Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

Abstract

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Regulation of plasminogen activation on cell surfaces and fibrin

“…Initiation of fibrinolysis after accumulation of plg was also demonstrated on the surfaces of activated platelets in an in vitro system under either shear [53] or a static condition [16]. Plg was bound to the surface of platelets through glycoprotein IIb/IIIa, especially when platelets were activated to express PS on their surfaces [53,[68][69][70][71]. As a result, PS exposed on platelet surfaces contributes to thrombin generation and subsequent fibrin formation, and also triggers plg activation by assembling plg and tPA on their surfaces [16].…”

Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood

“…4 Although many thrombotic and bleeding disorders (eg, clotting factor and platelet function defects) can be diagnosed through end point assays such as clot times 9 and light transmission aggregometry, 36 respectively, it is quite likely that others are overlooked 37 because they are best characterized by alterations in rate constants or because the defect involves platelet-fibrin interactions (eg, platelet nonmuscle myosin IIa disorders) rather than impaired platelet secretion or plateletplatelet interactions. 38 There are also substantial differences between the avidity of activated platelets for fibrin vs fibrinogen, and it is fibrin, but not fibrinogen, that promotes fibronectin assembly and expression of phosphatidylserine on the platelet surface, [39][40][41] which amplifies platelet-dependent generation of thrombin and fosters recruitment of additional platelets to sites of fibrin formation, among other differences. 42 Lastly, the contribution of RBCs and other components of whole blood to contraction cannot be assessed using bulk end point assays of plasma.…”

Platelet Hyperactivity inTNFSF14/LIGHTKnockout Mouse Model of Impaired Healing

“…In addition, increase in platelet integrin a IIb b 3 that supports the binding of activated platelets to the fibrin network could also increase PS exposure on the platelet surface. 27 An important structure of the platelet membrane is the presence of invaginations known as OCS that provide the capability to increase the surface to volume ratio of the membrane upon activation of platelets. The OCS provides the vehicle for external microenvironmental components to interact with the interior of the platelet, while the platelet contents, including the granules, exit through the OCS upon activation.…”