Expression of matrix metalloproteinase‐9 in human platelets: regulation of platelet activation in in vitro and in vivo studies

Abstract

1 The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2 In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3 MMP-9-gold labeling was observed on the plasma membrane, a-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml À1 ) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml À1) inhibited phosphoinositide breakdown, intracellular Ca 2 þ mobilization, and thromboxane A 2 formation in human platelets stimulated by collagen (1 mg ml À1). In addition, activated MMP-9 (21 and 90 ng ml À1 ) significantly increased the formation of nitric oxide/cyclic GMP. 4 Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nM). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml À1). Activated MMP-9 (1 mg g À1 ) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5 These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A 2 formation, thereby leading to inhibition of intracellular Ca 2 þ mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

“…ADAM17 is a major sheddase for GPIb␣, 20 and the cytoplasm and ␣-granules of platelets contain MMP9. 45 The broad-range metalloproteinase inhibitor GM6001 preserved GPIb␣ expression on the surface of FlnA-null platelets stored 24 hours at 37°C, as did a selective MMP9 inhibitor (data not shown). How FlnA-null platelets increase ADAM17 and MMP9 expression and GPIb␣ cleavage is unclear.…”

Concluding remarks

“…The sensitivity and specificity for predicting the likelihood of a donor being in the breast cancer group compared with the control group (no breast disease) was 78 and 89% respectively. While there may be questions and issues associated with the use of serum for measurement of MMPs because of their generally higher levels, 14,15 and the effects of preanalytical processing, [3][4][5] our results strongly suggest that there are still detectable differences in the MMP levels in serum that are apparently sufficient to allow distinction between different breast disease categories with statistical confidence. Our paper is important because it represents one of the few (if not the only one) to measure 4 MMP2 and 4 MMP9 variables in >300 serum samples collected from a population in which plasma has been analyzed.…”

Platelet Dysfunction in Asphyxiated Newborn Piglets Resuscitated with 21% and 100% Oxygen

“…Oxidative stress can contribute to the activation of MMPs (10,12). Similar to oxidant-induced increases in MMP activity (13,14), we speculate that 100% reoxygenation (to a lesser extent, 21% reoxygenation) causes the activation and release of MMP-9, which contributes to platelet aggregatory dysfunction (8,9). The nonsignificant difference in MMP-9 activities between 100% and 21% reoxygenated groups may be related to the small number of animals used.…”

“…In contrast to MMP-2, MMP-9 inhibits platelet aggregation when released from platelets (8,9). We previously reported that the plasma MMP-9 activity was not different, whereas the plasma MMP-2 activity increased gradually over the first day of extracorporeal membrane oxygenation in critically ill newborns (26).…”