Coagulation Activation Induced by Platelet-Derived Microvesicles is Mediated by Kallikrein-Dependent Activation of Factor IX
ISTH Academy. Key N. Jul 10, 2019; 273908; OC 71.2 Topic: Contact Pathway
Prof. Nigel Key
Prof. Nigel Key
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OC 71.2

Coagulation Activation Induced by Platelet-Derived Microvesicles is Mediated by Kallikrein-Dependent Activation of Factor IX

D. Noubouossie1, M. Piegore1, M. Henderson1, A. Ilich1, P. Ellsworth1, M. Mooberry1, R. Pawlinski1, I. Welsby2, M. Hoffman3, D.M. Monroe1, N.S. Key1
1University of North Carolina, Medicine, Chapell Hill, United States, 2Duke University, Anesthesiology, Durham, United States, 3Duke University, Blood Bank and Pathology, Durham, United States

Main Topic: Coagulation and Anticoagulation
Category: Contact Pathway

Background: Platelet transfusion may be associated with thrombosis, inflammation and increased mortality. The mechanisms underlying these adverse outcomes are unclear.
Aims: To elucidate how microvesicles(MVs) isolated from platelet concentrates(PLTC-MVs) activate coagulation.
Methods: PLTC-MVs were isolated from discarded platelet apheresis units by double centrifugation(2500g;15 minutes). PLTC-MVs were visualized, sized and counted using electron microscopy and nanoparticle tracking analysis. Phosphatidylserine exposure was also measured. To assess their ability to initiate thrombin generation(TG), PLTC-MVs were re-suspended in re-calcified MV-free normal pooled plasma(MVfNPP), or in MV-free plasmas deficient in factor XII(MVfDef-XII), XI(MVfDef-XI), IX(MVfDef-IX) or VII(MVfDef-VII). Antibodies against tissue factor(antiTF-Ab), corn trypsin inhibitor(CTI) or soybean trypsin inhibitor(STI) were used to block TF, FXIIa or kallikrein, respectively. FXII, prekallikrein (+high molecular weight kininogen(HK)) or FXI(+HK) activation was detected in buffer using fluorogenic substrate sensitive to FXIIa, FXIa or kallikrein. FIX activation in buffer was assessed using an ELISA that detects FIXa-antithrombin complexes(FIXa-AT).
Results: The mean concentration of PLTC-MVs was 9.66 x 1012 particles/ml; mean diameter was 73.7nm with 90% of particles ≤100nm(Fig1B); mean phosphatidylserine activity was 428 nM(Fig1C). PLTC-MVs triggered TG in MVfNPP despite the presence of inhibitory antiTF-Abs, and in MVfDef-VII(Fig1D). No TF activity was detected on PLTC-MVs(Fig1E). PLTC-MV-induced TG was unchanged in MVfDef-XII and MVfDef-XI, but was abolished in MVfDefIX(Fig1F). PLTC-MVs directly activated FXII(Fig1G) and PK(Fig1H), but not FXI(Fig1I) in buffer. PLTC-MVs activated FIX in buffer in a PK(+HK)-dependent manner(Fig2A). Purified kallikrein similarly activated FIX in buffer(Fig2B) and in plasma(Fig2C). In contrast to kaolin(Fig2D-F), PLTC-MV-induced(Fig2G-I) TG was insensitive to CTI, FXII or FXI deficiency, but was abolished by STI.
Conclusions: PLTC-MVs activate the contact system leading to TG via a noncanonical kallikrein-FIX pathway. These results should be confirmed by using molecules that block kallikrein more specifically than STI. Our findings support that PLTC-MVs may support hemostasis, but also promote thrombotic and inflammatory reactions to platelet transfusion.



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