Soluble dimeric glycoprotein VI (sdGVI) is a hemin scavenger and protects hemin-induced platelet aggregation

Vascular injury is associate with tissue hemorrhages and extravasation of plasma components and blood cells. Thereafter, erythrocytes undergo hemolysis with liberation of hemoglobin and generation of free iron-containing heme B and hemin. Hemin is a potent activator of platelets that in turn promotes thrombosis and thus, prevents further blood loss at site of tissue injury. Uncontrolled hemin-dependent thrombosis is associated with enhanced tissue inflammation which favors sustained tissue bleeding.  
The glycoprotein VI (GPVI) has been identified as hemin-binding receptor on the platelet plasma membrane. We hypothesized that the soluble form of GPVI (sGPVI) scavenges free hemin and therefore prevents hemin-induced platelet activation. To explore this hypothesis, isolated platelets were treated with various concentrations of hemin (3.1 to 25 µM) and platelet activation was analyzed with flow cytometry. We found that platelet activation (α_IIIbβ₃ integrin activation and P-selectin expression) was increased in response to hemin in a concentration-dependent manner. Similar results were obtained in aggregation experiments using light aggregometry of isolated washed platelets. Interestingly, at higher concentrations of hemin (> 6.25µM) platelet plasma membrane destruction occurs with enhanced formation of plasma membrane microvesicles. To test whether soluble GPVI (sGPVI) interferes with hemin-induced platelet activation, hemin was preincubated with a high-affinity dimeric recombinant GPVI receptor sdGPVI-Fc (Revacept) or Fc control before adding to isolated platelets. In the presence of 100 µg/ml fibrinogen and 1 mM Ca²⁺ aggregation was evaluated by light aggregometry. We found that 1 mg/ml sdGPVI-Fc but not 1 mg/ml Fc control inhibited hemin-induced platelet aggregation (AUC sdGPCI-Fc vs. AUC Fc control: 76.6 ± 62. vs. 214.4 ± 59.6, mean ± SD, p < 0.021). Similar results were obtained when platelet activation (α_IIIbβ₃ integrin, P-selectin) was analyzed in response to hemin.
Our results confirm that GPVI is a novel receptor for hemin. The soluble form of GPVI (sdGPVI) binds to hemin and inhibits hemin-induced platelet aggregation and plasma membrane destruction and thus, thrombo-inflammation. The results may help to explain why in patients treated with sdGPVI (Revacept) with coronary or carotid artery disease after interventional therapy reveal reduced bleeding events as shown previously in clinical phase 2 studies. Moreover, our results may help to explain why patients with CCS following PCI and enhanced bleeding complication show an increase of sGPVI levels.