Abstract
Background
Red cell microparticles (RMP) have come to recent attention as putative mediators of hemostasis. We reported that RMP improve hemostatic defect of blood samples of thrombocytopenia and thrombocytopathy and augment platelet function. To investigate possible mechanisms of this activity, we measured the effect of RMP on shear-dependent platelet adhesion and aggregation in whole blood.
Methods
RMP were produced by high-pressure extrusion of washed, packed RBC. The RMP produced in this way are similar to natural circulating RMP in phenotype and most functional assays. Blood was collected in citrate Vacutainers from normal healthy staff volunteers, and first 3 mL discarded to minimize artifact of platelet activation due to tissue factor. It was tested within 2 hours of drawing. Variable shearing rates were applied by a cone-and-plate device, the DiaMed Impact-R, which yields photomicrographs of objects adhering to the plate, and data including percent surface coverage (SC), number objects adhering (OBJ) per mm2, and mean aggregate size (AS) in μm2. Initially, 4μL of either RMP (1.0 x 108/μL) or saline were mixed with 126μL of whole blood obtained as above, and incubated in a microcentrifuge tube for 60s. The mixtures were pipetted to a well of the device and run at a selected shear rate (range 900s-1 to 2700s-1) for 2 minutes. Blood was then carefully drawn off and the wells were washed gently with deionized water. Wells were stained with May-Grünwald stain for 60s, and excess removed. When dry, micrographs of the well surfaces were taken. The most promising shear rates (1125 s-1, 1575s-1, 1800s-1, 2025s-1) were retested with higher numbers of RMP in the 130μL volume.
Results
Addition of RMP vs. saline (control) induced increased AS over a range of shear rates. For example, 4μL RMP (1x108/μL) running at 2250s-1 increased AS by 34.8%, from 59.2 ±25.1μm2 to 79.8 ±24.7μm2 (n=15, p=0.01). To investigate effect of RMP concentration at fixed shear rate of 1575s-1, we found that 8μL RMP induced increase of 12.0 μm2, 12μL RMP by 24.8 μm2, 16μL by 26.7 μm2, and 20μL by 26.3 μm2; p<0.05 and n=4 replicates for all. As seen by the trend, this effect on AS reached a plateau at 20 μL RMP. With fixed volume of RMP added and varying shear rate, we found the largest RMP-induced increase in AS occurred at 1125s-1 shear rate: 16μL of RMP increased aggregate size to 172% of control (from 41.3 ±10.2μm2 to 71.0 ±2.6μm2; n=3, p=0.02). With 1/4 as much RMP (4 μL), the peak effect occurred at 1125s-1. We did not observe any significant differences in SC at any shear rate or RMP volume. OBJ was generally lower as AS increased, but this did not reach significance.
Conclusion/Discussion
RMP increased the size of platelet aggregates under shear, indicating enhanced platelet adhesion by RMP. This effect varied with shear rate: larger amounts of RMP had maximum effect at lower shear rates; smaller amounts of RMP required higher shear to exert maximum effect. These effects are seen at physiologically relevant shear rates (200 - 2000s-1 or higher in pathology such as hypertension or valve defects). Due to the absence of endothelia from this model, high shear was required to induce maximum adhesion. Previous study has indicated the important role of GPIIb/IIIa in platelet adhesion and aggregate size under high shear [Varon, et al; Am Heart J, 1998]. We postulate that RMP may be acting in a GPIIb/IIIa dependent manner to enhance aggregate size. This was consistent with the findings that RMP-induced augmentation of platelet aggregation at relevant levels of ADP or AA concentrations in heparinized blood [Jy, et al; Thromb Haemost, in press]. We are continuing to investigate conditions affecting the interactions between RMP and platelets.
Disclosures:
No relevant conflicts of interest to declare.
Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface
