Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

Abstract

The fibrin(ogen) receptor, integrin a IIb b 3 , has a well-established role in platelet spreading, aggregation and clot retraction. How a IIb b 3 contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used a IIb b 3 blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca 2? responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in a IIb b 3 signaling similarly enhances thrombininduced Ca 2? rises and PS exposure. These responses are reduced in a IIb b 3 -deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of a IIb b 3 to tissue factor-induced platelet Ca 2? rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on a IIb b 3 activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca 2? signal generation, and furthermore that a considerable part of Syk activation relies on a IIb b 3 signaling. These results hence point to a novel role of Syk in integrindependent thrombin generation.

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

“…Moreover, Weiss and Lages [36] implied that α IIb β 3 may play an important role in maintaining a sufficiently elevated [Ca 2+ ] i level. In support of our findings, van der Meijden and colleagues [37] demonstrated a constitutive role of α IIb β 3 in [Ca 2+ ] i signaling and platelet PS exposure. In addition, they also showed a significant role of α IIb β 3 -dependent signaling via Syk kinase in TF-evoked platelet procoagulant activity in plasma.…”

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow

“…The locally produced thrombin fulfills its multiple roles, including fibrinogen cleavage and evoking persistent intracellular signaling in platelets to stimulate phosphatidylserine exposure and enhancement of procoagulant activity. 41 Early evidence for the assembly of the tenase complex of FVIIIa and FIXa came from experiments using only procoagulant phospholipid membranes. 42,43 Our localization studies of the (activated) coagulation factors on platelet thrombi indicate that both FIX(a) and FXa concentrate on phosphatidylserine-exposing platelets, as expected, because both contain Gla-domains.…”

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

“…In the activations, 0.2 mM Gly-Pro-Arg-Pro (GPRP) was added to prevent formation of large fibrin fibers. 19 Platelet sub-populations were distinguished by probing with Rhod-A14 (10 mg/mL), AF647-annexin A5 (1:200), AF488-factor V (20 nM), OG488-factor Xa (100 nM), AF647-fibrinogen (100 mg/mL) and FITC-PAC-1 (1.25 mg/mL). After staining for 5 min, samples were analyzed with a FACScan flow cytometer (BD Accuri Cytometer).…”

“…27 In the present study, we investigated this again by assessing the determinants of high fibrin(ogen) binding and PS exposure of platelets in tissue factor-activated blood plasma. For this purpose, plasma samples were defibrinated to prevent massive clot formation, 19 and then reconstituted with washed platelets. After triggering coagulation with tissue factor and calcium, the …”