Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion

Abstract

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9⌬ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin ␤3 phosphorylation and PtdIns(3,4)P 2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9⌬ platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl 3 -induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outsidein signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9⌬ mice. IntroductionImportant morphologic changes occur in platelets during their activation at sites of vascular injury. The cells lose their resting discoid shape to become spheroid and contracted, emitting membrane blebs and longer extensions. [1][2][3][4] Once in contact with a surface, the spheroid platelets extend long filopodia and finally spread over it by emitting thin, sheet-like lamellipodia. 1,2 Myosin activation plays a central role in the cytoskeletal rearrangements underlying these changes in morphology. Myosin becomes activated after phosphorylation of the myosin regulatory light chain (RLC), which results from both calcium-regulated myosin lightchain kinase activity and Rho kinase-regulated myosin phosphatase activity. [5][6][7][8] Activated myosin assembles into short filaments through the myosin heavy chain and interacts mainly with central actin filaments. Myosin has been proposed to participate in several platelet contractile functions such as platelet spheration, contraction and stress-fiber formation, and fibrin clot retraction. Platelet spheration and contraction, as observed in the aggregometer, closely correlate with phosphorylation of the RLC 9,10 and are prevented when RLC phosphorylation is inhibited. 6,7,9,10 Myosin has also been shown to be associated with stress fiber-like structures in spreading adherent platelets. 11 In addition, myosin could play a role in platelet secretion, as it is decreased by inhibition of myosin RLC phosphorylation. 5,[12][13][14][15] Finally, a role of myosin in clot retraction has long been suspected in view of the necessity for a contractile force and was ...

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

“…29 The most recent findings described by Leon et al demonstrated that megakaryocyte-restricted MYH9 knockout mice lack platelet contractile phenomena, including clot retraction, platelet shape change, and stress-fiber formation on a fibrinogencoated surface, whereas the heterozygous knockout mice have no abnormalities. 30 Our results substantiate previous findings that mutant NMMHC-IIA does not translocate to the lamellipodia in surface-activated platelets. We conclude that the overall defect in platelets with mutant NMMHC-IIA is haploinsufficiency.…”

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…Whether these platelets contain full-length FlnA or a truncated form, as shown for talin (Priddle et al, 1998), remains to be investigated. The high efficiency of gene inactivation is similar to that described for the widely used megakaryocytespecific Pf4-Cre transgenic mouse and posits the GATA1-Cre mouse as a strong alternative for studies in megakaryocytes and platelets (Léon et al, 2007;Petrich et al, 2007;Tiedt et al, 2007;Hitchcock et al, 2008;Wen et al, 2009).…”

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…The runx1 KO mice exhibited thrombopenia (2-3×10 5 platelets per ml), contrasting with mice negative for Cre-recombinase (~1×10 6 platelets per ml). The floxed myh9 strain was crossed with PF4-Cre mice to obtain mice with deletion of the MYH9 exon 1 in the MK lineage, as described previously 11 . Lin − cells were purified from the fetal liver (E14.5) after incubation with a mixture of rat monoclonal antibodies against lineage antigens (Gr1, TER119, CD11b, B220 and anti-CD3, 15 µg ml − 1 each, Pharmingen, CA, USA) and depleted with Dynabeads coupled to a mouse antibody against rat immunoglobulin (Dynal, Norway).…”

“…MYH9 has been considered as the only non-muscle myosin II heavy chain expressed in MK and platelets 10 . Therefore, we studied MK-restricted myh9 knock-out (KO) mice (myh9 f/f(PF4 Cre + ) ) to examine the role of MYH9 in MK ploidization 11,12 , expecting an increase in MK ploidy. Surprisingly, no significant difference in the in-vivo MK ploidy level between myh9 f/f(PF4 Cre + ) (17.11N, n = 6) and control mice (myh9 f/f(PF4 Cre-) ; 16.53N, n = 4, P = 0.22, t-test, ploidy measured in MK ≥ 8N) was seen (Fig.…”