Differential contributions of monocyte- and platelet-derived microparticles towards thrombin generation and fibrin formation and stability

Abstract

Background: Microparticles (MPs) are sub-micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood.

Objective: To determine how MPs promote thrombin generation and modulate fibrin density and stability.

Methods: Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP-1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high-speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability.

Results: MPs from monocytes (M-MPs) and platelets (PMPs) had similar shapes and diameters (100-300 nm). M-MPs had TF activity (16.7 ± 2.4 pm TF per 10(6) MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M-MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min(-1) , respectively), 38% higher fibrin network density and higher clot stability (3.8-fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8-fold lower prothrombinase activity than M-MPs. PMPs supported contact-dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7- and 1.3-fold, respectively) when mixed with THP-1-derived MPs.

Conclusion: MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.

Figure 1. M-MPs and PMPs have similar size distributions

Representative bivariate flow cytometry plots (forward and side scatter) of (A) TRAP-derived PMPs and (B) M-MPs. (C) TEM of representative TRAP-derived PMPs and M-MPs. Scale bar is 200 nm.

Figure 2. M-MPs, but not PMPs, promote thrombin generation in a TF-dependent manner

(A) TF activity (±SEM) was determined by factor Xa chromogenic substrate cleavage (n=5). *P<0.02 by ANOVA versus PMPs. (B) Prothrombinase activity (±SEM) was determined by thrombin chromogenic substrate cleavage (n=3–4). MP counts were determined by Annexin V-positive events in flow cytometry. *P<0.01 by ANOVA versus all PMPs. (C–D) Thrombin generation supported by 5000 PMP/μL or 1000 M-MP or THP-MP/μL was measured in the presence of anti-TF or control IgG. (C) Thrombin lag time and (D) peak (±SEM, n=3–6). Data from PMP from TRAP-stimulated platelets are shown; PMP from TRAP/convulxin- or A23187-stimulated platelets were similar. “Buffer” is HBS/BSA. TF is 1 pM (Innovin). *P<0.05 by ANOVA versus buffer controls. ^#P<0.05 by paired Student’s t test between anti-TF and IgG controls.

Figure 3. M-MPs initiate fibrin formation

MPs were spiked into re-calcified MDP and fibrin formation was followed by turbidity. (A) Representative fibrin formation curves. (B) Onset and (C) rate of fibrin formation. For conditions in which clots did not form, onsets were censored at 120 minutes and rates at 0 mOD/min. Data show mean (±SEM, n=4–6). TF is 1 pM (Innovin). *P<0.0001 by ANOVA versus buffer controls.

Figure 4. M-MPs, but not PMPs, increase fibrin network density

Clots were formed by incubating MPs (5000 PMP/μL and 1000 M-MP/μL, final) in re-calcified MDP containing Alexa488-conjugated fibrinogen, and imaged by confocal microscopy. (A) Micrographs of fibrin networks after clots were fully-formed (180 minutes). (B) Fibrin fiber density shown as mean percent positive grids (±SEM, n=3–5). TF is 1 pM (Innovin). *P<0.05 by ANOVA versus buffer controls.

Figure 5. M-MPs increase clot resistance to fibrinolysis

MPs (1000 M-MP or THP-MP/μL and 5000 PMP/μL) were spiked into re-calcified MDP in the presence of tPA and fibrin formation and lysis were followed by turbidity. (A) Representative turbidity curves. (B) Time to peak and (C) peak turbidity change. Data show mean (±SEM, n=4–7). TF is 1 pM (Innovin). *P<0.05 by ANOVA versus buffer controls.

Figure 6. PMPs increase thrombin generation and the rate of fibrin formation during TF-initiated clotting

PMPs were mixed with THP-MPs or Innovin and spiked into re-calcified MDP, and thrombin generation and fibrin formation were measured. (A) Peak thrombin and (B) fibrin formation rate in the absence of tPA. (C) Time to peak and (D) peak turbidity in the presence of tPA. Data show mean (±SEM, n=3–4). TF is 1 pM (Innovin). *P<0.02 by Student’s t test versus 0 PMP/μL.