Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

Immunoreactive calmodulin in supernatant fraction of platelet homogenates from humans, guinea pigs, rats, and rabbits

Immunoreactive Calmodulin (CM) was measured in the supernatant fraction of homogenates of platelets obtained from humans, rats, guinea pigs and rabbits, using a commercial RIA kit. The levels (microgram/g wet weight of platelets) of immunoreactive CM were 6.8 +/- 0.5, 6.9 +/- 0.4, 8.6 +/- 1.8 and 9.7 +/- 3.1 (mean +/- SEM) for rat, human, rabbit and guinea pig, respectively. There was no statistically significant difference in values between these four species. The refractoriness of rat platelets to aggregate to certain agonists such as platelet activating factor (PAF) cannot be explained on differences in amount of immunoreactive CM.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets

The binding characteristics of [3H]U46619 to washed human platelets were studied. [3H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a Kd of 11 +/- 4 nM (n = 4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a Kd of 20 +/- 7 nM and a Bmax of 9.1 +/- 2.3 fmole/10(7) platelets (550 +/- 141 binding sites per platelet) (n = 4). A number of compounds that act as either agonists or antagonists of the TXA2/PGH2 receptor were tested for their ability to inhibit the binding of [3H]U46619 to washed human platelets. The Kds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [3H]U46619 binds to the putative human platelet TXA2/PGH2 receptor.     

Platelet release products modulate some aspects of polymorphonuclear leukocyte activation.

The aim of this research was to evaluate in vitro interactions between platelets and polymorphonuclear leukocytes. The effects of supernatant from thrombin-activated platelets and two platelet release products (adenosine triphosphate and beta-thromboglobulin) were tested on the following features of polymorphonuclear leukocytes activation: opsonized zymosan and phorbol myristate acetate stimulated chemiluminescence, release of membrane bound calcium, NADPH-oxidase activity, and membrane fluidity (fluorescent polarization). The results showed that the addition of platelet supernatant to polymorphonuclear leukocytes induces a significant activation of cells. On the other hand, after three hours of preincubation of polymorphonuclear leukocytes with platelet supernatant, a decreased response of polymorphonuclear leukocytes to stimulation with phorbol myristate acetate, a significant decrease in NADPH-oxidase activity, and a lowered membrane fluidity were observed. Adenosine triphosphate modulated only opsonized zymosan stimulated chemiluminescence, with and without preincubation with polymorphonuclear leukocytes. Beta-thromboglobulin caused a decrease of the chemiluminescent response of polymorphonuclear leukocytes, using both agonists, with and without preincubation with polymorphonuclear leukocytes. Moreover beta-thromboglobulin only caused a decrease of the polymorphonuclear leukocytes membrane fluidity without preincubation with the cells. These results support the thesis that platelets have a "time-related" modulating activity on polymorphonuclear leukocytes.     

Release of a membrane surface glycoprotein from human platelets by phosphatidylinositol specific phospholipase(s) C.

Phosphatidylinositol (PI) specific phospholipase C (PIase C) treatment of human platelets caused release of a surface glycoprotein in the medium. Human blood platelets were isolated by low speed centrifugation and surface glycoproteins were labelled with periodate/[3H]borohydride procedure. Intact surface-labelled platelets were treated with PIase C purified from culture filtrates of Staphylococcus aureus (SA) or Bacillus thuringiensis (BT). After PIase C treatments platelets were spun at low speed, pellet and supernatant were separated. The supernatant was further centrifuged at high speed (140,000 x g) for 30 min. The resulting supernatant and the pellet from low speed were subjected to SDS-PAGE analysis. Protein patterns were obtained by fluorography. Release of a specific glycoprotein of approx. 150 kDa in the medium was observed due to the PIase C treatment. Prolonged incubation of platelets in 0.25 M sucrose and depletion of NaCl concentrations also affected the release of this glycoprotein. BT-PIase C released more approx. 150 kDa protein than SA-PIase C. Western blot experiment with a monoclonal antibody (mAB), epitope SZ2, reactive to human platelet surface glycoprotein Ib (GPIb) complex, confirmed that released 150 kDa glycoprotein reacted with mAB of GPIb. The release of this protein by PIase C was not inhibited by proteinase inhibitors (EDTA, PMSF and leupeptin). Treatment of human platelet membranes with PIase C also caused release of this glycoprotein as evidenced by reactivity to GPIb-mAB. These studies demonstrate that PIase C treatment causes release of 150 kDa glycoprotein from human platelet membrane surface. It is suggested that 150 kDa glycoprotein is anchored to PI in human platelets and that this glycoprotein represents the GPIb complex.     

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca]i in human platelets in vitro

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.     

Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen

A platelet membrane glycoprotein, 61 kDa, has been identified, which binds specifically to insoluble collagen. The detection of this protein was accomplished by incubating radiolabeled Triton-solubilized platelet supernatant with insoluble collagen, and, after washing the collagen pellet, extracting the 61-kDa glycoprotein from the pellet with sodium dodecyl sulfate buffer. The optimal conditions for specific binding were incubation of 120 micrograms of total platelet supernatant protein with 2 mg of collagen at 4 degrees C for 0.5 h in 0.5 ml of the incubating buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.2% Triton, pH 7.4). The 61-kDa glycoprotein is cleaved by trypsin into a major peptide (44 kDa) and a smaller peptide(s) linked together by disulfide bonds in a molecule which still binds to collagen. When intact platelets are treated first with trypsin and then with dithiothreitol, the 44-kDa peptide is released and was shown to bind to collagen. We conclude that the 61-kDa glycoprotein is a platelet membrane protein which specifically interacts through its extracellular domain with insoluble collagen, and, thus, must be considered as a possible component of the initial platelet-matrix adhesion process which leads to platelet aggregation in vivo.     

Model for the regulation of platelet volume and responsiveness by the trans-membrane Na+/K(+)-pump.

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.     

Homologous Desensitization of Muscarinic Cholinergic, Histaminergic, Adrenergic, and Serotonergic Receptors Coupled to Phospholipase C in Cerebellar Granule Cells

Cultured cerebellar granule cells express phospholipase C-coupled muscarinic cholinergic, histaminergic, alpha 1-adrenergic, and serotonergic receptors. In an attempt to study desensitization of these neurotransmitter receptors, cells were prestimulated with saturating concentrations of carbachol, histamine, norepinephrine, or serotonin during the labeling of cells with myo-[3H]inositol and then rechallenged with various receptor agonists for their ability to elicit accumulation of [3H]inositol monophosphate in the presence of lithium. Prestimulation with each of these receptor agonists was found to cause a time-dependent desensitization to subsequent stimulation with the desensitizing agonist. Thus, prestimulation for 0.5, 4, and 18 h decreased carbachol response to 87 +/- 4, 52 +/- 2, and 40 +/- 1% of the control, respectively; histamine response to 37 +/- 2, 24 +/- 2, and 18 +/- 2%, respectively; norepinephrine response to 55 +/- 5, 14 +/- 1, and 10 +/- 1%, respectively; and serotonin response to 36 +/- 1, 18 +/- 1, and 9 +/- 2%, respectively. In all cases, the responses mediated by receptors which were not prestimulated remained virtually unchanged, thus indicating homologous desensitization. Dose-response studies indicate that the desensitization was associated with a major reduction in the maximal extent of agonist-induced responses. The basal accumulation was markedly enhanced following 0.5- and 4-h prestimulation, but returned to near normal after 18-h pretreatment. Biologically active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate, rapidly attenuated basal phospholipase C activity, as well as the responses mediated by carbachol, histamine, norepinephrine, and serotonin, suggesting that activation and translocation of protein kinase C might play a role in the desensitization of phospholipase C-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.     

Specific binding of phospholipid platelet-activating factor by human platelets

The binding of the phospholipid platelet-activating factor 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) to washed human platelets was more than 80% complete within 2 min, which coincided with the time of initiation of platelet aggregation by AGEPC. Scatchard plot analysis of the binding of [3H]AGEPC to platelets without and with an excess of unlabeled AGEPC revealed two distinct types of binding sites. One platelet site for AGEPC exhibited a high affinity (KD = 37 +/- 13 nM, mean +/- SD), was saturable, and had a low maximal capacity of 1399 +/- 498 (mean +/- SD) molecules of AGEPC/platelet. The other platelet site demonstrated a nearly infinite binding capacity, consistent with nonreceptor uptake of AGEPC into cellular structures. The specificity of the high-affinity binding site for AGEPC was assessed by comparing the capacity of several analogues of AGEPC to inhibit the binding of [3H]AGEPC to platelets and to induce platelet aggregation. An ether linkage in position 1, a short-chain fatty acid in position 2, and a choline moiety in the polar head group proved to be critical both for the binding of [3H]AGEPC to platelets and for the initiation of platelet aggregation. Exposure of platelets to AGEPC for 5 min at 37 degrees C functionally deactivated the exposed platelets to subsequent stimulation by AGEPC, as assessed by diminished aggregation, and concomitantly reduced the specific binding of [3H]AGEPC. Evaluation of the time course of the events of deactivation revealed the loss of an aggregation response to AGEPC after 90 sec at 37 degrees C, despite the retention of up to 50% of the specific binding sites for AGEPC.     

pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.     

A subpopulation of platelets responds to thrombin- or SFLLRN-stimulation with binding sites for factor IXa

Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500-600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4-20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 +/- 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (approximately 10%) of PAR 1-stimulated platelets expose approximately 6000 factor IXa binding sites/platelet.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.