Platelet von Willebrand's antigen II: active release by aggregating agents and a marker of platelet release reaction in vivo

von Willebrand's antigen II (vW AgII), a plasma and platelet antigen immunologically and biochemically distinct from factor-VIII-related antigen (VIIIR:Ag), is decreased in von Willebrand's disease. vW AgII is released from platelets during aggregation and clotting. The release of platelet vW AgII was studied in washed platelets following aggregation by thrombin, collagen, and adenosine diphosphate (ADP). Total platelet vW AgII was 3.39 U/10(9) platelets. Thrombin and collagen yielded release of 85% and 86% of platelet vW AgII, respectively, at the highest concentrations. Release of platelet vW AgII was correlated with the release of 5-hydroxytryptamine (5HT). Release of vW AgII by collagen and thrombin was inhibited by metabolic inhibitors. In addition, collagen release of vW AgII was inhibited by aspirin. In clinical syndromes associated with intravascular platelet destruction, marked elevations of plasma vW AgII were noted. Thus, vW AgII is released by a metabolically active process from platelets during aggregation. In addition, vW AgII appears to be a marker of intravascular platelet destruction.     

Potassium uptake and release by human blood platelets

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01-0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.     

Histidine-rich glycoprotein is present in human platelets and is released following thrombin stimulation

Histidine-rich glycoprotein, and alpha 2-glycoprotein in human plasma, has been shown to interact with heparin, with the high-affinity lysine- binding site of plasminogen, with divalent cations, and is associated with the rosette formation between erythrocytes and lymphocytes. A specific enzyme-linked immunosorbent assay for histidine-rich glycoprotein has been developed and used to demonstrate that histidine- rich glycoprotein is present in human platelets. Histidine-rich glycoprotein was detected and quantified in detergent extracts of washed human platelets, with a mean level of 371 ng/10(9) platelets. Plasma histidine-rich glycoprotein, either in the platelet suspending medium or on the surface of the platelets, accounted for less than 3.4% of the detectable platelet histidine-rich glycoprotein. Histidine-rich glycoprotein was also demonstrated in human bone marrow megakaryocytes by immunofluorescence. The extent of histidine-rich glycoprotein release from platelets was dependent on the thrombin dose and correlated directly with the extent of serotonin release. The platelet and plasma histidine-rich glycoprotein were similar by immunochemical analysis. Anti-histidine-rich glycoprotein IgG did not inhibit platelet aggregation. Histidine-rich glycoprotein released by platelets following thrombin stimulation may play a significant role in modulating inflammatory events in the microenvironment of the platelet plug.     

Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.     

Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles

Activation of human platelets considerably enhanced their ability to accelerate factor Va inactivation by activated protein C (APC). The anticoagulant activity of platelet suspensions was markedly dependent on the kind of agonist used to activate platelets. APC-catalyzed factor Va inactivation in free solution was characterized by an apparent second-order rate constant of 2 x 10(5) (mol/L)-1 (seconds)-1. Nonstimulated platelets (2.4 x 10(8)/mL) and platelets stimulated with adenosine diphosphate or adrenalin accelerated factor Va inactivation fourfold. Rates of factor Va inactivation were increased 11-fold by thrombin-stimulated platelets, 29-fold after platelet stimulation with the Ca(2+)-ionophore A23187. At low platelet concentrations (3 x 10(7)/mL) only background levels of anticoagulant activity were observed in platelet suspensions that were nonstimulated or stimulated with thrombin or collagen. However, when such reaction mixtures were stirred during the activation procedure, platelet anticoagulant activity was increased more than 10-fold. Independent of platelet stimulation and stirring conditions, exogenously added purified plasma protein S increased platelet-dependent factor Va inactivation approximately twofold. Addition of a neutralizing antiprotein S antibody had little effect on the anticoagulant activity of platelets. This indicates that, under the reaction conditions tested, platelet-released protein S did not contribute to factor Va inactivation. Approximately 25% of the anticoagulant activity of stimulated platelet suspensions appeared to be associated with microparticles that were released on platelet activation. Such microparticles may provide an important source of anticoagulant activity. A similar distribution of procoagulant, ie, prothrombinase, activity between platelets and microparticles was observed for the same platelet suspensions. Because platelet stimulation and stirring also had the same overall effects on the ability of platelets and platelet microparticles to promote prothrombin activation and factor Va inactivation, it appears likely that the generation of potential platelet anticoagulant and procoagulant activities is coupled to the same platelet stimulation reactions.     

Platelet sialic acid and platelet survival after aggregation by ADP

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.     

Effect of heterologous antibody on human platelets

The effect of heterologous anti-human platelet antibody on human platelet function was examined in the presence and absence of whole plasma as an in vitro model for antibody-induced immune damage to cells. Heterologous IgG anti-human platelet antibody mediated platelet aggregation and released serotonin from both platelets in plasma and from platelets isolated by gel filtration and increased the availability of platelet acid phosphatase in a dose-response fashion. Anti-platelet antibody failed to release beta-glucuronidase (lysosomal enzyme marker) or cause lactic dehydrogenase loss (cytolysis). The effect of the antiplatelet antibody on platelets proceeded in the absence of complement. The active molecule in the anti-platelet antiserum was isolated in the IgG fraction and all three indicators of platelet injury were mediated by purified monomeric IgG. Thrombin was not required for the antibody-mediated effects, as three thrombin inhibitors failed to block the reaction. EDTA was an effective inhibitor, suggesting a cation requirement; however, as little as 38 muM calcium was sufficient for effective platelet aggregation and release. The inability of acetylsalicylic acid to inhibit the effect of the antiplatelet antibody suggests that heterologous antibody (IgG) induced platelet alteration proceeds by a different mechanism than that mediated by ADP and epinephrine and does not involve endogenous platelet prostaglandin synthesis.     

Human platelets contain and release TWEAK

The multifunctional cytokine, TWEAK (TNF-like weak inducer of apoptosis), is a member of the TNFα superfamily. TWEAK is found in a broad range of cell types and has been linked to cell growth and survival, angiogenesis and other inflammatory processes. These functions and their importance in inflammatory diseases have made TWEAK an attractive pharmaceutical target, particularly for immunotherapy with monoclonal antibodies (mAbs). Immunotherapy targeting another TNFα family member, CD154, was associated with thrombosis in clinical trials. Subsequent studies identified platelets, which contain CD154, as a possible contributing factor to thrombosis in these trials. Since clinical trials with anti-TWEAK mAbs have already begun, we considered it important to determine whether platelets contain TWEAK. Using a variety of immunologic methods we found that, upon activation, human platelets expose TWEAK antigen and release it in soluble form (sTWEAK). By flow cytometry we determined that human platelets activated by TRAP (Thrombin Receptor Agonist Peptide) and other agonists expose TWEAK antigen (22% median positivity) and release TWEAK positive microparticles. The presence of TWEAK on platelets was confirmed by confocal microscopy. By ELISA, we found that sTWEAK is released by activated platelets. Finally, western blot analysis revealed TWEAK protein (～34 kDa) in washed platelet lysates. The finding that human platelets contain TWEAK raises important questions about its possible functions in normal physiology, as well as in inflammatory diseases and their treatment.     

Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H.

The alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.     

Subcellular localization of thrombopoietin in human blood platelets and its release upon thrombin stimulation

Thrombopoietin (TPO) is a major regulator of platelet production. The concentration of circulating TPO seems to be determined by its binding and internalization by megakaryocytes and platelets. To elucidate the platelet compartments involved in TPO metabolism, we investigated intraplatelet TPO by post-embedding immunoelectron microscopy, incubated platelets with recombinant human (rh)TPO coupled to colloidal gold and visualized the TPO uptake using electron microscopy. TPO concentrations were measured in 12 platelet concentrates (PC) before and after stimulation with thrombin and after disruption of platelets by freezing-thawing. In resting platelets, immunogold labelling revealed a prevailing cytoplasmic localization of TPO antigen and minor labelling within the surface-connected canalicular system (SCCS); storage granules were devoid of labelling. In tracer experiments, TPO-gold was observed on the plasma and SCCS membranes and within the cytoplasm. Upon thrombin stimulation, endogenous TPO was still detected within the cytoplasm by immunolabelling, and tracer experiments revealed TPO-gold within the cytoplasm and on fibrin fibres. After thrombin stimulation of PC, the plasma TPO levels increased to an average of 535%, and after platelet lysis to an average of 1625% compared with plasma values in unstimulated PC. We conclude that platelets contain releasable immunoreactive TPO within the SCCS and within their cytoplasm, but not within granular compartments. Stored immunoreactive TPO is released upon thrombin stimulation, but only to a minor degree.     

Thrombin increases the adhesion of washed human platelets to collagen

Thrombin stimulates the adhesion of washed human platelets to fibrillar collagen. This phenomenon occurs also when platelets, before thrombin stimulation, are resuspended in the presence of prostaglandin E1 to minimize the release reaction. Enzymatic activity of thrombin is not necessary for the enhancement of platelet adhesiveness, since phenylmethylsulphonylfluoride inhibited thrombin is effective in this respect. Detachment of thrombin from thrombin treated platelets by the use of hirudin restores normal platelet adhesiveness to collagen.     

Human platelet 20S proteasome: inhibition of its chymotrypsin-like activity and identification of the proteasome activator PA28. A preliminary report

Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.5-8.0 and 5.0-5.5. These two activities were markedly inhibited by a 10 micromol/l concentration of two structurally unrelated proteasome inhibitors: lactacystin/beta-lactone or benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal, but not by ebelactone B, an inhibitor of lysosomal cathepsin A/deamidase. The chymotryptic-like activity of the 20S proteasome against succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was also significantly inhibited in platelets, after exposure of platelet-rich plasma to 10 micromol/l lactacystin and benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal for up to 60 min. This indicates that these inhibitors can enter platelets and selectively inhibit 20S proteasome activity. We also demonstrated for the first time by Western blot analysis that human platelets contain a proteasome activator, PA28, which is known to play a key role in antigen processing by significant stimulation of the proteasomal chymotryptic-like activity. Since the platelet 20S proteasome was also present in a latent form, this suggests that its activity may be regulated in vivo in human platelets. All these results can therefore be beneficial in future studies on the role of the 20S proteasome in platelet biology.     

Protease and cyclooxygenase inhibitors synergistically prevent activation of human platelets.

Thrombin induces platelet aggregation and formation of a fibrin clot in platelet-rich plasma; leupeptin, a protease inhibitor, partially inhibits platelet aggregation, but it does not inhibit fibrin clot formation. Indomethacin does not inhibit either thrombin-induced platelet aggregation or fibrin clot formation. However, when the two drugs are given together, a synergistic inhibition of thrombin-induced platelet aggregation occurs, while fibrin clot formation remains unaffected. Thrombin-induced stimulation of the release of serotonin in washed human platelets is also synergistically inhibited by the combined actions of leupeptin and indomethacin. Thrombin and collagen, added simultaneously, induce full platelet aggregation and release of serotonin. Neither leupeptin nor indomethacin inhibits platelet responses elicited by both agonists; however, when leupeptin and indomethacin are given together, a synergistic inhibition of thrombin- and collagen-induced response is observed. These findings might be relevant in prophylaxis and treatment of thromboembolic disease.     

The presence and release of alpha 2-antiplasmin from human platelets

An antigen immunochemically indistinguishable from plasma alpha 2- antiplasmin, the primary plasmin inhibitor, was detected in human platelets. By radioimmunoassay, 33-114 ng alpha 2-antiplasmin antigen was quantitated in the detergent-soluble extract of 10(9) washed human platelets from 10 normal donors with a mean level of 62 +/- 24 ng/10(9) platelets. Plasma alpha 2-antiplasmin, either in the platelet suspending medium or on the surface of the platelets, could account for less than 8% of the antigen present in the platelet extracts. When stimulated with thrombin, the platelets released alpha 2-antiplasmin antigen without cell lysis, and greater than 85% of the alpha 2- antiplasmin antigen was released at a high thrombin dose. At a lower dose of thrombin, alpha 2-antiplasmin and platelet factor 4 were partially released without concomitant secretion of serotonin. No alpha 2-antiplasmin antigen was detected in extracts or red blood cells, polymorphonuclear leukocytes, and adherent and nonadherent mononuclear cells. Thus, the platelet is the only peripheral blood cell containing significant amounts of alpha 2-antiplasmin.     

Platelet-derived collagenase inhibitor: characterization and subcellular localization.

Purified human platelets were found to contain a collagenase inhibitor that is immunologically, functionally, and chromatographically identical to that produced by human skin fibroblasts. None of the other formed elements of the blood (erythrocytes, granulocytes, mononuclear cells) possessed detectable quantities of this protein. Virtually all the collagenase inhibitor contained within platelets was released following platelet activation with thrombin. Similarly, platelet activation accompanying blood clotting also resulted in the release of this protein, the ratio of plasma to serum inhibitor levels being approximately equal to 0.5. When platelets were subjected to subcellular fractionation, essentially all of the platelet-associated collagenase inhibitor was found to be located in the alpha-granule. Studies with radiolabeled inhibitor failed to detect uptake of inhibitor by platelets. Furthermore, immunologically reactive protein of similar quantity to that found in platelets was identified in human megakaryocyte lysates. Thus, the data suggest that the collagenase inhibitor is endogenously produced and stored within platelet alpha-granules. The platelet-derived collagenase inhibitor was antigenically identical to the collagenase inhibitor from human skin fibroblasts in double immunodiffusion and, like its fibroblast counterpart, inhibited collagenase on a 1:1 stoichiometric basis. When subjected to several of the chromatographic procedures utilized to purify the fibroblast protein, the platelet inhibitor behaved in an indistinguishable manner. Platelet factor 4, previously reported to be a collagenase inhibitor, was found to be immunologically unrelated to the platelet-derived collagenase inhibitor. Furthermore, platelet factor 4 displayed no collagenase inhibitory activity. Although the function of platelet-derived collagenase inhibitor is unknown, such a protein released by activated platelets may serve to regulate collagen turnover during the early stages of the inflammatory process.     

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.     

Arachidonic acid metabolism by platelets of differing size

S ummary . The relationship between mean platelet volume (MPV) and platelet arachidonic acid metabolism was examined by studying the ability of human platelets of different size to incorporate and metabolize tritiated arachidonic acid ([³HIAA). Platelet phospholipids were labelled with [jH]AA and the platelets were then fractionated into size-dependent subpopulations by counterflow centrifugation. The incorporation of [jH]AA increased through the fractions proportional to the MPV. After thrombin stimulation the per cent of total jH-radioactivity released from the platelets decreased as the MPV increased. However, fractionation of the released %-radioactivity by HPLC (high performance liquid chromatography) demonstrated that MPV had no significant influence on the per cent of total platelet 3H-radioacti-vity released as cyclooxygenase products or as HETE (12-hydroxyeicosatetraenoic acid) but that the release of unmetabolized [3H]AA decreased as MPV increased. In separate experiments using unlabelled platelets the absolute release of thromboxane BL (TXB2) after collagen- and thrombin-induced aggregation was measured by radioimmunoassay and was found to increase in proportion to the MPV. These results demonstrate that the release of arachidonic acid metabolites is qualitatively similar in platelets of different size. However, the absolute ability of platelets to incorporate arachidonic acid, convert it to active metabolites and release them is proportional to their volume. The ability of platelets to release unmetabolized arachidonic acid varies inversely with their MPV.     

Diglyceride lipase: a pathway for arachidonate release from human platelets.

We provide evidence that the mechanism for arachidonate release from stimulated human platelets involves two enzymes: a phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) and a diglyceride lipase. After incubation of platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl-2-arachidonoyl diglyceride per 10(9) platelets, was isolated. Arachidonate was released from this substrate by the action of diglyceride lipase located in the particulate fraction of platelets. The enzyme has a pH optimum of 7.0, is stimulated by calcium ions and reduced glutathione, and liberates 31 nmol of fatty acid per min per mg of platelet particulate protein. The diglyceride lipase has sufficient activity to account for the 5-10 nmol of arachidonate released per 10(9) platelets upon thrombin stimulation. That only arachidonate is released upon thrombin stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 position. The lipase activity found in platelet membranes can also hydrolyze the 1-position fatty acid. Stearate is not released when intact platelets are stimulated with thrombin, and the fate of this fatty acid remains to be elucidated.     

Platelets release active matrix metalloproteinase-2 in vivo in humans at a site of vascular injury: lack of inhibition by aspirin

When stimulated in vitro, human platelets release matrix metalloproteinase-2 (MMP-2) that, in turn, potentiates platelet activation. The present study investigated if MMP-2 is released from activated platelets in vivo in humans and whether aspirin inhibits this release. MMP-2 levels were measured by zymography, immunoblotting, flow-cytometry and an activity assay system, in plasma prepared from blood emerging from a skin wound inflicted for the measurement of the bleeding time (shed blood) and simultaneously from venous blood in 27 healthy human volunteers. In a subgroup, the same measurements were carried out before and 1 h after aspirin intake. MMP-2 was significantly higher in shed blood than in venous blood and increased progressively, consistent with ongoing platelet activation. A significant correlation was evident between platelet number and MMP-2 in shed blood; platelet MMP-2 content in shed blood was lower than that of platelets in venous blood. The level of active MMP-2 released by activated platelets in vivo was within the range of concentrations that potentiate platelet activation. Aspirin did not reduce MMP-2 release in vivo. In conclusion, MMP-2 is released from platelets in vivo in humans at a localised site of vessel wall damage in amounts sufficient to potentiate platelet aggregation; aspirin does not reduce this release.     

Identification of and partial characterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1

Vitronectin (VN; = complement S-protein), a plasma glycoprotein that is also associated with extracellular sites, was identified in washed human platelets contaminated with less than 0.05% of plasma VN. A specific enzyme-linked immunosorbent assay (ELISA) for VN has been developed and was used to detect and to quantitate VN in detergent extracts of washed platelets with 8.1 +/- 4.6 micrograms/10(9) platelets (n = 10), representing about 0.8% of the plasma VN pool. Platelet and plasma VN were similar by immunochemical criteria using Western-blot analysis, although platelet VN was mainly found as partially proteolyzed polypeptide. Total release of platelet VN occurred at optimal doses of Ca-ionophore 23187 or thrombin, whereas no VN was released by platelet treatment with digitonin or Staphylococcus alpha-toxin. During stimulation of washed platelets with various concentrations of thrombin, the nearly concomitant release of VN and plasminogen activator inhibitor-1 (PAI-1) together with platelet factor 4 indicated the association of VN with inner-platelet storage granules. Furthermore, platelet VN and PAI-1 in Ca-ionophore releasates comigrated during ultracentrifugation in high mol wt fractions of sucrose density gradients, indicating a possible association of both components. Complex formation of platelet VN and PAI-1 was verified by a sensitive enzyme-linked immunosorbent assay (ELISA) and accounts at least in part for a high molecular form of platelet VN. The identification of platelet VN and its binding to platelet PAI-1 raises the possibility that VN, in contrast to other adhesive proteins, may participate in localized regulatory functions of blood coagulation and fibrinolysis in platelet-matrix interactions and the protection of the matrix against proteolysis.