Platelet-Associated Matrix Metalloproteinases Regulate Thrombus Formation and Exert Local Collagenolytic Activity

Objective Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. Approach and Results Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibrillar collagen fibers within hours, with loss of the majority of the platelet adhesive properties of collagen as a consequence. This collagenolytic activity was redundantly mediated by platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 but occurred independently of platelet -granule release (Nbeal2(-/-) mice). The latter was in line with subcellular localization experiments, which indicated a granular distribution of MMP-1 and MMP-2 in platelets, distinct from -granules. Whereas MMP-9 protein could not be detected inside platelets, activated platelets did bind plasma-derived MMP-9 to their plasma membrane. Overall, platelet MMP activity was predominantly membrane-associated and influenced by platelet activation status. Conclusions Platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 differentially modulate acute thrombus formation and at later time points limit thrombus formation by exerting collagenolytic activity

Epigenetic regulation of F2RL3 associates with myocardial infarction and platelet function

DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2–2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner

Phenotyping of <i>Myo5a</i>^−/− mice and platelets.

<p>(A) Schematic of the targeting vector used to disrupt the <i>Myo5a</i> genomic locus between exons 2 and 3. The cassette contains the lacZ reporter gene, the neomycin (neo) resistance gene, and a polyadenylate (pA) sequence which causes transcription termination. Flp recombination target (FRT) sites are included on either side of the vector along with a loxP site at the 3′ end. In (B), the difference in coat pigmentation of <i>Myo5a</i>^−/− mice compared to wild-type (WT) is shown. (C) Offspring ratios of knockouts (−/−), heterozygotes (+/−) and wild-types (+/+) from heterozygote breeding pairs were compared from 162 mice. Blood platelet count (D) and mean platelet volume (E) were measured in <i>Myo5a</i>^−/− and wild-type (WT) mice (n = 18 and 25, respectively). (F) Immunoblots showing the expression of myosin Va protein in lysates from human, wild-type (WT) mouse and <i>Myo5a</i>^−/− mouse platelets. Note the absence of a detectable myosin Va band in the knockout platelets. (G) Platelet surface levels of the indicated proteins were analysed by flow cytometry. Data (mean +/− SEM, n = 4–7) are mean fluorescence intensity levels.</p

Normal Ca²⁺ signalling in <i>Myo5a</i>^−/− platelets.

<p>Wild-type and <i>Myo5a</i>^−/− platelets were loaded with the Ca²⁺-sensitive dye Fura-PE3. (A) Ionomycin (1 µM) was added in presence of the calcium chelator EGTA (200 µM). (B) Platelets were stimulated with the indicated concentrations of CRP in the presence of 1 mM CaCl₂. The left hand panels show representative traces (n = 3). Data in the right hand panels (mean +/− SEM, n = 3) are expressed as area under the curve (AUC).</p

Loss of myosin Va does not affect platelet spreading on fibrinogen.

<p>Wild-type and <i>Myo5a</i>^−/− mouse platelets were dispensed onto fibrinogen-coated coverslips for 1 h. Where indicated, platelets were stimulated with the PAR4 agonist AYPGKF (300 µM) for 1 min prior to adhesion. Cells were stained with TRITC-phalloidin. In (A), representative images are shown (n = 3). (B) Platelet morphological subtypes were counted and fractions (mean +/− SEM, n = 3) were calculated.</p

Loss of myosin Va does not affect platelet dense, α-granule or lysosome secretion.

<p>Wild-type and <i>Myo5a</i>^−/− platelets were stimulated with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. (A) ATP release from dense granules was assessed luminometrically. Data (mean +/− SEM, n = 4–7) are levels of released ATP. (B) P-selectin expression as a result of α-granule secretion was measured by flow cytometry, after agonist stimulation for 10 min. Data (mean +/− SEM, n = 5–7) are shown as fold increase over basal. (C) Time course of P-selectin expression induced by AYPGKF (300 µM). Data (mean +/− SEM, n = 4) are levels of FITC fluorescence intensity. (D) Lysosome secretion, as assessed by LAMP1 flow cytometry, was determined after agonist stimulation for 10 min. Data (mean +/− SEM, n = 4) are shown as fold increase over basal.</p

No difference in integrin α_IIbβ₃ activation in myosin Va-deficient platelets.

<p>Wild-type and <i>Myo5a</i>^−/− mouse platelets were stimulated for 10 min with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. JON/A binding to the activated form of integrin α_IIbβ₃ was measured by flow cytometry. Data (mean +/− SEM, n = 5) are shown as fold increase over basal.</p

<i>Myo5a</i>^−/− platelets have normal subcellular morphology.

<p>(A) TEM images (4800×, scale bar: 1 µM) show representative images of WT and <i>Myo5a</i>^−/− mouse platelets (n = 4). Black arrows: dense granules. White arrows: α-granules. (B) Platelet dense and α-granules were quantified per field of view (10 fields of view per preparation) and data (mean +/− SEM, n = 4) are shown as the number of granules per platelet visible in the section. This number is for comparison between genotypes only, as granules above or below the thin section plane will not be visible, and so the number will be an underestimate of the total granule count per platelet.</p

No enhanced expression of myosin Vb, Vc and VI in <i>Myo5a</i>^−/− platelets.

<p>Immunoblots showing the expression of myosin Vb, Vc, and VI in lysates from human, wild-type mouse (WT) and <i>Myo5a</i>^−/− mouse platelets. Lysates from mouse liver, pancreas or kidney were used as positive controls for myosin Vb, Vc, and VI, respectively. GAPDH served as loading control. Images shown are representative of three independent experiments.</p