Search PubMed⌕ Search

Biomedical subjects

A R Gear

Publications and source records attributed to A R Gear.

At least 37 records · Page 2Linked to original sources
PubMed1994

Role of cyclic nucleotides in rapid platelet adhesion to collagen.

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4-fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2-fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.

Adenylyl Cyclases↗
PubMed1994

Platelet adhesion, shape change, and aggregation: rapid initiation and signal transduction events.

Blood platelets are essential for hemostasis, and knowledge of their function is important for understanding both normal and pathologic situations. A number of approaches have been used to evaluate platelet adhesion, aggregation, and secretion, and within the last 10 years much interest has been directed to the biochemical mechanisms and signal transduction events occurring during these various phases of function. New information has come from development of technologies to evaluate the changes occurring immediately after platelet activation and consistent with the speed needed for hemostasis in the arterial circulation. Use of rapid flow and mixing technologies as seen in quenched-flow, continuous-flow, and stopped-flow devices has revealed that platelet aggregation, shape change, adhesion, and secretion begin within 1 s and may be nearly complete by 5 s. Biochemical changes such as in protein phosphorylation, calcium release, and phospholipid hydrolysis are clearly evident in hundreds of milliseconds. Therefore, it is necessary to understand these early events in signal transduction and to assess alterations that may occur in diseases such as diabetes.

Blood Platelets↗
PubMed1993

Platelet adhesion to collagen via the alpha 2 beta 1 integrin under arterial flow conditions causes rapid tyrosine phosphorylation of pp125FAK.

Adhesion of human platelets to collagen under arterial flow conditions mediated by the alpha 2 beta 1 integrin increased tyrosine phosphorylation of several proteins, one of which was the focal adhesion tyrosine kinase, pp125FAK. Tyrosine phosphorylation of pp125FAK did not occur in non-adherent flowing platelets or in platelets attached to poly(L-lysine). Neither adhesion nor tyrosine phosphorylation was affected by pretreatment of platelets with GRGDSP peptide or by anti-alpha IIb beta 3 monoclonal antibody P2. Adherent platelets retained their discoid shape, suggesting that induction of pp125FAK precedes platelet spreading. The tyrosine kinase inhibitor erbstatin decreased tyrosine phosphorylation in non-stimulated platelets and blocked platelet adhesion. These results suggest that pp125FAK plays an important role in platelet adhesion to collagen via the alpha 2 beta 1 integrin.

Amino Acid Sequence↗
PubMed1993

Sub-second oscillations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate during platelet activation by ADP and thrombin: lack of correlation with calcium kinetics.

The hypothesis that ADP and thrombin liberate Ins(1,4,5)P3 in blood platelets, with kinetics consistent for releasing Ca2+ within 2s, was tested by quenched-flow techniques. Both agonists stimulated transient and equal synthesis of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 near 200 ms and later short-lived peaks, which were not correlated with the slower steady increase in intracellular [Ca2+] between 0.5 to 2 s detected by Indo-1. Shear forces alone caused transient liberation of these inositol phosphates within 0.5 s and up to 4 s, yet failed to increase intracellular [Ca2+].

Adenosine Diphosphate↗
PubMed1993

The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor.

The role of protein kinase C (PKC) in platelet activation by thrombin was assessed using a PKC inhibitor Ro 31-7549/001 (R2) which, in vitro, shows more selectivity for PKC than other kinase inhibitors. During early (1.5 s) thrombin-induced platelet activation, when phosphorylation of 47 kDa protein (pleckstrin) and myosin light chain by PKC and myosin light chain kinase, respectively, are most readily differentiated, R2 suppressed phosphorylation of pleckstrin more effectively than myosin light chain. R2-inhibited dense granule secretion (measured 0-10 s using quenched-flow techniques) with a dose dependency similar to that for inhibition of pleckstrin phosphorylation, supporting a role for PKC in this process. R2, at 0.5 microM inhibited 47 kDa protein phosphorylation by more than 60%, but had only minimal effects on the kinetics (0-3s) of ADP-induced primary aggregation. At this same concentration, R2 potentiated the thrombin-induced rise in cytosolic calcium during early (0-15 s) activation as measured in the presence or absence of external calcium. These data support the hypothesis that activation of PKC during early platelet function helps regulate cytosolic calcium levels by limiting calcium release into the cytosol.

Blood Proteins↗
PubMed1992

High-speed platelet adhesion under conditions of rapid flow.

The recognition of exposed collagen by circulating platelets is an initial step in the formation of the hemostatic plug or a thrombus after vascular injury. Theoretical calculations of the speed of platelet function required for effective hemostasis have suggested very short reaction times. However, it is not known how fast platelets can adhere to collagen under arterial flow conditions or which membrane proteins are involved. We have used a continuous-flow, microaffinity column linked to a resistive-particle counter to detect platelet adhesion. Adhesion of human platelets to native type I collagen was extremely rapid, with exponential half-times as short as 240 ms, and was nearly complete by 2 s. This RGD-independent process was not associated with platelet aggregation or secretion. The monoclonal antibody 6F1 directed against the glycoprotein Ia/IIa complex inhibited adhesion, suggesting that this complex plays an important role in the initial phases of platelet-collagen interaction under flow conditions. In addition, divalent cations were required for adhesion, as indicated by inhibition with EDTA in plasma and the dependence on Mg2+ for washed platelets.

Amino Acid Sequence↗
PubMed1992

Adhesion efficiency, platelet density and size.

We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low-density (1.040 < d < 1.065 g/ml), intermediate-density (1.065 < d < 1.070 g/ml) and high-density (1.070 < d < 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous-flow, micro-affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGI2). In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial-flow conditions. The greater efficiency of high-density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.

Antigens, CD↗
PubMed1991

Radiographic contrast agents and platelet function: a quenched-flow study.

Radiographic contrast media (RCM) may alter platelet behavior at concentrations achieved during cardiac angiography. We used quenched-flow aggregometry coupled to single-particle counting to study the influence of RCM on the kinetics of platelet aggregation (less than 5.0 sec) induced by adenosine diphosphate (ADP, 2.86 microM). At a concentration in platelet-rich plasma (PRP) of 5 per cent RCM by volume, platelet aggregation was inhibited by diatrizoate, iopamidol and ioxaglate either directly or following incubation of each contrast agent with PRP for 20 minutes. Diatrizoate inhibited more than did iopamidol or ioxaglate (56 +/- 6, versus 39 +/- 3 and 40 +/- 9 per cent respectively; P less than 0.003, p less than 0.009, n = 20 normal subjects). A small reduction (about 16 per cent) in aggregation velocity occurred within 5 seconds of exposure of PRP to all 3 RCM and the onset time (t) or lag period before aggregation begins was significantly prolonged by diatrizoate (p less than 0.03). The RCM vehicles alone (iodinated moiety removed, osmolality readjusted) had no effect on the ADP-induced aggregation. Platelet counts fell significantly after incubation with diatrizoate (12%; p = 0.04). Our data therefore show that early platelet aggregation was inhibited by 3 commonly-used ionic and nonionic contrast agents. Inhibition was apparently caused by the iodinated contrast molecule, began within seconds of platelet-RCM contact and was independent of vehicle composition. Since diatrizoate inhibited aggregation more than iopamidol or ioxaglate, its use may be of additional value during angiographic procedures in clinical situations involving enhanced platelet activation.

Adult↗
PubMed1989

Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.

Blood Platelets↗
PubMed1989

Endothelium-dependent potentiation of human platelet aggregation.

The endothelium produces many substances which have the potential to alter hemostasis. We examined the influence of a substance(s) released by cultured bovine aortic endothelial cells on very early events (less than 5 sec) in platelet function, using a new quenched-flow approach. Contributions of the endothelial cell surface to alterations in platelet function were avoided by separating the endothelium and platelets. Superfusates from endothelial cells were allowed to interact with platelets for several seconds, followed by challenge with ADP to induce aggregation. Single-particle counting provided a sensitive index of aggregation kinetics. In this system, unstimulated endothelial-cell superfusate had no effect on aggregation induced by ADP. Stimulation of the endothelial cells with bradykinin produced an unstable factor(s) that potentiated ADP-induced platelet aggregation, was not affected by hemoglobin or cyclooxygenase inhibitions, and had no platelet aggregatory activity on its own. Nitric oxide inhibited aggregation and its effect was blocked by hemoglobin. The identity of the potentiating factor is unknown, but it does not appear to be platelet activating factor, thrombin, ADP, thromboxane, nitric oxide, or endothelium-derived relaxing factor.

Adenosine Diphosphate↗
PubMed1989

Atrial natriuretic factor in essential hypertension.

We measured circulating levels of immunoreactive atrial natriuretic factor (ANF) in 10 patients with untreated, uncomplicated mild to moderate essential hypertension and in 15 normotensive controls. ANF concentrations were significantly higher in the hypertensive group than in the control group (38.4 +/- 6.9 pg/ml versus 18.3 +/- 1.8 pg/ml, p less than 0.02). A positive correlation between ANF levels and systolic, diastolic and mean blood pressure was noted in the total study population (p less than 0.008, r = 0.52; p less than 0.005, r = 0.55; p less than 0.02, r = 0.46, respectively). Thus, plasma ANF concentrations are elevated in essential hypertension and may result from increased intraarterial pressure.

Adult↗
PubMed1989

Platelet calcium and quenched-flow aggregation kinetics in essential hypertension.

Abnormal platelet function may contribute to the complications of essential hypertension. We have studied the kinetics of platelet aggregation induced by adenosine diphosphate (ADP) or epinephrine, plasma beta-thromboglobulin, and basal, cytosolic, and free calcium, as correlates of platelet function. Fifteen untreated patients with essential hypertension and without detectable atherosclerosis, 18-40 years old, were compared with 30 matched normotensive control subjects. Maximal rates of platelet aggregation (Vmax) with ADP and epinephrine were significantly higher in patients than in control subjects (p less than 0.03), as assessed by quenched-flow aggregometry. However, significance was lost when Vmax was corrected for the platelet count. Paradoxically, the activation constants (Ka) for ADP were higher in patients than in control subjects (p less than 0.03). With ADP as the inducing agent, onset time (t) or lag period before aggregation begins was longer in patients than in control subjects (p less than 0.02). beta-thromboglobulin levels, an index of in vivo platelet activation, were not significantly different between the two groups (p = 0.13). The mean platelet cytosolic free calcium concentration was higher in patients (213 +/- 19 nM) than in control subjects (172 +/- 14 nM), but this difference was not statistically significant (p = 0.07). However, there was a close correlation between the free calcium level and systolic, diastolic, and mean blood pressure (p less than 0.003, p less than 0.04, p less than 0.004, respectively). No difference in platelet volume between the two groups was found. Our data suggest that platelets in the early stages of essential hypertension display an overall increased aggregation potential but a diminished sensitivity to ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1988

Application of spray-freezing to the study of rapid platelet reactions by a quenched-flow approach.

A spray-freezing approach coupled to quenched-flow techniques has been developed to study rapid (0.3-5.0 s) biochemical and morphological changes during platelet activation. Platelet-rich plasma (PRP) pumped through the quenched-flow system was atomized and quenched within 50 ms in liquid propane at -196 degrees C. Analysis of platelet size and morphology by scanning electron microscopy (SEM) indicated that platelets can be sprayed without morphological damage. The spray-frozen platelets can be subjected to freeze-fracture, freeze-substitution, or freeze-drying procedures for later analysis by electron microscopy. Quenched-flow techniques have permitted investigation of rapid increases in cytosolic calcium protein phosphorylation and morphological changes which occur within 0.5 s after exposure to an activator. The combination of quenched-flow and spray-freezing methodologies should prove useful for studying equally rapid changes in protein distribution in activated platelets by means of electron microscopy coupled with immunocytochemical approaches.

Blood Platelets↗
PubMed1988

Potentiation of platelet aggregation by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.

Atrial Natriuretic Factor↗
PubMed1988

Effect of epinephrine on rapid ADP-induced aggregation, protein phosphorylation, and cytoplasmic calcium dynamics of platelets: a quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation with a low concentration of adenosine 5'-diphosphate (ADP) (0.5 mumol/L), epinephrine (15 mumol/L), or a combination of the two. Compared with ADP alone, the combination nearly doubled the rate of aggregation as measured by the loss of single particles. Our aim was to determine whether the aggregation, protein phosphorylation, and cytoplasmic calcium responses to this potentiating combination of ADP and epinephrine were analogous to those caused by a maximal concentration of ADP (10 mumol/L) and also to determine whether cyclooxygenase was involved in this potentiation. Phosphorylation of myosin light chain (MLC) and the 47 kd protein were analyzed during the first 5 seconds of platelet response and were related to the progress of aggregation. Unlike aggregation, ADP-induced phosphorylations of both MLC and the 47 kd protein were inhibited rather than potentiated by epinephrine. Pretreatment of the platelets with indomethacin did not affect aggregation or MLC phosphorylation, but increased 47 kd phosphorylation at 0.6 seconds and eliminated it at 3 seconds. Cytoplasmic free calcium ([Ca2+]i) of indo-1-loaded platelets were monitored with our continuous-flow fluorescence approach. Although epinephrine itself did not change [Ca2+]i, it potentiated the [Ca2+]i rise induced by a low dose of ADP. A comparison of these results with platelet activation caused by 10 mumol/L ADP indicates that MLC and 47 kd phosphorylations are not correlated with [Ca2+]i dynamics and are not required or directly involved in platelet aggregation.

Adenosine Diphosphate↗
PubMed1988

Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1.

The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single-particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).

Adenosine Diphosphate↗