Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

Mechanism and characteristics of platelet activation by haematin

The mechanism as well as some characteristics of haematin-induced human platelet aggregation were investigated. Haematin-induced platelet aggregation required the presence of devalent cations; Mg2+, and to a lesser extent, Co2+, were just as effective as Ca2+ in supporting the aggregation. Mono- and trivalent cations were ineffective. Verapamil inhibited the aggregation. The aggregation was accompanied by thromboxane formation which could be abolished by aspirin. The release of adenine nucleotides was only slightly inhibited by aspirin. The rate of aggregation and the ultrastructure of the aggregated platelets were comparable between control and aspirin-treated samples. It is concluded therefore that haematin-induced aggregation is not dependent on platelet prosta-glandin synthesis. Haematin induced binding of fibrinogen to platelets, and failed to aggregate thrombasthenic platelets. These findings indicate that haematin may induce platelet aggregation by promoting influx of divalent cations in association with increased fibrinogen binding and release of adenine nucleotides.     

Activation of protein kinase C in platelets by epinephrine and A23187: correlation with fibrinogen binding

Activation of protein kinase C (PKC), as revealed by phosphorylation of a 47 kd protein (p47), occurs in platelets stimulated by some agonists (eg, thrombin or phorbol esters). It is not known if activation of PKC occurs with pairs of agonists, such as epinephrine and A23187, that do not individually phosphorylate p47, nor is it known what role the concentration of cytoplasmic Ca++ ([Ca++]i) plays in these events. We stimulated aequorin-loaded platelets with subaggregating concentrations of epinephrine and A23187, neither of which by itself phosphorylated p47. The combination of agonists resulted in p47 phosphorylation, an increase in platelet-bound fibrinogen, and aggregation, but only if the concentration of each agonist was sufficient to increase [Ca++]i if it was added separately. Subaggregating concentrations of A23187 alone released platelet fibrinogen and increased platelet membrane binding of [3H]-phorbol dibutyrate, but these were not enhanced by epinephrine. Epinephrine and A23187 did not increase production of diacylglycerol. Thus, epinephrine and A23187 together activate PKC by a mechanism that does not require phospholipase C or enhanced binding of PKC to the plasma membrane; PKC activation in turn is correlated with enhanced platelet fibrinogen binding and aggregation. These events require an initial elevation of [Ca++]i above a threshold.     

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

Summary . A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of β-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B₂ is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A₂ aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI₂ inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and β-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Caz+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Immunocytochemical localization of fibrinogen during thrombin-induced aggregation of washed human platelets

Because thrombin aggregates afibrinogenemic platelets and platelets from patients with the gray platelet syndrome and because antibodies to fibrinogen inhibit thrombin-induced aggregation only at low concentrations of thrombin, the role of fibrinogen in the formation of thrombin-induced aggregates was investigated further with human platelets washed and resuspended in Tyrode-albumin solution containing apyrase, either with or without added Ca2+ (2 mmol/L). Samples for immunocytochemical assessment of fibrinogen distribution were taken at several times (up to five minutes) after aggregation induced by 0.5 U/mL of thrombin. Glutaraldehyde-fixed samples were embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. By 10 seconds, small aggregates formed, and granules were centralized; alpha granules were heavily labeled with immunogold, but the platelet surface was not. As large aggregates formed, granule swelling or fusion occurred, and in some areas granule material seemed to be in contact with the exterior. In these experiments with no added fibrinogen, there were some clusters of gold particles on the platelet surfaces remote from sites of granule discharge, but there were large areas where platelets were in close contact with little or no fibrinogen detectable between them. No fibrin was visible up to five minutes after the addition of thrombin, which indicated that fibrinogen from the granules does not readily become available for fibrin formation in the ambient fluid. Similar results were obtained in media with and without added Ca2+. Thus at least some aggregation in response to thrombin can occur without the participation of released fibrinogen, and much of the granule fibrinogen appears to remain localized at sites where granules fuse with the plasma membrane or the open canalicular system. Incubation of unstirred samples with thrombin for ten minutes resulted in the formation of small aggregates, extensive gold label in regions connected to the exterior of the platelets, but very little gold labeling of the platelet membrane and no visible fibrin formation. When the platelets were aggregated in the presence of external fibrinogen, the morphological changes within the platelets were the same, but fibrinogen rapidly became associated with the entire platelet surface, and visible fibrin formed within 30 seconds in the medium containing 2 mmol/L Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.     

Mechanism of action of adenosine on intracellular calcium and platelet aggregation]

Adenosine inhibits platelet aggregation and elevates the levels of cytoplasmic Ca2+ induced by thrombin, 0.3 U/ml). When given at the maximal (100 microM) concentration, adenosine completely inhibits the aggregation, but only partially (by 55%) suppresses the growth of Ca2+, blocking both its entry and intracellular depot mobilization. Adenosine is likely to affect intracellular Ca2+, by activating adenylate cyclase, since 2',5'-didesoxyadenosine (1 mM) prevents the effect of adenosine, by inhibiting the enzyme, whereas the phosphodiesterase inhibitor papaverine (1 microM) potentiates its effect. When stimulated with adrenaline, 1 microM, adenosine and dibutyryl-cAMP are also able to inhibit platelet aggregation in the absence of cytoplasmic Ca2+ growth.     

Platelet aggregation induced by type IIb platelet von Willebrand factor

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.     

The Morphology of Thrombin-activated Platelets with Reference to Different Fibrinogen Concentrations as Revealed by Computer-aided Three-dimensional Reconstruction

Washed human platelets incubated in different concentrations of fibrinogen were activated by thrombin and aggregated in the presence of Ca(2+) or did not aggregate when EDTA was present. They were analyzed by transmission electron microscopy and computer-aided three-dimensional reconstruction. The volumes and surface areas of the reconstructed models were calculated. The quotients of the values calculated for the whole platelet and the surface-connected canalicular system were taken as measures of the degree of surface invagination. Increasing the concentrations of fibrinogen reduced the values of the quotients indicating enhanced internalization of surface membranes, and tended to smoothen the outer surfaces to obtain spherical shapes. The invaginations are much more pronounced in platelets that did not aggregate in the presence of EDTA suggesting that aggregation fixes some membrane areas that otherwise would be redistributed to the platelet's inner compartments.     

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.     

Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.     

Platelet activation by sustained exposure to low-dose plasmin.

Plasmin has been reported to activate and inhibit platelet function depending on dose and exposure temperature. The present study examines the induction of fibrinogen-dependent platelet aggregation following prolonged (60 min) platelet exposure to very low doses of plasmin (0.05 CU/ml) at either 22 or 37 degrees C. Maximum aggregation [mean +/- SD, 60 +/- 19 light transmission units (LTU); n = 43] occurred following platelet exposure to plasmin at 22 degrees C, but significant platelet aggregation (28 +/- 4 LTU, n = 3) also occurred following plasmin treatment at 37 degrees C. Plasmin-induced platelet aggregates appeared microscopically larger than aggregates of adenosine diphosphate (ADP)-activated platelets, and were less reversible. Aggregated plasmin-treated platelets also expressed more procoagulant activity than platelets aggregated with ADP, as reflected by shortening of the plasma kaolin recalcification time. Aggregation of platelets exposed to very low doses of plasmin was not accompanied by dense or alpha-granule secretion, and was unaffected by ADP antagonists or aspirin. Partial inhibition of platelet aggregation, however, was achieved with metabolic inhibitors, PGE1, and inhibitors of phosphoinositide 3-kinase or protein kinase C. Although fibrinogen was required for plasmin-treated platelet aggregation, [125I]-fibrinogen binding comprised only 58 +/- 3% (n = 3) of fibrinogen binding associated with ADP aggregated platelets. This was consistent with observed decreases in reptilase-induced fibrin clot retraction. Taken together, these data suggest that sustained exposure of platelets to very low plasmin doses leads to platelet activation and thus may contribute to thrombotic complications in vivo.     

Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases

Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN- platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.     

Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood.

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.     

Adhesion of platelets to surface-bound fibrinogen under flow

After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.     

Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

The effects of activation of plasminogen by streptokinase and tissue-type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation-specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.