Platelets in hemostasis and thrombosis:Novelmechanisms of fibrinogen-independent platelet aggregation and fibronectinmediated protein wave of hemostasis

Yan Hou,Naadiya Carrim,Yiming Wang,Reid C.Gallant,Alexandra Marshall, Heyu Ni,5,✉

1Department of Laboratory Medicine,Keenan Research Centre for Biomedical Science,Li Ka Shing Knowledge Institute, St.Michael's Hospital and Toronto Platelet Immunobiology Group,Toronto,M5B 1W8,Ontario,Canada;

2Jilin Provincial Center for Disease Control and Prevention,Changchun,Jilin,130062 China;

3Department of Laboratory Medicine and Pathobiology,University of Toronto,Toronto,Ontario M5S 1A1,Canada;

4Canadian Blood Services,Toronto,Ontario M5B 1W8,Canada;

5Department of Medicine and Department of Physiology,University of Toronto,Toronto,Ontario M5S 1A1,Canada.

Platelets in hemostasis and thrombosis:Novelmechanisms of fibrinogen-independent platelet aggregation and fibronectinmediated protein wave of hemostasis

Yan Hou1,2,△,Naadiya Carrim1,3,4,△,Yiming Wang1,3,4,Reid C.Gallant1,3,Alexandra Marshall1, Heyu Ni1,3,5,✉

1Department of Laboratory Medicine,Keenan Research Centre for Biomedical Science,Li Ka Shing Knowledge Institute, St.Michael's Hospital and Toronto Platelet Immunobiology Group,Toronto,M5B 1W8,Ontario,Canada;

2Jilin Provincial Center for Disease Control and Prevention,Changchun,Jilin,130062 China;

3Department of Laboratory Medicine and Pathobiology,University of Toronto,Toronto,Ontario M5S 1A1,Canada;

4Canadian Blood Services,Toronto,Ontario M5B 1W8,Canada;

5Department of Medicine and Department of Physiology,University of Toronto,Toronto,Ontario M5S 1A1,Canada.

Platelets are smallanucleate cells generated from megakaryocytes in the bone marrow.Although plateletgeneration,maturation,and clearance are stillnotfully understood,significantprogress has been made in the last1-2 decades.In blood circulation,platelets can quickly adhere and aggregate atsites ofvascularinjury,forming the platelet plug(i.e.the firstwave ofhemostasis).Activated platelets can also provide negatively charged phosphatidylserinerich membrane surface thatenhances cell-based thrombin generation,which facilitates blood coagulation(i.e.the second wave of hemostasis).Platelets therefore play centralroles in hemostasis.However,the same process of hemostasis may also cause thrombosis and vessel occlusion,which are the most common mechanisms leading to heart attack and stroke following ruptured atherosclerotic lesions.In this review,we will introduce the classical mechanisms and newly discovered pathways of platelets in hemostasis and thrombosis,including fibrinogen-independent platelet aggregation and thrombosis,and the plasma fibronectin-mediated‘protein wave’of hemostasis that precedes the classical first wave of hemostasis.Furthermore,we briefly discuss the roles of platelets in inflammation and atherosclerosis and the potential strategies to control atherothrombosis.

platelets,thrombosis and hemostasis,integrinαIIbβ3,fibrinogen,fibronectin

Platelets:evolution,generation, and clearance

Platelets are small anucleate cells in the circulation, with a diameter of approximately 1-2μm.They were firstidentified in 1874 by Osler[1];however,itwas the Italian physician,Bizzozero,who in 1881 established the role ofplatelets in hemostasis and thrombosis in his seminalpublications[2-3].By staining the granules of platelets(Wright stain),it was later demonstrated thatthese anucleate cells are generated from megakaryocytes in thebone marrow[4].In thefollowing century,intensive investigation of the cellular and molecular mechanisms of platelets and megakaryocytes[5-7]enabled the developmentofa number oftherapeutic agents forthe treatment and prevention of thrombotic disorders[8-11].

In non-mammalian vertebrates,he mostasis is mediated by nucleated cells called thrombocytes[12]. Anucleated platelets evolved only in mammals,and to the bestofourknowledge,there is no animalspecies that have been found thus far to have an intermediate state between thrombocytesand platelets.In addition,platelets are more effective than thrombocytes in forming occlusive thrombiunderarterialshearstress[13].Since platelets havean averagelifespan ofonly 8-10 daysin humansand approximately 5 days in mice,they are constantly being produced from megakaryocytes in the bone marrow. During maturation,megakaryocytesundergo DNAreplications withoutcelldivision(a process called endomitosis),leading to generation ofpolyploid megakaryocytes. The abundantgenomic DNA in the polyploid megakaryocytes enhances their ability to synthesize proteins and package them into specific plateletgranules[7,14].

The exactmechanism ofplateletrelease from megakaryocytes is stillunderdebate.In vitro studies demonstrated that platelet formation begins at one pole of megakaryocytes,then the whole cell is disintegrated, resulting in the generation ofnumerous proplatelets[15]. However,a recentintravitalmicroscopy study revealed thatmegakaryocytes extend long protrusions into bone marrow sinusoids and release proplatelets from the tip of the protrusions under shear stress,suggesting that platelet generation in vivo is drastically differentfrom in vitro cell culture conditions[16].Proplatelets then undergo further division to generate mature platelets in vivo[17].In addition to bone marrow,new discoveries suggest that megakaryocytes can also mature in the lung and shed platelets into the pulmonary vasculature[18-19].Interestingly,a recent study suggested that platelets are capable of celldivision and progeny generation even withouta nucleus[20],although more evidence is required to confirm this finding.

The process and mechanism ofplateletclearance is also notwellunderstood,butitisassumed thatthisoccurs in the reticuloendothelialsystem by macrophages.Aged platelets may express more phosphatidylserine(PS), which may attractmacrophages for clearance[21-23]. During red blood cellclearance,oldercells may induce more autoantibody binding[24];however,whether this occurs in aged platelets and whether the Fc portion of the autoantibody interacts with Fc receptors on macrophages leading to phagocytosis stillremains to be determined.A more recentstudy demonstrated thatantibody opsonization can activate platelets,leading to platelet desialylation[25,26],amechanism also involved in clearance ofchilled platelets[27-29].These desialylated platelets can then be destroyed in the liver via hepatocyte Ashwell-Morellreceptors[21,25].Whetherthis novelplateletclearance pathway[25,30]plays a role in the clearance of aged plateletshas yetto be investigated.

Platelets in hemostasis and thrombosis

Hemostasis is a criticalphysiologicalprocess to stop bleeding.Plateletaccumulation at the site of injury is considered the firstwave ofhemostasis and the second wave of hemostasis is mediated by the blood coagulation pathway[31].Platelets play a centralrole in a series of sequential events during the platelet accumulation (i.e.platelet adhesion,activation,and aggregation) and are also actively involved in cell-based thrombin generation,which markedly amplifies the blood coagulation cascade.Thus,platelets contribute to both the first and the second waves of hemostasis[6,7,32-35].

Platelet adhesion

Plateletadhesion to the injured vesselwallcan occur atboth low and high shearconditions butare mediated through distinctmechanisms.Low shearrates(20-200/s) are observed in the venous system whilsthigher shear rates are found in arteries(300-800/s)and stenotic vessels(800-10,000/s)[36-37].Following vascular injury, subendothelial matrix proteins such as collagens are exposed to the blood components.Plasma von Willebrand Factor(VWF),originated from endothelial cells,megakaryocytes,and platelets,can then anchor onto the collagen.The VWF receptoron platelets[glycoprotein(GP)Ibα],via interaction with the immobilized VWF,subsequently initiates platelettethering to the site ofinjury[38-39].This binding isessentialforplatelet adhesion at high shear(e.g.coronary arteries), although the GPIbα-VWF interaction may also contribute to plateletadhesion atlow shear[40,41].Following platelettethering,GPVIand integrinα2β1 may interact with collagen and deliver activation signals to platelets[38,42-43].Stable adhesion is subsequently mediated by binding of several integrins to their ligands on the vessel wall(e.g.integrinαIIbβ3 to fibrinogen/fibrin and fibronectin,α5β1 to fibronectin or collagen,and α2β1 to collagen,etc.)[6,42,44-46].Atlow shear(e.g.veins) the interactions between platelet integrins and their ligands(e.g.αIIbβ3 to fibrinogen/fibrin or fibronectin etc.)may directly initiate plateletadhesion[6,47].

In the last decade there have been significant advances in in vivo models of platelet adhesion and thrombus formation using intravital microscopy.VWF knockout(-/-)mice demonstrate decreased platelet adhesion[39,48],a phenotype that,interestingly,is notas severe as the GPIbα-/-mice,suggesting that GPIbαhas additionalhemostatic function[49].Mice lacking GPVI presentwith prolonged bleeding times[50]and similarly, mice deficientinα2 orβ1 integrins also have delayed thrombus formation,although these deficiencies are mild compared to GPIbα-/-mice[51].

Platelet activation and granule secretion

The primary interactions between platelet surface receptors(e.g.GPIbα,integrins)and their ligands (e.g.VWF,collagen,fibrinogen/fibrin,fibronectin, etc.),can lead to plateletactivation[7,38,52,53].In addition, following vascular injury,the coagulation system is activated[11,54-55],which generates the mostpotentplatelet activation factor,thrombin.Through cleavage of protease-activated receptors(PARs)and binding to GPIbα,thrombin activates platelets[56-59].

Plateletactivation exposes PS on the membrane surface thatdrives the cell-based thrombin generation[34,35]and facilitates further platelet activation[53,60-61]. Activation signals induced by thrombin,collagen,or ligands ofadhesion receptors with the addition ofshear stress,can lead to plateletgranule release.Plateletadhesion molecules,P-selectin[62],integrins,VWF,fibrinogen,fibronectin[63-64],vitronectin[65],multimerin[60], plateletfactor4,and approximately 300 otherproteins are contained within theα-granules[66].Dense granules release adenosine di-phosphate(ADP),which supports the second waveofplateletaggregation following integrin activation[67].The release of Ca2+from the endoplasmicreticulumand thedensegranules via the Ca2+sensor, stromalinteraction molecule(STIM)1,and the Ca2+channel,Orai,isalso a significantcontributorto platelet activation[68-69].There are many positive feedback loops during plateletactivation/granule release.Notably, ADP,likely via interaction with its receptors on platelets,initiatescell-based thrombin generation and further plateletactivation/granule release[61].These secretion eventsactassecondary messengersand,in combination with thegeneration ofthromboxane(Tx)A2and reactive oxygen species,amplify the activation process and integrinαIIbβ3 inside-outsignaling,which in turn recruits more platelets foraggregation[70-74].

Platelet aggregation:fibrinogen-dependent and-independent aggregation

Following plateletactivation,integrinαIIbβ3 binds fibrinogen and otherligands(i.e.fibrinogen-dependent and-independent pathways[31,39,61,75-76]),which leads to platelet aggregation.It is notable that following the engagementofligands,integrinαIIbβ3 can deliveroutside-in signals,which further enhance platelet activation,cytoskeleton rearrangement,and granule secretion.These signal events facilitate hemostatic plug and thrombus formation.

For more than half a century,fibrinogen was considered required forplateletaggregation[61].Through interaction withαIIbβ3 via itsγchain C-terminus, fibrinogen bridges adjacent activated platelets[22,77]. However,data from Ni etal.demonstrated thatthrombus formation still occurred in fibrinogen-/-mice and in VWF and fibrinogen double knock-out(DKO)mice[39], indicating that fibrinogen was not indispensable for this process.Further studies demonstrated that DKO platelet aggregation occurred in vitro in platelet-rich plasma and gel-filtered platelets withoutanti-coagulant treatment(i.e.in a more physiologicalcondition compared to anti-coagulated blood used in clinic and research).In contrast,integrinβ3-/-mice exhibit no significant platelet aggregation,which indicates an essential role forαIIbβ3 in platelet aggregation and suggests the existence of other unidentifiedαIIbβ3 ligand(s)[61].

In VWF-fibrinogen DKO mice,fibrinogen-/-mice,and fibrinogen C-terminalγchain mutantmice[77],aswellas in afibrinogenemic patients[22,78],plateletfibronectin(an αIIbβ3 ligand)contentwas increased 3-5 fold due to enhanced internalization of plasma fibronectin(p Fn) by integrinαIIbβ3.Conditional p Fn-/-mice have impaired thrombus growth atarterialshear[79],implying thatfibronectin may be a compensatoryαIIbβ3 ligand thatsupports plateletaggregation.Unexpectedly,however,further depletion of pFn in VWF-fibrinogen DKO mice enhanced,instead ofabolishing,plateletaggregation[31,75].These results suggestthatp Fn can switch between supporting and inhibiting plateletaggregation, depending on the presence offibrinogen/fibrin[31,79].

AnotherαIIbβ3 ligand,vitronectin,plays a dualrole; aggregation is enhanced by granule-released vitronectin butis inhibited by plasma vitronectin[65].These studies suggested thatlikely neitherfibronectin norvitronectin are theαIIbβ3 ligand thatmediates fibrinogen-independentplateletaggregation.Cadherin 6 containsa canonical"RGD"(arginine-glycine-aspartic acid)integrinbinding motifand increases its expression on platelets afterplateletactivation[76].Whilstcadherin 6 contributes to plateletaggregation,clearly otherplasma and platelet proteinsexistthatcan also mediate and facilitate fibrinogen-independentplateletaggregation[61].However,what they are and how they contribute to thrombosis and hemostasis in differentpathophysiologicalconditions requires further study.

Platelet-mediated cell-based thrombin generation and blood coagulation

In addition to their central roles in the platelet adhesion,activation,and aggregation(the first wave of hemostasis),platelets also contribute to coagulation pathway(the second wave of hemostasis).The blood coagulation cascade can be activated by either the extrinsic(tissue factor)or the intrinsic(contact activation)pathways in thrombosis[54,80].Thrombin,a vital product of the coagulation cascade,converts fibrinogen to fibrin,the end product of the coagulation cascade.

Besides these two classicalcoagulation pathways,the exposure of PS on platelets,following platelet activation,markedly potentiates thrombin generation by inducing a negatively charged surface that harbors the coagulation factors[34].Interestingly,in a study of platelet aggregation in fibrinogen and VWF DKO mice, Yang et al.found that ADP can induce thrombin generation that is required for platelet aggregation in the DKO mice[61].Recently,GPVI was also identified as a novel fibrin receptor involved in potentiating thrombin generation[81].Thrombin initiates robustdownstream signaling,through PAR1,PAR4[82]and GPIbα,leading to plateletactivation and further PS exposure,a positive feedback loop forthrombin generation and blood coagulation[57,83-84].Thus,there are many interactions between the firstwave(plateletaccumulation)and the second wave(blood coagulation)of hemostasis,which synergistically contribute to the arrestof bleeding.

Platelets and the"protein wave"of hemostasis:new discoveries

One of the mostrecentstudies revealed a novelconcept of a′protein wave′of hemostasis,where pFn deposition on the injured vesselwalloccurs priorto platelet accumulation(the first wave of hemostasis)and contributes to hemostasis[31,85].In mice lacking fibrinogen,further depletion of pFn markedly increased the mortality rate due to uncontrolled bleeding.Increased bleeding time was also observed in pFn conditional-/-mice,treated with heparin and other anti-coagulants, suggesting thatpFn is importantfor hemostasis in both genetic and drug-induced deficiencies ofblood coagulation.We observed that the p Fn deposition onto the injured vessel wallcan occur independently of fibrinogen,VWF,β3 integrin,and platelets.Itseems thatthe pFn-collagen interaction may play an importantrole in this process[31,85].pFn,likely via the covalentbinding to fibrin,increases the diameter of fibrin fibers and enhances the mechanical strength of the clot and this mechanism likely contributes to the pFn deposition onto the injured vessel walls of normal individuals where fibrin exists.Interestingly,in the absence of fibrin(a product of fibrinogen),pFn switches its function from promoting to inhibiting platelet aggregation. As fibrin is mainly formed atthe bottom of the hemostatic plug close to the vessel wall,pFn may support hemostasis at the base of the thrombi(likely through the formation of a pFn-fibrin complex)and switch to inhibiting excessive thrombus growth at the apical surface ofthrombi.Through this mechanism,pFn servesto control bleeding,while preventing excessive thrombus growth and vessel occlusion.Further investigation of the interaction between platelets and circulating/deposited pFn may reveal novel therapeutic targets for thrombotic disorders,as well as usage of pFn for transfusion to control bleeding disorders,particularly those patients in association with anti-coagulant therapy.Itwould also be interesting to investigate whether the markedly increased platelet fibronectin content in fibrinogen-/-mice and afibrinogenemic patients can be released onto the injured vessel wall and contribute to the protein wave of hemostasis(Fig.1).

Conclusion and future directions

Theprimary physiologicalfunction ofplateletsisto stop bleeding upon vascularinjury.Platelets,via theircontributionsto the"protein wave"and to theclassicalfirstand second waves ofhemostasis,play key roles in the arrestof bleeding[31].Thrombocytopenias caused by eithergenetic deficiencies[86]or autoimmune[87-89]and alloimmune responses lead to bleeding disorders[21,32-33,90-96].However, the same plateletaccumulation and coagulation may lead to thrombosis.Thrombotic eventsoccuratthesiteofa ruptured atheroscleroticplaqueand can resultin heartattack or stroke,the leading causes of mortality and morbidity worldwide.

In addition to thrombosis,the late stage of atherothrombosis,recent studies demonstrated that platelets are actively involved in the initiation of atherosclerosis[97,98].Plateletsaresensitive to environmentalchanges, such asfood products[99-101],lipids[102],and advanced glycation end products in diabetes[103-104],which may affect atherosclerosis.Furthermore,aswedemonstrated,platelets can respond to fibrinogen level changes.Through interaction with integrinαIIbβ3,platelets can use their residualmRNA to de novo synthesize P-selectin and otherproteins[62,105],which may also affectinflammation and directly orindirectly affectatherosclerosis and the stability of atherosclerotic plugs[106-108].

Fig.1 At the site of vascular injury plasma fibronectin deposition occurs even before platelets adhere.Platelets may release their internalized plasma fibronectin from intracellular granules.Platelet receptors then bind physiological ligands,such as VWF and collagen,activating integrinαIIbβ3 and resulting in fibrinogen binding and subsequentplateletaggregation.Thrombin is generated on the negatively charged plateletsurface and further activates platelets and contributes to the coagulation cascade.In a growing hemostatic plug/thrombus,the fibrin and fibronectin matrix is usually formed at the interface between the injured vessel wall and the platelet plug.

In this review,we described the conceptof fibrinogen/VWF-independent platelet aggregation,which was first noted in the early 2000s[61],and provided insightinto multiple and diverse interactions between platelets and their environment.Despite considerable efforts[62,76],the'x'ligand(s)of integrinαIIbβ3 has yet to be uncovered.These concepts are an example of how diverse platelets can be and demonstrate the need for further investigation into platelet interactions. Furthermore,whilst platelets play a pivotal role in hemostasis and thrombosis,they are also versatile cells and are involved in multiple functions,including inflammation,immune responses,lymphatic vessel development,angiogenesis,tumor metastasis,as well as atherosclerosis[106-109].Further elucidations of platelet versatilities willprovide insights into development of new methods to control not only thrombosis and hemostasis butalso inflammation,cancer,and immunologicaldisorders.

Acknowledgement

This work was supported in part by Canadian Institutes of Health Research(MOP 119540),National Natural Science Foundation of China-Canadian Institutes of Health Research(China-Canada Joint Health Research Initiative Program),Heartand Stroke Foundation of Canada(Ontario).This work was also supported by equipment Funds from St.Michael’s Hospital,Canadian Blood Services,and Canada Foundation for Innovation. Naadiya Carrim is a recipientof a Postdoctoral Fellowship from Canadian Blood Servicesand Health Canada. Yiming Wang is a recipientof a Ph.D.Graduate Student Fellowship from Canadian Blood Services and Meredith&Malcolm Silver Scholarship in Cardiovascular Studies,University of Toronto.Yan Hou is a recipient of a State Scholarship Fund from the China Scholarship Council.

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△These authors contributed equally to this work.

✉Corresponding author:Heyu Ni,MD,PhD Professor,Department of Laboratory Medicine and Pathobiology,Department of Medicine,and Departmentof Physiology,University of Toronto;Scientistat Canadian Blood Services;Platform Director for Hematology,Cancer,and Immunological Diseases,St.Michael's Hospital,Room 420,LKSKI-

Keenan Research Centre,209 Victoria Street,Toronto,Ontario,M5B 1W8,Canada.Tel:416-847-1738;,E-mail:nih@smh.ca.

Received 26 August2015,Accepted 12 October 2015,Epub 30 October 2015

R331.1+43 Document code:A

The authors reported no conflict of interests

©2015 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.29.20150121