--- list the five factors required for platelet aggregation from handout "platelet aggregation require platetet stimulation by one or more specific agonist and the presence of specific proteins on the plaatelet surface and in blood plasma" agonists: thrombin, a serine protease - most important agonist thrombin receptor - a 425 AA platelet membrane protein ADP is the classical platelet aagonist - secreted by stmiulated platelets from storage sites in dense granules, also from damaged tissue and red cells possible ADP receptor also requires divalent cations like Ca++ or Mg+ also requires accessory molecule, usually fibrinogen, sometimes vWF fibrinogen receptor on platelet surface, on the GPIIb/IIIa complex so...to me it sounds like you need platelet agonist, divalent cation, accessory molecule...and I'm not sure what the other two are. -- for final - 7 questions will be given and you have to answer five. Today: Joel Bennett, prof of medicine at HUP we're going to talk about how platelets work, compare/contrast adhesion and aggregation. slide: how platelets work. they are anucleate fragments of megakaryocytes that circulate through blood. they tend to be at periphery of blood column b/c red cells push them out. when vascular lining is damaged, subendothelium is exposed and platelets stick to that. platelets will adhere to the subendothelium then, they will aggregate - they get sticky for eachother and glom onto eachother to form the hemostatic plug, to stop you from bleeding. if your platelets won't aggregate, you bleed. so platelet function haas four parts: adhesion aggregation secretion procoagulant activity platelet adhesion: a passive process. it is initiated by endothelial damage and exposure of subendothelium. if blood isn't flowing fast, platelets stick to collagen, fibronectin, vitronectin, or laminin. if they see these things, they stick to them. however, under high shear conditions of rapid blood flow, platelets also need vWF present to stick to those things. platelets have receptors for all these proteins - fibronectin receptors, collagen receptor, - a2b1 binds both of those, a6b1 binds laminin... some patients are a2b1 deficient and have bleeding disorders. the vWF receptor is GP1b-IX vWF is a multimeric protein, synthesized by endothelial cells and megakaryocytes; circulates in multimers from 0.5 to >20 kDa. each monomer has binding sites for the receptor. normally though platelets do not interact with vWF. the abx ristocetin makes people thrombocytopenic by causing vWF to bind to GP1b-IX on platelets. (and so was taken off the market) what in vivo allows platelets to interact w/vWF? not sure. just shear effect? maybe. or, maybe when vWF binds subendothelium, it changes its conformation and allows the interaction? slide of vWF: looks like diagram, allegedly. anyway, platelets have tons of GP1b-IX on them, and they just stick all over the vWF. GP1b-IX is a complex protein - noncovalent complex of two GPs; N terminus of 1b fraction is what binds. slide of GP1b-IX - little balls connected by a rod. a very rigid protein extending from the surface (ha ha). very CHO rich. so very stiff, sticking up from platelet surface (ROFL). under shear stress, may distort, and better bind vWF. maybe. who knows. anywy, it's energy independent. shear, vWF, and platelets will stick. tht's all you need. so adhesion is passive under low shear also under high shear need vWF. now - platelet aggregation: not passive. dead platelets do not aggregate there is a ligand involved - it's in blood, not endothelium. there's a lot of biochemistry in this. there are some requirements: 1. fibrinogen requirement: fibrinogen must be present before washed or gel filtered platelets will aggregate in response to ADP or epinephrine. Afibrinogenemic patients often have prolonged bleeding time and defective platelet aggregation that is corrected by infusions of fibrinogen. fibrinogen associates with aggregting platelets so platelet function in vitro is studied with an aggregometer - a spectrophotometer which uses ADP added to platelet solutions to cause aggregation, and you measure how much light passes through - more aggregation, more light gets through. with ADP only, you get first wave aggregation. adding more ADP or more potent stimuli you get a second wave also. you also see secretion happening with second wave. platelets aggregate like this: fibrinogen receptors on platelet surfaces are stimulated to expose binding site for fibrinogen. fibrinogen binds multiple receptors - crosslinking the platelets. Merck has a drug to interfere with the platelet/fibrinogen interaction (Aggrestat). 2 [I'm saying this, not him] fibrinogen receptor must be available to bind the fibrinogen! process of how platelets aggregate there are agonist receptors agonist binds receptor on platelet surface receptor is coupled to a g protein downstream action initiates signalling causing change in conformation of fibrinogen receptor so tht in presence of calcium, fibrinogen will bind. two subunits: GPIIb and GPIIIa IIb has regions that look like calmodulin - we think they bind calcium. when fibrinogen binds, it crosslinks the second Ca++ binding region (??) IIIa has cysteine rich repeats. only one subunit. the RGD binding region of IIIa is also there and may be a fibrinogen binding site. so maybe part of IIb and part of IIIa together form the fibrinogen binding site. this is very soft data. not proven. fibrinogen receptor is an integrin - an adhesive protein with an alpha subunit which is a two chain molecule. integrins are put into families based on beta subunits. beta1 family has collagen receptor, fibronectin receptor, laminin receptor. integrins on wbc have beta2 subunits - this family has ICAM1, C3BI, etc - involved in leukocyte aggregation. kids with mutant b2 subunits get recurrent infections b/c they can't phagocytose - this is LAD phenotype. the b3 family has fibrinogen receptor aand vitronectin receptor. there's also a beta 4 family, b5, b6, b7 whatever - there are a bunch. so they've been trying to understand for scientific and therapeutc reasons how this is activated. IIbIIIa is present =- thousands of molecules per platelet - but what activates it so it can interact with fibrinogen? wwe think a conformtional change occurs... agonist interacts iwth platelet with IIbIIIa is on it IIbIIIa-->IIbIIIa* then in presence of calcium, fibrinogen will bind agonist + IIbIIIa ---> IIbIIIa* + fibrinogen with calcium present what activates IIbIIIa? G proteins get activated... phospholipae C gets activated, setting off IP3/DAG thing calcium fluctuations all these biochemical pathways are present but the true mechanism of activation is unclear. so a model was put forth... hinge model - for b3 integrin activity this suggests IIbIIIa is on surface, in inactive state omething in cell keeps it inactive in presence of ATP it changes, allows b3 to open up, so it can bind. well, this model isn't quite right but it's worthwhile to remember (why?) ok, so they tried to study this. they wanted to mimic the platelet system in vitro IIbIIIa is like integrins in leukocytes. so maybe you could express IIbIIIa in a lymphocyte? they tried this. they put IIbIIIa cDNA into B cell, grew them in culture they got cells expresssing IIbIIIa on their surface initial assay they used was to put fibrinogen on there, label cells with hot methionine, and add something to activate the cells, and some calcium. they incubated for half an hour, washed it, and cells that didn't stick came off... then they added something to dissolve the cells, and quantified the methionine. this worked. now he's talking about PMA but I do not know what PMA is. there was more adherence in the presence of it, though. but how platelets really work is to bind soluble fibrinogen. labelling fibrinogen with fluorescent label and using flow cytometry - they found cells with no agonist took up less fibrinogen than cells exposed to PMA agonist. it didn't matter what agonist you used. cells would bind soluble fibrinogen. platelets and lymphocyte have cytoskeletons. some of the skeleton is associated with the membrane - integrins are also associated with membrane skeleton. membrane skeletons contain spectrin, other things, filamentous actin - polymer of actin monomers. f-actin has polarity. can add monomers rapidly at one end,and only slowly at the other end. drug can interfere with this. cytochalasins, fungal metabolites, prevent the growth of actin filaments by binding the fast end. so they took the lymphocytes and incubated with cytochalasin. not sure why. if you put cytochalasin on a fibroblast on dish it comes of dish. no surprise - more you add, fewer lymphocytes would stick. but, the unstimulated cells surprised them - incubating with decreasing concentrations of cytochalasin caused more and more of it to stick. eh? did I get some of this backwards? low concentrations of cytochalasin activates the integrins, is the point. what about soluble fibrinogen? fibrinogen binding to lymphs exposed to cytochalasin - yuo could inhibit fibrinogen binding to stimulated cells; and you could make it bind to the lymphocyte just by exposing to cytochalasin.what? this suggeted that maybe the cytoskeleton is constraining IIbIIIa and if you disrupt skeleton in some way, you allow IIbIIIa to be active. what about in platelets? they took the platelets, incubataed with cytochalasin, added hot fibrinogen. spun it through oil to form pellet. then looked for hot fibrinogen in the pellet. they found that if you use increasing concentrations of cytochalasin, you get about half amount of fibrinogen binding as you would using ADP. you can make platelets aggregatethis way. looks like cytochalasin is acting like platelet agonist. ok. is this an artifact of cytochalasin? they tried latrunculin A (which i will call LTA) which is a toxin from red sponges. LTA inhibits actin polymerization by binding actin monomers and keeping them from binding onto the f-actin. so LTA added to platelets acted like cytochalasin. it's a great platelet activator, you get about 50% of fibrinogen binding you would by using ADP. so disrupting cytoskeleton acts like platelet agonist and promotes aggregation. turns out, when platelets are aggregated, they bind the Ab PAC1. platelets exposed to LTA or cytochalasin will bind PAC1 too :) activated platelets have IIbIIIa* on them. there are Ab that recognize IIbIIIa* and bind it. so they were able to show that cytochalasin also causes IIbIIIa* to form. a problem - people have measured actin turnover in platelets anad there is very little. but stimulated platelets have a lot of actin turnover. so they looked at ability of various inhibitors to prevent this process. if you inhibit metabolism at least for two hours...if you add prostaglandin to inhibit platelet function or ADP antaagonists or whatever you can wipe out cytochalasin effect. so mybe you are generating a bit of ADP, causing a bit of actin turnover, killing that with what you add, and activating the integrin platelets secrete ADP so maybe we're just showing that these things cause pltaelet secretion? no. seems to be direct effect. when platelets are stimulated, causes calcium flux. maybe that's it. maybe this is part of LTA effects. using ca++ chelators, we wiped out ADP response, LTA response, cytochal. response, but adding calcium returns the reponse. this suggests a metabolic process involving a calcium flux that promotes actin turnover and if you inhibit the turnover, you make platelets mumble mumble. what? summary: in nonstimulated platelets, actin or actin associated protein in membrane keeps IIbIIIa in inactive state adding agonist induces increase in platelet cytosolic calcium, initiating actin filament turnover, maybe via activating proteins like cofilin or gelsolin increased actin turnover allows IIbIIIa to assume the active conformation required to bind fibrinogen so, 1. energy metabolism, 2. agonist, 3. fibrinogen, 4. (divalent cation) calcium, 5. IIbIIIa ---