Shear stimulated red blood cell microparticles: Effect on clot structure, flow and fibrinolysis.

BACKGROUND: Microparticles (MPs) have activity in thrombus promotion and generation. Erythrocyte microparticles (ErMPs) have been reported to accelerate fibrinolysis in the absence of permeation. We hypothesized that shear induced ErMPs would affect fibrin structure of clots and change flow with implications for fibrinolysis. OBJECTIVE: To determine the effect of ErMPs on clot structure and fibrinolysis. METHODS: Plasma with elevated ErMPs was isolated from whole blood or from washed red blood cells (RBCs) resuspended in platelet free plasma (PFP) after high shear. Dynamic light scattering (DLS) provided size distribution of ErMPs from sheared samples and unsheared PFP controls. Clots were formed by recalcification for flow/lysis experiments and examined by confocal microscopy and SEM. Flow rates through clots and time-to-lysis were recorded. A cellular automata model showed the effect of ErMPs on fibrin polymerization and clot structure. RESULTS: Coverage of fibrin increased by 41% in clots formed from plasma of sheared RBCs in PFP over controls. Flow rate decreased by 46.7% under a pressure gradient of 10 mmHg/cm with reduction in time to lysis from 5.7 ± 0.7 min to 12.2 ± 1.1 min (p < 0.01). Particle size of ErMPs from sheared samples (200 nm) was comparable to endogenous microparticles. CONCLUSIONS: ErMPs alter the fibrin network in a thrombus and affect hydraulic permeability resulting in decelerated delivery of fibrinolytic drugs.

Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

A Flow Induced Autoimmune Response and Accelerated Senescence of Red Blood Cells in Cardiovascular Devices.

Red blood cells (RBCs) passing through heart pumps, prosthetic heart valves and other cardiovascular devices undergo early senescence attributed to non-physiologic forces. We hypothesized that mechanical trauma accelerates aging by deformation of membrane proteins to cause binding of naturally occurring IgG. RBCs isolated from blood of healthy volunteers were exposed to high shear stress in a viscometer or microfluidics channel to mimic mechanical trauma and then incubated with autologous plasma. Increased binding of IgG was observed indicating forces caused conformational changes in a membrane protein exposing an epitope(s), probably the senescent cell antigen of band 3. The binding of immunoglobulin suggests it plays a role in the premature sequestration and phagocytosis of RBCs in the spleen. Measurement of IgG holds promise as a marker foreshadowing complications in cardiovascular patients and as a means to improve the design of medical devices in which RBCs are susceptible to sublethal trauma.

Possible erythrocyte contributions to and exacerbation of the post-thrombolytic no-reflow phenomenon.

BACKGROUND: Reperfusion injury often occurs with therapeutic intervention addressing the arterial occlusions causing acute myocardial infarction and stroke. The no-reflow phenomenon has been ascribed to leukocyte plugging and blood vessel constriction in the microcirculation. OBJECTIVE: To assess possible red cell contributions to post-thrombolytic no-reflow phenomenon. METHODS: Blood clots were formed by recalcifying 1 ml of citrated fresh human venous blood and then lysed by adding 1,000 units of streptokinase (SK) at several intervals within 1 hour. Red cell deformability was tested by both a microscopic photometric and a filtration technique, viscosity by a cone and plate viscometer, and erythrocyte aggregation by an optical aggregometer. RESULTS: Two sampling methods were devised for the microscopic photometric test, both of which indicated increases of erythrocyte stiffness after being lysed from the clot by SK. In accompanying experiments, the viscosity, aggregation and filterability of the post-lytic erythrocytes were assessed. Results indicated increased viscosity in Ringer's, decreased aggregation index and filterability through a 5 µm pore size Nuclepore membrane. CONCLUSION: Findings demonstrated that post-lytic changes in red cell deformability do occur which could contribute to the no-reflow phenomenon.