Platelets and Microtubules: EFFECT OF COLCHICINE AND D2O ON PLATELET AGGREGATION AND RELEASE INDUCED BY CALCIUM IONOPHORE A23187

We examined the role of microtubules in platelet aggregation and secretion (release reaction) induced by the calcium ionophore A23187 (0.8-5 muM). At these concentrations, platelet aggregation was preceded by a lag period of approximately 1 min. Colchicine (an agent that disrupts microtubule assembly-disassembly) was shown to bind to platelet microtubules by employing [(3)H]colchicine at a concentration that is specific for microtubules in other tissues (10 nM). Colchicine prolonged the lag period, inhibited the secondary wave of platelet aggregation, and inhibited the release reaction (release of [(14)C]serotonin). Platelets were next incubated with 20-60% D(2)O, an agent that stabilizes microtubules. D(2)O overcame colchicine-induced inhibition of the lag period, aggregation, and release. D(2)O alone enhanced platelet aggregation by 59+/-14% (SEM) and shortened the lag period by 43+/-10%. We conclude that functioning microtubules are required for platelet aggregation and release, and that microtubules of platelet preparations are functioning submaximally.     

A synthetic peptide derived from the sequence of a type I collagen receptor inhibits type I collagen-mediated platelet aggregation.

A synthetic peptide-1, an 18 amino acid residue peptide derived from a hydrophilic domain of a cloned platelet type I collagen receptor, was used to study the role of the receptor on types I and III collagen-induced platelet aggregation and the release of ATP. The peptide inhibits the type I, but not the type III, collagen-induced platelet aggregation and the release of ATP in a dose-dependent manner. The [125I]peptide-1 specifically binds to type I collagen-coated microtiter wells in a dose-dependent manner (with Kd = 10 nM). The binding of [125I]peptide-1 can be inhibited by an excess of unlabeled peptide-1 suggesting that the binding is specific. The labeled peptide-1 does not bind to type III collagen-coated microtiter wells. Results from an enzyme-linked immunosorbent assay show that the peptide reacts with the poly- and monoclonal antibodies raised against the purified platelet type I collagen receptor (Mr 65 kD). The peptide also inhibits the adhesion of platelets on type I collagen matrix and rabbit aortic segments in a dose-dependent manner. These results suggest that the reactive site of the platelet receptor for type I collagen resides in this portion of the molecule.     

Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction.     

Human platelet activation by C3a and C3a des-arg

C3a liberated from C3 by treatment with C3 convertase (or by trypsin) induced aggregation of gel-filtered human platelets and stimulated serotonin release. At concentrations of 10(-10) M to 8 X 10(-12) M, C3a induced aggregation when added alone to platelets. However, at lower concentrations (2 X 10(-12) M) C3a did not aggregate platelets directly but exhibited highly significant synergism (two-way analysis of variance P less than 0.0001) with ADP in mediating platelet aggregation and release of serotonin. Removal of the C-terminus arginine from C3a abolished anaphylotoxin activity but did not affect the platelet- stimulating activity of the peptide. C3a and C3a des-arg were equally reactive in mediating platelet aggregation and release of serotonin. Further C3a and C3a des-arg exhibited synergism with ADP of equal significance in both aggregation and the release reaction. The concentrations of C3a required for the platelet-stimulating activity involve relatively small number of molecules per platelet (4,000-10,000 for the synergistic reaction with ADP). These data suggest the possibility of a C3a (C3a des-arg) receptor on human platelets. This premise is strengthened by the demonstration ultrastructurally of C3a on the platelet membrane subsequent to C3a stimulation.     

Effect of insulin treatment in streptozocin-induced diabetic rats on in vitro platelet function and plasma von Willebrand factor activity and factor VIII-related antigen

Diabetes mellitus is associated with altered platelet function and endothelial damage, but their relationship remains unclear. We examined the effect of short-term metabolic control with insulin in 14- and 28-day streptozocin-induced diabetic rats on alterations in in vitro platelet aggregation and serotonin release. Endothelial damage was assessed by plasma concentrations of von Willebrand factor activity (VIIIR:WF) and factor VIII-related antigen (VIIIR:Ag). Insulin was administered for 5 or 7 days at 9 or 21 days, respectively, after streptozocin. Enhanced platelet aggregation responses to adenosine diphosphate (ADP) and thrombin occurred after both durations of diabetes. Insulin therapy returned ADP-induced, but not thrombin-induced, responses to normal. Enhanced thrombin-induced platelet release of serotonin occurred at both times. Collagen-induced platelet release was enhanced in 28-day diabetic rats. Insulin therapy returned these responses to normal. Plasma concentrations of VIIIR:WF and VIIIR:Ag were elevated in 28-day, but only VIIIR:WF was elevated in 14-day diabetic rats. Insulin therapy reduced the elevated levels of VIIIR:Ag in 28-day diabetic rats, but had little effect on either parameter after the shorter duration of diabetes. In summary, Enhanced platelet aggregation and increased release of serotonin occur shortly after the induction of diabetes by streptozocin in adult rats. These platelet changes precede alterations of endothelial function, as determined by plasma VIIIR:WF and VIIIR:Ag levels. Platelet changes respond more rapidly to insulin therapy than do endothelial changes in diabetic rats. The duration of diabetes before insulin therapy does not affect these relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased platelet serotonin transporter sites and increased platelet inositol triphosphate levels in patients with unipolar depression: effects of clomipramine and fluoxetine

BACKGROUND: The central serotonergic system has been implicated in the pathophysiology of depression and in the mechanism of the action of antidepressant drugs. The human platelet has been proposed as a peripheral model of central serotonergic neurons. METHODS: Six peripheral serotonergic parameters were determined simultaneously in 27 patients with unipolar depression before and after 2, 4, and 12 weeks of clomipramine or fluoxetine treatment according to the psychiatrist. RESULTS: In patients with depression versus matched control subjects, platelet [3H]paroxetine binding sites were found to be significantly decreased (2.10 +/- 0.70 versus 3.88 +/- 0.77 fmol/10(9) platelets; P = .0001), platelet serotonin (5-HT) content was found to be significantly decreased (1.90 +/- 1.52 versus 2.74 +/- 1.12 nmol/10(9) platelets; P = .001), and platelet inositol triphosphate levels were found to be significantly increased (2.85 +/- 0.70 versus 1.85 +/- 0.77 fmol/10(9) platelets; P = .0001). No significant difference between patients and control subjects was found for platelet [3H]-lysergic acid diethylamide ([3H]LSD) binding sites, aggregation tests with 5-HT or adenosine diphosphate and plasma 5-HT levels. Treatment with both clomipramine and fluoxetine gradually further reduced the density of platelet [3H]paroxetine binding sites and induced a dramatic decrease in platelet and plasma 5-HT levels. With clomipramine, the decreased blood 5-HT levels are associated with increased platelet [3H]LSD binding sites and aggregation responses. After 12 weeks, nonresponders to both treatments had platelet inositol triphosphate levels that were still increased (2.81 +/- 0.75 fmol/10(9) platelets) when responders levels were not different from those of control subjects (1.41 +/- 0.45 versus 1.70 +/- 0.25 fmol/10(9) platelets). CONCLUSIONS: Drug-free patients with depression had simultaneously decreased 5-HT transporter (5-HTT) sites and overstimulated phosphoinositide signaling systems. Clomipramine and fluoxetine treatments, which further decreased the density of 5-HTT sites, allowed platelet inositol triphosphate levels to return to normal values only in responders.     

Nitric oxide released from activated platelets inhibits platelet recruitment.

Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.     

Platelet Antibodies in Patients With Migraine

The reported decrease of platelet serotonin receptors in patients with migraine could be due to an autoimmune reaction. We, therefore, examined sera from 42 migraineurs without aura, 26 migraineurs with aura, and 107 headache-free blood donors for platelet-reactive antibodies using the platelet adhesion immunofluorescence test, the NIH-lymphocytotoxicity test, and the monoclonal antibody-specific immobilization of platelet antigens test. IgG antibodies against non-HLA class I platelet antigens were found in 9.5% of patients with migraine without aura, 7.6% of patients with migraine with aura, and in 7.5% of controls; IgM antibodies were found in 11.9% of patients with migraine without aura, in 30.8% of patients with migraine with aura, and in 13.1% of controls. Most antibodies ware directed against glycoprotein complexes IIb-IIIa (fibrinogen receptor) or IB-IX (thrombin receptor). Two patients with migraine without aura but no patient with migraine with aura nor any control subject had IgG antibodies of unknown specificity. One patient (2.4%) with migraine without aura and two patients (7.7%) with migraine with aura, as well as 2 controls (1.9%) had IgM antibodies not further specified. The differences in frequency of platelet antibodies of known or unknown specificity in patients with migraine without aura and migraine with aura and controls were not statistically significant. Therefore, our data do not support the hypothesis of a pathophysiologically relevant autoimmune reaction against platelet serotonin receptors in the majority of patients with migraine. We can not exclude the occurrence of antibodies against neuron-specific serotonin receptors.     

Platelet collection using the IBM 2997 cell separator

Platelets were collected using the dual-channel module on the IBM 2997 Blood Fraction Separator. We carried out 320 procedures to harvest platelets for therapeutic purposes and yielded 5.1 +/- 1.5 X 10(11) platelets (mean +/- SD). Infusion into previously unsensitized recipients with hypomegakaryocytic thrombocytopenia achieved increments at 1 hr of 19 +/- 7.3 X 10(9)/liter/m2 (mean +/- SD) and at 24 hr of 15 +/- 6.3 X 10(9)/liter/m2. The only consistent donor reaction was mild hypocalcaemia, easily corrected by calcium gluconate infusion. Changes in donor packed-cell volume and white cell count were not statistically altered (p greater than 0.05) but donor platelet counts fell from 216 +/- 43.1 X 10(9)/liter to 162.5 +2- 41.7 X 10(9)/liter (mean +/- SD) (p less than 0.01). Additional plateletphereses were carried out in seven normal volunteers, using the same technique, in order that the function of the harvested platelets could be studied. Following radiochromium labelling and reinfusion into the same donors, normal in vivo recoveries were obtained at 10 min (59.4 +/- 3.4%; mean +/- SD) and platelet mean life span was also normal (218 +/- 12 hr; mean +/- SD). Furthermore, in vitro platelet factor III availability and aggregation patterns of the harvested platelets did not differ from control values and their ultrastructural appearance was normal.     

Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.4, and 1.0 mg/kg of purified AP-2 IgG, and three animals were given 1.0 mg/kg of the F(ab)2 fragment of AP-2. Five additional animals were given 10 mg/kg LJ-CP8 IgG. At the highest dose, radiolabeled AP-2 IgG bound to an average of 33,000 sites on the circulating platelets. Serial measurements included platelet count, bleeding time, platelet aggregation (induced by ADP, collagen, and gamma-thrombin), and 111In-platelet deposition onto Dacron vascular grafts. Bleeding times were markedly prolonged after injection of 1.0 mg/kg AP-2 IgG (19.2 +/- 3.4 min), 1.0 mg/kg AP-2 F(ab)2 (16.5 +/- 1.8 min), and 10 mg/kg LJ-CP8 (greater than 30 min) vs. control studies (4.6 +/- 0.2 min), and remained prolonged for 48 h. With each antibody platelet aggregation was initially reduced or absent, with partial recovery over 48 h in a manner that was inversely related to dose. AP-2, both whole IgG and F(ab)2 fragment, but not LJ-CP8, caused a dose-dependent reduction (20-46%) in the circulating platelet count over 24 h. Neither AP-2 nor LJ-CP8 caused a reduction in intraplatelet platelet factor 4, beta-thromboglobulin, or [14C]serotonin. Graft-associated platelet thrombus formation was reduced by 73% (1.0 mg/kg AP-2 IgG and 10 mg/kg LJ-CP8) and 53% (1.0 mg/kg AP-2 F(ab)2) relative to control values. In contrast, neither heparin (100 U/kg) nor aspirin (32.5 mg/kg twice a day) showed antithrombotic efficacy in this model. Thus, antibodies that functionally alter the platelet GP IIb/IIIa complex may produce immediate, potent, and transient, antihemostatic, and antithrombotic effects.     

Two novel monoclonal antibodies to VWFA3 inhibit VWF-collagen and VWF-platelet interactions

BACKGROUND: The interaction of collagen-von Willebrand factor (VWF)-GPIb is essential for platelet adhesion, especially under high shear conditions. VWF, which acts as a bridge between platelets and exposed subendothelium, interacts with collagen through its A3 domain, which is a new target for the antithrombotic agent. OBJECTIVE: To develop functional blockers that specifically inhibit VWF-dependent adhesion of platelets to collagen under high shear stress. METHODS: To develop murine antihuman VWF A3 monoclonal antibodies (mAbs) by standard hybridoma technology, and characterize their abilities to block interactions between VWF A3 and collagen as well as platelet function. RESULTS: Thirty anti-VWF-A3 mAbs were obtained. Among them, two mAbs, designated as SZ-123 and SZ-125, were found to inhibit VWF-collagen type III interaction. SZ-123 and SZ-125 inhibited the binding of purified human VWF (1.5 or 3 mug mL(-1)) to human placenta collagen type III (IC(50) = 0.07 +/- 0.02 and 0.15 +/- 0.03 mug mL(-1), respectively) or to calf skin collagen type III (IC(50) = 0.48 +/- 0.06 and 0.51 +/- 0.07 mug mL(-1), respectively) coated on plates. Under flow shear condition (1000 s(-1)), SZ-123 and SZ-125 inhibited platelet adhesion on human placenta collagen- or calf skin collagen-coated surfaces. Both mAbs also inhibited platelet aggregation induced by ristocetin, botrocetin or bovine plasma. CONCLUSIONS: SZ-123 and SZ-125 inhibited VWF-collagen and VWF-platelet interactions.     

Quinine- and Quinidine-dependent Antiplatelet Antibodies: REQUIREMENT OF FACTOR VIII-RELATED ANTIGEN FOR PLATELET DAMAGE AND FOR IN VITRO TRANSFORMATION OF LYMPHOCYTES FROM PATIENTS WITH DRUG-INDUCED THROMBOCYTOPENIA

The requirement of Factor VIII-related antigen (VIIIR:Ag) for platelet damage by quinine-and quinidine-dependent antibodies was studied in platelet-rich plasma (PRP) of four patients with severe von Willebrand's disease (vWd) (Factor VIII deficiency). Platelet factor 3 availability, platelet aggregation, and release of [¹⁴C]serotonin from labeled vWd-PRP by drug-dependent antibodies were significantly reduced in comparison with PRP from normal controls. Addition of purified VIIIR:Ag restored levels of platelet damage to that of normal PRP. In vWd-PRP, platelet damage by two human antiplatelet sera, not dependent on drugs, and by a rabbit antiplatelet serum did not differ from that in normal PRP. PRP from patients deficient in Factor VIII coagulant activity, Factor IX, or Factors II, VII, IX, and X behaved like normal PRP.     

Mechanisms of platelet-neutrophil interactions and effects on cell filtration in septic shock

ABSTRACT-We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock. Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers. Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock. Cell adhesion was assessed by filtration through 5-microm pore filters and by flow cytometry. Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIla, P-selectin, ICAM-2, CD11a, CD11b, and CD18. The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 +/- 1.0 mmHg vs. 14 +/- 1.0 mmHg; P< 0.05). The difference between the Pi of cells suspended in SPPP or NPPP (deltaPi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the APi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01). The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies. The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 +/- 130 Ifu vs. 295 +/- 35 Ifu; P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil. We conclude that platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.     

Castration reduces platelet thromboxane A2 receptor density and aggregability

BACKGROUND: Exogenously administered testosterone upregulates platelet thromboxane A2 (TXA2) receptors and increases aggregation response to thromboxane mimetics in healthy male volunteers. However, the biological impact of endogenous testosterone on platelet TXA2 receptor expression, especially in older men at risk of coronary artery disease, is unclear. AIM: To investigate the impact of reduction in circulating testosterone on platelet TXA2 receptor expression in older men. DESIGN: Cross-sectional case-control study. METHODS: We studied surgically and/or medically castrated men with prostate cancer (group A, n = 8, aged 71 +/- 8 years) and age-matched, uncastrated urology patients (group B, n = 7, aged 67 +/- 9 years). Plasma testosterone was measured by radioimmunoassay. Platelet TXA2 receptor expression was assessed by radioligand binding studies using radioactive 125I-BOP. Platelet aggregation responses to TXA2-mimetic I-BOP, and to thrombin, were also studied. RESULTS: Group A had significantly lower plasma testosterone than group B (16 +/- 5 ng/dl vs. 308 +/- 47 ng/dl, p<0.001). Platelet TXA2 receptor density (B(max)) but not affinity (K(d)) was lower in group A (0.50 +/- 0.12 vs. 1.01 +/- 0.17 pmol/mg protein, p = 0.03). Maximum platelet aggregation response to I-BOP (E(max)), but not sensitivity (EC50) was lower in group A (53 +/- 2% vs. 63 +/- 2%, p = 0.003 ANOVA). In vitro, high concentrations of hydroxyflutamide (100 microM) competitively inhibited U46619-induced platelet aggregation in washed platelets, without affecting the binding of 125I-BOP to platelet TXA2 receptors. DISCUSSION: Endogenous testosterone regulates platelet TXA2 receptor B(max) and the E(max) aggregation response to thromboxane mimetic I-BOP. Blockade of androgen receptors or inhibition of testosterone production may reduce platelet aggregation responses. Preliminary evidence suggests the presence of functional androgen receptors on human platelets, which may regulate TXA2 receptor expression.     

Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.     

Inhibition of human and rabbit platelet activation by Ketotifen

The effects of Ketotifen and disodium cromoglycate were investigated on human and rabbit platelet activation. Ketotifen inhibited dose-dependently human and rabbit platelet aggregation. The paf-acether pathway was the most markedly influenced by Ketotifen in human and rabbit platelets (IC50 = 38.8 +/- 7.7 microM and 7.2 +/- 4.5 microM respectively) as compared to adenosine diphosphate (IC50 greater than 100 microM and 79 +/- 19 microM) and to arachidonic acid (IC50 greater than 100 microM and 98 +/- 28 microM). Similar concentrations of Ketotifen inhibited the ATP release from human platelets induced by paf-acether. Disodium cromoglycate up to 5 x 10(-4) M did not inhibit platelet aggregation induced by paf-acether, adenosine diphosphate and arachidonic acid.     

Platelets,Endothelium-Dependent Responses and Atherosclerosis

Basal release of endothelium-derived relaxing factor (EDRF) and prostacyclin from intact vascular endothelium may inhibit continuously platelet aggregation. If local platelet aggregation occurs, platelet-derived adenine nucleotides stimulate the release of EDRF. Stimulated EDRF release may override the direct vasoconstrictor effects of other platelet products such as thromboxane and serotonin resulting in local vasodilatation. In addition, stimulation of EDRF release by adenine nucleotides may inhibit further platelet adhesion and aggregation by a feedback mechanism. Thus, intact vascular endothelium may play an important role in the defense against platelet deposition and vasospasm. In atherosclerosis, basal and stimulated release of EDRF is markedly reduced. Endothelial dysfunction will impair this protective mechanism and will favour vasoconstriction and further platelet disposition. Occurrence of occlusive thrombus formation in patients with coronary artery disease may be pathophysiologically related to this impairment of endothelial defense.     

Platelets, endothelium-dependent responses and atherosclerosis.

Basal release of endothelium-derived relaxing factor (EDRF) and prostacyclin from intact vascular endothelium may inhibit continuously platelet aggregation. If local platelet aggregation occurs, platelet-derived adenine nucleotides stimulate the release of EDRF. Stimulated EDRF release may override the direct vasoconstrictor effects of other platelet products such as thromboxane and serotonin resulting in local vasodilatation. In addition, stimulation of EDRF release by adenine nucleotides may inhibit further platelet adhesion and aggregation by a feedback mechanism. Thus, intact vascular endothelium may play an important role in the defense against platelet deposition and vasospasm. In atherosclerosis, basal and stimulated release of EDRF is markedly reduced. Endothelial dysfunction will impair this protective mechanism and will favour vasoconstriction and further platelet disposition. Occurrence of occlusive thrombus formation in patients with coronary artery disease may be pathophysiologically related to this impairment of endothelial defense.     

Assessment of functional group B streptococcal antibodies in intravenous human immunoglobulin preparations

Five different intravenous human immunoglobulin preparations were assessed for their opsonic activity against types 1a, 1b, II, III group B streptococci (GBS) by a chemiluminescence test. Opsonic antibodies against the four GBS types were present, but there was no significant difference between the preparations. The presence of complement increased significantly the opsonic activity for types BII and BIII. Moreover, antibodies against the four GBS types were detected by ELISA with the whole bacteria as antigen. For each type of specific antibody, a close correlation was found between results obtained by ELISA and chemiluminescence in the absence of complement r=0·81, and in the presence of complement r=0·67. These data support the presence of protective antibodies GBS in intravenous immunoglobulins and the agreement of modified ELISA to their investigation.     

Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase.

Human platelets that were preincubated with porcine elastase aggregated spontaneously upon the addition of fibrinogen. Maximal aggregation to fibrinogen was observed with platelets pretreated with an elastase concentration of 111 micrograms/ml, and half-maximal aggregation occurred after treatment with 11 micrograms/ml elastase. Binding of radiolabeled fibrinogen to elastase-treated platelets was specific, saturable, and showed a single class of 48,400 +/- 9,697 fibrinogen-binding sites per platelet with a dissociation constant of 6.30 +/- 1.48 X 10(-7) M. ATP, apyrase, and the stimulators of platelet adenylate cyclase forskolin, prostaglandin E1, prostacyclin, and N6, 2''-O-dibutyryl cyclic AMP did not inhibit the fibrinogen-induced aggregation of elastase-treated platelets. EDTA completely blocked the initiation of aggregation and reversed the fibrinogen-induced aggregation of elastase-treated platelets. Monoclonal and polyclonal antibodies directed against glycoproteins (GP) IIb and IIIa completely blocked the fibrinogen-induced aggregation of elastase-treated platelets. Immunoprecipitates with these antibodies obtained from detergent extracts of surface-radiolabeled, intact, and elastase-treated platelets contained the glycoproteins IIb and IIIa. We conclude that surface proteolysis by low concentrations of elastase can expose fibrinogen-binding sites associated with GPIIb and GPIIIa on the platelet surface, resulting in spontaneous aggregation upon the addition of fibrinogen. These findings may be relevant to hemostatic changes observed in patients with increased levels of circulating elastase.