavenger receptor SP, oxLDL, VLDL,

oxPL, collagen type V Many unctions

L- ” Ig superamily Fibrinogen? L- soluble orm

correlated with DIC

PEAR ” Multiple EGF-like domain

protein —

Phosphorylated aferplatelet contact

Negative regulation

VPAC Platelet plasma membrane G protein-coupled

receptors PACAP

PECAM- ” Ig superamily PECAM-, collagen,

glycosaminoglycans

GB-b ” ” —

PGI receptor (IP) ” G protein-coupled

receptors PGI

Prostacyclin releasedrom endothelial cells

PGD receptor ” ” PGD

PGE receptor (EP) ” ” PGE

: A comparison o endothelial and platelet properties (adapted rom Warkentin et al., []).

Endothelium Platelets

Nucleus Yes No

mRNA Lots Little but active

Cell dimensions

Highly variable: up to M

length in large vessels

Diameter∼M, volume –L

(inversely proportional to platelet count)

Lie span Long (months to years) Short (– days)

Daily production Not known . ×

Circulating Few Most (normally / sequestered in spleen; may become

sequestered on activated endothelium)

Diagnostic markers Indirect and not clinically

useul CBC, peripheral smear, platelet unction studies

Origin Bone marrow Bone marrow

Storage granules Weibel-Palade bodies granules, dense granules, lysosomes

Partial list o storage components vW, P-selectin, multimerin vW, P-selectin, multimerin, brinogen, PDGF, GF-, IL-,

VEGF, angiopoietin, RANES, PF, ADP, AP, serotonin

the technique are very poor. And overall in case o low platelet count the result is very difficult to interpret. Tusthis technique was almost replaced by ow cytometry assay [, ], which is ar more convenient andperect. Expression o several platelet markers suchas CDp (P-selectin) and CDis now being well studied using ow cytometry technique [,]. PAC , a monoclonal (Figure ) antibody with specicbinding to the brinogen receptor o activated platelets, isnow widely used to assess platelet activation. Platelet-boundantibodies were detected either by uorescein isothiocyanate(FIC) or by phycoerythrin (PE) [, ].

As platelets are devoid o any nucleus, proteomics aidsnew therapeutic targets o many diseases with platelet dys-unction []. Afer obtaining platelet-rich plasma, plateletsare isolated and simultaneously disrupted to analyze theirproteomic contents. D or D gel electrophoresis is involvedto separate the protein ractions where mass spectrometry can analyze the proteins. Further investigation involves theusage o Western blotting which will ultimately validatethe obtained data. A researcher group ound alteration incytoskeleton as well as signicant differences in ILK, GAPDHand PK in platelets rom arterial thrombosis []. Another


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: Clinical assessment o platelet unctions.

Platelet aggregometry

Photo-optical platelet aggregometry [] Platelet-rich plasma is taken to check the platelet aggregation.

Impedance platelet aggregometry [] Platelet aggregation is checked by using the whole blood by electrical impedance.

Light-scattering platelet aggregometry [] It is a combination o laser light scattering and aggregometry to monitor platelet

microaggregate ormation.Point-o-care or platelet unction tests

Ultegra rapid platelet unction assay (RPFA)[]

RPFA is a simple and ast, automated point-o-care device that monitors GPIIb-IIIainhibition. Tis test is based on platelet agglutination rom interaction betweenunblocked GPIIb-IIIa receptors and brinogen-coated beads.

Platelet-activating clotting test (PAC) assay []

Te PAC assay, HemoSAUS, measures AC without a platelet activator, comparing itwith ACs obtained with increasing concentrations o platelet-activating actor (PAF).

Platelet unction analyzer (PFA-) []

PFA- exposes platelets within citrated whole blood to high shear stress through acapillary tube, ollowed by an aperture in a membrane coated with collagen and eitherADP or epinephrine. Te platelets adhere and aggregate until the aperture is occluded,and the time to this closure is recorded.

Plateletworks test []

Te Plateletworks is a point-o-care test that uses a Coulter counter to measureplatelet-count ratio, red blood cell count, hemoglobin, and hematocrit. Platelet count ismeasured in a control sample in which aggregation is prevented by EDA and comparedwith the platelet count in an agonist-stimulated (ADP or collagen) sample.

Clot signature analyzer [] Tis test uses nonanticoagulated whole blood and can measure several aspects o platelet

unction and clotting properties.

Tromboelastography [] Tis device measures clot strength and gives a global assessment o hemostasis.

group ound signicant alteration in cytoskeleton, differentialexpression o Coronin-B, PSB, and pleckstrin, and upregu-lation o proteins associated with energy metabolism. Tus,proteomics assay could implicate a new horizon in plateletstudy [].

.. Symptoms of Platelet Dysfunctionand Clinical Assessment.Diagnosis o platelet unction relies on clinical ndingso both detailed medical history and amily history. Someplatelet disorders are hereditary where most o them areacquired due to various diseased conditions. An alterationin platelet unction includes prolonged or excessive bleed-ing which is the primary screening procedure to assessplatelet dysunction. Change in platelet number and meanplatelet volume are another indicating markers in plateletdysunction. Some o the common symptoms include []the ollowing:

(i) unexplained or extensive bruising particularly associ-ated with sof tissue hematoma,

(ii) epistaxis, particularly lasting more than minutes orcausing anemia,

(iii) menorrhagia, particularly i present since menarche,

(iv) gingival bleeding,

(v) heavy and prolonged bleeding childbirth,

(vi) bleeding ollowing invasive procedures (e.g., dentalextraction, tonsillectomy, and adenoidectomy).

Clinically, platelet unction is assessed either by checkingplatelet aggregation by taking the whole blood or platelet-rich

plasma rom the patients to the pathology lab or by point-o-care (POC) platelet unction test. Te tests are summarizedin (able ).

3. Platelet Dysfunction inDisease Pathophysiology

In recent times, platelets have emerged to be important mark-ers or disease pathophysiology. Tey are multiunctionalblood particles and very important clinical targets or many disease pathophysiology (Figure ). Being important inam-matory markers, they play important roles in atherosclerosisand cardiovascular disorders which are correlated with type diabetes. Tey have proound role in tumor biology aswell as allergic inammation. Trombin, an agonist releasedby platelets has proound role in inammation [–],angiogenesis [] and embryonic development [, ].

4. Platelet Dysfunction in Cardiovascular

Disorder (CVD) and Diabetes

Diabetes mellitus is heterogeneous, multiactorial, polygenicdisease characterized by deect in insulin’s secretion (the betacell secretory deect) and action (insulin resistance) [].ype which is the most prevalent orm is basically a liestyledisorder now becoming a major global threat. Obesity is themajor cause o diabetes in the adults []. Te most prevalentdiabetic macrovascular complication is cardiovascular disor-der []. BMI was signicantly and linearly associated withsystolic blood pressure, asting glucose levels, plasma totalcholesterol, VLDL cholesterol, and LDL cholesterol levels andwas inversely and linearly associated with HDL cholesterol


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Endothelial cell

PMP

F

MMP

PMP EMP

Monocyte

CD62P PMP

CD11b

CD54

PSGL-1

AA

PKC MAPK

COX-2

F : Mechanism o vascular changes by platelet-derived microparticles (PMPs). PMPs activate monocytes by a reaction between P-selectin and PSGL- (P-selectin glycoprotein ligand-). Activated monocytes induce expression on the cell surace o tissue actor (F) andCDb. Activated monocytesalso induce release o monocyte-derived microparticles (MMPs). PMPsinduce COX- production in endothelialcells. PMPs enhance expression o CD (ICAM-) on the endothelial surace. Activated endothelial cells also induce release o endothelialcell-derived microparticles (EMPs), enhancing adhesion between endothelial cells and monocytes. Finally, monocytes induce migration o endothelial cells, resulting in vascular changes. Abbreviations: arachidonic acid (AA); protein kinase C (PKC); mitogen-activated proteinkinase (MAPK) (adapted rom Nomura, []).

level [], having a direct correlation in developing DM.Obesity is a key eature o metabolic syndrome, reecting theact that the syndrome’s prevalence is driven by the strongrelationship between body mass index (BMI) and increasingadiposity [].

Our research group previously ound hyperglycemia-induced oxidative stress in structural unctional alterations o hemoglobin and red blood cells[, ]. But there are limitedreports to elucidate the altered behavior o platelets in di-erent diseases, majorly ocusing on diabetes and associatedcardiovascular disorders which are major threats to society in recent times.

.. Hyperaggregation in Platelets. One o the most commonchanges in platelet behaviour in diseased condition, such asdiabetes, is platelet hyperaggregation. In response to vari-ous agonists, hyperaggregation in platelets was reported inpatients with both type and type diabetes mellitus [ ,]. Hyperaggregation is associated with generating more -dehydro-thromboxane B

2 which is the important end prod-

uct o thromboxane pathway as discussed earlier (Figure ).DM has been reported to increase the production o thisproduct as well as prothrombin accelerating aggregation; thiscan increase the chances o CVD among diabetic patients,as hyperaggregated platelets have a tendency to block the

blood vessels. By contrast, anticoagulant markers, such asactivated protein C, protein C activation peptide, and solublethrombomodulin (M), were depressed in DM, urtherincreasing the chances o CVD [].

.. Alterations in Tromboxane Production. Tromboxane isan important product which plays proound role in plateletaggregation. Studies showed that thromboxane production isenhanced in diabetes subjects compared to controls increas-ing platelet aggregation, indicating a higher risk o CVD[–]. o initiate aggregation, platelets rom patients withDM can synthesize greater amount o xB

2 but it requires

less arachidonate, the precursor o thromboxane pathway and collagen, than rom normal nondiabetic controls [].xA

2 production has been positively correlated with asting

plasma glucose and HbAc; higher blood glucose increasesthe production o xA

2 which is an important product

in thromboxane pathway. Several studies showed reducedxA

2 production in improved glycaemic controls [, ,

]. Data suggests that -iso-PGF, a marker involvedin lipid and arachidonate peroxidation; is correlated withxA

2 biosynthesis which could be a link between glycaemic

control, oxidative stress, and platelet activation []. Tusalteration in thromboxane pathway can be a link betweenplatelet dysunction and diabetes, obesity, and CVD.


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Primary hemostasis

Secondary hemostasis

Vascular injury

Vasoconstriction

Platelet plug formation

Coagulation (brin formation) brinolysis

Repair process

Wound healing

F : Pathway illustrating hemostasis.

Arachidonic acid

Cyclooxygenase

Prostaglandin G2(PGG2)

Prostaglandin H2(PGH2) Prostacyclin (PGI2)

xA2-synthase

Tromboxane A2 (xA2)

Tromboxane B2 (xB2)

11-Dehydro-xB2 -dinor-xB22,3

F : Tromboxane biosynthesis pathway.

.. Changes in Platelet Membrane Fluidity. Change in mem-brane uidity has been assigned to the impairment o plateletunctions since membrane uidity can modulate cell unc-tion, and reduced membrane uidity in cholesterol- enrichedplatelets is associated with platelet hypersensitivity to differ-ent agonists []. Due to glycation o membrane proteins,platelet membrane uidity has been grossly impaired indiabetes. Decreased membrane uidity o these platelets is

attributed to an increased cholesterol-phospholipid molarratio in platelet membranes. Reduced membrane uidity is associated with hypersensitivity to thrombin in intactplatelets rom diabetic subjects [, ].

.. Altered Expression of Platelet Agonists. Te number andadhesiveness o several platelet specic surace glycoproteinreceptors (GP) are signicantly enhanced in diabetic subjects.CD ligand on platelets has been correlated with HbAcconcentrations. An upregulation o the CD-CD ligandsystem has been observed in patients with DM [] alongwith an increase in GPIIb/IIIa, vW, GPIa/IIa, P-selectin(CD), and CD [–]. Patients suffering rom ischemic

heart disease (IHD) and depression concurrently may haveabnormal platelet activation resulting in an increased risk o thrombosis. Mean PF and -G plasma levels in the IHDgroup with depression were ound to be signicantly higherthanthose o the control and IHD groups []. Moreover high

vW value may increase the chance o CVD [] which can

also act as an index or disease prediction.Hyperglycemia induces downregulation o SIR andupregulation o PAF-R in endothelial progenitor cells whoseassociation is linkedwith vascular complications[]. Plateletactivity is correlated with nitric oxide (NO) synthesis, whichis impaired in acute coronary syndrome (ACS). Signicantdifferences o eNOS gene polymorphism and expressionhave been observed between diabetic patients with ACSand patients with ACS but no history o diabetes [].Platelet FcgammaRIIA is signicantly overexpressed in type diabetes whose interaction with collagen may alter plateletadhesion.

Several candidate gene polymorphisms have been associ-ated with platelet unctions. Polymorphisms o these genesmay develop CAD, CVD, and ACS in both diabetic andnondiabetic subjects. Some o these genes are glycoprotein

Ia/IIa (21) (—C>, G>A—, —C>, A>G),glycoprotein lb (VNR A-D), and glycoprotein IIb/IIIa(substitution o proline or leucine at position ) [].However, alpha integrin C gene polymorphism plays acrucial role in arterial thrombosis [].

.. Changes in Mean Platelet Volume (MPV). MPV ismachine generated average size o a platelet, whose valueis enhanced in subjects with DM. An explanation o thisphenomenon is that larger platelets are more reactive and can

express more surace receptors[, ]. Increased MPV valueis associated with diabetic retinopathy. Event o myocardialinarction is positively correlated with MPV [].

.. Alterations in Intracellular Ionic Environment. Platelethyperactivity is associated with reduced Na+/K+ APase and

increased Ca2+ APase activity which can increase intra-

cellular Ca2+ concentration, thus increasing platelet activity []. Moreover a decrease in intracellular Mg+ concentrationin diabetic platelets may provoke an accelerating effect inplatelet activity []. Oxidative stress is associated withsuperoxide anion production and nitric oxide (NO) synthesisas well as production o reduced glutathione. Hyperglycemia

exerts oxidative stress which can urther implement alteredplatelet unctions [].

.. Changes in Mitochondrial Membrane Potential. Studieshave established that plasma glycated haemoglobin (HbAc)level is positively correlated with platelet AP content, sug-gesting that hyperglycemia promotes platelet mitochondriato generate more AP, but decreases platelet mitochondrialpotential [].

.. Effect of Antidiabetic Drugs on Platelet Dysfunction.Apart rom aspirin which is commonly used in diabetes mel-litus, many widely used antidiabetic drugs also result in


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Subendothelium

Collagen vWf

GPVI GPIb-IX-V

GPIIb/IIIaFibrinogenGPIIb/IIIa

GPIIb/IIIaFibrinogenGPIIb/IIIa

ADP

Adhesion

Shape change

Primary aggregationSecretion

Secondary aggregation

xA2

F : Afer an injury in the vessel wall, activation o platelets begins to start. It involves its adhesion to the subendothelium surace.Interaction between receptors like GPIb-V-IX, GPIa-IIa, and subendothelial compounds like vW and collagen triggers the release o plateletgranule contents accelerating aggregate ormation.

Vasoconstrictive substances

Cytokines

ADP

Activation

Ca2+Platelet Conformationalchanges

GPIb-IX-V(GPIb)

G P I I b / I I I a

G P I I b

/ I I I

a

GPIIb/IIIaFVW

A1

A1

A3

A3

RGDRGD

RGD

RGD

Te greater the shear rate, the greater therole of vWf in platelet adhesion

Collagen VI Collagen

Extracellular matrix

F i b r i n o g e n

↑

Collagen I–III

F : Diagram il lustrating the role o von Willebrand actor (vW) in platelet adhesion. High ow rates induce conormational changesin vW allowing interaction o its A domain with matrix collagen. Tis induces a conormational change in the A domain, thereby allowinginteraction with glycoprotein (GP) platelet receptor Ib-IX-V. Tis interaction stimulates calcium release, subsequent platelet activation, andsubsequent conormational change o the brinogen receptor (GPIIb/IIIa), whichcan interactwith brinogen and vW to avor the interactionbetween platelets (platelet aggregation process). ADP indicates adenosine diphosphate; RGD and Arg-Gly-Asp are amino acid sequences(adapted rom Badimon et al., []).

platelet malunction. Te majority o these drugs are pre-sented hereunder.

... Metformin. Metormin, an oral antidiabetic, rst linedrug o biguanide class, is particularly useul or overweightand obese diabetic patients. Chakraborty et al. reported thatmetormin is particularly useul to restore the antioxidantstatus o cells hampered in type diabetes []. Metormintreatment also decreases ROS generation and creates mito-chondrial membrane hyperpolarization lowering the risk o CVD []. Different researchers concluded that metormincan stabilize platelet by reducing platelet density, -G, andplatelet superoxide anion production [–]. Metorminalso helps to reduce PAI- in plasma, thus putting benecialeffects on brinolysis. By reducing FVII and FXIII, met-ormin also plays an important role in coagulation [].

... Sulfonylurea. Sulonylurea derivatives are a class o antidiabetic drugs which act by increasing insulin releaserom the beta cells in the pancreas. Gliclazide, a sulony-

lurea derivative, can check blood glucose level by its reeradical scavenging ability. It also coners benecial effectson the hemorrheologic abnormalities by reducing plateletabnormalities in diabetic patients by increasing prostacy-clin synthesis []. Gliclazide can reduce platelet aggre-gation, enhance brinolysis, inhibit the surace expressiono endothelial adhesion molecules, and inhibit neutrophilendothelial cell adhesion [, ].

... Tiazolidinediones. Tiazolidinediones or glitazonesactivate PPAR via FFA and eicosanoids. When activated,the receptor binds to DNA in complex with the retinoid Xreceptor (RXR) modiying the transcription o certain genes


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C D 6 2 P E

CD61FICCD62P expression 45%

-1

1

1

10

10

100

100

1000

1000−1

( 1 2 8

×

1 2 8 )

(a)

CD61FICCD62P expression 17%

C D 6 2 P E

-1

1

1

10

100

1000

10 100 1000−1

( 1 2 8

×

1 2 8 )

(b)

F : Flow cytometric detection o P-selectin in (a) diabetic patient and (b) nondiabetic patient (adapted rom Saad et al., [ ]).

Platelet

functions

Hemostasis and thrombosisAdhesionActivationSpreadingSecretion

AggregationProcoagulant activity

Clot retractionissue repair

Maintenance/regulation of vascular tone

Uptake of serotonin when restingRelease of serotonin, thromboxane, prostaglandins

Upon activation

umor biology

umor growthumor killing

umor metastasisHost defense

Phagocytosis/internalisation of virusesand bacteria

Killing of bacteria

Superoxide production

InflammationAtherosclerosis

Allergic asthmaRenal disease

Platelet-leukocyteinteractions

Chemotaxis

Release of platelet microbicidal proteins

F : Te multiunctional platelet (adapted rom Harrison, []).

in several tissues including vascular tissue []. Plateletshave proound role in inammation due to their release o theproinammatory and proatherogenic mediators: CD lig-and (CDL) and thromboxanes (Xs). Studies showed thatplatelet incubation with rosiglitazone prevented thrombin-induced CDL surace expression and release o CDLand thromboxane B

2(xB

2) []. Administration o rosigli-

tazone has signicantly reduced von Willebrand actor inone study []. Platelets rom patients with type diabetesmellitus lack platelet endothelial cell adhesion molecule-.Rosiglitazone treatment has been ound to increase platelet

SERCA- expression and Ca2+-APase activity. Moreover,it has inhibiting effect on SERCA- tyrosine nitration, thus

normalizing platelet Ca2+ concentration. Rosiglitazone alsoreduces -calpain activity, normalizes platelet endothelialcell adhesion molecule- levels, and partially restores plateletsensitivity to nitric oxide synthase inhibition [].

... Acarbose. Acarbose blocks carbohydrate digestion by inhibiting enzyme glycoside hydrolases. Acarbose is knownto decrease platelet-monocyte aggregate ormation by reduc-ing postprandial hyperglycaemia in diabetic patients [].


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.. Effects of Insulin on Platelets. Insulin resistance and asso-ciated metabolic syndrome jointly correlate with prothrom-bic state in diabetes mellitus to put a cumulative effect in theprogression o vascular complications []. Insulin has beenreported to have antiplatelet effects. Researchers have identi-ed a direct correlation between insulin-induced attenuation

o the thrombin-induced Ca

2+

response and platelet aggrega-tion [, ]. Diabetic platelet shows an altered Ca2+ home-ostasis []. yrosine phosphorylation o platelet plasma

membrane Ca2+-APase may serve as positive eedback to

inhibit membrane Ca2+-APase and increase intracellularcalcium during platelet activation [].

Reports suggest that in nondiabetic patients with acuteischemic heart disease binding o PGI

2 with insulin is less-

ened []. But insulin administration has normalized plateletactivity to PGI

2 in coronary heart diseases both in vitro and

in vivo [, ]. Vitamin E deciency plays a crucial rolein platelet aggregation, where -tocopherol supplementationin response to ADP decreases xA

2 []. Although several

studies concluded the benecial role o Vitamin E supple-mentation in diabetic patients with CVD, large scale study ails to conclude the minimization o myocardial inarctionoccurrence in diabetic patients []. Insulin therapy may improve platelet sensitivity to NO, thus providing a suitableplatorm in treating CVDs among diabetic patients.

5. Platelet Dysfunction in Other Diseases

.. Heart Disease. Not only diabetes related cardiovasculardisorder but also platelet dysunction has been reported inother heart diseases. It has been reported in many cases thatpatients with congestive heart ailure (CHF) have increased

risk o venous thromboembolism, stroke, and sudden death[]. J. Mehta and P. Mehta rst reported that patientswith CHF have signicantly higher number o circulatingplatelet aggregates than the normal subjects []. In onestudy, patients with lef ventricular dysunction have shownincreased number o brin D-dimer, brinogen and vW levels compared to healthy controls []. Patients with acutedecompensated heart ailure (AHF) show more abnormal-ities o platelet activation than stable CHF patients andhealthy controls []. In heart ailure, endothelial nitricoxide (NO) production is much lower where oxidative stressand NO degradation rate are much higher. Platelet hasbeen shown to produce less bioactive NO in patients with

heart ailure, mainly due to the deect in the the plateletl-arginine/NO/guanylyl cyclase pathway []. Tus alteredplatelet activity can give major insights into heart disease andcan be an interesting eld to explore.

.. Renal Disease. Complex hemostatic disorders have beenound in patients with end stage renal disease (ESRD) thatmay be in orm o bleeding diatheses. Platelet dysunctionsresult due to the presence o toxic products in the circulat-ing blood. Dialysis improves this complication; however, itdoes not eliminate the risk o hemorrhage. Some commonpathological eatures include thrombocytopenia, glomerularthrombosis, and thrombi in small arteries and glomerular

capillaries []. Both intrinsic platelet abnormalities andimpaired platelet-vessel wall interaction can contribute toplatelet dysunction []. In renal ailure, the normal acti-

vation process o the platelets to orm aggregates is impaired.

.. Cancer and umorigenesis. Platelet plays versatile role in

cancer progression. Te procoagulant environment providedby platelets can secure the coagulation o cancer cells,protecting them rom immune system, thus prompting theormation o tumors []. Platelets acilitate tumor cellmigration and invasiveness, prompting metastasis. It hasbeen reported in both breast and ovarian cancer that plateletsincrease the invasiveness o cancer cells which can induce theurtherprogressiono the disease [–]. Moreover, tumorcells also have the ability to aggregate platelets [], urtherincreasing the chance o inducing metastasis. Activation o platelets and regulation o other cells have been controlled by thrombin by means o G protein-coupled protease-activatedreceptors (PARs) []. Researchers have shown that throm-

bin signaling also contributes hugely to the progression o tumorigenesis and angiogenesis [].

.. Alzheimer’s Disease. Platelet dysunction has been alsoimplicated in Alzheimer’s disease (AD), which is the mostcommon orm o dementia. Platelet can store a huge amounto amyloid precursor protein (APP); recent nding showsthat platelet APP metabolism may accumulate A in thebrain and its vasculature through the blood brain barrier[]. Platelet -granules store RANES, an inammatory signaling molecule whose secretion rom PBMC has beenreported to increase in AD []. Te uidity o the hydro-carbon region o platelet membranes rom the AD patients is

signicantly higher than that o control group []. Plateletsecretases activities and COX enzyme activity (which is alsoa component in APP secretion pathway) have been impairedin platelets o AD patients, thus making this blood particle asuitable marker o this disease [].

.. Liver Disease. Quantitative and qualitative plateletdeects, hyperbrinolysis, accelerated intravascular coagula-tion, and decreased synthesis o clotting and inhibitor actorsare well observed in both acute and chronic liver diseases[]. As most coagulation actors except vW are synthesizedby liver parenchymal cells, and liver’s reticuloendothelialsystem plays crucial role in the clearance o activation

products, a deect in liver unction may alter hemostasis[, ]. Not only unctions but platelet numbers also havebeen reported to decrease in liver diseases []. It has beenreported that platelet transusion can improve liver unctionin patients with chronic liver disease and cirrhosis [].Platelets morphological parameters have been also reportedto alter in liver diseases [], thus providing a platorm orplatelet research in liver diseases.

6. Conclusion

Biomarkers are now emerging with immense scientic andclinical value through the whole journey o the disease


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process.Beore diagnosis, markers canbe used orprediction,screening, and risk assessment. During diagnosis, markerscan determine staging, grading, and selection o initial drugtherapy. During treatment, markers are used to monitorthe prognosis o therapy or to select additional treatmentneeded. Platelet indices indicating differential dysunction,

hyperactivation, aggregation, or adhesion are capable o expressing the characters o disease pathogenesis. Accuratedetermination o platelet indices is cost effective and theirimpaired unction can be correlated with the inammationinvolved in diseased state. Tus, an attempt to identiy different platelet biomarkers is o signicant impact on theglobal scene o clinical application and development.

Conflict of Interests

Te authors declare that there is no conict o interestsregarding the publication o this paper.

Acknowledgement

Te authors acknowledge Council o Scientic and IndustrialResearch, Government o India, or providing research el-lowship to Ms. K. Ghoshal.

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