Clinical and functional consequences of platelet membrane glycoprotein polymorphisms

The last several years have seen an abundance of studies of genetic risk factors for vascular disease, and platelet glycoprotein (GP) polymorphisms have been a primary focus of this area of research. This article reviews GP receptor polymorphisms, particularly those on GPIa-IIa (integrin alpha2beta1), GPIb-IX-V, GPIIb-IIIa (integrin alpha(IIb)beta3), and GPVI, and summarizes clinical and functional studies that have attempted to clarify their roles in human disease. Our focus is on recent work relevant to thrombotic and hemostatic processes and advances in pharmacogenetics. We consider issues affecting our ability to derive firm conclusions from these studies, and discuss future directions in this rapidly evolving area.     

Transformed rat arterial smooth muscle cells induce platelet aggregation

Atherogenesis is characterized by a proliferation of arterial smooth muscle cells that may be of transformed nature. Platelets are implicated in the progression of atherosclerotic lesions through thrombosic complications. The present study was designed to investigate whether transformed arterial smooth muscle cells (SMC) could specifically aggregate platelets. We used rat transformed arterial SMC lines, V6- and V8-lines, that we had previously established. Experiments were performed with an in vitro homologous rat system. Suspensions of SMC were added without any other aggregating agent to rat heparinized platelet-rich plasma (PRP) in a coagulo-aggregometer. The effect of transformed V6-line and V8-line SMC was compared to that of their normal parental counterparts, V6- and V8-parent cells. Suspensions of transformed SMC induced, in a dose-dependent manner, an immediate and reversible ADP-like platelet aggregation. The amplitude of platelet aggregation was much higher with addition of transformed cells than of the corresponding control SMC (7.39 ± 0.75 cm vs. 0.85 ± 0.62 cm with 2 × 10⁶ SMC, V6-line vs. V6-parent cells, respectively). ADP-like aggregation did not significantly differ between the two transformed V6- and V8-lines. ADP-like platelet aggregation was also obtained with supernatants of transformed SMC suspensions, the amplitude being higher with supernatants than with cell suspensions (21.0 ± 3.64 cm vs. 6.8 ± 1.22 cm with 1.0 × 10⁶ V8-line cells, supernatant vs. cell suspension, respectively). The transformed SMC-induced aggregation of platelets was inhibited by apyrase (125 μM) and iodoacetate (25mM) and thus was ascribable to ADP released by the SMC. In addition, all suspensions of SMC, normal or transformed, but not their supernatants, induced plasma clotting after variable coagulation times. Coagulation was inhibited by hirudin (25 to 100 U/ml) and phospholipase A2 (10 U/ml) indicating thrombin generation through activity of the SMC membrane tissue factor. The present results show that transformed arterial smooth muscle cells may directly aggregate platelets via a release of ADP and this could be of pathophysiological relevance for thrombosis associated with atherosclerosis.     

Hemodynamic and functional consequences of radial artery removal for coronary artery bypass grafting

Five years after surgery the echo-Doppler characteristics of the forearm circulation and the transcutaneous oxygen and carbon dioxide pressures of the operated and control arm were determined at rest and under conditions of hand exercise in 34 patients who received a radial artery graft for myocardial revascularization. Doppler measurements showed the ulnar compensation to radial artery removal, and transcutaneous measurements demonstrated a moderate degree of exercise-induced hand ischemia on the operated site.     

Aprotinin attenuates platelet accumulation in ischaemia-reperfusion-injured porcine skeletal muscle.

This purpose of this study was to evaluate the effect of aprotinin, a serine protease inhibitor, in ischaemia- and reperfusion-injured myocutaneous flaps and skin flaps. Flap survival, microcirculatory platelet accumulation, and regional blood flow were investigated in seventeen pigs which had been subjected to 8 h of ischaemia and 18 h of reperfusion. The pigs were randomly assigned to aprotinin treatment (n = 9) or saline (n = 8). In-vitro studies were performed to investigate the influence of aprotinin on the activated partial thromboplastin time. The survival of skeletal muscle correlated positively with the concentration of aprotinin (P = 0.02) and could not be explained by regional changes in blood flow. Platelet accumulation was decreased in aprotinin-treated muscle (P = 0.04). In-vitro (n = 10), 100 kallikrein inactivator units/ml aprotinin prolonged the activated partial thromboplastin time both in plasma (P = 0.001) and in blood (P = 0.002), suggesting an anticoagulant rather than a procoagulant effect. In conclusion, aprotinin at high concentrations may be beneficial for the survival of skeletal muscle and provides protection from platelet accumulation in the microcirculation of skeletal muscle exposed to ischaemia and reperfusion injury.     

Disseminated intravascular coagulation associated with Staphylococcus aureus septicemia is mediated by peptidoglycan-induced platelet aggregation

Disseminated intravascular coagulation (DIC) may complicate severe septicemia caused by Staphylococcus aureus. S. aureus can induce spontaneous platelet aggregation in vitro, the rapidity and degree of which correlates with the severity of DIC in patients with sepsis. Purified peptidoglycan from DIC isolates aggregated human platelets in the presence of staphylococcal protein A with significantly shorter aggregation times than did peptidoglycan from non-DIC isolates. Purified teichoic acid from DIC and non-DIC isolates failed to aggregate platelets in vitro, or in vivo in guinea pigs but inhibited the peptidoglycan-induced aggregation in a dose-response manner. These studies suggest that peptidoglycan may mediate S. aureus-induced spontaneous platelet aggregation in vitro and DIC in vivo. The variability among strains of S. aureus to induce DIC and platelet aggregation may depend on the unique composition of their peptidoglycan and perhaps also the extent of exposure or availability of cell wall teichoic acid.     

Inhibition of intravascular platelet aggregation by endothelium-derived relaxing factor: reversal by red blood cells

Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet-inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.     

Measurement of growth rate of laser-induced intravascular platelet aggregation and the influence of blood flow velocity.

The growth rate of platelet microthrombi induced by laser injury in ear chamber arterioles on conscious rabbits was measured. A growth rate constant was obtained for each microthrombus by plotting its volume change on a semilogarithmic plot. In most instances each platelet microthrombus formed at any one injury site conformed to a similar growth rate pattern. There was a direct positive correlation between the mean growth rate constant and the number of emboli counted for each laser injury. Blood flow velocity had a variable effect on the number of emboli from sites of laser injury. At velocities above 2.5 mm sec^?1 the number of emboli remained relatively constant; between 1.0 and 2.5 cm sec^?1 the number of emboli tended to increase and there was a greater variation at this level; below 1.0 mm sec^?1 the number of emboli decreased. The number of platelets participating in formation of a microthrombus was expressed as a percentage of the number of available platelets to give a measure of platelet aggregability in vivo. Platelet aggregability varied with alterations in blood flow velocity. At blood flow velocities between 1.0 and 2.5 mm sec^?1 the percentage of platelets participating varied between 20 and 50%, but at velocities above 3.0 mm sec^?1 the percentage decreased to less than 20%. The results suggested that the number of emboli was an indirect measurement of the growth rate of microthrombi forming at sites of laser injury. Further, both the number of emboli and the percentage platelet aggregability in vivo were not significantly affected by changes in blood flow velocity over the range normally found in rabbit ear chamber arterioles.     

Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.     

Hemodynamic and myocardial consequences of scorpion venom

The hemodynamic effects of scorpion venom (Leiurus quinquestriatus) and the mechanism of heart failure were investigated in two groups of anesthetized spontaneously breathing dogs. The effects of different adrenergic and cholinergic blocking agents on the venom-induced hemodynamic changes were also evaluated. In one group the venom was given before autonomic nervous system blocking agents and in a second group propranolol, atropine, phentolamine and hexamethonium alone or in different combinations were given before the venom. Complete autonomic nervous system blockade was induced in two animals to evaluate a possible direct myocardial effect of venom.The venom, a powerful arrhythmogenic agent stimulating the autonomic sympathetic nervous system and adrenals, induced dramatic hemodynamic increases in left ventricular systolic and diastolic pressures, pulmonary and systemic arterial pressures and left ventricular contractility. The hemodynamic data show clearly for the first time that pulmonary edema in severe scorpion envenomation is cardiac in origin, thus emphasizing the importance of the abnormal left ventricular hemodynamics. Heart failure is most probably the result of the interaction of several mechanisms that include a catecholamine-induced decrease in left ventricular compliance and increased impedance to left ventricular emptying and cardiac arrhythmias, all of which may impede left ventricular filling. The combination of propranolol and phentolamine was the most effective blocking agent in reversing the venom-induced hemodynamic changes. However, atropine was effective only when the venom-induced cholinergic effects dominated the clinical picture.     

Hemodynamic and myocardial metabolic consequences of PEEP

The cardiac effects of positive end expiratory pressure (PEEP) were examined in 50 patients six hours after elective coronary bypass surgery. Increasing the level of PEEP from 5 to 10 to 15 cm H2O decreased cardiac index (evaluated by thermodilution), stroke index and left ventricular end diastolic volume index without a change in left ventricular ejection fraction (evaluated by nuclear ventriculography). Right ventricular end diastolic volume index remained unchanged. Coronary sinus blood flow (measured by the continuous thermodilution technique) and myocardial oxygen and lactate consumption were unchanged with the application of 15 cm H2O PEEP. In 21 patients, volume loading (250 ml [mL] of plasma) was performed at 5 cm, and again at 15 cm H2O PEEP. Volume loading produced a similar increase in cardiac volumes and cardiac index at 5 and 15 cm H2O PEEP. Right and left ventricular performance and left ventricular systolic function were not altered by PEEP (by analyses of covariance). Coronary sinus blood flow and myocardial oxygen consumption increased with volume loading at 5 and 15 cm H2O of PEEP, but myocardial lactate utilization tended to increase at 5 cm, and decrease at 15 cm H2O PEEP (p = 0.08). Of the 33 patients who underwent complete hemodynamic and metabolic measurements, 16 increased cardiac lactate utilization at 15 cm H2O PEEP and 17 decreased cardiac lactate utilization at 15 cm H2O PEEP. PEEP decreased cardiac index, perhaps by reducing left but not right ventricular volumes. Volume loading during PEEP restored cardiac index and revealed no depression in myocardial performance or systolic function. With the application of PEEP, myocardial metabolism was maintained in half the patients, but ischemic metabolism was observed in the other half.     

Hemodynamic consequences of neuropeptide Y-induced obesity

BackgroundAcute central nervous system administration of neuropeptide Y (NPY) elicits variable hemodynamic responses. Chronic intracerebroventricular (ICV) administration of NPY produces obesity in rats. Obesity has been shown to increase arterial pressure.MethodsIn this study we examined the chronic hemodynamic effects of NPY-induced obesity. Sprague-Dawley rats were implanted with radiotelemetry transmitters to continuously record heart rate and arterial pressure in the conscious state. Neuropeptide Y or vehicle was delivered into the third cerebral ventricle by osmotic minipumps over 2 weeks. Three groups were studied: vehicle, NPY-treated (free-fed), and NPY-treated (pair-fed to vehicle-treated rats).ResultsNeuropeptide Y increased food intake and body weight in free-fed animals, and substantially augmented visceral adiposity in both free- and pair-fed rats. Despite increased adiposity, chronic ICV administration of NPY in conscious unstressed rats did not increase arterial pressure. Neuropeptide Y decreased heart rate, suggesting a sympathoinhibitory effect.ConclusionsObesity induced by 2-week ICV administration of NPY does not increase arterial pressure, perhaps indicating inhibition of sympathetic outflow that may oppose the pressor effect of adiposity.     

Mechanisms of platelet aggregation

Platelet aggregation is initiated by receptor activation coupled to intracellular signaling leading to activation of integrin alphaIIbbeta3. Recent advances in the study of platelet receptors for collagen, von Willebrand factor, thrombin, and adenosine diphosphate are providing new insights into the mechanisms of platelet aggregation.     

Changes in the distribution and organization of platelet actin induced by diamide and its functional consequences

Exposure of blood platelets to diamide (azodicarboxylic acid-bis-dimethylamide) results in oxidation of sulphydryl groups present in the cytoskeleton and other proteins. This results in dramatic changes in functional behaviour of the cells. The distribution and level of organization of the major cytoskeletal protein actin has been studied analytically by the DNase-I inhibition assay and morphologically by electron microscopy (EM) of Triton X-100 treated platelets adherent to EM grids. Exposure to diamide results in a redistribution of actin within the cell reflected in an increase in cytoskeletal F-actin and a concomitant decrease in cytosolic actin. The magnitude of these changes depends upon the concentration of diamide and the time of exposure. Diamide also alters platelet aggregatory functions in response to certain stimuli. Treatment of normal human platelets with 0.1 mM diamide proceeds via disaggregation (5 min exposure to diamide), inhibition of aggregation (30 min exposure), to finally a normalization of the aggregation response after 60-120 min incubation with diamide. In parallel with the return to full functional response the distribution of F-actin between the cytoskeleton and cytoplasmic compartments returns to the control pattern. Incubation of the platelets with 0.5 mM diamide for 60 or more minutes leads to total inhibition of the aggregatory ability. In these cells the cytoskeleton associated F-actin remains significantly elevated and the structural organization of the cytoskeleton is markedly altered. In contrast to the network of filaments subadjacent to the surface membrane seen in unstimulated platelets, the cytoskeleton now shows electron dense zones in the more central parts of the cytoplasm. This diamide-induced structural reorganization of platelet cytoskeletal elements, associated with the inhibition of functional responses, emphasizes the dynamic nature of the membrane-cytoskeletal axis and its importance in the expression of shape changes and aggregatory phenomena in response to surface stimuli.     

Effect of heparin on platelet count and platelet aggregation

The in vitro effect of heparin on platelet aggregation was studied in three groups: in 26 subjects recently treated with heparin, in 18 subjects on maintenance hemodialysis, and in 20 normal controls. With the aid of Technicon H6000, platelet counts and platelet aggregations were compared in whole blood samples collected in ethylenediaminetetraacetic acid (EDTA) and in heparinized tubes. Although there was no significant difference between platelet count of heparinized and EDTA blood in the control group, the dialysis group and the group recently treated with heparin showed significantly lower platelet counts and more platelet aggregation in heparinized tubes than in EDTA tubes. We speculate that the majority of subjects exposed to heparin develop an antibody or a proaggregator which can aggregate or agglutinate platelets in the presence of heparin and causes destruction of platelets; but only in a small percentage of subjects receiving heparin is this reaction severe enough to cause thrombocytopenia.     

Collagen age and platelet aggregation

Rat tail tendon collagen-initiated platelet aggregation exhibits a collagen age-dependent lag time. This lag time is an inverse function of the previously determined rate of fibril formation of collagen, and corresponds to the elapsed time necessary to form a collagen fibril of requisite size under the platelet aggregation conditions chosen. Such fibers exhibit native spacing and appear to be 45 to 90 A in diameter. Fibers preformed to that size (less than 2 min for 21- to 1,100-day-old collagen), no matter what the age of the collagen, give rise to identical platelet aggregations. Fibers formed after more prolonged incubation, greater than or equal to 20 min, have impaired platelet aggregating ability.