PLATELET-ENDOTHELIAL CELL INTERACTIONS

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An estimated 10 to 20% of all patients with hemophilia A or B have unexplained prolonged bleeding times together with elevation in levels of circulating immune complexes (CIC's). We hypothesize that the CIC's cause functional endothelial cell injury leading to increased production of prostacyclin (PGI2) and prostaglandin E2 (PGE2), the net effect of which is to impair platelet plug formation. To evalutate this hypothesis, objectives of this work are 1) to observe and quantify, for such patients as well as normal subjects, platelet adhesion-aggregation upon local injury sites on cultured endothelial cell monolayers grown to confluence on collagen-coated cover glasses and exposed to controlled conditions of whole blood flow, 2) to assess parallel endothelial cell function and possible injury by downstream measurement of production of PGI2, PGE2 and intracellular enzymes (lactate dehydrogenase, LDH), and 3) to relate findings to bleeding time and, for the patient group, clinical severity of bleeding episodes. Key to the first of these aims is the use of an in vitro flow chamber in which a monolayer of cultured endothelial cells forms one bounding surface. Thickness of the flow path (254Mum) and rate of flow from a well-stirred, constant temperature blood reservoir by means of a roller pump allow for control of shear stress and shear rate at the blood-monolayer interface. The chamber also accomodates a video microscope whereby platelets adherent to or aggregated upon injury sites can be imaged in whole blood by either transmitted light or EPI-fluorescent microscopy. Endothelial cell function and possible injury will be monitored by means of a special chamber feature: inclusion of a magnetically stirred flow region immediately downstream from the monolayer. In this flow region, blood samples for PGI2 and PGE2 (measured by specific RIA's) and LDH permit calculation (concentration flow rate) of rate of production of PGI2, PGE2 or LDH by the monolayer as a whole. Direct observation by phase contrast microscopy (after displacing blood by buffer, but before chamber disassembly) yields complimentary information concerning endothelial cell detachment and morphologic change. Relationships will be sought between platelet adhesion-aggregation and monolayer function, on the one hand, and bleeding time and a score for severity of bleeding episodes, on the other. Independent experimental variables will include concentration of CIC's, size (30 to 150Mum) of cell monolayer injury, shear rate, shear stress and duration of blood flow.