Clinical Hemorheology and Microcirculation

Recent evidence suggests that shear-induced platelet aggregation (SIPA) is an important mechanism of thrombosis at arterial bifurcations or stenotic lesions. We measured SIPA using newly-developed equipment in patients with cerebral ischemia, and also studied the effects of various pharmacological agents on SIPA in vitro and ex vivo. Platelet aggregation was induced by high shear stress (108 dyne/cm²), which is dependent on von Willebrand factor and on glycoproteins IIb/ IIIa and Ib. In vitro studies, SIPA was inhibited by agents that increase the activity of adenylate cyclase as well as inhibitors of platelet ionized calcium, ADP receptor and Gj protein. but it was not affected by inhibitors of cyclooxygenase, lipoxygenase, thromboxane A₂ synthetase, or platelet-activating factor. An increase of SIPA was observed in patients with atherothrombotic stroke and transient ischemic attacks, but not in those with lacunar stroke. SIPA was more often increased in patients who showed an increase of larger von Willebrand factor multimers on SDS agarose gel electrophoresis. Oral aspirin did not inhibit SIPA, but oral ticlopidine significantly inhibited it. The above results indicate that SIPA is mediated or regulated by cyclic AMP, cytoplasmic calcium, and ADP, and also indicate that SIPA is increased in some subtypes of cerebral ischemia, and can be corrected by ticlopidine but not by aspirin.

A technique based on Fraunhofer diffraction by suspended particles is applied in this paper to develop a numerical process to assess information about the erythrocyte deformability distribution over a population of more than six millions cells. The ability to evaluate the full distribution rather than just a mean deformability of erythrocytes allows better assessment of the erythrocyte rheologic capabilities. In order to determine both standard deviation and mean of erythrocyte population deformability, the experimental profile of intensities was matched with a theoretical one. This theoretical profile has been drawn by combining theoretical intensities corresponding to the dimensions of each deformed cell class and assuming a normal distribution. The method has been verified by analyzing mixtures of known proportions of normal erythrocyte and hardened spherocytes. Normal and pathological erythrocytes population were also analyzed and their respective deformability distributions are presented.

We tested the accuracy and the usefulness of the MT90 (falling ball) viscometer as an alternative to more expensive and sophisticated devices for physiological and clinical investigations. This instruments measures viscosity from the velocity of a ball falling through a syringe filled with less than 1 ml of blood or plasma at a shear rate of 1000 s-1, i.e. the range of the so-called ‘newtonian’ behavior of blood. We postulated that this shear rate allows us to measure RBC rigidity in a completely disaggregated structure. Four indices of RBC rigidity derived from high shear rate viscometry were used: μ45r (i.e. blood viscosity at corrected hematocrit 45% divided by plasma viscosity); ‘Tk’ (Dintenfass); ‘k’ (Quemada); ‘μ(RBC)’ (Breugel and coll.). All were strongly correlated to each other (r=0.98 p<0.01) and easily detected in vitro rigidification of RBCs with (a) ionophore A23187 (10⁻⁴M); (b) heating at 56°C during 10 min; (c) 30 min incubation in hypercalcemic-hyperosmolar buffer, when used together with Hanss' hemorheometre. At this shear rate, modifications of blood viscosity induced by changes (within a physiological range) of hematocrit and RBC rigidity were correctly described by both Quemada's and Dintenfass's equations for blood viscosity. ‘k’ values given by the equation of Quemada at this shear rate correlated with RBC rigidity indices calculated with the Carri-Med rheometer using both Quemada's and Wang's equations. Correlations between these indices of rigidity and those given by the hemorheometre were found only for in vitro experiments of RBC stiffening, while in clinical situations viscometric and hemorheometric indices gave rather different results. The coefficient of variation for viscometric measurements was 2% (plasma viscosity) and 3% (blood viscosity). We conclude that this viscometer gives results consistent with other methods. We suggest that it is useful and accurate for physiological and clinical studies.

The performance of nickel and polycarbonate micropore filters (both with 5μm pore diameters) were compared using two different blood filtration methods in control and diabetic subjects. Significant differences were detected between these subject groups with nickel filters but not with polycarbonate filters. The coefficients of variation with both filtration methods were lower with the nickel filters, possibly due to their better pore geometry. It is concluded that Veco nickel filters are superior to Nucleopore polycarbonate filters when assessing blood filtration in clinical studies.

This study determined whether the extent of atherosclerotic plaque buildup, smoking history, or serum cholesterol level could be correlated with red cell membrane deformability as determined by the micropipette aspiration technique. Smoking and non-smoking subjects 40 to 70 years old were grouped by atherosclerotic plaque thickness. A 50% increase (p≤0.01) in red cell membrane shear elastic modulus was seen in the advanced atherosclerosis group (plaque thickness = 2.0–4.0 mm). This increase was independent of the smoking history of the subjects. An increase in shear elastic modulus indicates a decrease in red blood cell membrane deformability. Other variables tested including age and serum cholesterol level were not significantly correlated with red blood cell shear elastic modulus. Such changes in red cell deformability may play an important role in the pathophysiology of vascular occlusion and cerebral ischemia in patients with carotid artery atherosclerosis.

Plasma fibrinogen is an independent risk factor of atherosclerosis. By altering lipid and plasma fibrinogen concentrations, bezafibrate modifies several factors which potentially affect progression of atherosclerosis. 23 male patients were randomized after successful PTCA. The bezafibrate group (n=11), in addition to diet, was given 200 mg bezafibrate three times daily. In the control group (n=12) there was usual care only. All patients received acetylsalicylic acid. Reassessment was after 6 months. Bezafibrate patients had a significant reduction of plasma fibrinogen from 340 ± 92 to 224 ± 29 mg/dl (p<0.05). The difference in fibrinogen concentrations within the 6 months was also significant when the two groups were compared (p<0.05). Patients of the bezafibrate group had a significant decrease of plasma triglycerides from 297 ± 303 to 134 ± 64 mg/dl (p<0.05) and a significant increase in HDL-cholesterol (30 ± 13 to 48 ± 11 mg/dl, p<0.05), apolipoproteins A1 (116 ± 19 to 138 ± 18 mg/dl, p<0.05) and apolipoproteins A2 (41 ± 8 to 56 ± 7 mg/dl, p<0.05). Changes in HDL-cholesterol and apolipoproteins A1 and A2 were also significant (p<0.05) when the two groups were compared after 6 months. Restenoses were found in 11 of the 23 patients (47.8%), 4 restenoses in the 11 bezafibrate patients and 7 restenoses in the 12 usual care patients. A potential efficacy of bezafibrate in the prevention of restenosis may be related to the drug's effects on plasma fibrinogen.

In order to clarify some aspects of cell involvement in the mechanism of tissue injury during ischaemia, haemoreological parameters and polymorphonuclear leukocyte (PMN) function were studied in patients affected with peripheral obliterative arterial disease (POAD) undergoing treadmill test and isotonic ischaemic exercise at an upper limb. In POAD patients, ischaemia was associated with an impairement in haemorheological parameters, in regional as well as in systemic blood. During treadmill test, cytosolic free calcium levels in resting PMNs from systemic blood increased significantly, while no significant change was observed in the response to fMLP stimulation. Superoxide anion generation from PMNs increased only for sub-maximal doses of fMLP (0.01 μM). Conversely, isotonic ischaemic exercise was accompanied by a significant increase in cytosolic free calcium levels in resting and fMLP-activated PMNs in the blood from the ischaemic area, as well as in superoxide anion generation from 1 μM fMLP-stimulated cells. Moreover, an increase in PDGF and ATP plasma levels was also observed, as an expression of platelet activation, possibly inducing PMN priming. Our data support the view that PMN activation is associated with rheological impairement and tissue damage occurring in the course of ischaemic events.

At a constant haematocrit ratio the yield shear stress (YSS) depends on the level of fibrinogen and on the influence of other plasma constituents, erythrocyte membrane changes on the red cells and fibrinogen interaction. The study of the influence of the H.E.L.P. system on the value of YSS was carried out in 9 patients with vascular disease (acute stroke - 3 patients -, multi-infarct dementia - 3 patients -, peripheral arteriosclerotic vascular disease - 2 patients - and coronary heart disease - 1 patient) in the model of diluted plasma media varying in fibrinogen concentration. The H.E.L.P. system application significantly decreased the value of YSS (p<0.001), which was related to the parallel reduction of the fibrinogen level (p<0.001) and plasma viscosity (p<0.001). The significant coefficients of correlations were found out between following parameters: YSS and fibrinogen (p<0.001), YSS and plasma viscosity (p<0.001) and fibrinogen and plasma viscosity (p<0.001) .