Low adhesion and interaction forces of Myh9 mutant platelets lead to impaired clot retraction and unstable thrombus formation

Objective Once a vessel is injured, platelets adhere and prevent blood loss. The protein non-muscle myosin heavy chain IIA, encoded by the MYH9 gene, is a contractile protein, which binds to filamentous actin and generates forces in cells. Heterozygous defects in the human gene lead to different autosomal dominant syndromes, which are characterized among others by macrothrombocytopenia and a mild to moderate bleeding tendency. We hypothesized that reduced platelet force generation is responsible for the increased bleeding risk in MYH9 patients.

Material and Methods To investigate this, we analyzed mouse lines each with one point mutation in the Myh9 gene at the positions 702, 1424, or 1841. These mouse lines have been described to recapitulate defects found in patients. We characterized the basic platelet function using flow cytometric, immunoblotting, clot retraction and flow chamber assays. Further, we tested the biophysical properties of the mutant platelets with real-time deformability cytometry, atomic force spectroscopy, and microposts arrays.

Results Myh9 mutant mice displayed a macrothrombocytopenia, but only slightly altered glycoprotein expression. Activation of αIIbβ3 integrin and P-selectin surface exposure of mutant platelets were comparable to controls in response to all tested agonists. Mutant platelets were stiffer, showed more F-actin content, but the G- to F-actin ratio was unaltered. The capacity to assemble actin after activation was reduced in Myh9 mutant platelets. In addition, phosphorylation of the myosin light chain after activation with thrombin was strongly reduced. In line with this, biophysical analysis revealed that Myh9 mutant platelets generate lower adhesion forces to collagen, lower interaction forces between platelets and reduced traction forces when spread on fibrinogen-coated microposts. Clot retraction of mutant samples was delayed and less effective, further reflecting less force generation of mutant platelets. Finally, we observed less contracted and more instable thrombi, when blood of Myh9 mutant mice was perfused ex vivo over collagen fibers.

Conclusion We can show that Myh9 mutant platelets generate and interact at lower forces. These data suggest that reduced platelet-substrate and platelet-platelet forces lead to the increased bleeding tendency found in MYH9 patients. We are currently testing multiple drugs to stabilize the clot in order to prevent bleeding.

Publication History

Article published online:
18 June 2021