Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

Abstract

Key Points• Under physiological flow rates, plasminogen primarily accumulates on fibrin(ogen), emanating from platelets and initiates fibrinolysis.• Plasminogen is localized to defined "caps" on the surface of PS-exposing platelets in a fibrin(ogen)-dependent manner.The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and GlyPro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PSexposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding "cap." These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. (Blood. 2015;125(16):2568-2578 IntroductionPlatelet accumulation is central to the hemostatic response. Platelets are activated in vivo by numerous agonists of varying potency, including thrombin, collagen, adenosine 59diphosphate, and thromboxane A2. Platelets exhibit a nonuniform response to activation, with distinct populations forming with different surface characteristics.1 Aggregating platelets are characterized by a spherical shape, binding of fibrinogen, and expression of the active integrin a IIb b 3, and predominantly function in clot retraction. Highly activated platelets are observed on collagen fibers 1 and in the core region of a thrombus nearest the vascular injury.2 These platelets are characterized by membrane exposure of phosphatidylserine (PS), a rounded balloon-like structure, sustained increase in cytosolic Ca 21, and binding of coagulation factors. 3,4 PS-exposing platelets, also termed procoagulant platelets, substantially enhance the activity of the prothrombinase complex, 5,6 and subsequent thrombin and fibrin formation. 7 An additional subpopulation of platelets, termed "coated" platelets, are generated in response to strong dual agonist stimulatio...

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

“…PS-positive platelets display a cap of FXII on the activated surface that we have recently shown to be rich in fibrin(ogen) and plasminogen. 58 We also show that platelet-derived polyP is retained on the membrane of PS-positive platelets, but unlike FXII, fibrinogen, and plasminogen, it is homogenously distributed. Inhibition of fibrin polymerization attenuates the association of polyP with the platelet surface, consistent with its known affinity for fibrin, 30 but does not completely abrogate binding.…”

“…Nevertheless, we also show that polyP can translocate from the "hot-spots" of PS-positive platelets into the surrounding fibrin network. FXII also decorates fibrin strands, accentuating the importance of platelet-bound fibrin in localizing FXII and plasminogen 58 in the vicinity of activated platelets. The release of polyP from stimulated platelets could stimulate activation of FXII on fibrin and enhance the plasminogen activator capacity of aFXIIa; in this sense, fibrin will be acting as a surface for its own destruction, as it does in tPA-mediated plasminogen activation.…”

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting

“…The same nonuniformity of surface distribution was recently reported for platelet-derived factor XIII (FXIII) 16 and plasminogen. 17 The "caps" are convexities of the balloon-shaped platelet membranes with a diameter of ;2 mm (compared with 4 to 6 mm diameter of a "ballooned" platelet) present in the quantity of strictly 1 per procoagulant platelet. 9 A recent report on the dynamics of platelet shape change during activation suggested that the "caps" are original bodies of platelets remaining after formation of the "balloons.…”

Glu- and Lys-forms of plasminogen differentially affect phosphatidylserine exposure on the platelet surface

“…Lysplasminogen under these conditions had no effects. The relationship between plasminogen binding to cells and phosphatidylserine exposure has been earlier described in the literature [9][10][11]. O'Mullane et al showed that in the presence of apoptotic factors (thrombin is considered as one of them) exogenous Glu-plasminogen considerably increases phosphatidylserine exposure on the surface of cells of monocyte culture U 937 [12].…”