Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

Abstract

C oated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α IIb b 3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α IIb b 3 . Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α IIb b 3 . Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α IIb b 3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α IIb b 3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α IIb b 3 .

Integrating platelet and coagulation activation in fibrin clot formation

“…The platelet‐dependent fibrin formation is in particular triggered by TF, and contributes to the formation of a densely packed thrombus core . Extensive fibrin formation can also be observed on coated platelets and, here, relies on transglutaminase (FXIII) activity and α IIb β 3 binding …”

“…The PS‐exposing platelets are often confused with coated platelets . The latter form a subpopulation, also arising after strong agonist stimulation, which is characterized by the formation of a covalent coat, containing transglutaminase‐anchored platelet‐derived proteins .…”

“…The latter form a subpopulation, also arising after strong agonist stimulation, which is characterized by the formation of a covalent coat, containing transglutaminase‐anchored platelet‐derived proteins . Conceptually, it is now believed that, after initial PS exposure, FXIII activation by thrombin is required to de‐encrypt the transglutaminase activity, and to allow cross‐linking of multiple proteins including fibrin at the platelet surface . Both transglutaminase and integrin α IIb β 3 interactions are required for a platelet control of fibrin formation.…”

Formation and Relevance of Platelet Subpopulations

“…Mattheij et al present a combined role for αIIbβ3 and FXIII in the formation and orientation of the formed fibrin network [77]. Abaeva et al also show a decrease in fibrinogen binding to PS exposing platelets in a patient with FXIII deficiency, but interpret this as a non-significant reduction [74], whereas FXIII knockout in mice caused no reduction in the generation of coated platelets [76,77].…”

“…This hypothesis was strengthened by the fact that stimulation with thrombin and collagen-related peptide (CRP) in the presence of GPRP (a peptide inhibiting fibrin polymerization) [79,80] reduced fibrin(ogen) binding whereas ancistron (which is claimed to induce fibrin polymerization) would increase the fibrin(ogen) binding also in the absence of thrombin [74]. Mattheji et al describe that on Annexin V binding platelets, FXIII is spread out over the entire platelet [77]. However, judging from the presented images, the localization of FXIII resembles the cap structure described by Abaeva et al [74].…”