Endothelial dysfunction seems to be a key factor in the development of several complications observed early after hematopoietic stem cell transplantation (HSCT). The conditioning regimen and many other factors associated with the procedure are responsible for this endothelial damage. The effects of immunosuppressive agents on endothelial function have not been explored in detail. We evaluated the effects of 3 drugs commonly used in HSCT: 2 calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus (TAC), and an inhibitor of mTOR, sirolimus (SIR). We also evaluated the effect of the combination of TAC and SIR (TAC+SIR), which is used increasingly in clinical practice. Microvascular endothelial cells (HMEC-1) were exposed to these drugs to evaluate changes in (1) intercellular adhesion molecule (ICAM)-1 expression on the cell surface, assessed by immunofluorescence labeling and expressed as the mean gray value (MGV); (2) reactivity of the extracellular matrix (ECM) toward platelets, upon exposure of the ECM to circulating blood; and (3) whole-blood clot formation, assessed by thromboelastometry. Studies were conducted in the absence and presence of defibrotide (DF) to assess its possible protective effect. The exposure of HMEC-1 to CSA and TAC+SIR significantly increased the expression of ICAM-1 (157.5 ± 11.6 and 153.4 ± 9.5 MGV, respectively, versus 105.7 ± 6.5 MGV in controls [both P < .05]). TAC applied alone increased ICAM-1 slightly (120.3 ± 8.2 MGV), and SIR had no effect (108.9 ± 7.4 MGV). ECM reactivity increased significantly only in response to CSA (surface covered by platelets of 41.2% ± 5.4% versus 30.1% ± 2.0%, P < .05). DF attenuated all these changes. No significant changes in the viscoelastic properties of clot formation were observed in any condition with blood samples incubated in vitro. In conclusion, CSA and TAC+SIR had a proinflammatory effect, but only CSA exhibited an additional prothrombotic effect. Interestingly, DF exerted clear protective anti-inflammatory and antithrombotic effects on the endothelium.

Our data indicate that rFVIIa promotes a procoagulant activity at sites of vascular damage. This mechanism could explain the beneficial hemostatic effect of rFVIIa in patients with thrombocytopenia or with Glanzmann's thrombasthenia.

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We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.