BACKGROUND. EMP have emerged as sensitive biomarkers of endothelial dysfunction. Little is known of their function. EMP interact with leukocytes and platelets and exert prothrombotic and proinflammatory effects. Recently we reported that vWf is expressed in EMP. The present study extends this observation to characterize EMP-bound vWf (EMP/vWf), specifically their multimer size and proaggregatory activity in ristocetin-induced platelet aggregation.
METHODS. EMP, released from human renal microvascular endothelial cells 24hr following stimulation with TNF-α, were isolated by centrifugation (10,000g x 30min) and washed twice for vWf multimer analysis and proaggregatory activity assay. vWf multimer size was analyzed by 0.8% argarose gel electrophoresis and Western blotting. The proaggregatory activity of vWf was assessed flow ctyometrically by a two-step method: first, washed platelets were incubated with either EMP or soluble vWf such as plasma or Humate P (a commercial vWf multimers) in the presence of ristocetin for 10 min, then disappearance of free platelets as they formed small aggregates was measured. Second, the formed platelet aggregates were diluted 20-fold with buffer and degree of disaggregation was determined at intervals to assess the stability of PltAg.
RESULTS. (i) Electrophoresis showed that vWf bands from normal plasma do not exceed 1,000 kDa and Humate P about 2,000 kDa while vWf bands on EMP extends much higher than 2,000 kDa. The protein banding pattern of EMP/vWf also differed from free soluble vWf (plasma) or humate P, the latter both showing clear ladder-like banding while EMP/vWf often appeared as a continuous smear of sizes in the high molecular weight regions, probably due to covalent bonds to transmembrane structures. (ii) Incubation of EMP (1 x 107 in 100 μL) with humate P (0.3 U) for 2hr (rotary shaker, 100 rpm) followed by washing the EMP revealed that significant additional vWf was adsorbed to the EMP: the vWf in the supernatant was decreased by 30% – 40%, and Western blotting showed additional high MW band on the treated EMP. This demonstrates that EMP express unoccupied sites capable of binding free vWf. (iii) Incubating EMP/vWf with washed platelets in the presence of ristocetin resulted in formation of platelet aggregates (PltAg). When PltAg were diluted 1:20, the PltAg began dissociating into free platelets in a time-dependent manner. The time for 50% dissociation of PltAg was 15–20min with soluble vWf (plasma or Humate P), but was prolonged to 45–60min with EMP/vWf. We attribute this mainly to the presence of ULvWf on EMP. (iv) Plasma from 4 acute TTP patients showed elevated EMP and enhanced formation of PltAg resistant to disaggregate. While 5 vWD plasma showed the opposite effect and their deficiency was corrected by added EMP.
CONCLUSIONS. A new role of EMP is indicated in the present study. EMP-bound vWf is functionally active, inducing platelet aggregates of greater stability than free vWf in a ristocetin-based assay. EMP are capable of adsorbing additional vWf from plasma, indicating presence of extra binding sites. EMP enhance the stability of aggregates probably through ULvWf on the EMP, or to additional platelet-EMP-platelet adhesion molecules, or both. Through their high-affinity interaction with ULvWf, EMP likely play a role in vWf-dependent thrombotic disorders such as TTP and other platelet mediated thrombosis.