Myosin-Actin Interaction Provides Contractile Force for Calcium-Independent Platelet Internal Transformation.

Abstract

An important component of platelet shape change is internal transformation resulting in movement of alpha granules (G) and dense bodies (DB) to platelet centers surrounded by rings of actin microfilaments (MF) and microtubules (MT). We have previously demonstrated that the circumferential microtubular coil remains intact during internal transformation. Also, the MT coil will remain at the cell periphery with G and DB randomly distributed following activation if the platelets are pretreated with cytochalasin B, while MF form a tight knot in the cell center. Thus MF are critical components of internal transformation. We postulated that myosin-actin interaction provided the energy to effect internal transformation, and therefore, evaluated the role of myosin in this process. The possible contribution of the p38 MAP kinase pathway was also evaluated since it was reported to play a role in shape change. We studied human, aspirin-treated, platelets suspended in Hepes buffer (134mM NaCl, 6mM KCl, 1mM MgCl2, 5mM NaHCO3, 5.5mM glucose, 0.5mM EDTA, 20mM HEPES, pH 7.4), without added calcium. These platelets were stirred in an aggregometer with low concentration (25–50nM) thromboxane A2 analogue, U46619, that does not elevate cytosolic calcium or activate protein kinase C. These platelets do not aggregate or secrete. Shape change (aggregometry and transmission electron microscopy [EM]) and protein phosphorylation [Phos] (SDS-PAGE, phosphospecific antisera, chemiluminescence) time courses were observed +/− inhibitors in 8 studies. U46619 stimulated aggregometer shape change that was confirmed by internal transformation and fillopodial expression on EM. These changes were accompanied by rapid Phos of myosin light chain (MLC), and slower, more sustained Phos of p38 MAPK, and its indirect substrate, heat shock protein 27 (HSP 27). Shape change was abolished by TP receptor antagonist, SQ29548, but not affected by ADP receptor antagonists, A3P5P and MeSAMP, nor 50 μM BAPTA pretreatment. The Rho-kinase inhibitor, Y27632 (10 μM) completely blocked MLC Phos, shape change and internal transformation when added before U46619, and it reversed shape change and MLC Phos when added 30 sec. after agonist. The myosin inhibitor, BDM, (20 mM) abolished shape change and internal transformation, but it also blocked MLC Phos. The specific myosin II ATPase inhibitor, blebbistatin (150–200 μM) blocked and reversed aggregometer shape change and impaired internal transformation, with little effect on MLC Phos. Specific p38 MAPK inhibitor, SB203580 (10 μM), abolished HSP 27 Phos, but it had no effect on MLC Phos, and it did not inhibit shape change or internal transformation. These results demonstrate that calcium-independent platelet internal transformation is dependent on myosin activation and interaction with actin, but independent of P38 MAPK and HSP 27activation. The data support the concept that myosin-actin interaction provides the force required for platelet internal transformation via MF mediated constriction of the MT band. They also indicate that myosin interaction with actin is necessary for fillopodial expression during calcium-independent shape change.