TMEM16F-Mediated Platelet Membrane Phospholipid Scrambling Is Critical for Hemostasis and Thrombosis but not Thromboinflammation in Mice—Brief Report

Abstract

Objective-It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. Approach and Results-We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. Conclusions-Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.

Use of microfluidics to assess the platelet-based control of coagulation

“…These studies indicate that as long as the flow rate is high enough (i.e., the thrombin formed is sufficiently diluted), adhered and activated platelets are required for a local buildup of fibrin fibers. Interestingly, inability of platelet PS exposure (e.g., blood from Scott patients or anoctamin-6 deficient mice) severely delayed the formation of fibrin [20,36]. This corresponds with in vivo studies [37,38] and supports the concept of platelet-based coagulation.…”

Integrating platelet and coagulation activation in fibrin clot formation

“…Exposure of the negatively charged phospholipid PS at the membrane surface has shown to be a controlling process in hemostasis, as recently observed in mice carrying platelets with deficient PS exposure . Yet, upon injury or activation, also the endothelium and other vascular cells can provide a PS‐exposing surface.…”

“…On the other hand, a lack of fibrin in the patients’ thrombi reduces the stability and increases embolization . In platelets from Scott syndrome patients or anoctamin‐6 deficient mice, with defective PS exposure, fibrin formation is also impaired …”

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo

“…The first-described patient, Mrs. M.A. Scott, had a relatively severe bleeding phenotype: she was found to have an isolated defect in PF3a (Introduction) (112); impaired PS exposure upon platelet activation, thereby resulting in deficient procoagulant activity and abrogated fibrin formation at sites of vascular damage (3,113); and diminished EV formation (Agonist-Induced Phosphatidylserine Exposure) The genetic defect in four of the six known Scott syndrome patients for whom mutational analysis is available, as well as in canine Scott syndrome, in German Shepherd dogs, involves homozygous and heterozygous variants in the TMEM16F gene (47,49,(114)(115)(116)(117)(118), resulting in an absence of expression of the TMEM16F protein (Scramblase and TMEM16F) Knockout of TMEM16F in genetically modified mice recapitulates the Scott syndrome phenotype (119)(120)(121)(122).…”

“…There are certainly indications that the procoagulant platelet might be a useful target in reducing thrombosis. Firstly, knockout of TMEM16F in platelets of genetically modified mice (Pathologies of Phosphatidylserine Exposure) decreases platelet thrombus formation in vitro on collagencoated coverslips under flow conditions and in models of arterial and venous thrombosis (119)(120)(121)(122). Secondly, there is evidence that in clinical conditions of thrombosis, specifically coronary artery disease, and essential thrombocythemia, the procoagulant platelet response is increased, and that increased levels of procoagulant platelets are associated with increased risk for recurrent infarction in lacunar and non-lacunar stroke and predict incident stroke after transient ischemic attack (131,(133)(134)(135)(136).…”