Abstract

Objective— In platelets, as in most mammalian cells, the anionic phospholipids such as phosphatidylserine are present only in the inner leaflet of the membrane bilayer. During platelet activation, phosphatidylserine moves from the inner to the outer leaflet of the membrane bilayer. The transbilayer movement of phosphatidylserine is responsible for platelet procoagulant activity as the exposed phosphatidylserine provides high affinity binding sites for the assembly of the prothrombinase and tenase complex. Externalization of anionic phospholipids in platelet is accompanied by the release of phosphatidylserine-rich microvesicles. These microvesicles account for the procoagulant activity of plasma by providing an efficient catalytic surface.

Lactadherin, also known milk fat globule-EGF 8, is a 45 kDa glycoprotein secreted by macrophages. Lactadherin contains EGF-like domains at the amino terminus and two C-domains at the carboxy terminus that share homology to the phosphatidylserinebinding domains of blood coagulation factors V and VIII. Lactadherin binds to apoptotic lymphocytes and phosphatidylserine-expressing red blood cells via the C-domains and anchors them to macrophage integrins via its RGD sequence in the EGF domain. We have examined the role of lactadherin in clearance of phosphatidylserine-rich platelet-derived microvesicles.

Methods and Results—Platelet-derived microvesicles were labeled with the fluorophore BODIPY-maleimide and incubated with THP-1 cell derived macrophages. The extent of phagocytosis was quantified by measuring the intracellular fluorescence by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at ~ 5 ng/ml. A monoclonal antibody to lactadherin and a carboxy terminal fragment of lactadherin inhibited lactadherin-dependent phagocytosis. Lactadherin-deficient mice had increased number of microvesicles in their plasma and generated more thrombin compared to their wild type littermates. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet microvesicles. Finally, in a in vivo model of light/dye-induced endothelial injury/ thrombosis model, lactadherin-deficient mice, showed enhanced thrombus formation (5.93 ± 0.43 min) compared to their wild-type littermates ( 9.80 ± 1.14 min; P=0.01, n=9 in each group) in the cremastric venules.

Conclusion— Our studies show that lactadherin mediates the clearance of PS expressing platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.

Disclosures: No relevant conflicts of interest to declare.

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